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Animal Reproduction Science xxx (2009) xxx–xxx
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Time related changes in luteal prostaglandin synthesis and
steroidogenic capacity during pregnancy, normal and
antiprogestin induced luteolysis in the bitch
Mariusz Pawel Kowalewski a,∗,1, Hakki Bülent Beceriklisoy b,2, Selim Aslan b,
Ali Reha Agaoglu b, Bernd Hoffmann a
a
b
Clinic for Obstetrics, Gynecology und Andrology of Large- and Small Animals, Justus-Liebig-University Giessen, Germany
Clinic for Obstetrics and Gynecology, Faculty of Veterinary Medicine, University Ankara, Turkey
a r t i c l e
i n f o
Article history:
Received 24 August 2008
Received in revised form 10 December 2008
Accepted 11 December 2008
Available online xxx
Keywords:
Dog
Pregnancy
Corpus luteum
Prostaglandins
Antiprogestin
Steroidogenesis
a b s t r a c t
In nonpregnant and pregnant dogs the corpora lutea (CL) are the
only source of progesterone (P4) which shows an almost identical
secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained
that physiological luteal regression is devoid of a functional role of
the PGF2␣-system and seems to depend on the provision of StAR.
Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2␣, may be indicative for different
regulatory mechanisms. To assess this situation and by applying
semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant
and prepartal dogs and of mid-pregnant dogs treated with the
antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin
E2 synthase (PGES), prostaglandin F2␣ synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein
(StAR), 3␤-hydroxysteroid-dehydrogenase (3␤HSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were
determined by RIA. CL were collected via ovariohysterectomy from
pregnant bitches (n = 3–5) on days 8–12 (Group 1, pre-implantation
∗ Corresponding author at: Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß- und Kleintiere mit Tierärztlicher
Ambulanz, Justus-Liebig-Universität Giessen, Frankfurter Strasse 106, D-35392 Giessen, Germany. Tel.: +49 641 99 38704;
fax: +49 641 29328.
E-mail address: [email protected] (M.P. Kowalewski).
1
Present address: Texas Tech University Health Sciences Center, Dept. of Cell Biology and Biochemistry, 3601 4th Street,
Lubbock, TX 79430, United States.
2
Present address: Clinic for Obstetrics and Reproductive Diseases, Faculty of Veterinary Medicine, University of Adnan
Menderes, Aydin, Turkey.
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.12.011
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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period), days 18–25 (Group 2, post-implantation period), days
35–40 (Group 3, mid-gestation period) and during the prepartal
progesterone decline (Group 4). Additionally, CL were obtained from
groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h
after the second treatment with Aglepristone. Expression of Cox2
and PGES was highest during the pre-implantation period, that of
PGFS and FP during the post-implantation period. EP4 and EP2
revealed a constant expression pattern throughout pregnancy with
a prepartal upregulation of EP2. 3␤HSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it
was matched by the course of P4 concentrations. Expression of the
PR was higher during mid-gestation and prepartal luteolysis than
in the two preceding periods. After application of Aglepristone the
overall mRNA-expression resembled the situation during prepartal
luteolysis except for EP2, which remained unchanged.
These data suggest that – as in the nonpregnant bitch – also in
the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand
induction of luteolysis by the PR blocker Aglepristone points to a
role of luteal P4 as an autocrine factor in a positive loop feedback
system controlling the availability of P4, StAR and 3␤HSD.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
In pregnant and nonpregnant bitches the corpora lutea (CL) are the only sources of progesterone
(P4) (Concannon et al., 1989), with the course of P4-concentrations in peripheral blood being virtually identical shortly until parturition, when it declines around day 60 of pregnancy to basal levels
preceding the onset of fetal expulsions (Concannon et al., 1978). In nonpregnant dogs P4-levels continue to decrease gradually and reach anoestrus levels <1 ng/ml between days 60 and 90 (Feldman and
Nelson, 1987). During the ﬁrst 20–30 days after ovulation the newly formed CL are independent of
gonadotropic support (Concannon et al., 1987); thereafter in the second half of dioestrus or gestation,
removal of pituitary gland support results in luteolysis as LH and in particular prolactin have become
essential luteotropic factors (Concannon, 1980; Okkens et al., 1990). Availability of prolactin and LH
increases during this period, commencing with the decline of P4 (Gräf, 1978; Hoffmann and Schneider,
1993). Hence luteal regression occurs in spite of an increased gonadotropic support (Hoffmann et al.,
1996).
In the nonpregnant bitch luteal function is independent of a luteolysin of uterine origin since normal ovarian function was observed after hysterectomy (Hoffmann et al., 1992). In pregnant bitches,
however, the immediate prepartal P4 decrease coincides with an increase of PGF2␣ (Concannon et
al., 1988; Nohr et al., 1993) of unknown origin suggesting a functional role in relation to parturition,
also because luteal life-span can be terminated by exogenous application of PGF2␣, though relatively
high dosages or repeated treatments are necessary and strong side effects can occur (Romagnoli et al.,
1991; Concannon and Hansel, 1977).
As observed in other species among the many factors involved in the control of luteal function
prostaglandins seem to be of particular importance. Thus in ruminants and the pig onset of cyclic
luteolysis is triggered by a well timed release of PGF2␣ from the endometrium, while luteal PGF2␣,
which is expressed in many species (reviewed by Wiltbank and Ottobre, 2003), seems to contribute
to structural luteolysis (Diaz et al., 2002; Hayashi et al., 2003). Small amounts of uterine PGF2␣ acting
via induction of luteal Cox2 stimulate the intraluteal production of PGF2␣ (Diaz et al., 2002). On the
other side, PGE2 has shown to be a very potent luteotropic factor in several species. It stimulates luteal
P4 secretion via a cAMP-mediated pathway as was shown for cattle, rabbits and humans (Kotwica et
al., 2003; Marsh and LeMaire, 1974; Boiti et al., 2001). Its luteotrophic efﬁciency has been shown to be
comparable to that of LH (Weems et al., 1997).
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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In a previous paper we addressed the capacity of canine CL from nonpregnant bitches to produce prostaglandins; luteal Cox1 and Cox2 expression was clearly shown with expression of Cox1
resembling the pattern of a housekeeping gene and that of Cox2 being strongly cycle related with
a peak on both, the mRNA and protein level, at the beginning of dioestrus (Hoffmann et al., 2004;
Kowalewski et al., 2006a). In a follow up study expression of the main downstream enzymes, PGES
and PGFS, leading to the formation of PGE2 and PGF2␣ was investigated (Kowalewski et al., 2008a,b).
While PGFS was not or only weakly expressed, expression of PGES resembled that of Cox2-mRNA; it
was highest at the beginning of the CL-phase and dropped signiﬁcantly thereafter (Kowalewski et al.,
2008a).
Expression of mRNA encoding for the PGE2 receptors, EP2 and EP4, was colocalized with PGES to
luteal cells (Kowalewski et al., 2008a) and the spatio-temporal expression pattern of Cox2, PGES, EP2
and EP4 in the canine CL during dioestrus suggested that the locally produced PGE2 might act as a
luteotropic factor during the initial period of formation of the canine CL, explaining its apparent independence of gonadotropic support (Kowalewski et al., 2008a). Other than for PGFS, expression of the
prostaglandin F2␣ receptor (FP) was clearly demonstrated in the CL of nonpregnant bitches with the
mRNA-expression levels increasing from early to late dioestrus, explaining the receptivity of canine CL
to exogenous PGF2␣ (Kowalewski et al., 2008b). In order to further characterize the functional stages of
the CL, expression of steroidogenic acute regulatory (StAR) protein and of 3␤-hydroxysteroid dehydrogenase 4/5-isomerase (3␤HSD) was assessed. StAR is responsible for transport of cholesterol from
the outer to the inner mitochondrial membrane where it serves as substrate for the side-chain-cleavage
enzyme (P450scc). 3␤HSD catalyzes the synthesis of P4 from pregnenolone. The expression-pattern
observed for both factors on both, the protein and mRNA-level, suggests that P4 production might be
controlled by the provision of substrate at the level of cholesterol transfer to the inner mitochondrial
membrane rather than the enzyme availability (Kowalewski et al., 2006b; Kowalewski and Hoffmann,
2008).
All these observations support our previous hypothesis that luteal regression in the nonpregnant
dog is not an actively regulated process but rather a permissive one (Hoffmann et al., 2004; Kowalewski
et al., 2008b). Other than that, the prepartal increase of PGF2␣ in pregnant dogs (Concannon et al.,
1988; Nohr et al., 1993) is suggestive of an active luteolytic system which might originate in either the
CL of pregnancy or the utererine/placental compartment or both. The present paper addresses a likely
involvement of the CL of pregnancy in such a system. To get the respective information, expression
of Cox2, PGFS, PGES and the respective PGF2␣ and PGE2 receptors (FP, EP2 and EP4) as well as the
expression of the P4 receptor (PR), was assessed in CL of pregnant bitches.
Functional loss of P4 activity leads to abortion/parturition in the dog and can be induced by application of a P4 receptor blocker, e.g. Aglepristone (Fieni et al., 1996; Hoffmann et al., 1999; Hoffmann
and Schuler, 2000). Interestingly this treatment also induces luteolysis and it was speculated that this
effect might result from an interaction of the antiprogestin with P4 receptors expressed in the canine
CL (Hoffmann et al., 2004).
Thus in order to test which steps within the cascade of endocrine changes leading to abortion/parturition would be affected by antiprogestin treatment, the same parameters as in normal
pregnancies were assessed following treatment with Aglepristone.
2. Materials and methods
2.1. Animals, tissue sampling and preservation
All animal experiments performed were approved by the respective authorities [permit no II 25.319c20-15c GI 18/14 and VIG3-19c20/15c GI 18,14 (Gießen) and permit no Ankara 2006/06 (Faculty of
Veterinary Medicine, University of Ankara)].
2.1.1. Normal pregnancy
Four groups (n = 3–5) of clinically healthy, pregnant bitches of various breeds, aged 2–8 years, were
formed with ovariohysterectomy (OHE) scheduled for the following days of pregnancy:
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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Group 1, pre-implantation, days 8–12, n = 5;
Group 2, post-implantation, days 18–25, n = 5;
Group 3, mid-gestation, days 35–40, n = 5;
Group 4, prepartal progesterone decline, n = 3.
In Group4 P4 was determined in 6 h intervals beginning on day 58 of pregnancy. OHE was performed when P4 levels continued to decrease in two consecutive measurements below the level of
3 ng/ml.
2.1.2. Induced abortion
In a second study 10 bitches were treated with the antiprogestin Aglepristone between days 40 and
45 of pregnancy, using the dose recommended for induction of abortion [10 mg/kg bw, 2 times 24 h
apart]. OHE was performed 24 h (n = 5) and 72 h (n = 5) after the second injection.
In all bitches the day of insemination/mounting (day 0) had been recorded; pregnancy was conﬁrmed by either ultrasound (Groups 2–4; dogs where abortion was induced) or detection of embryos
in uterine ﬂushings (Group 1).
Immediately after surgery the corpora lutea were trimmed off the surrounding tissue, incubated
overnight in RNAlater® (Ambion Biotechnologie GmbH, Wiesbaden) for RNA preservation and then
stored at −80 ◦ C until further use as described earlier (Kowalewski et al., 2006a).
2.2. Determination of progesterone (P4)
Determination was by an established in house radioimmunoassay as described (Hoffmann et al.,
1973).
2.3. Reverse transcription (RT), semi-quantitative Real Time (TaqMan) polymerase chain reaction
(PCR) and data evaluation
Expression proﬁles of Cox2, PGES, PGFS, FP, EP2, EP4, PR, 3␤HSD and StAR were assessed
using semi-qantitative Real Time (Taq Man) RT-PCR. Nucleotide sequences of previously published
TaqMan-systems were applied (Kowalewski et al., 2006a,b, 2008a,b; Kowalewski and Hoffmann,
2008). The primers were ordered from MWG Biotech AG, the 6-carboxyﬂuorescein (6-FAM) and
6-carboxytetramethyl-rhodamine (TAMRA) labelled probes were from Eurogentec, B-4102 Serain,
Belgium. For primers and TaqMan probes sequences, see Table 1. Total RNA was isolated using
Trizol® -Reagent and subsequently treated with DNase, according to the manufacturer’s instructions
(Gibco-BRL, Life Technologies, Karlruhe and Roche Molecular Biochemicals, Mannheim, respectively).
Our previously described protocol was applied (Kowalewski et al., 2006a) and the reactions were
carried out in an automated ﬂuorometer ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany). Brieﬂy: 100 ng of DNase-treated total RNA was reverse-transcribed using
the GeneAmp Gold RNA PCR Kit (PerkinElmer Applied Biosystems GmbH, Weiterstadt, Germany).
Samples were analyzed in duplicates. 25 ␮l of reaction mixture contained 12.5 ␮l TaqMan® qPCR MasterMix (Eurogentec, B-4102 Serain, Belgium), 300 nM of each primer and 200 nM TaqMan Probe and
5 ␮l of cDNA. Ampliﬁcation was carried out as follows: denaturation for 10 min at 95 ◦ C followed by
40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 60 s.
Relative quantiﬁcation was done by normalizing the signals of target genes with the GAPDH signal
and using the comparative CT method (CT method) according to the instructions of the manufacturer of the ABI PRISMTM 7000 Sequence Detector.
2.4. Statistics
To test for an effect of the observational group on the mRNA-levels of the target genes, a parametric analysis of variance (ANOVA) was applied. Multiple comparison post-tests were performed
in case of p < 0.05. Those were, Tukey–Kramer Multiple Comparison Test during the course of pregnancy and the Dunnett’s Multiple Comparison Test to test the effect of the antiprogestin treatment
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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Table 1
List of primers used for Real Time (TaqMan) PCR.
Primer
Accession numbers
Primer sequence
Product
length (bp)
Cox2-for
Cox2-rev
Cox2-Taq Man Probe
AY044905
5 -GGA GCA TAA CAG AGT GTG TGA TGT G-3
5 -AAG TAT TAG CCT GCT CGT CTG GAA T-3
5 -CGC TCATCATCC CAT TCT GGG TGC T-3
88
PGFS-for
PGFS-rev
PGFS-TaqMan Probe
AY875970
5 -AGG GCT TGC CAA GTC TAT TGG-3
5 -GCC TTG GCT TGC TCA GGA T-3
5 -TCC AAC TTT AAC CGC AGG CAG CTG G-3
74
FP-for
FP-rev
FP-TaqMan Probe
DQ138060
5 -ACC AGT CGA ACA TCC TTT GCA-3
5 -GGC CAT CAC ACT GCC TAG AAA-3
5 -CAT GGT GTT CTC CGG TCT GTG CCC-3
86
PGES-for
PGES-rev
PGES-TaqMan Probe
EF063141
5 -CTG TCA TCA CCG GCC AAG T-3
5 -CCT GGT CAC TCC GGC AAT A-3
5 -ACG CCC TGA GAC ACG GAG GCC T-3
99
EP2-for
EP2-rev
EP2-TaqMan Probe
AF075602
5 -CAC CCT GCT GCT GCT TCT C-3
5 -CGG TGC ATG CGG ATG AG-3
5 -TGC TCG CCT GCA ACT TTC AGC GTC-3
78
EP4-for
EP4-rev
EP-TaqMan Probe
AF177934
5 -AAA TCA GCA AAA ACC CAG ACT TG-3
5 -GCA CGG TCT TCC GCA GAA-3
5 -ATC CGA ATT GCT GCT GTG AAC CCT ATC C-3
96
3␤HSD-for
3␤HSD-rev
3␤HSD-TaqMan Probe
AY739720
5 -GGG TAC TCA GCT CCT GTT GGA A-3
5 -GCC ACC TCT ATG GTG CTG GTA T-3
5 -TGC CCA GGC TAG TGT GCC GAT CTT-3
78
StAR-for
StAR-rev
StAR-TaqMan Probe
EF522840
5 -CGA GGC TCC ACC TGT GTG T-3
5 -CCT TTC TGC TCA GGC ATC TC-3
5 -CTG GCA TGG CCA CAC ATT TC-3
65
PR-for
PR-rev
PR-TaqMan Probe
AF177470
5 -CGA GTC ATT ACC TCA GAA GAT TTG TTT-3
5 -CTT CCA TTG CCC TTT TAA AGA AGA-3
5 -AAG CAT CAG GCT GTC ATT ATG GTG TCC TAA CTT-3
GAPDH-for
GAPDH-rev
GAPDH-TaqMan Probe
AB028142
5 -GCT GCC AAA TAT GAC GAC ATC A-3
5 -GTA GCC CAG GAT GCC TTT GAG-3
5 -TCC CTC CGA TGC CTG CTT CAC TAC CTT-3
113
75
on the mRNA-expression of target genes. Due to the uneven distribution of the Real Time data for
the StAR mRNA-expression during the course of pregnancy the Kruskal–Wallis Test (a nonparametric
ANOVA) followed by Dunn’s Multiple Comparisons Test was applied. Numerical data were presented
as the mean ± standard deviation. For all tests the statistical software program, GraphPad3 (GraphPad
Software Inc., San Diego, CA, USA) was used.
