1. health and fitness
2. disease

BOVINE CONCEPTUS PRODUCTS INVOLVED IN
PREGNANCY RECOGNITION1
R M. Roberts, P. V. Malathy, T. R. Hansen,
C. E Farin and K. hakawa2
University of Missouri3, Columbia 65211
ABSTRACT
Maternal recognition of pregnancy in cows occurs around 15 to 16 d of gestation, and
conceptus secretory proteins have been implicated in triggering maternal responses that
lead to a prolongation of corpus luteum function. The major protein component of these
secretions during this critical period is bovine trophoblast protein-1 (bTP-l), which consists
of several variant forms of molecular weight between 22,000 and 24,OOO and isoelectric
points between 6.3 and 6.8. These diverse forms appear to arise as the result of both
translation of multiple mRNA and differences in glycosylation. Several cDNA for b p - 1
have been cloned and their nucleotide sequences determined. The inferred amino acid
sequence of the protein has indicated that bTP-1, like its ovine counterpart OW-1,belongs
to an unusual 172-amino acid residue long alpha interferon (IFN-a) family, the so-called
IFN-an. Evidence to date suggests that the bTP-1 genes may represent a separate subclass
with approximately five genes within the 15- to 20-member IFN-an gene family. bTP-1
has the antiviral and antiproliferative properties typically associated with an IFN-a and can
bind to IFN-a receptors. A cDNA probe representing the 3'-untranslated end of bTP-1
mRNA has been used to screen dot blots and Northern blots of total cellular RNA from
bovine conceptuses of different ages and for in situ hybridization to mRNA on tissue
sections. The expression of bTP-1 mRNA appears maximal between d 15 and 19, remains
high until at least d 25 of pregnancy and can be detected as early as d 12. Recombinant
bovine IFN-a11, a 166-amino acid IFN,can mimic at least some of the actions of bTP-1. In
particular, intrauterine infusion or intramuscular injection results in extended interestrous
interval in cows.
(Key Words: Bovine Trophoblast Protein-1, Cattle, Embryo, Interferon, Pregnancy.)
Introduction
The theme of this symposium is the use of
molecular biology in understanding reproduction. In this paper we have chosen to review
studies on the protein we consider to be the
antiluteolytic factor secreted by the bovine
conceptus around the critical period of gestation from d 15 to 17 when a conceptus must be
present in utero if luteal lifespan of the dam is
to be maintained. This protein was originally
'Journal Series Number 10,893 from the Missouri Agricultural Experiment Sta!ion. Research from this laboratory
was supportedby NE3 Grant HD21896.
2Present address: 'Ihe Women's Research Institute,
Wichita KS 67214.
&t.
of Anim. Sci. and Biochern.
28
identified by two-dimensional polyacrylamide
gel electrophoresis as the major polypeptide
product secreted in vitro by bovine conceptuses after they had been flushed from the
uterus during the pen-implantation period of
early pregnancy (Bart01 et al., 1985). It was
later named bovine trophoblast protein-1
(bTP-1) by Helmer et al. (1987) when they
showed that it cross-reacted immunologically
with the principal secretory product of the
peri-implantation ovine conceptus, ovine trophoblast protein-1 (oTP-1). o p - 1 had been
purified some years earlier from culture m e
dium in which ovine conceptuses had been
cultured (Godkin et al., 1982) and has been
clearly implicated as a major factor controlling
maternal recognition of pregnancy in the ewe
(Godkin et al., 1984a,b; Roberts et al., 1985;
Bazer et al., 1986; Thatcher et al., 1986).