3. Results
3.1. Normal pregnancy
3.1.1. Expression studies
A signiﬁcant effect of time was observed in the expression of Cox2 and PGES (p < 0.0001 and
p < 0.0022, respectively) with expression being highest during the pre-implantation period and significantly lower (p < 0.001 and p < 0.01) thereafter (Figs. 1A and 2A).
A signiﬁcant effect of time was also observed for the expression of PGFS mRNA (p < 0.04) which
was lowest at the pre-implantation period, highest (p < 0.05) in the post-implantation and slowly
decreasing thereafter towards parturition (Fig. 1C).
A similar expression pattern was observed for FP, however, the changes were statistically not
signiﬁcant (Fig. 1E).
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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Fig. 1. Expression of Cox2, PGFS and FP as determined by Real Time (TaqMan) PCR during pregnancy and normal luteolysis (A, C,
E) and during Aglepristone induced luteolysis (B, D, F; compared with the mid-gestation group.). RGE: relative gene expression
(mean ± standard deviation); bars with different asterisks differ with p < 0.001 (A, D) and p < 0.05 (C).
Expression of EP2 but not EP4 showed an effect of time (p < 0.01), with a constantly low expression
until prepartal luteolysis when an upregulation was observed (Fig. 2C and E).
PR revealed a signiﬁcant effect of time (p = 0.04) with a biphasic expression pattern showing an
increased mRNA expression during the second half of gestation (Fig. 3A).
Expression of StAR and 3␤HSD was time dependant (p = 0.04 and p = 0.002, respectively),
showing a constant decrease from the pre-implantation period until prepartal luteolysis (Fig. 3C
and E).
3.1.2. Progesterone concentrations
Mean progesterone concentrations were: 35.71 ± 7.9 ng/ml in the pre-implantation period,
29.73 ± 13.23 ng/ml in the post-implantation period, 13.32 ± 8.66 ng/ml at mid-gestation and
2.07 ± 0.99 ng/ml during the prepartal progesterone decline; the effect of time was highly signiﬁcant
(p < 0.0019).
3.2. Induced abortion
3.2.1. Expression studies
For all parameters the expression patterns were compared with the mid-gestation group. Following
treatment with the antiprogestin expression of StAR and 3␤HSD decreased signiﬁcantly (p < 0.01 and
p < 0.05, respectively) resembling the situation in prepartal luteolysis (Fig. 3D and F), The expression
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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Fig. 2. Expression of PGES, EP2 and EP4 as determined by Real Time (TaqMan) PCR during pregnancy and normal luteolysis (A, C,
E) and during Aglepristone induced luteolysis (B, D, F; compared with the mid-gestation group.). RGE: relative gene expression
(mean ± standard deviation); bars with different asterisks differ with p < 0.01 (A, C).
of PR, PGES, EP2, EP4, Cox2, and FP (Figs. 3B, 2B, D and F and 1B and F) was not affected; as in normal
prepartal luteolysis but more pronounced (p < 0.001) was the decrease in PGFS (Fig. 1D).
3.2.2. Progesterone concentrations
The mean progesterone concentration before the ﬁrst treatment was 15.11 ± 6.7 ng/ml. Mean concentration at the second Aglepristone treatment 24 h later was at 13.61 ± 8.2 ng/ml. It had decreased
(p < 0.01) to 5.1 ± 2.7 ng/ml 24 h later and was at 2.33 ± 1.44 ng/ml, respectively 1.2 ± 0.6 ng/ml 48 and
72 h later.
4. Discussion
Establishment and maintenance of pregnancy in the dog depends on the secretion of P4 from
the CL—the only source of this hormone in nonpregnant and pregnant animals. Luteotropic factors
securing CL-function during the second half of pregnancy, respectively dioestrus are prolactin and
LH (Concannon, 1980; Okkens et al., 1990) and only recently some evidence was obtained that luteal
PGE2 acting via autocrine/paracrine mechanisms might support the developing CL during the ﬁrst
two to three weeks of dioestrus (Kowalewski et al., 2008a). On the other side our investigations in the
nonpregnant, dioestric dog have revealed no hints of an active luteolytic principle which would involve
uterine (Hoffmann et al., 1992) or CL-derived PGF2␣ (Kowalewski et al., 2008b). Clearly the decrease
of P4-synthesis results from a decreased expression of StAR (Kowalewski and Hoffmann, 2008) and
3␤HSD (Kowalewski et al., 2006b). Though the mechanisms governing StAR- and 3␤HSD-expression
in the CL of the bitch still remain open, the hypothesis was developed that the luteal regression in the
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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Fig. 3. Expression of PR, 3␤HSD and StAR as determined by Real Time (TaqMan) PCR during pregnancy and normal luteolysis (A,
C, E) and during Aglepristone induced luteolysis (B, D, F; compared with the mid-gestation group.). RGE: relative gene expression
(mean ± standard deviation); bars with different asterisks differ with p < 0.04 (A), p < 0.01 (C, F) and p < 0.05 (D, E).
nonpregnant dog is not an actively regulated but rather a passive, permissive process (Kowalewski et
al., 2008b).
Other than in nonpregnant animals, there is no information available on the intraluteal expression of
the prostaglandins system in the CL of pregnant dogs. Yet there is strong evidence, that in the pregnant
animal prepartal luteolysis is actively regulated through PGF2␣ mediated mechanisms, since there is
an immediate prepartal increase of PGF2␣ concomitant to the decline of P4 (Concannon et al., 1988;
Nohr et al., 1993), which – apart from being responsible for the induction of labour – might also lead
to luteolysis. The origin of this PGF2␣ increase is, however, unknown and both, the uterine/placental
compartment as well as the CL might contribute. This would resemble the situation in the cattle, where
luteal PGF2␣ is supposed to contribute to structural luteolysis (Diaz et al., 2002; Hayashi et al., 2003)
in conjunction with the upregulation of placental Cox2 (Schuler et al., 2006).
The present study has clearly shown that Cox2 and PGES, similarly to the nonpregnant dog
(Kowalewski et al., 2006a, 2008a), are upregulated at the phase of CL formation (pre-implantation
period), with PGE2 possibly acting as a luteotropic factor. While the respective receptors for PGE2, EP2
and EP4, are expressed on a rather constant level from days 15 to 65 in nonpregnant dogs, expression
of EP2, but not EP4, is upregulated in pregnant dogs during prepartal luteolysis. At present no functional interpretation can be given, however, an increased availability of EP2 might render the CL more
susceptible for PGE2 of extra luteal origin. Other than in nonpregnant animals, where only weak or no
PGFS expression was detectable in the CL during the dioestrus (Kowalewski et al., 2008b), luteal PGFS
mRNA-expression was relatively high throughout pregnancy. The observed PGFS-levels were upregulated from the pre-implantation to the post-implantation period followed, however, by a gradual
decrease towards parturition, making it most unlikely that the CL of late pregnancy is the source of the
prepartal PGF2␣ increase. Similar changes though not signiﬁcant, were observed for FP, an observation
Please cite this article in press as: Kowalewski, M.P., et al., Time related changes in luteal
prostaglandin synthesis and steroidogenic capacity during. . . . Anim. Reprod. Sci. (2009),
doi:10.1016/j.anireprosci.2008.12.011
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9
pointing towards a likely functional role of PGF2␣ as an autocrine/paracrine factor during early- and
mid-gestation.
Following application of Aglepristone on days 40–45 of pregnancy, a time point distinctly apart from
the onset of physiological prepartal luteolysis, a signiﬁcant downregulation of StAR and 3␤HSD was
observed after 24, respectively 72 h after the second injection, paralleled by a concomitant decrease
of P4 from 13.61 ± 8.2 ng/ml to 5.1 ± 2.7 and 1.2 ± 0.6 ng/ml and a signiﬁcant decline in the expression
of PGFS.
The mechanisms underlying these processes are still obscure; yet it might be speculated that interference of the antiprogestin with luteal P4-receptors, the expression of which is increased during
mid-gestation and prepartal luteolysis (Fig. 3A), might be a triggering effect.
Thus, except for the upregulation of EP2 during prepartal luteolysis, the gross overall expression
pattern of mRNA encoding for the prostaglandins system in antiprogestin treated dogs resembled that
during the prepartal withdrawal of P4.
Thus our data do suggest that – as in nonpregnant bitches – also in pregnant bitches PGF2␣ of luteal
origin is not a factor modulating luteolysis.
In conclusion, our data suggest that the blocking of PR by the antiprogestin Aglepristone triggered
downregulation in the expression of StAR, 3␤HSD and PGFS, pointing towards an involvement of luteal
P4 as an autocrine factor within a positive loop feedback system. Similarly as in the nonpregnant bitch
also in the pregnant bitch PGF2␣ of luteal origin seems not to be involved in the mechanisms leading
to regression of the CL. However, as in the nonpregnant bitch the expression of FP renders the CL
susceptible to extraluteal PGF2␣. Our ﬁrst and not yet published results make the placenta a likely
candidate for the origin of the prepartal PGF2␣ increase. The high expression of PGES observed in the
pre-implantation period agrees with previous observations in the nonpregnant bitch and stresses the
role of CL derived PGE2 as an autocrine luteotropic factor during the period of CL formation in the dog.
Acknowledgements
With support of the German Research Foundation. The authors also greatly appreciate the support
of Virbac, Germany, in providing the Aglepristone and ﬁnancial support of the Aglepristone associated
studies.
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Theriogenology 66 (2006) 1560–1567
www.journals.elsevierhealth.com/periodicals/the
Dopamine agonists, anti-progestins, anti-androgens,
long-term-release GnRH agonists and anti-estrogens
in canine reproduction: A review
C. Gobello *
Small Animal Clinic, Faculty of Veterinary Medicine, National University of La Plata, Argentina
Abstract
Over the last 10 years, new drugs have been applied to canine reproduction, widening the spectrum of therapeutic possibilities
for diseases that were previously surgically treated, and facilitating better control of the estrous cycle and fertility. Some are not
approved for use in dogs; their use is experimental and further clinical trials are necessary. Dopamine agonists such as cabergoline,
bromocriptine or metergoline are ergoderivative alkaloids that exert an anti-prolactinergic effect via stimulation of D2 pituitary
receptors or inhibition of central serotoninergic ones. Their main indication is suppression of lactation. Anti-prolactinergic
compounds have also been successfully used for pregnancy termination and shortening of interestrous intervals. Anti-progestins,
(e.g. mifepristone and aglepristone) are synthetic steroids that bind with high affinity to progesterone (P4) receptors, preventing P4
from exerting its biological effects. Anti-progestins have been indicated in P4-dependent conditions, such as pregnancy termination,
induction of parturition and the medical treatment of pyometra. Several groups of drugs have been described to have antiandrogenic properties through different mechanisms of action: progestins, receptor binding anti-androgens (e.g. flutamide),
competitive enzyme inhibitors (e.g. finasteride), aromatase inhibitors, and GnRH agonists. Their main application is medical
treatment of benign prostatic hyperplasia. Long-term release formulations of GnRH agonists (e.g. leuprolide or deslorelin acetate)
postponed puberty and reversibly suppressed reproductive function in male and female dogs for periods exceeding 1 year. Antiestrogens (e.g. clomiphene and tamoxifen citrate) are synthetic non-steroidal type I anti-estrogenic compounds that competitively
block estrogen receptors with a combined antagonist-agonistic effect. In dogs, their action is more agonistic than antagonistic.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Dopamine agonist; Anti-estrogen; Anti-progestin; Anti-androgen; Long-term-release GnRH agonist; Dog
1. Introduction
Over the last 10 years, new drugs have been applied
to canine reproduction. They have expanded the
spectrum of therapeutic possibilities for diseases that
were previously surgically treated, and they permit
better control of the estrous cycle and fertility in
general. Due to differences in availability of these drugs
* Tel.: +54 221 4252155; fax: +54 221 4257980.
E-mail address: [email protected].
0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.02.005
in various countries, the frequency of application and
the general knowledge of these compounds vary
considerably among practitioners.
Although most of these drugs have already been used
for years in other species, only few are widely marketed
or approved for use in dogs. The aim of this article is to
briefly review the main pharmacological properties,
reported trials and possible indications of new groups of
drugs, e.g. dopamine agonists, anti-progestins, antiestrogens, anti-androgens, and long-term-release preparations of gonadotrophin releasing hormone (GnRH)
agonists for use in canine reproduction. Several of the
C. Gobello / Theriogenology 66 (2006) 1560–1567
drugs discussed are not approved for use in dogs and/or
as described; such use should be considered experimental, with the owner required to sign a release form
where applicable.
2. Dopamine agonists
Dopaminergic agonists are ergotine-derivative alkaloid compounds that exert an anti-prolactinergic effect.
Two of the most widely used dopamine agonists in dogs
are bromocriptine and cabergoline, which have a direct
action on D2-dopamine receptors of the lactotrophic
cells of the anterior pituitary gland. Metergoline,
another ergot alkaloid, is a serotonin antagonist,
exerting dopaminergic effects at high doses [1,2].
The ability of dopamine agonists to inhibit prolactin
(PRL) secretion makes them optimal for milk suppression, either during overt pseudopregnancy episodes or
in the post-partum period [3–7] and they are marketed
with that indication in several countries. It is well
known that PRL is a required luteotropic hormone
during the second half of canine luteal phase [8].
Therefore, anti-prolactins can also be used to suppress
luteal function in progesterone (P4) dependent conditions such as pyometra, unwanted pregnancy [9–11] and
mammary tumors.
Different combinations of either natural or synthetic prostaglandins (PG) F2a and dopamine agonists
have been reported to efficiently terminate gestation
from 25 days after the luteinizing hormone (LH)
surge, without substantial side effects [12–14]. This
drug combination was also used for medical treatment
of various stages of spontaneous pyometra, with a
success rate of 82% [15]. Dopamine agonists (e.g.
cabergoline, 5 mg/kg/day po) have also been recommended for pre-surgery treatment of canine mammary
tumors, and for reducing the incidence of mammary
tumors by treating overt pseudopregnancy (associated
with mammary tumors [16]), as well as for reducing
mammary size before mammectomy [17]. Finally,
treatment with PRL inhibitors shortened the interestrous intervals in bitches, either by advancing luteal
regression or by reducing the anestrus period [7,18–
20]. Although they were effective in inducing estrus in
bitches with prolonged anestrus [21,22], precise
mechanisms of action of dopamine agonists leading
to estrus induction in anestrus bitches are not well
understood. As estrous induction by dopamine
agonists does not depend on decreasing serum PRL
concentrations [7,23], perhaps they directly stimulate
the hypothalamic-pituitary axis or exert a peripheral
action on the ovaries.