J. Anim. Sci. 1990. 68:(Suppl. 2):28-38
BOVINE CONCEPTUS INTERPERONS
The Establlshment of Pregnancy In Cattle
29
whether bTP-1 is the antiluteolytic substance
of the cow. Total conceptus secretory proteins
from this period proved to be capable of
causing several days of estrous cycle extension
(Knickerbocker et al., 1986) in nonpregnant
cattle. However, bTP-1 has proven difficult to
purify, and only recently has sufficient homogeneous product been isolated to prove that
intrauterine administration of bTP-1 alone
could cause an increase in interestrous interval
matcher et al., 1989). It should be emphasized, however, that intrauterine administration
of neither OW-1nor of bTP-1 is capable of
inducing more than a 3- to 4-d extension of the
estrous cycle. Conceivably, the proteins either
need to be administered continuously rather
than acutely or some additional embryonic
component is required to provide a more
extended effect.
The luteolytic substance in domestic livestock species is now generally regarded to be
prostaglandin F2a (PGFA, which is produced
by the uterine endometrium. A large body of
evidence has been gathered to support this
contention (Horton and Poyser, 1976; Hansel
and Convey, 1983; Thatcher et al., 1986) and
will not be reviewed here. Episodic spikes of
PGF2u released from the uterus are evident in
nonpregnant cows and heifers preceding
luteolysis but are generally absent during
pregnancy (see Thatcher et al., 1986). Introduction of dialyzed bovine conceptus secretory
products, of which bTP-1 is the major component, into the uterus of nonpregnant cows
between d 15 and 21 of their estrous cycles
significantly reduced the amount of PGF2a in
vena cava plasma compared with controls
(Knickerbocker et al., 1986). Moreover, the
treatment also reduces the frequency of PGFza
pulses. These results strongly suggest that
bovine secretory proteins, and by inference
bTP-1, attenuate endometrial PGF production.
Experiments with endometrial cultures have
confinned that either total bovine conceptus
secretory proteins or purified bTP-1 itself
cause a decrease in the amount of P G F p
produced but leave prostaglandin
production unaffected (Gross et al., 1988; Helmer et
al., 1989). The latter studies have indicated
that bTp-1 may induce an intracellular inhibitor of prostaglandin synthesis.
The establishment of pregnancy in any
eutharian mammal requires complex interchange of biochemical information between
the mother and conceptus to ensure that
progesterone production by the ovary is
maintained and that the uterus continues to be
receptive for the embryo. It is also clear that
there must be an appropriate degree of
synchrony maintained between the uterus and
the conceptus it nourishes and protects or else
the pregnancy is lost (Pope, 1988).
Three sets of experiments defined d 15 to
17 of gestation as critical to maintenance of
the corpus luteum of pregnancy in cattle. First,
interestrous interval was extended when conceptuses were removed from cows on d 17 or
d 19 of pregnancy, whereas removal at d 15
had no effect (Northey and French, 1980).
Second, intrauterine infusion of d 17 to 18
conceptus homogenates into nonpregnant re
cipients from d 14.5 to 18 post-estrus lengthened interestrous intervals (Northey and
French, 1980). Similar effects were observed
when freeze-killed d-16 conceptuses were
placed in utero (Dalla Porta and Humblot,
1983). Third, successful pregnancies resulted
from transfer of embryos to synchronous
recipients up to d 16 of pregnancy, but not
beyond (Betteridge et al., 1980). Together,
these results indicated that some signal is first
produced in critical amounts between d 15 and
17 of pregnancy that results in prolongation of
the functional lifespan of the corpus luteum.
These observations are comparable to a more
extensive series of studies made on the sheep
by Moor and Rowson (1966a,b), Rowson and
Moor (1967), Martal et al. (1979) and Ellinwood et al. (1979) that indicated that the
antiluteolytic substance was produced for a
limited (d 13 to 21) period of development and
was most probably proteinaceous. These experiments on the sheep underpinned the
discovery and purification of oTP-1 (Godkin et
al., 1982) and the demonstration that this
substance alone could prolong interestrous
interval when it was infused into the uteri of
cyclic ewes (Godkin et al., 1984b Vallet et al.,
1988).