1561
In an early report, bromocriptine was used in four
bitches (20 mg/kg bid po) from 1 to 5 days after the LH
surge to the beginning of the next proestrus. The
interestrous interval was significantly reduced in treated
versus control bitches (123 23 versus 245 8 days,
respectively) [18]. A pregnancy rate of more than 60%
was reported [24] in bitches that were mated during
bromocriptine-induced estrus. In an early study, using
metergoline (12.5 mg im per bitch every 3 days, starting
between 78 and 161 days after proestrus) the
interestrous interval was significantly shorter than in
the control group (144 versus 207 days, respectively).
Ten treated bitches in this protocol ovulated and nine
became pregnant, although the high dose of metergoline
(1 mg/kg) provoked vomiting [25]. In another trial,
metergoline (0.1 mg/kg bid po) administered to seven
bitches 100 days after ovulation significantly decreased
serum PRL concentrations, but did not affect the
interestrous interval [26].
Oral treatment with cabergoline (5 mg/kg/day po) in
bitches with prolonged anestrus resulted in estrous
induction after 5–18 days of treatment and in pregnancy
in all 28 bitches that were treated. [21]. Administration
of the same dose of cabergoline from 30 days after the
LH surge reduced the interestrous intervals from 216 to
66.5 days. However, none of these bitches became
pregnant, probably due to insufficient uterine endometrial regeneration. Nevertheless, this study demonstrated that estrus cannot only be induced in anestrus,
but also in diestrus [19]. The duration of cabergoline
treatment was shorter in late versus early anestrus
(means, 6 versus 20 days, respectively) and normal
hormonal characteristics and fertility were obtained on
induced cycles [20].
3. Anti-progestins
Anti-progestins are synthetic steroids that bind with
great affinity to P4 receptors, preventing P4 from
exerting its biological effects [27]. Some anti-progestins
also have the ability to interact with different binding
affinity for glucocorticoid receptors [27]. In dogs, the
anti-progestins mifepristone (RU 486) and aglepristone
(RU 534) have been used for experimental and clinical
purposes, including pregnancy termination and management of pyometra. Aglepristone is available in the
veterinary market of some American and European
countries, with an indication for pregnancy termination.
Aglepristone acts as a true P4 antagonist at the uterine
level, without initially decreasing serum P4 concentrations. Mifepristone also terminated pregnancy in the
bitch within 3–4 days, without side effects [28,29].
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C. Gobello / Theriogenology 66 (2006) 1560–1567
Aglepristone (10 mg/kg sc given twice, 24 h apart)
was tested for pregnancy termination in 104 bitches at
15–55 days after mating. Treatment before 25 days
resulted in resorption in all animals, whereas later
administration induced abortion within 7 days in 96% of
the cases [30]. Typical signs of parturition were
associated with abortion, and vaginal discharge
persisted for 3–5 days afterwards. Clinical monitoring
over more than 18 months did not reveal any treatment
sequelae [30]. In a more recent study in five beagle
bitches receiving the same therapeutic protocol 30 days
after ovulation, abortion was induced within 4–7 days
after the start of treatment. Plasma PRL concentrations
were elevated during treatment, whereas plasma P4
remained unchanged [31]. In 10 bitches treated in early
or mid-pregnancy, aglepristone did not modify plasma
concentrations of P4, PGF2a, oxytocin or cortisol
within 24 h after administration, but increased PRL
concentrations within 12 h of administration [32]. The
interestrous intervals following treatments were shortened by 1–3 months in the three studies [30–32].
Other clinical applications described for antiprogestins are induction of parturition and medical
treatment of pyometra. In four bitches, mifepristone
induced signs of parturition after 50 days of pregnancy,
although a caesarean section was necessary [33]. In
another study, aglepristone (15 mg/kg sc) was administered 58 days of pregnancy in 10 bitches. Starting 24 h
later, the bitches were injected sc with either oxytocin
(0.15 IU/kg) or the PG alfaprostol (0.08 mg/kg) every
2 h until the end of parturition. On average, parturition
started 32 h after aglepristone administration. Peripheral
P4 concentrations increased after aglepristone injection
such that bitches whelped at high P4 concentrations.
Plasma concentrations of oxytocin and cortisol were
unchanged during the first 24 h after aglepristone
administration, whereas PG and PRL concentrations
increased after 4 and 20 h, respectively [34]. Induction of
parturition with aglepristone and PGs was also reported
in one bitch with prolonged pregnancy [35].
In preclinical studies and early clinical trials,
endometritis and pyometra complex were successfully
treated with anti-progestins [36,37]. Aglepristone
(10 mg/kg sc) was used successfully in bitches with
pyometra that had P4 concentrations >3.2 nmol/L and
normal ovarian function [38]. The combination of
aglepristone and PGF2a has shown good results for
the medical treatment of canine endometrial hyperplasia–pyometra complex [39,40]. In another combineddrug trial with the same dose of aglepristone and PGF
analog cloprostenol (1 mg/kg sc every other day from
days 3 to 15), clinical signs, blood parameters and uterine
diameter consistently improved to normal values,
independent of the initial P4 concentrations throughout
days 15 or 28 of treatment. In 20% of bitches treated,
pyometra recurred at the next estrous cycle [40]. In
another study, the recurrence rate was 18.9% at 1 year
after aglepristone treatment [41]. To prevent recurrent of
pyometra, it is often suggested that successful medical
treatment of pyometra should be followed by breeding at
the subsequent cycle (in bitches intended for breeding).
4. Anti-androgens
Several groups of compounds have been described to
have anti-androgenic properties (through different
mechanisms of action), such as progestins, receptor
binding anti-androgens, competitive enzyme inhibitors,
aromatase inhibitors, and GnRH analogues. Estrogens
and anti-estrogens have also been shown to have antiandrogenic effects in dogs [42,43]. The main clinical
application of anti-androgens in dogs is benign prostatic
hyperplasia. Other possible indications are management
of testosterone (T) dependent behavioral problems and
reversible suppression of fertility in male dogs.
Different progestins may have different anti-androgenic potency, depending on their anti-gonadotrophic
and androgenic receptor binding effects. Administration
of medroxyprogesterone acetate (3 mg/kg sc) decreased
prostate size [44]. Doses ranging from 3 to 4.8 mg/kg
reduced serum T concentrations for 5–13 weeks after
treatment without affecting semen quality or libido [45].
Similar results have been found after megestrol acetate
(2 mg/kg po for 7 days), whereas a higher dose (4 mg/
kg) produced sperm abnormalities [46]. Cyproterone
acetate, a potent anti-androgenic progestin used for the
treatment of prostatic disorders in men, has also been
tested in dogs [27,47]. Other progestins, like delmadinone [48] and clormadinone acetate are known to be
effective in the treatment of benign prostatic hyperplasia in this species [49].
Flutamide, a pure androgen receptor blocker, inhibits
androgen uptake and nuclear binding by binding to the
androgen receptor [27]. Flutamide has yielded good
results in treating prostatic hyperplasia without
alteration of semen or libido [50]. Aromatase inhibitors, such as formestane, exert their anti-androgenic
effect by inhibiting the conversion of androgens to
estrogens in peripheral tissues [51]. Finasteride is a
synthetic steroid that prevents conversion of T into
dihydrotestosterone (DHT) by inhibition of the type II
5a-reductase [52]. Five beagle dogs with benign
prostatic hyperplasia that were treated with finasteride
(1 mg/kg po, q 24 h for 21 weeks) had decreased size
C. Gobello / Theriogenology 66 (2006) 1560–1567
of the prostate (30% of the initial value) and complete
lack of an ejaculate by the end of the treatment period.
Fertility was restored after a 20–22 weeks recovery
phase [53].
A dose–response study of finasteride (0.1, 0.25, or
0.5 mg/kg po every 24 h for 7 days) in three normal
intact male dogs caused a significant decrease of DHT
without changing T serum concentrations, libido and
spermatogenesis [54]. More recently, using the same
drug (0.1–0.5 mg/kg po every 24 h) for 16 weeks in nine
client-owned dogs with spontaneous benign prostatic
hyperplasia resulted in decreased serum DHT concentrations and a 43% reduction of prostatic volume.
Finasteride treatment diminished ejaculate volume
without affecting other semen characteristics, libido,
serum T concentrations, or fertility [55].
5. Long-term-release GnRH agonists
Similar to the actions of native GnRH, synthetic
GnRH agonists like nafarelin, leuprolide, deslorelin,
buserelin, and goserelin, stimulate production and
release of gonadotrophins from the pituitary. However,
GnRH agonists, when used at sustained doses,
reversibly inhibited the pituitary gonadal axis after
the initial period of stimulation. Inhibition is produced
by down-regulation of anterior pituitary GnRH receptors [56]. Continuous administration of, or long term
release formulations of, GnRH agonists reversibly
suppressed reproductive function in male and female
dogs for periods exceeding 1 year in some studies [56–
59]. The previous disadvantage of these compounds was
the need for frequent administration over prolonged
intervals; a substantial advance was development of
slow-release formulations that can be easily implanted
subcutaneously.
A single leuprolide acetate injection (1 mg/kg sc)
administered to five male dogs decreased ejaculate
volume and was accompanied by an increase in
morphologically abnormal spermatozoa. An initial rise
in LH and T serum concentrations, followed by a
marked decline to below-normal concentrations for
6 weeks, was also described in this study. Twenty weeks
after treatment, spermatogenesis had returned to normal
[58].
In a more recent study, 30 mature dogs received sc
implants of deslorelin acetate (doses ranged from 0.08
to 0.79 mg/kg), and 11 were re-implanted either before
or after the suppression period. Their T concentrations
decreased to <1 ng/mL within a mean of 17 days after
implantation in all the treatment groups and remained at
this level from 3 months to 2.7 years. The duration of the
1563
inhibitory effect seemed to be dose related and
restoration to initial conditions became evident, based
on scrotal circumference, T concentrations, semen
quality, and fertility [60]. In another report, 0.5–1 mg/
kg of the same GnRH agonist implanted in five dogs
significantly decreased serum T and prostate size for 32
and 48 weeks, respectively [61]. Recently, in seven
dogs that were implanted with 6.6 mg of buserelin, T
and estradiol decreased to basal concentrations within
15 days of implantation and remained there for a mean
of 233 days. Testicular and prostatic size were
reversibly reduced and no semen could be collected
21 days after implantation [62].
The GnRH agonist [D-Trp6, des-Gly-NH210] GnRH
ethylamide administered sc daily to prepubertal male
and female dogs for 23 months decreased steroid
hormone concentrations and delayed puberty, with
normal fertility following a recovery period [63]. In
three female dogs, sc implantation (in proestrus) of
an osmotic pump releasing natural nafarelin acetate
(18 mg/kg/day) suppressed estrous cycles for 18 months.
Cyclicity was restored 3–18 weeks after cessation of
treatment. When the same drug was administered to
anestrous bitches, an infertile ovulatory estrus was
induced, 1–2 week after the start of treatment.
Postponement of pubertal estrus was obtained in three
bitches in the same study during 18 months of treatment
[56]. In a more recent report, pregnant, diestrous and
anestrous bitches were treated with doses ranging from
0.1 to 2.4 mg/kg of deslorelin acetate in sc implants.
Treatment prolonged interestrous intervals up to 27
months, independent of the stage of the cycle at
implantation. When serum P4 was <5 ng/mL, an estrous
cycle was induced 4–8 days after implantation. Six of
nine bitches that were mated after recovery of the
treatment became pregnant [60]. In a study in which
azagly-nafarelin was administered sc to six bitches, there
was no estrous behavior and low concentrations of
FSH and P4 for 1 year of treatment [64].
In one trial, the expected initial estrous response
induced in anestrous bitches after implantation of 6 mg
deslorelin was suppressed by a 21 or 14-day treatment
with 2.2 mg/kg megestrol acetate po, started 2 or 1 week
before implantation, respectively [65]. Administration
of the same dose of megestrol for 8 days, starting the
day before deslorelin implantation, prevented estrous
response in four of eight bitches [59].
Systematic re-implantation of long term release
GnRH agonists could offer an option for dogs
diagnosed with hormone-dependent diseases that have
unacceptable risk factors for anesthesia or surgery.
In this context, goserelin was used successfully for
1564
C. Gobello / Theriogenology 66 (2006) 1560–1567
12 months in the treatment of canine mammary gland
tumors [66].
Considering the initial stimulating effect of GnRH
agonists, these drugs have been used for estrus
induction in bitches. A single sc injection of a sustained
release formulation of the potent GnRH agonist,
leuprolide acetate (100 mg/kg), followed by fertirelin on the first day of induced estrus, provoked
behavioral estrus in 100% of bitches, with a pregnancy
rate of 50–100% [67]. In a recent study with six bitches,
removal of a 2.1 mg deslorelin implant at the time of
the preovulatory LH surge induced 100% estrus and
50% pregnancy [68], whereas with the same protocol,
one of the eight bitches remained pregnant to term if
implants were not removed [69].
6. Anti-estrogens
Pharmacologic groups of compounds that inhibit or
modify estrogens are classified as anti-estrogens. Some
of these are GnRH analogs and aromatase inhibitors
that inhibit estrogen synthesis. Another anti-estrogenic
drug group is estrogen receptor blockers [70].
Clomiphene and tamoxifen citrate are synthetic nonsteroidal type I anti-estrogenic compounds which
competitively block estrogen receptors with a combined antagonistic-agonistic effect. Type I estrogen
antagonists partially inhibit the action of estrogen
agonists, but due to their own agonistic properties they
also induce, to some extent, estrogenic responses. The
manifestation of these different actions depends on
each species, organ, tissue and cell type considered
[27]. Thus in women, tamoxifen has anti-estrogenic
activities on the mammary gland and agonistic effects
on the uterus [71]. The exact mechanism of this duality
is incompletely understood, but may depend on the
variable expression of specific cellular estrogen
receptors [27]. In humans, estrogen receptor blockers
have been used for treatment of male and female
infertility and breast cancer [70–72].
Little is known about the effect of receptor blocker
anti-estrogens in dogs. Most studies have been carried
out using tamoxifen, which seems to act more like an
agonist than antagonist [73–76], limiting its use in
female dogs. In that regard, signs of proestrus were
induced in two of four bitches after 10 days of
clomiphene citrate (12.5–25 mg po) [77].
Although tamoxifen should be efficacious in preventing or terminating canine pregnancy when administered during proestrus, estrus or diestrus, it had
estrogen-like side effects. Endometris, pyometra and
ovarian cysts developed in nine of 20 bitches treated
with tamoxifen (1 mg/kg po bid) [73]. In one study in
which tamoxifen was administered (2.5–10 mg po bid)
to seven bitches with inoperable or metastatic mammary carcinoma, tamoxifen reduced tumor burden in
five animals. Unwanted side effects were vulvar
swelling, vaginal discharge, urinary incontinence, and
pyometra [75]. In another report, 18 bitches diagnosed
with mammary tumors were treated with tamoxifen in
addition to mastectomy and ovariectomy. Ten bitches
showed estrogenic effects and no anti-tumor activity
could be documented [76]. Additional studies are
needed to establish the potential benefit of anti-estrogen
therapy for canine mammary tumors, particularly in
combination with hormone receptors assays.
In a study in which seven male beagle dogs were
treated with tamoxifen (2.5 mg po every 24 h for 28
days), the drug decreased testicular size and libido.