After it was realized that bTP-1 constituted
the major in vitro secretory component of d-17
bovine conceptuses (Figure 1) and that its
Multlple Forms Of bTP-1
production remained elevated for at least the
bTP-1 was shown by Bartol et al. (1985) to
week following its first detection (Bartol et al.,
1985), attempts were made to determine consist of at least seven to eight different
30
A
c3
I
X
I-
I
Figure 1. (A) Two-dimensional electrophoretic analysis of totalsecretory proteins from a d-18 bovine conceptus by twodimensional electrophoresis and fluorography. "be cooceptus had been cplhued for 24 h in medium supplemented with L[%Ileacine. For details of the e n t a l procedures see Helmer et al. (1987). The horizontal axis represents isoelectric
point @H), the vertical axis molecular weight.(B)Analysis of material immunopmcipitable from bovine conceptus secretory
proteins by an antiserum to 0°F'-1. Roteins were aualyd by t w d i e n s i o d polyylamide gel electrophoresis and
fluorography in a manner identicalto Figure l(A). After providing conceptuses with G [ H]leucine for 24 h, samples of
culture medium were mixed with anti-o"F'-1 a n t i s m and immune.complexes collected on Rotein A-Sepharose. For
txperimentaldetails, see Helmer et al. (1987). "%e various isoforms of bW-1 are visible as a cluster of products of pI between
6.8 and 6.5 and fall mainly into two molecular weight classes (Mr22,OOO and 24,000). T h i s figure is reproduced from Helmer
et al. (1987) with permission.
polypeptides clustered over a range of isoelectric points and molecular weights. All of these
forms could be collected by indirect immunoprecipitation by employing an antiserum
to 0°F'-1 (Figure 1; Helmer et al., 1987). Thus,
bTP-1 appears to be of higher molecular
weight and to constitute a more diverse p u p
of components than 0°F-1, which has usually
been observed as a cluster of three or four
isoelectric variants of molecular weight about
18,000 (Godkin et al., 1982).
Cell-free translation of total poly(A)+
mRNA from d 17 to 19 bovine conceptuses,
however, gave rise to translation products that
BOVINE CONCEPTUS INTERFERONS
PH
81 0.0
1.6
7.0
0.6
0.0
6p
6.0
110,oQo91.00077-
U
a
lL14Q-
$
:%
77,000.
¶O*
11-
Figure 2. Two-dimensional SDS-PAGE and autoradiography of [3’S] methionine-labeledproteins synthesized in
the presence ofbovine conceptusmRNA. In vitro translation
products synthesized with whcat germ lysate in thepresence
of d-18 bovine conceptus polyadenylated mRNA and
[35S]methioninewere analyzed by two-dimensional SDSPAGE (12.5%gels) and autoradiography. They were analyzed either as total translation products (top panel) or following immunoprecipitation with anti-OW-1 antiserum
(bottom panel). Arrows indicate the location of prsbW-1
on both panels. The figure is reproducedfrom Anthony et al.
(1988) with permissioa
appeared smaller than the h a l mature products (Anthony et al., 1988). Nevertheless,
several distinct isoforms were still evident
Figure 2), an observation that led to the
suggestion that bTP-1 most probably arose
from multiple species of rnRNA, and possibly
from multiple genes. Translation of the mRNA
in presence of microsomal membranes from a
dog pancreas, which are capable of adding Nlinked carbohydrate chains to nascent polypeptides as well as removing NH2-tenninal signal
sequences, increased the size of the translation
products and sequestered them within the
membrane vesicles (Anthony et al., 1988).
This result strongly suggested that bTP-1
became glycosylated cotranslationally as it
crossed the membranes of the rough endoplasmic reticulum. That carbohydrate chains were
31
indeed present on bTp-1 has been proved in a
number of ways: 1) by providing conceptuses
with radioactive precursors to carbohydrate
chains (e.g., D-[3~glucosamine or D[3lcilmannose) and identifying the label in bTp1 (Anthony et al., 1988); 2) by inhibiting bTP1 glycosylation in vitro with tunicamycin and
demonstrating a reduction in molecular size of
the secreted product (Helmer et al., 1988); 3)
by removing the carbohydrate chains with
endoglycosidases (Anthony et al., 1988); and
4) by demonstrating that bTP-1 binds to plant
lectin columns (Helmer et al., 1988). It was
concluded fmm these experiments that bTP-1
was a glycoprotein and that part of the
diversity in molecular weight forms evident in
Figure 1 was the result of variability in the
complexity and possibly in the number of
carbohydrate chains present on the molecules.
By contrast, there has been no evidence that
0°F-1 is a glycosylated molecule (Anthony et
al., 1988). Whether this carbohydrate on bTp-1
is important to either its biological activity or
stability remains to be determined.
The Molecular Cloning of bTP-1 mRNA
The ability to generate cDNA has frequently become important because it can
provide a shortcut to inferring the primary
structure of the protein it represents. Sequencing a DNA, for example, is much quicker and
more straightforward than determining an
amino acid sequence, a procedure that can
often be very laborious. A cDNA can, in
addition, provide a useful probe for studying
gene expression in any tissue of interest. It
can, when appropriately tailored, also be
utilized to generate a recombinant protein
product in a heterologous cell, such as a
bacterium.
The molecular cloning of bTP-1 and oTP-1
was conducted simultaneously in this laboratory from cDNA libraries consmcted with
poly(A)+ mRNA isolated from d 17 to 19
bovine and d 15 to 16 ovine conceptuses. The
procedure entailed preparing cDNA copies of
the total complement of A
in the tissue,
copying the first cDNA strands to yield cDNA
duplexes, and introducing these doublestranded structures into the P-galactosidase
gene of bacteriophage hgtll (Huynh et al.,
1985). Phage that expressed a whole or part of
the bTp-1 (or oTP-1) protein were recognized
by screening many thousands of bacteriophage
32
ROBERTS ET AL.
plaques with a rabbit antiserum to oTP-1. Such 1985; Tamrn et al., 1987). The apparent
positive plaques were picked from the plates involvement of IF" in reproduction was
and recornbinant phage were isolated by completely unsuspected.
several rounds of "plaque purification." The
However, the majority of the IFN-a so far
cDNA inserts were removed, placed into a hown are 166, not 172, amino acids in length,
bacterial plasmid and amplified, and their although a single 165-residue species (human
nucleotide sequences were determined (Im- ET-&) has been described (see DeMaeyer
akawa et al., 1987, 1989).
and DeMaeyer-Guignard, 1988). In 1985,
All of the bTP-1 recombinant plaques however, Capon et al. (1985) screened human
recognized by the antiserum contained rela- and bovine genomic DNA libraries under nontively short inserts, the majority of which were stringent conditions with IFN-a probes and
from the 3'-end of the coding region and hence described a new family of IFN genes that
represented the COOH-terminus of the protein appeared to code for 172 residue polypeptides
(Imakawa et al., 1989). One possibility was after their signal sequences had been cleaved.
that this end of the molecule contained the The same year Hauptmann and Swetly (1985)
dominant epitopes on bTP-1. Alternatively, employing similar procedures cloned cDNA
larger fusion proteins might have been more representing such "long" IFN-afrom a cDNA
susceptible to proteolytic degradation and were library derived from virally induced human
not, therefore, present. Whatever the explana- leukocytes. The former group name these
tion, these short cDNA were employed to novel interferons IFN-an to distinguish them
rescreen the library for longer recombinant from the 166-amino acid long IFN-a~.By
clones, and several full-length or nearly full- contrast, Hauptmann and Swetly (1985) emlength cDNA were isolated and sequenced. ployed the entirely new designation IFNThe latter clearly represented multiple species omega (a)because they felt that the degree of
of rnRNA, which, though all differed slightly nucleotide similarity (-75%) and amino acid
from each other in nucleotide sequence, were sequence homology ( 4 0 % ) deserved a sepaabout 1 kb in length. All contained a single rate classification. It should be emphasized that
585-base open reading frame that coded for IF" nomenclature is still in a state of flux and
polypeptides of 195 amino acids. By applying requires further standardization, in part be
the rates of von Heinje (1986) rules, it was cause of the multiplicity of genes so far
clear that the first 23 residues represented a discovered. In the human, for example, at least
hydrophobic signal sequence and that the most 18 distinct LFN-al and IFN-an genes have
likely region for signal peptidase cleavage was been described. To complicate matters further,
before the cysteine residue designated as Cys' scientists in the U.S. classified these I F N q
in Figure 3. Thus we assume mature bTP-1 to genes alphabetically (A,B,C, etc.), but Eurobe 172 amino acids in length.