During treatment, tamoxifen also decreased prostatic
volume and T concentrations. One spermatogenic cycle
after treatment, sperm count and volume decreased to
low values, while motility and morphology deteriorated; all of these parameters returned to pre-treatment
values the next spermatogenic cycle. No clinical or
haematological side effects were observed and fertility
was restored at the end of the study [74].
7. Conclusions
Progress in our understanding of physiology of
canine reproductive has enhanced the possibility of
utilizing pharmacological agents that were previously
used only in other species. Although most of these drugs
have already had an impact on canine reproductive
therapy, only a few indications for dopamine agonists,
long-term-release GnRH agonists and anti-progestins
have been approved to date for use in dogs. Thus, further
clinical randomized controlled trials are necessary to
better refine indications and treatment regimens and to
enhance the expected clinical outcome before the use
of these drugs can be widely recommended.
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Theriogenology 66 (2006) 797–803
www.journals.elsevierhealth.com/periodicals/the
Treatment of growth hormone excess in dogs with the
progesterone receptor antagonist aglépristone
S.F.M. Bhatti a,*, L. Duchateau b, A.C. Okkens c, L.M.L. Van Ham a,
J.A. Mol c, H.S. Kooistra c
a
Department of Small Animal Medicine and Clinical Biology, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
b
Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, B-9820 Merelbeke, Belgium
c
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine,
Utrecht University, Yalelaan 8, NL-3508 TD Utrecht, The Netherlands
Received 2 December 2005; received in revised form 18 January 2006; accepted 20 January 2006
Abstract
Acromegaly or hypersomatotropism in dogs is almost always due to progestin-induced hypersecretion of GH originating from
the mammary gland. The aim of this study was to investigate whether aglépristone, a progesterone receptor antagonist, can be used
to treat this form of canine acromegaly. In five Beagle bitches hypersomatotropism was induced by administration of MPA for over
1 year. Subsequently, aglépristone was administered. Blood samples were collected before MPA administration, immediately
before, during, and 3.5 and 5.5 weeks after the last administration of aglépristone for determination of the plasma concentrations of
GH and IGF-I. In addition, blood samples for the determination of the 6-h plasma profile of GH were collected before MPA
administration, before aglépristone administration, and 1 week after the last aglépristone treatment.
MPA administration resulted in a significant increase of the mean plasma IGF-I concentration, whereas analysis of the pulsatile
plasma profile demonstrated a trend (P = 0.06) for a higher mean basal plasma GH concentration and a higher mean AUC0 for GH.
Treatment with aglépristone resulted in a significant decrease of the mean plasma GH and IGF-I concentrations. Analysis of the
pulsatile plasma profile showed a trend (P = 0.06) for a lower mean basal plasma GH concentration and a lower mean AUC0 for GH
1 week after the last aglépristone treatment compared with these values before aglépristone administration. Three and a half and 5.5
weeks after the last aglépristone administration the mean plasma IGF-I concentration increased again.
In conclusion, aglépristone can be used successfully to treat dogs with progestin-induced hypersomatotropism.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Acromegaly; Hypersomatotropism; Insulin-like growth factor-I; Medroxyprogesterone acetate; Progesterone receptor blocker
1. Introduction
Acromegaly is characterized by bony and soft tissue
overgrowth due to excessive growth hormone (GH)
secretion. The syndrome is known to occur in humans,
* Corresponding author. Tel.: +32 9 2647687; fax: +32 9 2647791.
E-mail address: [email protected] (S.F.M. Bhatti).
0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.01.052
dogs, and cats. However, the pathogenesis differs
among these species. Acromegaly in humans and cats is
commonly caused by a somatotroph adenoma of the
pituitary gland [1], whereas in dogs the GH excess
usually originates from an extra-pituitary site [2]. In
dogs, endogenous progesterone secreted during the
luteal phase of the estrous cycle or exogenous
progestins such as medroxyprogesterone acetate
(MPA) used for estrus prevention may promote
798
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
hypersecretion of GH from foci of hyperplastic ductular
epithelium of the mammary gland [2–4]. In contrast to
the pulsatile secretion pattern of GH in healthy dogs [5–
7], the plasma GH profile in bitches with progestininduced acromegaly is not pulsatile [8]. In addition,
progestin-induced GH hypersecretion cannot be stimulated with GH-releasing hormone (GHRH) and aadrenergic agonists, nor can it be inhibited by
somatostatin [8,9]. The progestin-induced increase in
plasma GH concentrations are associated with
increased plasma concentrations of insulin-like growth
factor-I (IGF-I) [2].
The physical changes of progestin-related hypersomatotropism in dogs tend to develop gradually and
consist of prominent skin folds, abdominal distension,
and widening of the interdental spaces [10]. Due to the
insulin-antagonistic action of GH, hyperglycemia and
eventually diabetes mellitus may occur [11]. Ovariectomy is the treatment of choice in female dogs with
spontaneous progesterone-induced acromegaly. Plasma
GH concentrations rapidly return to normal after
ovariohysterectomy [12]. However, in dogs with
acromegaly due to progestin administration the detrimental effects of the depot progestins may continue for
a long time after cessation of administration [10,13].
Progesterone receptor blockers such as aglépristone
(RU 46534) and mifepristone (RU 38486) are competitive antagonists of the progesterone receptor [14,15].
Aglépristone is the first progesterone receptor blocker
licensed for veterinary use and has been used efficiently
to terminate pregnancy [16,17] and to induce parturition
[18]. Furthermore, it is successfully used for the
treatment of fibroadenomatous mammary hyperplasia
in cats [19–21] and may be a useful adjunct in the
medical treatment of endometritis and pyometra in the
dog [22].
The presence of progesterone receptors in mammary
gland tissue of dogs [23] allows for a targeted endocrine
therapy with progesterone receptor blockers in dogs
with progestin-induced hypersomatotropism. The aim
of this study was therefore to investigate whether the
progesterone receptor antagonist aglépristone can be
used to treat canine acromegaly.
2. Materials and methods
2.1. Dogs
Five intact Beagle bitches were housed with outdoor
access, fed on a commercial dog food once a day, and
given water ad libitum. The ages and body weights of
the dogs ranged from 3 to 9 years (mean 5 years) and 9.0
to 10.3 kg (mean 9.5 kg), respectively. The dogs were
accustomed to the laboratory environment and procedures such as collection of blood samples.
2.2. Treatments
The five Beagle bitches were treated with the
synthetic progestin depot preparation Depo-Promone1
(medroxyprogesterone acetate (MPA), Pharmacia Animal Health, Puurs, Belgium). MPA treatment was
started during anestrus and consisted of subcutaneous
injections in a dosage of 10 mg/kg body weight at 4week intervals for a total of 14 (three dogs) or 15 (two
dogs) administrations.
Five (=day 0) and six days (=day 1) after the last
MPA administration (=day 5), aglépristone (Alizin1,
Virbac Animal Health, Barneveld, The Netherlands)
was administered subcutaneously in a dosage of 10 mg/
kg body weight. One (=day 8), two (=day 15), and three
(=day 22) weeks later a single aglépristone treatment
was given in the same dose. Three randomly chosen
dogs received the first aglépristone treatment after the
14th MPA administration, and the other two dogs after
the 15th MPA administration so that these two dogs
could serve as control dogs for the three dogs that
received the aglépristone treatment first.
2.3. Blood sample collection
Blood samples for determination of the plasma
progesterone concentrations were collected 5 and 12
months after the start of the MPA treatment.
Blood samples for determination of the plasma
concentrations of GH and IGF-I were collected before
MPA treatment, at days 9, 8, 7, 5, 3, 2, 1,
and 0 (=immediately before aglépristone treatment and
after MPA treatment for over 1 year), at days 1, 3, 5, 7, 8,
11, 13, 15, 18, 20, 22, and 25 (=during aglépristone
treatment), and at days 46 and 60 (=3.5 and 5.5 weeks
after the last aglépristone treatment). On days of
treatment (MPA or aglépristone), blood samples were
collected prior to the drug administration.
Blood samples for determination of the pulsatile
plasma profiles of GH were collected at 15-min intervals
between 08:00 and 14:00 h before MPA administration,
before aglépristone administration, and 1 week after the
last administration of aglépristone (at day 28).
All blood samples were collected by jugular
venipuncture after an overnight fast, immediately
transferred to ice-chilled EDTA-coated tubes and
centrifuged at 4 8C for 10 min. Plasma was stored at
25 8C until assayed.
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
2.4. Assays
Plasma progesterone concentrations were determined with a previously validated radioimmunoassay
(RIA) [24]. The intra-assay and inter-assay coefficients
of variation were 8.8 and 7.1%, respectively. The
sensitivity of the assay was 0.005 ng/L.
Plasma GH concentrations were measured using a
commercially available RIA for porcine and canine GH
(PGH-46HK; Linco Research, St. Charles, MS). The
intra-assay coefficient of variation was 7.6% at a plasma
concentration of 4.4 mg/L. The sensitivity of the assay
was 1 mg/L.
Total plasma IGF-I was measured after acid–ethanol
extraction to remove interfering IGF binding proteins.
Plasma IGF was extracted using a mixture of 87.5% (v/
v) ethanol and 12.5% 2 M formic acid. Tubes containing 100 mL plasma and 400 mL of the ethanol–formic
acid mixture were mixed thoroughly and incubated for
30 min at room temperature. After centrifugation for
30 min at 5500 g at 4 8C, a 50 mL aliquot of the
supernatant was diluted 1:50 with assay buffer containing 63 mM Na2HPO4 (pH 7.4), 13 mM Na2EDTA, and
0.25% (w/v) BSA. The extraction efficiency amounted
to 92.5 5.7%. IGF-I concentrations were measured in
a heterologous RIA validated for the dog [25]. The intraassay coefficient of variation was 8.6% at a plasma
concentration of 100 mg/L. The sensitivity of the assay
was 10 mg/L. IGF-I antiserum AFP4892898 and human
IGF-I for iodination were obtained from the National
Hormone and Peptide Program (Harbor-UCLA Medical
Center, Torrance, CA).
2.5. Data processing and statistical analysis
To study the effect of MPA administration, the
plasma GH and IGF-I concentrations before and after
MPA treatment were compared using a mixed model
with dog as random effect and period (two levels: before
and after MPA treatment) as categorical fixed effect.
In order to assess the overall effect of aglépristone on
the plasma GH and IGF-I concentrations, a mixed
model was fitted with dog as random effect and period
(three levels: immediately before aglépristone, during
aglépristone, and 3.5 and 5.5 weeks after the last
aglépristone treatment) as categorical fixed effect. The
three periods were compared pair wise using Tukey’s
multiple comparisons technique.
To study the evolution of the GH and IGF-I
concentrations during the aglépristone period, a mixed
model was fitted with dog as random effect and time
799
since start of aglépristone treatment as continuous fixed
effect at a global significance level of 5%.
The plasma GH and IGF-I concentrations before MPA
treatment were compared with the concentrations 3 days
after the last aglépristone treatment (i.e. at day 25) using a
mixed model with dog as random effect and period (two
levels: before MPA treatment and 3 days after the last
aglépristone treatment) as categorical fixed effect.
To evaluate the effect of withdrawal of aglépristone
treatment, the two last measurements during aglépristone treatment (days 22 and 25) were compared with the
two measurements after aglépristone treatment (days 46
and 60) using a mixed model with dog as random effect
and period (two levels: days 22 and 25, and days 46 and
60) as categorical fixed effect.
The 6-h plasma profiles of GH were analyzed by
means of the Pulsar program developed by Merriam and
Wachter [26]. The program identifies secretory peaks by
height and duration from a smoothed baseline, using the
assay SD as a scale factor. The cut-off parameters G1–
G5 of the Pulsar program were set at 3.98, 2.40, 1.68,
1.24, and 0.93 times the assay SD as criteria for
accepting peaks 1, 2, 3, 4, and 5 points wide,
respectively. The smoothing time, a window used to
calculate a running mean value omitting peaks, was set
at 5 h. The splitting cut-off parameter was set at 0.5 and
the weight assigned to peaks was 0.05. The A-, B-, and
C-values of the Pulsar program, used to calculate the
variance of the assay, were set at A = 0, B = 7.2, and
C = 5. The values extracted from the Pulsar analysis
included the mean of the smoothed baseline, the pulse
frequency, and the area under the curve (AUC). The
AUC for GH was calculated above the zero-level
(AUC0) as well as above the baseline (AUCbase). The
difference in variables before MPA treatment, before
aglépristone administration, and 1 week after the last
aglépristone treatment (i.e. at day 28) were analyzed by
the signed rank test with dog as block.
All values are expressed as mean S.E.M. or
median. Statistical significance was defined at
P 0.05. Analyses were performed with SAS Version
9.1 for Windows (Insightful Corp., Seattle, USA).
2.6. Ethics of the study
This study was approved by the Ethics Committee of
the Faculty of Veterinary Medicine, Ghent University.
3. Results
During MPA administration none of the dogs showed
signs of estrus and the mean plasma progesterone
800
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
Fig. 1. Mean (S.E.M.) plasma concentrations of GH (a) and IGF-I (b) in five Beagle dogs before administration of MPA (before MPA) and after 1
year treatment with MPA (after MPA). Significant differences between periods are indicated with an asterisk.
concentration was low 5 months (0.2 0.2 ng/L) and
12 months (0.2 0.1 ng/L) after the start of the MPA
treatment. The mean body weight of the dogs on the day
of the last administration of MPA (12.4 0.7 kg) was
significantly higher (P < 0.02) than that on the day of
the first MPA administration (9.5 0.3 kg) (paired
Student’s t-test). Signs of acromegaly became apparent
in three of the five dogs after 6 months of MPA
treatment and consisted of prominent skin folds
especially on the head, an increase in the interdental
spaces, inspiratory stridor, and snoring.
MPA administration for over 1 year resulted in a
higher mean plasma GH concentration (2.3 0.5 mg/L)
compared to that before MPA treatment (1.9 0.3 mg/
L), although this difference did not reach statistical
significance (Fig. 1a). However, the mean plasma IGF-I
concentration after 1 year of MPA administration
(146 25 mg/L) was significantly (P = 0.003) higher
compared to that before MPA treatment (36 6 mg/L)
(Fig. 1b). Analysis of the pulsatile plasma GH profiles
after 1 year of MPA administration revealed a trend
(P = 0.06) for a higher mean basal plasma GH
concentration and a higher mean AUC0 for GH
compared to these values before MPA treatment
(Table 1; Fig. 2).
The administration of aglépristone caused no side
effects except a short-term skin irritation at the site of
the injection in one dog. The mean plasma GH
concentration immediately before aglépristone administration (2.3 0.5 mg/L) was significantly higher than
that during (1.7 0.3 mg/L; P < 0.0001) and 3.5 and
5.5 weeks after (1.8 0.3 mg/L; P = 0.018) the last
administration of aglépristone (Fig. 3a). Also the mean
plasma IGF-I concentration immediately before aglépristone administration (146 25 mg/L) was significantly higher than that during (108 27 mg/L;
P < 0.0001) administration of aglépristone (Fig. 3b).
In the weeks when aglépristone was administered,
analysis of the course of the circulating hormone
concentrations indicated a significant decrease in
plasma GH (P = 0.005) and IGF-I (P < 0.0001)
concentrations (Fig. 4a and b, respectively).
The plasma GH and IGF-I concentrations before
MPA treatment did not differ significantly from these
concentrations 3 days after the last aglépristone
treatment (i.e. day 25).
Analysis of the pulsatile plasma GH profiles revealed
a trend (P = 0.06) for a lower mean basal plasma GH
concentration and a lower mean AUC0 for GH 1 week
after the last aglépristone administration (i.e. day 28)
compared with these concentrations before aglépristone
treatment. The AUCbase for GH increased again after the
last aglépristone treatment compared with this concentration before aglépristone administration, although
Table 1
Area under the curve for GH above the baseline (AUCbase) (mg/L 6 h), AUC for GH above the zero-level (AUC0) (mg/L 6 h), GH pulse
frequency (peaks per 6 h), and basal plasma GH concentration (mg/L) in five Beagle dogs before MPA administration (before MPA), after 1 year of
MPA administration (=before aglépristone administration) (before A), and 1 week after the last aglépristone administration (after A)
Before MPA
Before A
After A
AUCbase
(mean S.E.M.)