pean p u p s used Arabic numbers (1,2,3, etc.).
The nucleotide sequences of the bTP-1 Compounding these difficulties, I F N q A does
cDNA showed around 90% identity with those not correspond to IFN-ql (DeMaeyer and
for OW-1.Remarkably, the 3'-ends were the DeMaeyer-Guignard, 1988). For the rest of
most conserved regions of the molecules, with this paper bTp-1 and oTP-1 will be referred to
90 to 95% preservation of nucleotide sequence, as IFN-an, although it is anticipated that this
whereas most divergence was noted in the classification may eventually change.
5'-untranslated region. It seems possible that
A comparison of the associated sequences
the 3'ends play some sort of regulatory role in of bTP-1 with a bovine lFN-an is shown in
bTP-1 and om-1 expression.
Figure 3. All of the IFN-a,whether 166 or 172
A complete search of gene data banks residues long, possess four highly conserved
quickly revealed that bTP-1 and 0°F'-1 were cysteines that are involved in two disulfide
related structurally to the alpha interferon bonds: Cys' is linked to Cysw, and CysZ9 is
(IFN-a)family of proteins that have been bonded to Cys139 (Wetzel, 1981). There are in
studied intensively because of their ability to addition a number of highly conserved regions,
induce an antiviral state in a diversity of cell including one stretch from
to ArglS0
types (Pestka et al., 1987; DeMaeyer and that is retained in bTP-1 and oTP-1. All of the
DeMaeyer-Guignard, 1988) and to behave as bTP-1 cDNA so far isolated possess a single
regulatoq cytokines with a variety of effects, potential site for N-glycosylation (Am7*),
particularly on the immune system (Rossi, whereas this sequence is absent in the bovine
33
BOVINE CONCEPTUS INTERPERONS
DTP-I
Bo Fh-
.
.
CAlCCCC~CMACTA~AlTCACCIUACQT~CCCAUCCC~lC~CAKCACCCCAKACCACCCACA~C~TCCCC
CACAGAACClLCCTCMCCTTCUCWCACCCTCTCTCACCUCCCCACUCCACCCTCA~ClTCCCC
I
R
.
R t
tt
t tt
s23
1
2
m e t a l a phe val l e u s e r l e u l e u met a l a l e u v a l l e u v a l s e r t y r g l y g l n g l y a r g ser leu g l y c y s t y r
ATC CCC TTC CTC C l C TCT CTA CTC ATC GCC CTC CTC CTC GTC ACC TAC GCC CAC CU C U T C l CTC CCT TGT TAC
ATC CCC TTC CTC CTC T C l CTA C l G ATC CCC CTG CTG CTC CTC A K TAT CCC CCC CC4 GU TCC CTC CCC TCT U C
pro
gly
t
asp
Z
1.