AUC0
(mean S.E.M.)
GH pulse frequency
(median)
Basal GH
(mean S.E.M.)
1 0.4
0 0.0
0.3 0.2
11.3 1.1
17.0 3.6
8.8 0.7
1
0
0
1.7 0.1
2.8 0.6
1.4 0.1
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
801
was significantly (P < 0.0001) lower compared with
that 3.5 and 5.5 weeks after withdrawal of aglépristone
(124 29 mg/L).
In the two control dogs that did not receive
aglépristone after the 14th MPA administration, the
mean plasma concentrations of GH and IGF-I before the
14th administration of MPA (1.6 0.1 and
105 28 mg/L, respectively) were not different from
those before the 15th administration of MPA (1.7 0.1
and 116 26 mg/L, respectively).
Fig. 2. The plasma profiles of GH in a 4-year-old Beagle bitch. Blood
samples were collected at 15-min intervals between 08:00 and
14:00 h, before MPA administration (&), after 1 year of MPA
administration (=before aglépristone administration) (dotted line),
and 1 week after the last aglépristone treatment (&). Significant
pulses, calculated by the Pulsar program, are indicated by an asterisk.
this difference did not reveal statistical significance
(Table 1; Fig. 2).
The mean plasma GH concentration at the end of the
period of aglépristone administration (i.e. days 22 and
25) (1.5 0.1 mg/L) did not differ significantly from
that at 3.5 and 5.5 weeks (i.e. days 46 and 60) after
withdrawal of aglépristone (1.8 0.3 mg/L). However,
the mean plasma IGF-I concentration at the end of the
period of aglépristone administration (88 22 mg/L)
4. Discussion
In three of the five Beagle dogs, signs of acromegaly
became apparent after 6 months of MPA administration.
In line with these changes, the mean plasma IGF-I
concentrations were raised. Moreover, analysis of the
pulsatile plasma profile showed a trend for a higher
mean basal GH concentration and a higher mean AUC0
for GH in the five Beagle dogs. These findings are
consistent with progestin-induced hypersecretion of GH
[3,27–29].
Previous studies identified foci of hyperplastic
ductular epithelium of the mammary gland as the site
of origin of GH excess induced by progestins [2,4]. The
expression of the gene encoding GH has been
Fig. 3. Mean (S.E.M.) plasma concentrations of GH (a) and IGF-I (b) in five Beagle dogs immediately before aglépristone (before A), during
aglépristone (during A), and 3.5 and 5.5 weeks after the last aglépristone administration (after A). Significant differences between periods are
indicated with an asterisk.
Fig. 4. Mean (S.E.M.) plasma concentrations of GH (a) and IGF-I (b) in five Beagle dogs at 1 week, 2 weeks, 3 weeks, and at day 25 during
aglépristone administration.
802
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
demonstrated in canine mammary gland tissue, and
sequence analysis has revealed that this gene is identical
to the pituitary GH gene [30,31]. Immunohistochemical
studies have demonstrated the intracellular co-localization of both the progesterone receptor and GH in
progestin-exposed, hyperplastic canine mammary
epithelial tissue, whereas immunoreactive GH could
not be demonstrated in progesterone receptor-negative
epithelial cells [23]. These observations are consistent
with the central role of progestins in GH gene
expression in the canine mammary gland and allow
for a targeted endocrine therapy with progesterone
receptor blockers in dogs with progestin-induced
mammary-derived GH hypersecretion.
To the authors’ knowledge, treatment of acromegalic
dogs with the progesterone receptor antagonist aglépristone (RU 46534) has not been reported before. The
results of the present study demonstrate that progestininduced elevations in circulating GH and IGF-I
concentrations decrease significantly during treatment
with aglépristone. At the end of the aglépristone
treatment period the plasma GH and IGF-I concentrations did not differ significantly from those before MPA
administration. Our findings are in agreement with
those of Watson et al. [8] who found that administration
of the antiprogestin mifepristone (RU 38486) resulted in
a decrease of plasma GH concentrations and normalization of plasma IGF-I concentrations in bitches with
progestin-induced acromegaly.
The mean basal plasma concentrations of GH and
IGF-I in the two control dogs that did not receive
aglépristone after the 14th MPA administration
remained high and did not decrease. This indicates
that indeed aglépristone is responsible for the lowering
of the plasma GH and IGF-I concentrations in the dogs
treated with the progesterone receptor blocker, and that
this lowering is not due to, for example, a waning effect
of MPA on GH and IGF-I secretion.
The 6-h pulsatile plasma profile of GH represents a
more sensitive way of analyzing the effects of different
treatments on the secretion of GH than the plasma GH
concentration itself. Analysis of the plasma GH profiles
revealed that the mean basal plasma GH concentration
and AUC0 for GH tended to decrease at the end of the
treatment with the progesterone receptor blocker
compared with these values before aglépristone
administration. In addition, the AUCbase for GH, i.e.,
the amount of GH secreted in pulses, increased again
during aglépristone treatment, although this difference
did not reveal statistical significance. Thus, treatment
with aglépristone resulted in partial restoration of the
normal pulsatile GH secretion. Higher dosages of
aglépristone may result in complete normalization of
the secretion pattern of GH.
Plasma IGF-I concentrations are generally regarded
as more sensitive indicators of the GH status than
plasma GH concentrations [32]. Consequently, the
significantly higher plasma IGF-I concentrations at days
46 and 60 compared with those at days 22 and 25
therefore suggest increased GH exposure, despite the
fact that analysis of the plasma GH concentrations did
not reveal a significant increase. The recurrence of IGFI hypersecretion after withdrawal of aglépristone
treatment is not surprising as all dogs received a depot
progestin preparation for a period of 1 year, and the
progestin effect of this depot preparation is much longer
than the duration of aglépristone treatment in the
present study. Similarly, in a cat with fibroadenomatous
mammary hyperplasia due to treatment with a depot
progestin preparation hyperplasia recurred 8 days after
discontinuation of aglépristone administration [19].
This indicates that treatment with an antiprogestin is
required as long as the action of the synthetic progestin
is present. Also in our three dogs with acromegalic
signs, no physical changes were visible during or after
treatment with aglépristone.
Due to the insulin-antagonistic action of GH,
progestin-induced hypersecretion of GH may also
result in hyperglycemia and eventually manifest
diabetes mellitus may ensue [11,29]. Disappearance
of these catabolic abnormalities depends on the
functional status of the pancreatic b-cells. If an
adequate population of functional b-cells is present
at the time the progestin effect is blocked, hyperinsulinemia, carbohydrate intolerance, and hyperglycemia
may be reversible after correction of the hypersomatotropism [13]. If the population of functional b-cells is
severely decreased, permanent diabetes mellitus can be
anticipated. Because the effects of depot progestins may
persist for several months, prevention or reversal of the
catabolic effects of progestin-induced GH excess is
especially important when, in case of hyperglycemia,
the depot progestin has been administered only recently.
The results of the present study illustrate that in these
cases aglépristone offers an effective treatment option.
In conclusion, administration of aglépristone significantly decreases plasma GH and IGF-I concentrations
in dogs with progestin-induced hypersomatotropism.
Acknowledgements
The authors are grateful for the technical assistance
of Mrs. J. Wolfswinkel, Mrs. E. Sprang-Timmermans,
Mr. F. Riemers, Mr. M. Coryn, and Mr. H.G.H. van
S.F.M. Bhatti et al. / Theriogenology 66 (2006) 797–803
Engelen. This study was financially supported by
Virbac Nederland B.V., Barneveld, The Netherlands.
The critical reading of the manuscript by Dr. A.
Rijnberk is highly appreciated.
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Theriogenology 66 (2006) 1709–1714
www.journals.elsevierhealth.com/periodicals/the
Morphology of canine placental sites after
induced embryonic or fetal death
K. Steiger a,*, E. Politt b, T. Hoeftmann b, A. Meyer-Lindenberg c,
H.-A. Schoon a, A.-R. Günzel-Apel b
a
Institute of Veterinary-Pathology, University of Leipzig, An den Tierkliniken 33, 04103 Leipzig, Germany
b
Institute for Reproductive Medicine, School of Veterinary Medicine, Hanover, Germany
c
Clinic for Small Domestic Animals, School of Veterinary Medicine, Hanover, Germany
Abstract
Although spontaneous and medically induced canine embryonic or fetal death and ‘‘resorption’’ are clinically well documented,
morphological studies of these processes are still missing. The objective of this study was therefore a detailed morphological
investigation of canine placental sites after embryonic or fetal death. In five pregnant beagle bitches, embryonic or fetal death was
induced by cloprostenol and cabergoline or by aglepristone. Two dogs served as untreated controls. Between Days 30 and 33 of
gestation, the bitches were ovariohysterectomized, placental sites were fixed and examined by different methods.
Morphological features of placental sites after both treatments were similar, finally leading to a complete disappearance of the
placental labyrinth. Although there was an increase in the number of cells in the glandular chambers (superficial endometrial
glands) expressing lysozyme after induced fetal death, signs of phagocytosis were absent in these cells, and no increased infiltration
of maternal stroma by macrophages (compared to normal placental sites at the same time of gestation) occurred. We inferred that
fetal and placental tissues were lysed, but no phagocytosis by genuine or ‘‘functional’’ macrophages was detectable. Further
investigations are needed for a more detailed understanding of the morphological processes occurring after embryonic or fetal death
in the dog.
# 2006 Published by Elsevier Inc.
Keywords: Dog; Fetal death; Resorption; Morphology; Placenta
1. Introduction
The administration of prostaglandin F 21 (or its
analogue cloprostenol) has been used for decades to
terminate unwanted pregnancies in dogs [1,2]. Since
1990, several reports were published about the
administration of progesterone-receptor antagonists,
such as mifepristone [3] or aglepristone [4,5] to induce
embryonic or fetal death in dogs. Independent of the
* Corresponding author. Tel.: +49 341 97 38 270;
fax: +49 341 97 38 299.
E-mail address: [email protected] (K. Steiger).
0093-691X/$ – see front matter # 2006 Published by Elsevier Inc.
doi:10.1016/j.theriogenology.2006.01.046
time of gestation at which such treatment starts, there
are still different opinions whether the use of luteolytic
agents (sometimes in combination with a dopamine
agonist) or antigestagenic drugs results in abortion
(defined as the expulsion of a conceptus incapable of
independent life) [2,6] or in a so-called resorption (loss
of the embryonic or fetal structure, without expulsion of
embryonic or fetal or placental components) [7,8]. To
our knowledge, there are no morphological studies on
placental and/or endometrial changes after induction of
embryonic or fetal death in dogs by those drugs. There
is only one ultrastructural study of decidual alterations
early after administration of mifepristone [9] and a
number of morphological investigations into decidual
1710
K. Steiger et al. / Theriogenology 66 (2006) 1709–1714
Fig. 1. Normal morphology of canine placenta on Day 30 of gestation: chorioallantoic membrane (C), placental labyrinth (PL) (inset),
necrosis zone (N), glandular chambers (GC), haemophagus zone (H),
supraglandular layer (S), deep endometrial glands (GD) and myometrium (M) (inset: H.–E.-stain; magnification 10).
alterations after intra- [10,11] or extra-amniotic
applications of prostaglandin [12] in women. After
induction of a second trimester abortion by extraamniotic administration of prostaglandins in humans,
no specific lesions of the placenta or decidua [12] were
observed.
Light-microscopic and ultrastructural morphology
of the canine placenta during mid-gestation was
described by several authors [13–15]. The canine
zonary placenta consists of the chorioallantoic membrane, the placental labyrinth (the area of direct
fetomaternal interaction, with fetal chorionic villi
contacting maternal blood vessels), the necrosis zone
(which develops earlier during gestation due to erosion
of the maternal epithelium and connective tissue by the
terminal segments of the syncytiotrophoblast), the
maternal glandular chambers (also named spongy zone,
which develops from the superficial endometrial
glands), the supraglandular layer (formed by connective
tissue) and the deep endometrial glands (Fig. 1). A
hemophagus zone develops at both edges of the
placental labyrinth (Fig. 1).
The objective of the present study was to investigate
the morphology of the canine placental labyrinth and
the glandular chambers after experimentally induced
embryonic or fetal death, in comparison to normal
placental morphology at corresponding stages of
gestation, and with special emphasis on the character
of processes after embryonic or fetal death.
2. Material and methods
In five clinically healthy pregnant beagles (between
1 and 3 yr of age) embryonic/fetal death was induced by
administration of luteolytic substances. Two dogs were
given 1 mg/kg of cloprostenol subcutaneously on Days
24 and 27 (ovulation = Day 0) and 5 mg/kg cabergoline
given per os once daily from Days 24 to 30. Three dogs
were given 10 mg/kg of aglepristone (an antigestagenic
drug) subcutaneously on Days 24 and 25. Two pregnant
beagles served as untreated controls. Starting on Day
20, all dogs were submitted to daily sonographic
examinations for assessment of embryonic or fetal
development and death, respectively. The dogs were
ovariohysterectomised between Days 30 and 34 of
gestation. As most of the cell differentiation and
organogenesis in the dog is complete by Day 30 of
gestation, the conceptuses removed will be referred to
as fetuses in this study. Numbers and sizes of
placentation sites and their localizations within the
uterine horns were recorded. For light-microscopical
investigations, placental sites (n = 41) were fixed in 4%
neutral-buffered formalin and embedded in paraplast.
Histological slides were stained with Hematoxylin–
Eosin (H.–E.). Additionally, enzyme histochemistry
[naphthol-AS-D chloracetate-esterase (CLAE) and
alpha-naphthylacetate-esterase (ANAE), acidic and
alkaline phosphatase] was conducted. For immunohistochemistry, primary monoclonal antibodies against Ki67 antigen and myeloid histiocytes as well as polyclonal
antibodies against lysozyme were used and routine
immunohistochemical techniques with a peroxidase–
anti peroxidase complex and diaminobenzidine (DAB)
as chromogen were followed, counterstained with
Papanicolaou’s technique. The antibodies used were
as follows: anti-Ki-67 antigen, clone MM1, Medac
GmbH, Hamburg, Germany; anti-myeloid histiocyte
antigen, clone MAC 387, Dako Diagnostika GmbH,
Hamburg, Germany; anti-lysozyme (Dako Diagnostika). Additionally, three samples from each placental
site (placental labyrinth, glandular chambers and deep
endometrial glands) were fixed in glutaraldehyde (3%)
and embedded in glycidether 100 for electronmicroscopy. In 0.3 mm (toluidinblue) slides, the area of
interest was selected, from which 60 nm ultra-thin
slides were cut, contrasted with uranyl-acetate and lead
citrate and examined by transmission-electron microscopy.
3. Results
Fetal death, especially visible as lack of fetal
heartbeat, was detected sonographically in 17 placentation sites of the five treated dogs, whereas 10 fetuses in
four of the dogs were alive on the day of ovariohysterectomy (Table 1). One of the two untreated controls
showed spontaneous fetal death in 3 of 11 placentation
K. Steiger et al. / Theriogenology 66 (2006) 1709–1714
1711
Table 1
Day (ovulation = Day 0) of ultrasonographic detection of fetal death in individual placentation sites in bitches given cloprostenol and cabergoline
treatment beginning on Day 24 or aglepristone on Days 24 and 25
Placentation
site number
1
2
3
4
5
6
7
8
9
10
11
Day of
ovariohysterectomy
a
Cloprostenol and cabergoline
Aglepristone
Dog 1
Dog 3
a
23
Alive
33
32
Alive
–
–
–
–
–
–
33
Dog 2
34
31a
Alive
Alive
–
–
–
–
–
–
–
34
29
28a
28
29
28
28
–
–
–
–
–
32
Untreated controls
Dog 4
Dog 5
Dog 6
Dog 7
Alive
Alive
Alive
30
28a
Alive
–
–
–
–
–
30
a
Alive
Alive
Alive
–
–
–
–
–
–
–
–
31
21 a
Alive
Alive
Alive
24 a
Alive
Alive
28
Alive
Alive
Alive
30
27
33
32
Alive
26a
Alive
–
–
–
–
–
33
No fetus or remnants of fetal material were visible at hysterectomy.
sites (Table 1). In six placentation sites of treated dogs,
no fetal structures were detectable macroscopically
(Table 1); only slight to moderate amounts of a
brownish, gelatinous material was present. Such areas
were frequently neighbouring intact placentation sites
with an apparently normal conceptus.
Maternal capillaries were lined by at least three
endothelial cells, with a considerable amount of
cytoplasm and large numbers of membranous organelles. The necrosis zone consisted of small amounts of
cellular debris between the placental labyrinth and the
glandular chambers.
3.1. Normal placental morphology during midgestation
3.1.2. Glandular chambers
The epithelial cells of the glandular chambers were
high columnar with light to moderate intracytoplasmatic vacuolisation. Single epithelial cells were
degenerated and desquamated into the necrosis zone
especially in the apical region and in the periphery of
chambers. Additionally, some syncytial cells and single
giant cells with one or two large nuclei occurred within
the epithelial layer. Some macrophages in the interstitium of the glandular chambers contained reaction
products of enzymes investigated histochemically
(acidic and alkaline phosphatase, CLAE, ANAE). In
single epithelial cells of the glandular chambers, only
reaction products of acidic phosphatase were detected.
Intracytoplasmatically, they expressed lysozyme moderately. Regarding ultrastructure, some epithelial cells
of the glandular chambers had apical protrusions
covered by fine, short microvilli. These protrusions
contained some round to oval (cristae-type) mitochondria and a few mucous vesicles, some of which fusing
with the surface cell membrane. There were some welldeveloped Golgi complexes and rough endoplasmatic
reticulum (rER), and some lipid droplets in the infraand supranuclear cytoplasm. The euchromatin-rich
nucleus was round to slightly oval. Individual cells,
mainly in apical regions of the glandular chambers
The histomorphology of the placentation sites in
untreated controls (in Dog 7, only those with a living
fetus at the day of ovariohysterectomy) corresponded to
physiological histological placental appearance during
mid-gestation, as described by other authors [13–15].
3.1.1. Placental labyrinth
Fetal capillaries with one or two thin endothelial
cells and a few organelles were embedded in fetal
connective tissue. In the single-layered flat to cuboidal
cytotrophoblast (the inner layer of the fetal trophoblast
with sparse cytoplasm and an oval, heterochromatinrich nucleus), single cells proliferated. Cells of the
syncytiotrophoblast had moderate amounts of an
eosinophilic cytoplasm and a heterochromatin-rich
nucleus, sometimes with a distinct nucleolus. Very
few cells showed signs of pyknosis. Between the
cytotrophoblastic cells as well as the cytotrophoblast
and the syncytiotrophoblast, desmosomes (approximately 6/intercellular contact) were present ultrastructurally. The intercellular spaces between cyto- and
syncytiotrophoblast were narrow (150–500 nm). Maternal decidual cells were not evident ultrastructurally.
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K. Steiger et al. / Theriogenology 66 (2006) 1709–1714
showed moderately electron-dense cytoplasm with high
apical protrusions, filled with mucus containing
vacuoles.
3.1.3. Endometrial stroma, deep endometrial
glands and myometrium
Variable (slight to moderate) infiltration of the
placental stroma by macrophages occurred in all
placental sites. There were no differences between
controls and treated dogs in endometrial stroma, deep
endometrial glands and myometrium, as assessed with
light microscopy.
3.2. Alterations of placental morphology in treated
dogs
The number of macrophages infiltrating the maternal
stroma did not increase after treatment.
3.2.1. Placental labyrinth
Quality and degree of alterations in the placental
sites of treated dogs depended on the time after fetal
death. Morphological alterations, however, after both
treatments were similar, but they occurred somewhat
earlier in dogs that had received aglepristone. In most
placentation sites (n = 3), where fetal death had been
detected sonographically on the day of ovariohysterectomy, the syncytiotrophoblast detached multifocally
from the cytotrophoblast and fibrinous extravasations
occurred in the resulting spaces (Fig. 2a). Within these
areas of detachment, the number of cytotrophoblast
cells expressing Ki-67 increased slightly (from 4/high
power field (HPF) in the normal placenta to 8/HPF). In
two of these altered placentation sites, extended areas
of coagulation necrosis were also observed, but no
thromboses were found in fetal or maternal blood
vessels. Under light microscopy, those parts of the
placental labyrinth being not affected by detachment
corresponded to that of the normal placenta; only the
number of proliferating cytotrophoblastic cells (Ki-67
antigen) increased (to 6 per HPF). However, ultrastructurally, intercellular spaces between cyto- and
syncytiotrophoblast were widened (up to 2 mm;
Fig. 2c). They were empty or contained small amounts
of slightly electron-dense granular material. Additionally, no or only a few desmosomes occurred
between the two cell types. Cytoplasm, organells and
nuclei of the endothelial cells of the maternal
capillaries were moderately swollen. In those placentation sites where fetal death was diagnosed by
sonography 2–4 d before ovariohysterectomy, the
number of cytotrophoblast cells expressing Ki-67
antigen obviously increased (up to 20/HPF), and
the cytotrophoblast was multifocally multilayered
(Fig. 2b). The cytoplasm of the underlying syncytiotrophoblast contained moderate amounts of very small
eosinophilic clods and vacuoles, nuclei were hyperchromatic with signs of pyknosis or karyorrhexis. Due
to degeneration and necrosis of the syncytiotrophoblast, the placental labyrinth was thinned. Because of
Fig. 2. (a) Detachment of syncytiotrophoblast (arrows) from cytotrophoblast (arrowheads) in a canine placental site where the fetus had died on the
day of ovariohysterectomy (H.–E.-stain; magnification 10); (b) proliferation of cytotrophoblast (arrowheads) and degeneration of syncytiotrophoblast 3 d after fetal death (H.–E.-stain; magnification 20); (c) loss of desmosomes and extended intercellular spaces (arrow) between cyto(CT) and syncytiotrophoblast (ST) in a placental site where the fetus had died on the day of ovariohysterectomy (transmission electron microscopy;
magnification 3000).
K. Steiger et al. / Theriogenology 66 (2006) 1709–1714
Fig. 3. Pleomorphic glandular chambers in a canine placenta 3 d after
fetal death, with numerous epithelial cells being desquamated
(arrows) into the necrosis zone (N) that extends into the lumina of
the glandular chambers (H.–E.-stain, magnification 20). Inset:
intracytoplasmatic lumen (L) lined by short microvilli (arrow) within
epithelial cells of glandular chambers after fetal death (transmission
electron microscopy, magnification 3000).
excessive cellular shrinkage in two of the placentation
sites, the two trophoblast cell populations were not
distinguishable. These phenomena were accompanied
by an increased amount of cellular debris in the
necrosis zone, reaching deep into the lumina of
glandular chambers. In three of four placental sites
where fetal death had been diagnosed more than 5 d
before ovariohysterectomy and in two placental sites
4 d after fetal death, no placental labyrinth was
detectable. However, great amounts of cellular debris
from the necrosis zone were noted on and within the
lumina of glandular chambers.
3.2.2. Glandular chambers
In placental sites with a living or recently deceased
fetus, epithelial cells of the glandular chambers had a
moderate increase in cytoplasmic vacuolisation and
higher numbers and quantities of apical protrusions
(visible already by light microscopy). As for ultrastructure, the cytoplasm of glandular epithelia contained very distinct rER and very numerous apical
vacuoles, mostly filled with mucous material, as well
as many small lipid droplets. Compared to controls,
the number of epithelial cells containing lysozyme
increased very slightly. In addition, the enzyme
appeared within the apical protrusions of several
cells. Glandular epithelia of those placentation sites,
where the fetus had died 2 d or more before
ovariohysterectomy, had a distinct pleomorphy and
the number of cells degenerating and desquamating
into the necrosis zone had clearly increased (from 20/
HPF in the controls up to 50/HPF; Fig. 3). Several
large intracytoplasmatic vacuoles, ultrastructurally
1713
corresponding to intracytoplasmatic lumina, lined
by short microvilli, represented a special finding in
the epithelia of the glandular chambers (Fig. 3).
Lysozyme was distinctly expressed at the cytoplasmatic borders of the vacuoles, and in total, the number
of lysozyme expressing cells increased obviously
compared to the controls. After the complete
disappearance of the placental labyrinth (i.e., after
about 5 d or more after fetal death), the number of
cells with large intracytoplasmic vacuoles decreased
again. No secondary and/or tertiary lysosomes were
detectable (besides single myelinosomes) in some of
the epithelial cells. By enzyme histochemistry, there
were no differences between the glandular chambers
of treated and untreated control dogs. The morphology
of the placentation sites of Dog 7 (control) with
spontaneous occurring fetal death corresponded to that
described after induced fetal death, but the alterations
seemed to proceed at a slower rate.
4. Discussion
Both protocols used for pregnancy termination in
this study caused similar morphologic changes of the
placentation sites. In humans, it is suggested that
antiprogestins induce an increased production of
prostaglandins in the decidua (due to reduced
concentrations of prostaglandin dehydrogenase in
these cells) [9,16,17]. Taking this into account, similar
placental alterations observed in this study might have
been caused by comparable mechanisms, i.e. a
decreased blood supply due to prostaglandins inducing
hypoxic degeneration of the syncytiotrophoblast.
Coagulation necrosis observed in two of the placentation sites early after fetal death was morphologically
consistent with infarcts, but no thromboses were
visible within placentation sites investigated, neither
in fetal nor in maternal blood vessels. The edematous
swelling of the maternal endothelium detected in this
investigation was the only morphological alteration of
blood vessels that might contribute to hypoxia. The
first placental morphological lesion after treatment
(before fetal death) was a loosening of intercellular
contacts between cyto- and syncytiotrophoblast.
Afterwards, if cells do not separate immediately
(and undergo coagulation necrosis), the syncytiotrophoblast degenerates, and reactively, the cytotrophoblast proliferates. Those parts of the placental
labyrinth that are not desquamated into the necrosis
zone had degeneration with cellular shrinkage, finally
leading to vanishing of the labyrinth. The distinct rER,
the high number of secretory vacuoles and the
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K. Steiger et al. / Theriogenology 66 (2006) 1709–1714
intraepithelial lumina in the epithelial cells of the
glandular chambers are signs of increased secretory
activity in treated dogs (beginning early after
treatment, before fetal death) compared to findings
in the normal canine placenta during mid-gestation
[15] and to the controls. Grether et al. [15] assumed
that mucus, secreted by glandular chambers, might be
harmful or even toxic to the trophoblast. Increased
production of glandular secretions after medication
might thus be conducive to the destruction of the
placental labyrinth. Enzymes secreted by the epithelial
cells of glandular chambers may contribute to this.
Enzyme histochemistry, however, did not reveal any
differences between treated dogs and controls. Only
the production of lysozyme (a ubiquitous enzyme
splitting up mucopolysaccharides and mucopeptides)
rises in glandular chambers after medication. The
functional relevance of the lysozyme secretion in the
controls and in the treated dogs as well remains
unclear, because this enzyme specifically destroys the
murein in bacterial cell walls. As bacterial infection
appears to be impossible during our experiments, the
secretion of lysozyme may represent just a nonspecific endometrial reaction during gestation with an
increase after induced fetal death. It remains to be
clarified, whether other enzymes that might destroy
fetal and/or placental material are co-secreted with
lysozyme.
That the numbers of macrophages infiltrating the
placental structures do not increase after fetal death,
may lead to the conclusion that phagocytosis by
genuine or functional macrophages does not play a
major role in the disappearance of the dead fetuses.
Additionally, no indications of active phagocytosis (i.e.
lysosomes) or any other kind of uptake were detected
(i.e. intercellular absorption) in the placental and
glandular cells investigated. However, abortion, i.e. the
expulsion of the whole conceptus (fetus together with
fetal membranes) can probably be excluded, because
affected placentation sites were usually neighbouring
intact ones.
In summary, active secretion of glandular epithelia
and degeneration of the placental structures, not
phagocytosis, appeared to be the major morphological
incidents after experimentally induced and spontaneous fetal death in dogs. However, additional
immunohistochemical and electronmicroscopical
investigations have to be carried out for a more
detailed understanding of morphological processes
after fetal death in dogs.
References
[1] Concannon PW, Hansel W. Prostaglandin F2alpha induced
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[3] Concannon PW, Yeager A, Frank D, Iyampillai A. Termination
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[6] Fieni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for
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[7] Onclin K, Verstegen JP. Practical use of a combination of a
dopamine agonist and a synthetic prostaglandin analogue to
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[8] Onclin K, Verstegen JP. Comparisons of different combinations
of analogues of PGF2 alpha and dopamine agonists for the
termination of pregnancy in dogs. Vet Rec 1999;144:416–9.
[9] Schindler AM, Zanon P, Obradoviv D, Wyss R, Graff P, Herrmann WL. Early ultrastructural changes in RU-486-exposed
decidua. Gynecol Obstet Invest 1985;20:62–7.
[10] Puri S, Aleem F, Schulman H. A histologic study of the placentas
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Placental histopathology of midtrimester termination. Obstet
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placenta and decidua after induction of abortion by extraamniotic prostaglandin. Histopathology 1978;2:145–51.
[13] Anderson JW. Ultrastructure of the placenta and fetal membranes of the dog. Anat Rec 1969;165:15–36.
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[15] Grether BM, Friess AE, Stoffel MH. The glandular chambers of
the placenta of the bitch in the second third of pregnancy (day
30–44): an ultrastructural, ultrahistochemical and lectinhistochemical investigation. Anat Histol Embryol 1998;27:95–103.
[16] Cheng L, Kelly RW, Thong KJ, Hume R, Baird DT. The effect of
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Animal Reproduction Science 60–61 Ž2000. 295–312
www.elsevier.comrlocateranireprosci
Receptor blockers — general aspects with respect
to their use in domestic animal reproduction
Bernd Hoffmann ) , Gerhard Schuler
Klinik fur
und Andrologie der Groß-und Kleintiere mit Tierarztlicher
Ambulanz der
¨ Geburtshilfe, Gynakologie
¨
¨
Justus-Liebig-UniÕersitat
¨ Giessen, Frankfurter Strasse 106, D-35392 Giessen, Germany
Abstract
Receptor blockers compete with the respective agonist for binding to a given receptor without
inducing complete signal transduction. In recent years, major interest has focused on sex-steroid
hormone receptor blockers Žantagonists.. Indications have been obtained that inadequate changes
in receptor conformation and subsequent failure of transcriptional activation are major events
preventing hormonal activity. However, various subtypes and variants of receptors and receptor
mutations have also been identified. Expression of antihormonal effects may vary depending on
the type of receptor the blocker is bound to. Hence, receptor blockers may also have an inherent
agonistic activity. Aglepristone w is the first antiprogestin registered for veterinary use with the
indication ‘‘interruption or prevention of pregnancy’’; similarly, these types of compounds were
successfully used for induction of parturition in the dog and cat and for conservative treatment of
pyometra in the dog. Moreover, application of antiprogestins has clearly demonstrated the role of
progesterone as a major factor controlling overt pseudopregnancy in dogs. With respect to farm
animals, parturition was induced in cows without an increased incidence of retained fetal
membranes. Other than antiprogestins, antioestrogens and antiandrogens are still in a more
experimental phase. In particular for use in humans, high-affinity blockers binding to the
oxytocinrvasopressin receptor are in development; they exert distinct tocolytic activities. Also,
the release of GnRH can be inhibited by respective antagonists; however, their use in reproduction
is still hampered by the high dose requirement and the side effects observed. q 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Hormone antagonists; Receptors; Sex steroids; Domestic animals
)
Corresponding author. Tel.: q49-641-993-8704; fax: q49-641-29-328.