*
*
27
7
leu ser g l u
CTG TCT GAG
TTC TCT CCC
pro
asp h i s
U C CAC
M C CAC
asn
met l e u
ATG CTA
GTC C l C
"a1
g l y a l a a r g g l u a s n l e u a r g l e u l e u a l a a r g met asn a r g l e u ser p r o h i s
GCT CCC AGG GAG M C CTC AGC CTC CTC CCC C U I ATG M C ACA C l C TCT CCT U T
GTT CCC ACC CAC M C CTC ACC CTC CTC GCC CAA AlC AGC A U CTC TCC CCT CCC
Val g l y
28
p r o cys leu g l n a s p a r g l y s a s p phe g l y
CCC TCl CTG CAC U C ACA MA U C T T T GCT
TTC TCT CTG CAG U C ACA A M U C TTT CCT
ala
phe
gln
gly gln
l e u p r o g l n g l u met val g l u g l y
CTT CCT CAC UG ATC CTC U C CCC
TTC CCC CAC U G ATC CTC GAG CTC
phe
val
53
a l a i l e s e r v a l l e u h i s g l u met l e u g l n g l n c y s leu a s n leu phe t y r
K l ATC T C l CTC CTC CAC GAG ATC CTC CAC CAC TCC CTC M C CTC TTC TAC
CCC A T 1 TCT CTC CTC CAT U C ATC CTC CAC CAC AGC TTC AAC CTC T l C U C
t
ser p h e
his
*
*
arg
arg
asn g l n l e u g l n
M C CM; CTC CAC
K C CAC TTC CAG
ser
phe
52
l y s asp g l n
M C U T CAC
C4G CCC CAC
g l u ala
77
t h r g l u h i s ser ser a l a a l a t r p
ACA CAC CAC TCC TCT CCT CCC TCC
AAA U G CCC TCC TCT CCT CCC TCG
lys
arg
78
asn thr t h r I C Y l e u g l u g l n l e u
AAC ACC ACC CTC CTC C4C CAC CTC
CAC ACT ACC CTC CTC GAG CAC CTC
asp
cys t h r g l y l e u g l n g l n g l n l e u g l u asp l e u asp a l a cys leu g l y
TCC ACT CCC CTC CAA CAG U C CTG CAG CAC CTG CAC CCC TCC CTC CCC
CTC ACT GGA CTC CAT CAC CAC CTC GAT U C CTC U T CCC TCT C l G GCC
t
leu
+
his
asp
la3
val
CTC
TTG
leu
met g l y a r g
ATG CCA AGG
CTC CC4 ACC
le"
met g l y g l u
ATC GU CAC
ACT C W UIC
thr
*
l y s a s p ser asp
AAA U C TCT U I C
CAA GAC TCT GCC
ala
9lu
met g l y p r o
ATG CCC CCC
ACC GK CCC
thr
i l e leu t h r v a l 1 1 s
A T 1 CTC ACT CTC AAG
ACA CTC CCC ATC AAG
thr
a l a met
127
l y s t y r phe g l n g l y $ l e
AAC TAC TTC CAC GGT ATC
PCG TAC TTC CAG CCC ATC
t
arg
153
172
ser s c r s e r t h r t h r I C " g l n l y s a r g l e u a r g 11s met g l y g l y a s p leu as" ser l e u
TCT T U TCA ACC ACC TTC U A MA AGG TTA A U AAC ATC CCT G U U T CTC AAC TCA C l T
TCT TU TCA ACC AGC TlG CAA C A A ACC TTA ACA ATC ATC U T C U U C CTG AAA TCA CCT
ser
g)u
met
asp
Iys
pro
t t
t
**
t
**
H
102
pro
CCA
CTC
1 eu
end
T U CATUCTCTCCCTUCT
T U FTCACTCTCACTUCT
t
end
***
t t
TTClCACCACTMTAAGUAClAU~ATMAAC~AC~CACC~CTUCC~KAlGACTCCl~AACTCATKCT~CC~UTCTlATCTCTTCT~U~TlATC
TTCTCACTACTP M T C A A l A T A C A T A M l T T T T ~ T C C C T C C A C C ~ C C A T C A C T C C C U A C T G A A U C T C C C C T l A l T T T A T l C T T T C C T ~ A ~ T T A ~
tt
R
t
t t
C1R.t
t
t
"
tt
t t
t
R
.