E-mail address: [email protected] ŽB. Hoffmann..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 1 2 9 - 9
296
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
1. Introduction
The removal of endocrine organs and study of the resulting effects has been a
classical approach in endocrine research. However, by such an operation in general not
only one but a whole array of hormonal factors is removed. Thus the observed responses
may be of multifactorial origin. In order to overcome this problem, more specific
approaches were followed in the past, like active and passive immunisation against
specific hormones, the development of competitive enzyme blocking agents, development of neurotransmitter-like agents blocking the release of a distinct hormone Žsee
Concannon et al., 1987., development of competitive receptor blockers Žsee below. and
— as the latest addition — the targeted disruption of genes encoding for mediators or
the respective receptors in knockout models.
This paper deals with receptor blockers and their use in domestic animal orientated
basic research, biotechnology and therapy. By taking into account data from own
studies, main attention will be given to those agents blocking the activity of sex steroid
hormones. However, reference will also be made to those agents blocking the activity of
peptide hormones of hypothalamic origin.
2. Steroid hormone receptor blockers
Knowledge about the mechanisms of action of receptor blockers for steroid hormones
is still incomplete. They exert their activity by binding to the steroid hormone receptor
without inducing transcription. The understanding of this phenomenon is based on the
knowledge of the processes induced by hormone–receptor interaction. Steroid hormones
are small lipophilic hormones able to pass the membranes of target cells. They bind to
intracellularly located receptor proteins and after decades of controversial discussion
there is now overwhelming evidence that these receptors are basically located in the
nucleus ŽYamashita, 1998.. Nuclear hormone receptors represent an evolutionary conserved class of transcription factors being present from flies to mammals Žfor review see
Beato et al., 1995.. According to the hormone they bind to, these receptors can be
classified into those binding to steroids Že.g. progestins, androgens, oestrogens., steroid
derivates Žvitamin D3., nonsteroids Že.g. thyroid hormone. and receptors for which no
ligands have been found yet Žorphan receptors.. Hormone binding leads to a conformational change, resulting in specific binding to palindromic DNA sequences Žhormone
responsive elements, HREs. as liganded receptor dimers ŽGronemeyer, 1992; Beato et
al., 1995; Tenbaum and Baniahmad, 1997.. Thus, one role of the hormone is to induce
DNA binding Žsee Fig. 1..
In general nuclear receptors possess a highly conserved DNA binding region separating the receptor into a variable amino ŽN. terminal and a higher conserved carboxy ŽC.
terminal part. The DNA binding region is essential for sequence-specific recognition of
the hormone response element of the target gene by the receptor. It consists of 66 amino
acids containing two zinc fingers. The N terminals of the receptor are the least
conserved parts. Length and sequence composition varies strongly among receptors.
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
297
Fig. 1. Steroid hormone receptors ŽR.: schematic representation of hormone and antihormone action Žfrom
Baniahmad; personal communication.. HREs hormone responsive elements, HSPs heat shock protein,
R s receptor.
However, a constitutionally active transcriptional activation function ŽAF-1. was demonstrated to be located in the N terminals for most of the analyzed receptors. The C
terminals of nuclear receptors harbour multiple functions such as hormone binding
Žligand binding., transcriptional activation ŽAF-2., transcriptional repression Žsilencing.,
nuclear translocation and dimerisation Žsee Fig. 2.. C-terminal receptor functions are
modelled upon hormone binding. Thus, in contrast to AF-1, the second transactivation
function is dependent on ligand binding ŽEvans, 1988; Gronemeyer, 1992; Tenbaum and
Baniahmad, 1997..
There is now ample evidence that a ligand may interact with various isoforms or
variations of a nuclear receptor. Thus progesterone may be bound to either progesterone
receptor A, B or C, most likely resulting in different transcriptional activities ŽKastner et
al., 1990; Gronemeyer et al., 1991; Vegeto et al., 1993; Wen et al., 1994; Wei et al.,
1996; Giangrande et al., 1997.. In particular for the classical oestrogen receptor ŽERa .
the formation of many variants and mutants has been described, mostly resulting from
the deletion of one or several complete exons by alternative splicing ŽHu et al., 1996;
Pfeffer et al., 1996; Friend et al., 1997; Murphy et al., 1997; Okada et al., 1998..
Cloning of another oestrogen receptor from a rat prostate cDNA library ŽKuiper et al.,
1996. led to the definition of the oestrogen receptor b, which has been demonstrated in
an increasing list of mammalian ŽMosselman et al., 1996; Tremblay et al., 1997; Pau et
al., 1998; Rosenfeld et al., 1999. and nonmammalian species ŽLakaye et al., 1998;
298
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
Fig. 2. Typical functional organization of nuclear hormone receptors. The bars indicate the localization of
receptor functions.
Tchoudakova et al., 1999.. In man, the genes for the two oestrogen receptors are located
on different chromosomes ŽGosden et al., 1986; Enmark et al., 1997.. However,
homogenity of the DNA binding domain between the two receptors is high Ž96%.; it is
distinctly less in the other domains ŽEnmark et al., 1997, Mosselman et al., 1996.. For
the oestrogen receptors a and b, a different but overlapping distribution pattern has
been described ŽEnmark et al., 1997; Kuiper et al., 1997, 1998; Shugrue et al., 1998;
Hiroi et al., 1999.. Thus, in many cases they may occur simultaneously in the same
target cell, possibly together with other receptor variants. In addition it has been shown
that one ligand may be bound with different affinities to the various receptor variants
ŽKuiper et al., 1997. or may exhibit different agonistrantagonist activities ŽBarkhem et
al., 1998.. Moreover, it must be assumed that heterologous dimerisation products may
form following binding of the ligand ŽCowley et al., 1997; Pace et al., 1997; Pettersson
et al., 1997.. Thus, depending on the expression of receptors in a target tissue, different
responses may be induced by the same ligand
Also steroid hormone antagonists show a high affinity for the respective nuclear
receptors with dissociation constants in the picomolar to lower nanomolar range
ŽCapony and Rochefort, 1978; Katzenellenbogen et al., 1981; Hurd and Moudgil, 1988;
Terakawa et al., 1988.. As with agonists, this binding induces dimerisation, which is
followed by binding to the HREs of the respective genes, however, without or only
slightly inducing transcription. This latter phenomenon is still poorly understood. Recent
investigations point to the role of a coactivator in the case of inducing transcription and
a corepressor in case of steroid hormone antagonists ŽBaniahmad et al., personal
communication, see Fig. 1.. In addition, the steroid hormone antagonist leads to
conformational changes of the receptor. Since inhibition of transcriptional activity by the
steroid hormone antagonist may vary, most antihormones have a partial agonistic
activity. In addition, expression of agonistic and antagonistic activity may vary between
hormone dependent tissues, depending on the spectrum of locally available nuclear
receptors. Based on these observations it can be explained why tamoxifen, an anticancer
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
299
drug used in human therapy, acts predominantly as a strict anti-oestrogen in the
mammary gland ŽGottardis et al., 1988; Jordan, 1992, 1997. while it shows clear
oestrogenic activity on the uterine endometrium ŽMalfetano, 1990; Daniel et al., 1996..
In conclusion, the characteristics of steroid hormone antagonists may be summarised
as follows.
Ø
Ø
Ø
Ø
They bind to the ligand-binding domain of a receptor.
Binding is competitive and reversible.
They induce a different conformation of receptors.
They reduce hormone dependent gene activation andror other receptor mediated
effects.
3. Observations following the use of hormone antagonists in laboratory and
domestic animal species
Steroid hormone antagonists exhibiting a competitive binding to the oestrogen,
androgen and progesterone receptor have been developed and are presently in use for
therapy in human medicine. To the knowledge of the authors, there is so far only one
registration of a sex hormone antagonist in veterinary medicine ŽAglepristone w , Virbac,
France.. However, apart from the application in laboratory animal species, there are data
from experimental use in large and small animals with apparent emphasis on the
application of antiprogestins. In view of the multitude of the experiments performed, this
paper will restrict itself to observations in domestic animal species.
4. Antiprogestins
The chemical structure of three antiprogestins, mifepristone ŽRU38486., aglepristone
ŽRU46534. and onapristone ŽZK98299., is shown in Fig. 3. They exhibit a high
structural similarity and it has been proposed that the hydrophobic side chain at C17 is
responsible for the high-affinity receptor binding while the additional aromatic ring at
C11 with a dimethyl-amino– group is responsible for the changes in receptor conformation leading to a suppression of transcription ŽBaulieu, 1985, 1987.. As shown in Table
1 for RU38486, apart from binding to the progesterone receptor, binding to the
glucocorticoid receptor and — to a lesser extent — also to the androgen receptor has
been observed. Binding affinity may vary between species with no binding observed to
the progesterone receptor of the chicken oviduct ŽSakiz et al., 1984.. No binding was
observed to sex-hormone-binding globulin and transcortin in the human, monkey and
chicken, confirming own observations in the dog ŽGerres, 1991.. Thus it must be
expected that the effects observed after treatment with an antiprogestin will vary
depending on receptor expression, the affinity to the receptor and the dose applied.
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B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
Fig. 3. Molecular structures of antiprogestins.
Moreover, effects will vary depending on the role of progesterone as an endocrine and
paracrine factor at a given stage of the reproductive cycle.
Table 1
Binding of RU38486 to steroid receptors and plasma proteins in different species Žfrom Baulieu, 1985.
qqq: K D s10y1 0 M; qq: K D s10y9 M; q: K D s10y8 M; y s no affinity.
P s progesterone.
Dsdexamethasone.
T s testosterone.
Progesterone receptor
Glucocorticoid receptor
Androgen receptor
Mineralocorticoid receptor
Sex-hormone-binding globulin
Transcortin
a
ratrrabbit
Žqqq. Ž 4 P. a
ratrmouse
Žqqq. Ž 4 D. a
rat qŽ1 4 T. a
rat Žy.
manrmonkey
Žqq. Ž 4 P. a
manrmonkey
Žqqq. Ž 4 D. a
chicken
Žqqq. Ž 4 D.q
manrmonkey Žy.
manrmonkey Žy.
chicken Žy.
chicken Žy.
In comparison to the affinity of the corresponding agonist.
chicken Žy.
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
301
5. Application of antiprogestins in the dog and cat
Different from other domestic animal species the course of the progesterone concentrations in peripheral plasma is virtually identical in pregnant and nonpregnant dogs.
They begin to increase prior to ovulation, reach a maximum during early dioestrus and
decrease thereafter, reaching baseline levels Žbelow 1 ng mly1 plasma. about 70–90
days after onset of dioestrus. In pregnant animals parturition is preceded by a change
from the more gradual to a precipitous decline prior to parturition Žlength of pregnancy
63 " 2 days.. Luteolysis in the dog is independent of a uterine luteolysin ŽHoffmann et
al., 1992.. However, depending on the dose regimen selected, application of prostaglandin F2 a ŽPGF2 a . during the second half of pregnancy may lead to a temporary or
permanent depression of luteal function resulting in abortion ŽRomagnoli et al., 1993..
Also in the cat, an induced ovulator, ovarian function is independent of the uterus. After
sterile matings cats become pseudopregnant and the resulting corpora lutea pseudograviditates show a life span of about 40 days ŽTsutsui and Stabenfeldt, 1993..
5.1. Effects of antigestagen treatment during early pregnancy and prior to parturition
Observations after treatment during early pregnancy are limited to the dog. There,
implantation occurs about 14–15 days after ovulation. As was shown by Fieni
´ et al.
Ž1996., parenteral treatment prior to implantation with two times 10 mg aglepristone
kgy1 bw 24 h apart completely blocked establishment of pregnancy. None of the 35
mated bitches became pregnant, confirming our own observations ŽHoffmann et al.,
unpublished data..
Also during the second half of pregnancy treatment with the antigestagen interfered
with pregnancy. Oral application of 20 mg RU38486 kgy1 bw induced abortion in two
bitches; no reactions were observed in two other dogs ŽLinde-Forsberg et al., 1992..
Concannon et al. Ž1990. report about abortions or resorptions in conjunction with vulval
discharge after oral application of 2–5 mg RU38486 kgy1 bw over several days.
Interestingly, in these studies luteolysis was observed after 40–45 days. Following
parenteral application of two times 10 mg aglepristone 24 h apart, Fieni
´ et al. Ž1996.
report about a 97.1% success rate in inducing resorptionrabortion up to day 55 post
mating.
In our own experiments repeated subcutaneous injections of 6 mg RU38486 kg y1
bw after day 56 of pregnancy led to an onset of parturition in the presence of unchanged
high progesterone concentrations. Otherwise, initial changes Ždrop in body temperature,
onset of cervical opening, maternal behaviour. resembled a normal parturition. However,
concerning the further cascade of events leading to expulsion of the fetuses, the prepartal
increase of PGF2 a was missing in the treated dogs and parturition had to be finished by
cesarean section ŽNohr et al., 1993; Hoffmann et al., 1996.. From these observations a
combined treatment with aglepristone and PGF2 a was developed to initiate parturition in
the dog ŽHoffmann et al., 1999; Riesenbeck et al., 1999.. Using a similar type treatment
regimen abortion could be induced in a 5-week pregnant cat suffering from fibroadenomatosis Žsee below.. These data clearly show that in dogs and cats progesterone is the
302
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
dominating factor throughout the entire length of pregnancy controlling uterine and
cervical function.
5.2. Effect of antigestagen treatment on gynaecological disorders
Pseudopregnancy is an inherent phase of the reproductive cycle of the nonpregnant
bitch. Depending on the appearance of clinical symptoms, it may be classified as
covered or overt ŽChakraborty, 1987.. Clinical symptoms associated with overt pseudopregnancy are mammary gland hyperplasia with or without secretion, increased aggressiveness, nesting behaviour and acceptance of dummy puppies. Symptoms may appear
as early as 30 days after ovulation and can last 30–90 days ŽArbeiter and Winding,
1977.. The underlying endocrine mechanisms are still poorly understood and in order to
test for the involvement of progesterone, in a controlled clinical study overtly pseudopregnant dogs were treated with the antiprogestin RU38486 ŽGerres and Hoffmann,
1994.. Treatments commenced on days 24, 35 and 43 after onset of prooestrous
bleeding, the antiprogestin was given by subcutaneous injections in a dose of 2 mg kgy1
bw in 2–3 day intervals until progesterone concentration had reached levels between 1
and 2 ng mly1 Ž3–6 nmol ly1 . plasma. Treatment with the antigestagen led to an earlier
onset of overt pseudopregnancy while clinical symptoms were less. Furthermore,
duration of pseudopregnancy was shortened when treatment commenced during the first
and second third of dioestrus ŽTable 2.. These results implicate a role for progesterone in
the onset and maintenance of overt pseudopregnancy. The advanced onset of overt
pseudopregnancy in the treatment group was interpreted as a result of the mimicked
progesterone withdrawal induced by treatment with the antiprogestin. The other observations of reduced clinical symptoms and duration suggest that maintenance of pseudopregnancy at least partly depends on progesterone.