t t
t
T A T l C C T T C T T C C A T C T M C A T A C l T M A A T A T T U U M T T T C T A A A C T l A C A l T l U T T T C T A C AT C T A I ~ ~ T T l C T ~ A M C P T C T ~ T A C C / r T T
~ T l C l T P I A T T I A T T C T T T ' A ~TTCCTCAIATT T A ~ T T ~ T C U T A T A A A A l A ~ ~ l l T C l l l P C A l l C l ~ T l T M U M ~ A C A ~ ~ A A U ~ T ~
.-t
t.t
rft
nmr
tftk
*mmm e * *
I*-
tft
* e * *
TTCTGTTATTMATTTGTCCTTTCTTCTATTTATTMATCAAACMAATUAAMMAMMMMAAMA
T T A T T T C ~ T T T C T A A I T T C T T T T A I T ~ATTCTCAAGGTUACTTCTTCAATT
T
t t t
t
tt
t * * t t
Figure 3. Comparison of bTP-1 and bIFN-an cDNA and their deduced polypeptide sequences. 'zhe deduced amino acid
sequence of bTP-1 is given above the nucleotide saqumce. Amino acid residues differing in bovine IF%an are indicated
below the nucleotide sequence.Asterisksmark differencesbetween the nucleotide sequences that do not result in a change in
an amino acid residue. Potential polyadenyhtionsignalsare undcrlimd. There is only a single sequence (Am7*Thr79Thr?
that provides a potential site for N-glycosylation of bTP-1. This figun is reproduced from Imakawa et al. (1989) with
permission.
IFN-an gene. Thus, it seems likely that the
diversity in molecular weight forms of bTF-1
noted during electrophoresis may relate to the
degree of complexity of the carbohydrate
chains rather than to the number of glycosylation sites (Helmer et al., 1988). However, the
presence of o-linked carbohydrate groups on
bTp-1 has not yet been ruled out.
In cattle there are known to be at least 15 to
20 IFN-an genes in addition to an impressive
number of IFN-~I(Capon et al., 1985; Velan
et al., 1985). However, evidence is beginning
to accumulate that the bTP-1 genes may
constitute a specific subset within this larger
grouping. The evidence for this contention is
as follows. First, all the bTP-1 cDNA so far
34
ROBERTS ET AL.
antiviral activity of bTP-1 or a significant loss
of biological potency during purification is
unclear. Certainly the various human IFN-q
exhibit a considerable range of antiviral
activities (DeMaeyer and DeMaeyer-Guignard,
1988), and IFN-afrom different species vary
markedly in stability (Piasecki, 1988).
Recently we have shown that bTP-1 could
displace an 1251-labeled recombinant bovine
IFN-a (rboIFN-all) from class I IFN receptors
in ovine endometrial membranes (J‘. R. Hansen and P. V. Malathy, unpublished data). The
bTP-1 was about one-seventh as effective on a
mass added basis as the unlabeled rboIFN-all,
which showed a Kd for the receptors of 6 x
lo-” M (Hansen et al., 1989).
bTP-1 at concentrations below 10-8 M is
also able to inhibit the incorporation of
[3Wthymidine into bovine leukocytes that
have been stimulated to divide by the plant
IFN-a.
mitogen Concanavalin A (P. V. Malathy, Y.
Niwano, K. Imakawa and R. M. Roberts,
bTP-1 is a BlologlcallyActive lntetferon
unpublished data). Such thymidine inhibition
The molecular cloning data discussed above assays have been widely used as tests for
CIMIYshowed bTP-1 and oTP-Ito be Closely immunosuppression, and it should be empharelated to the IFN-a,more distantly related to sized that IFN-a have a wide range of effects
the IFN-p (fibroblast IFN)and distinct from on cells of the immune system in addition to
IFN-y (immune IF“). The question arose, their ability to inhibit proliferation of certain
however, as to whether they had the biological classes of Tcells. Indeed, it seems feasible
that the trophoblast IFN may play some role in
activity of IFN-a.