Another gynaecological problem in the dog is the development of a pyometra
Žendometritis purulenta.. In respect to the importance of progesterone in controlling
uterine and cervical function and based on the observation that over 60% of the dogs
presented with pyometra are during the state of dioestrus with progesterone levels higher
than 1 ng mly1 plasma Ž) 3.18 nmol ly1 ., in a series of experiments the hypothesis was
tested that progesterone might be an important regulatory factor involved. An initial
pilot study ŽBlendinger et al., 1997. clearly demonstrated that treatment with RU38486
at a dose of 6 mg kgy1 bw twice on day 1 of treatment and once on days 2, 3 and 4 led
to a complete evacuation of uterine contents. These observations were confirmed in an
open clinical study comprising 41 bitches ŽLemmer, 1999.. However, successful treatments were bound to an undisturbed ovarian function and progesterone levels above 1
ng mly1 plasma at onset of treatment. As far as reported, dogs regained their breeding
capacity.
Fibroadenomatosis is a noncarcinogenic tumour of the mammary gland in cats
occurring spontaneously during pregnancy or pseudopregnancy. The enlargement of the
mammary gland is predominantly due to a gestagen-dependent proliferation of its
stromal component. Common therapy is ovariohysterectomy andror mastectomy. Treatment with the antiprogestin RU38486 given daily by subcutaneous injection over 5 days
not only resulted in abortion Žafter additional treatment with PGF2 a , see above., but also
Onset of treatment
Žday after onset of
pro-oestrous bleeding.
Onset
24
35
43
48.2"7.5
62.0"4.5
62.5"4.5
a
b
Control cycle
a
Treatment cycle
b
Duration Ždays.
Score
48.8"10.8
40.2"21.2
43.7"25.3
3.8"0.4
3.4"0.9
3.3"0.8
Day after pro-oestrous bleeding.
Mean maximum score of mammary gland development Ž0–4..
Onset a
Duration Ždays.
Scoreb
37.0"7.9, p- 0.05
47.0"1.6, p- 0.001
51.7"5.6, p- 0.01
26.2"7.7, p- 0.01
16.8"4.9, p- 0.05
21.0"6.9, n.s.
1.0"0.0, p- 0.001
1.2"0.4, p- 0.01
1.2"0.4, p- 0.01
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
Table 2
Effects of the antiprogestin RU38486 on onset and duration of overt pseudopregnancy and mammary gland development; values expressed as j "SD
ns s Not significant.
303
304
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
in a reduction of mammary gland hyperplasia. After further treatments on days 9, 14,
and 20 mammary gland enlargement was completely regressed on day 28 ŽBlendinger et
al., 1994.. These observations clearly confirm that fibroadenomatosis is a progestagenmediated disease in the cat and that blocking of progesterone at the receptor level is an
effective therapy. That was demonstrated in several clinical cases and also a male cat
which developed severe fibroadenomatosis after application of a depot progestagen for
hormonal castration was successfully treated with the antigestagen ŽHoffmann, unpublished data..
5.3. Effect on luteal function in the dog
Following treatment with RU38486 for induction of abortion Concannon et al. Ž1990.
and Linde-Forsberg et al. Ž1992. reported about an accelerated luteal regression.
Similarly, in our own studies when treating dogs with pyometra, progesterone declined
significantly during treatment from 18.3 ng mly1 on day 1 to 6.1 ng mly1 on day 6
ŽBlendinger et al., 1997. and from 16.8 nmol ly1 on day 1 to 6.82 nmol ly1 on day 14
and 4.9 nmol ly1 on day ) 21 ŽLemmer, 1999.. On the other hand, in a hysterectomized dog luteal function was not affected by subcutaneous applications of 2 mg
RU38486 kgy1 bw in 2-day intervals over the entire cycle ŽGerres, 1991.. This raises
the question whether the observed accelerated luteal regression in ‘‘intact’’ dogs is a
direct or rather indirect effect of the antiprogestin. The conclusion that it is a rather
indirect effect is supported by observations of Li et al. Ž1991a. in gilts, where it was
shown that the acute luteolytic effect of RU38486 depended on the presence of the
uterus andror conceptuses.
5.4. Other obserÕations
Antiglucocorticoid effects of RU38486 were clearly demonstrated in the human and
laboratory animals. Thus in both species prior treatments with RU38486 inhibited the
activity of dexamethasone ŽPhilibert et al., 1981, Baulieu, 1985.. In the dog, Wade et al.
Ž1988. report about significantly increased cortisol and ACTH concentrations following
oral application of 20 and 50 mg RU38486 kgy1 bw, respectively. However, as it was
indicated by unchanged cortisol levels when treating pseudopregnant dogs ŽGerres and
Hoffmann, 1994. and dogs with pyometra ŽLemmer, 1999. in our own studies using a
different dose regimen no interference with adrenal function became obvious. The only
side effects observed were labour-like pains after a dog with pyometra had been treated
with a single dose of 20 mg kgy1 bw.
6. Application of antiprogestins in large animals
Application of RU38486 induces premature parturition in cattle and sheep. In cattle
treatment with 2 mg RU38486 kgy1 bw at 8 h on days 277 and 278 resulted in
parturition 55 h later, whereas in vehicle-treated control cows parturition occurred after
210 h Ž p - 0.01.. Different from induction of parturition with glucocorticoids or PGF2 a
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
305
ŽAdams, 1969; Wagner et al., 1974; Johnson and Jackson, 1982., no increase in retained
fetal membranes was observed ŽLi et al., 1991b.. In sheep the interval from the first
treatment to the onset of lambing was 31 " 2 h in animals given 4 mg RU38486 kgy1
bw on day 144 while it was 121 " 27 h in the vehicle-treated controls Ž p - 0.01. ŽGazal
et al., 1993.. In both animal species progesterone levels started to decline following
treatment with the antiprogestin, though the prime source of progesterone in the sheep is
the placenta while it is the corpus luteum in the late pregnant cow ŽHoffmann, 1994..
Also in pigs oral treatment with 4 mg RU38486 kgy1 bw on days 111 and 112 of
pregnancy resulted in parturition on day 112.7 compared to day 114.7 in the control
group Ž p - 0.01.. In treated animals progesterone decreased abruptly from a pretreatment mean of 11 to less than 0.6 ng mly1 during the second day of RU38486 treatment;
preterm parturition coincided with an advanced relaxin peak with a maximum on day
112.1. Also in this study, hysterectomized gilts carrying persistent corpora lutea were
submitted to treatments with RU38486. Rather than a decrease of progesterone concentration an abrupt increase of progesterone and prolactin secretion was observed leading
to the conclusion that the acute luteolytic effect of RU38486 observed in pregnant pigs
depends on the presence of the uterus andror conceptuses ŽLi et al., 1991a.. Thus, it
may be concluded that luteal regression in cattle and the decrease of placental progesterone synthesis in sheep following treatment of pregnant animals with RU38486 may
also be an effect depending on the presence of the uterinerfetal compartment. Gonçalves et al. Ž1997. studied the effect of an antiprogestin Žonapristone. on in vivo and in
vitro fertilization. Studies were performed in oestrus synchronised, superovulated ewes.
Treatment with the antiprogestin 3 and 15 h after sponge removal had no effects on
synchronization of oestrus, ovulation and oocyte maturation; however, in vivo fertilization rate decreased significantly from 41% in the control group to 2–3% in the treatment
group Ž p - 0.001. due to sperm arrest in the cervix. Also fertilization of bovine oocytes
in vitro decreased significantly from 62.6% in the control group to 48% in the treatment
group. These observations clearly point to the role of progesterone in respect to control
of cervico-uterine Žtubal. functions and to sperm–oocyte interaction.
7. Antiandrogens and antioestrogens
The antioestrogen tamoxifen and the antiandrogen cyproterone acetate Žsee Fig. 4. are
established compounds used in human therapy. Indication for treatment with tamoxifen
is mammary cancer therapy; similarly, an indication for the application of cyproterone
acetate is a nonsurgically accessible prostate carcinoma. Other indications are male
hypersexuality and hyperandrogenisation in females. The phenylethylene derivatives
tamoxifen or chlomifen Žsee Fig. 4. are mixed antagonists–agonists of oestrogen action
and belong to the group of type I antioestrogens ŽClark, 1994; Klein-Hitpaß et al., 1998..
Type I oestrogen antagonists partially inhibit the action of agonists, but, due to their own
inherent weak agonistic properties, they also induce to some extent oestrogenic responses. The degree of agonistic or antagonistic activity depends on the species, organ,
tissue, or cell type that is being examined. Thus in the human tamoxifen exerts distinct
antioestrogenic activities in the mammary gland while it exerts agonistic activities on the
306
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
Fig. 4. Molecular structures of relevant antioestrogens and antiandrogens.
uterus ŽGottardis et al., 1988; Jordan, 1992, 1997.. The underlying mechanisms are not
yet fully understood; however, they may relate to the expression of cell-type-specific
oestrogen receptor variants, cell-type-specific arrays of cofactors or to different ways of
receptor–DNA interactions. Moreover, in the dog tamoxifen acts rather like an agonist
than antagonist ŽMorris et al., 1992., suggesting also species specific variations with
respect to the agonistic activities of type I antioestrogens.
The 7a-alkyl-amide analogues ICI 164,384 and ICI 182,780 may be considered pure
antioestrogens and are classified as type II antioestrogens. They lack agonistic activity
for uterine growth as shown in rats and mice and they are unable to induce the
progesterone receptor in the immature rat. Similarly with type I antioestrogens, and as
shown in experiments with transiently transfected mammalian cells, these type II
antioestrogens also induce receptor dimerisation and DNA binding. Since there seems to
be no significant reporter gene activation, they obviously prevent the activity of both
AF-1 and AF-2 Žfor review see Klein-Hitpaß et al., 1998..
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
307
Type I antioestrogens have been applied in domestic animals to further explore the
endocrine functions of estradiol-17b. Thus, Jacobs et al. Ž1988. could demonstrate that
the oestrogen provoked preovulatory LH release in heifers is inhibited by concomitant
treatment with tamoxifen. The same experiment showed that the blocking of endogenous
estradiol-17b prevented the endometrial PGF2 a-release leading to luteolysis. However,
Cloprostenol w -induced luteolysis was not prevented. Janowski et al. Ž1996. treated
prepartal cows with a dose of 360 mg tamoxifen in 4-h intervals starting on day 268 of
pregnancy. They observed no changes in respect to course of parturition and oestrogen
synthesis, raising the question on the biological role of prepartal oestrogen production in
cattle.
Cyproterone acetate Žsee Fig. 4. is the 1,2-cyclopropal derivative of chlormadinone
acetate. It exerts antiandrogenic and gestagenic activities with an inherent androgenic
activity. In laboratory animals cyproterone acetate inhibits development of accessory
sexual organs but has little effect on neuro-gonadal feedback mechanisms and hence
testosterone biosynthesis. Other than cyproterone acetate, cyproterone and flutamid, a
nonsteroidal receptor-binding antiandrogen Žsee Fig. 4., inhibit the negative feedback of
androgens which in consequence leads to increased LH and testosterone values ŽNeumann et al., 1984; Fuhrmann et al., 1998.. In rats, guinea pigs or mice no inhibiting
effect of cyproterone acetate on male sexual activity was observed, yet inhibition was
observed in dogs, rabbits and in ewes in which a male behaviour had been induced by
repeated treatment with testosterone Žreviewed by Fabre-Nys, 1982.. Daily treatment of
boars with 200 mg cyproterone acetate completely suppressed development of boar taint
ŽHorst and Bader, 1969.. However, based on the intrinsic hormonal activities of
cyproterone acetate, this effect is probably a result of the progestagenic rather than
antiandrogenic activity. Also in the dog it has been shown that cyproterone acetate has
an antigonadotrophic effect Žvan Sluijs, 1997.. This inherent diversity of hormonal and
antihormonal effects of cyproterone acetate makes it difficult to relate experimental
observations to distinct hormonal activities, which may explain the paucity of information on the use of these type antiandrogens in domestic animal species.
8. Peptide hormone receptor blockers
In respect to reproduction GnRH and oxytocin antagonists have been developed.
Briefly these two hormones comprise 10 ŽGnRH. and 8 Žoxytocin. amino acids, their
main synthesis is by hypothalmic neurons though synthesis in periphal organs like the
ovary Žoxytocin. and placenta ŽGnRH-like peptide. have been described ŽDocke,
¨
1994a,b.. They bind to membrane receptors inducing second messenger mediated
intracellular signal cascades. The GnRH and oxytocin receptors are members of the
large family of G-protein-coupled receptors and have seven transmembrane domains
ŽKimura et al., 1992; Flanagan et al., 1997; Salvatore et al., 1998.. They have been
identified in various organs and their specific inhibition would provide further information on the biological role of these two hormones in addition to the opening of new
therapeutic approaches. In conjunction with dog reproduction, Vickery et al. Ž1989. refer
to GnRH antagonists with different relative potencies measured on the inhibition of LH
release. Side effects observed concern the mast cell degranulating activity of these
308
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
compounds and the side effects to be expected from the release of histamine and other
mediators. Together with the high dose requirements, this asks for the development of
more potent and safer analogues. However, since similar effects may be observed by
downregulation of GnRH receptors following application of a continuously high dose of
GnRH, there is only a relative need for these type compounds to interfere with small
animal reproduction.
The oxytocin receptor gene is expressed in the myometrium, endometrium and
cervical epithelium and blocking the activity of oxytocin by an appropriate antagonist
might proof to be a valuable approach to control gynaecological or obstetrical disorders
Žreviewed by Goodwin and Zograbyan, 1998.. Concerning their application in domestic
animals, Fuchs et al. Ž1997. report about the affinity and specificity of the antagonist
w1-DŽCH 2 .5 , TyrŽME. 2 , Thr 4 , Tyr-NH 92 x ornithine vasotocin and its use in late pregnant
cows. The antagonist showed a similarly high binding to endometrial and myometrial
oxytocin binding sites and a 40-fold molar excess of antagonist inhibited the release of
prostaglandin induced by oxytocin in the bovine endometrium prior to parturition.
Similar observations in the sheep ŽJenkin et al., 1994. point to the role of these
compounds as tocolytic agents. Thus the oxytocin and vasopressin antagonist atosiban
has reached the phase III of clinical development for human use ŽBossmar, 1998..
9. Conclusions
As was demonstrated during the past 20 to 30 years hormone antagonists blocking the
activity of reproductive hormones have shown to be powerful tools in endocrine
research, therapy and biotechnology. Of particular interest are compounds which block
agonists by competitive binding to the respective receptor, thereby inducing complete
loss of receptor functions Žpure antagonists.. In respect to sex-steroid hormone antagonists the effects observed not only depend on the molecular structure of the antagonist
but also on the structure of the respective receptor. Thus for example, for the oestrogen
and progesterone receptor various subtypes, variants and mutations have been identified
by adequate cloning experiments and their distribution varies between cell types, tissues
and species. In spite of their highly conserved DNA-binding domain transcriptional
activation seems to be regulated differently, leading to a tissue Žcell.-specific expression
of — for example — oestrogenic activity. Accordingly, the phenomenon of antagonists
exhibiting a partial agonistic activity may at least partly be explained on interactions
with different subtypes of receptors. However, in spite of recent data explaining in more
detail the regulation of receptor activity, still only little is known about the receptor
inactivating mechanisms of action of hormone antagonists. Further insight into these
mechanisms together with a comprehensive mapping of receptors and receptor subtypes
are necessary to develop more specific and selective hormone antagonists.
In spite of the still existing limitations research and also therapeutical approaches in
domestic animal reproduction have benefited from the availability of hormone receptor
antagonists. This particularly concerns progestagen antagonists and their use in small
and large animals. There are also distinct indications for the application of androgen,
oestrogen and GnRH antagonists; however, particularly in respect to therapy further
research is necessary.
B. Hoffmann, G. Schulerr Animal Reproduction Science 60–61 (2000) 295–312
309
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