Because OW-1was most readily purified, it controlling interaction between the maternal
was a relatively simple matter to show that it immune system and the “foreign” conceptus at
had antiviral activity against a range of animal the placental interface.
viruses in bovine epithelial cells and that its
potency approached that of other IFN-a,
The Use of cDNA Probes to
including ones produced in bacteria by recomStudy Expression of bTP-1
binant techniques (Pontzer et aL, 1988; RobThe 3’-nontranslated region of the bTP-1 or
erts et al., 1989). oTP-1 also possessed the
antiproliferative properties typical of an IFN-a oTP-1 cDNA has provided a specific probe to
(Roberts et al., 1989; Niwano et al., 1989) and distinguish the expression of their mRNA from
could strongly inhibit the incorporation of those of other IFN-a.Such probes can be
[3~]thymidineinto mitogen-stimulated leuko- employed to detect mRNA when RNA is
“dotted“ directly onto nylon membranes or
cytes.
Fewer studies have been conducted with after the RNA is first subjected to electrophobTP-1. However, total bovine conceptus secre resis and then transferred to such membranes
tory proteins can protect cells against viral (Northern blots). Finally, a cDNA or a cRNA
lysis and had a specific antiviral activity of .5 can be employed to detect the presence of
x 104 units/mg of protein when tested against mRNA on tissue sections (in situ hybridizaVesicular Stomatitis Virus (VSV) in cultured tion) provided that sufficient care is taken to
bovine kidney cells (unpublished data). After preserve the integrity of the RNA during tissue
purification, the bTP-1 itself had an activity of fixation and embedding.
The fonner two methods are relatively
about 106 units/mg protein, a value
10- to 100-fold lower than that of many straightforward but less sensitive than in situ
recombinant IFN ( P e a et al., 1987). hybridization. They indicated that bTP-1
Whether this result reflects an inherently poor mRNA amounts per microgram tissue RNA
cloned and sequenced resemble OW-1cDNA
considerably more than they do the one fully
sequenced bovine IFN-an gene (i.e., there is
more conservation across species than within
species). Second, the 3‘ends of bTP-1 and
0°F-1 are remarkably alike yet differ significantly from the corresponding 3’-ends of the
transcription unit of the bovine IFN-an gene
(Figure 3). Finally, Southern blotting of bovine
DNA with a specific 3’end probe under highly
stringent conditions of hybridization and wash
has revealed far fewer (probably no more than
4 to 5 ) than the 15 or so genes noted by Capon
et al. (1985) (T. R. Hansen and R. M. Roberts,
unpublished results). One possibility, which
will be discussed again later, is that the bTP-1
family of genes are uniquely expressed in
trophoblast tissue because they have some
function that cannot be mimicked by other
BOVINE CONcEprzIS INTERPERONS
and mRNA concentrations fell off markedly
after d 17 of pregnancy, approximately 4 d
after mRNA expression per cell was maximal
(Hansen et al., 1988; Farin et al., 1989).
1400-
1ZOO-.
1000800-
35
I
Can Recombinant I F N q Mimic
the Actlon of bTP-l?
Eioq>,i)
400..
200..
0.
I
DAY OF PREGNANCY
P i p 4. Amount of bTP- 1mRNA relative to total cellular RNA in embryos during pregnancy. Total RNA from d
15, 17, 19, 21, 23 and 25 comxptwes was extracted and
samples (4 pg) spotted onto nylon membranes as described
byaansenetal. (1988).TheRNAwasallowed tohybridize
with a 32P-hbe1d cDNA (813 base pairs;nucleotides 241 to
1,035 of the bTP-1 cDNA in Figme 3). A standard curye was
preparedby using known amounts (15.6pg to 2 ng) of a fulllength synthetic bTP-1 mRNA prcpand in the pBS M13
transcriptionvector(seeHansenetal.,1988)anddotttdonto
the same membrane. Only one d- 15 conceptus provided sufficient RNA for analysis. RemainiOg values are means f
standard errors for RNA from three different conceptuses.
were maximal around d 15 to 19 and were
maintained at relatively high levels until at
least d 25 (Figure 4). In situ hybridization
studies generally confirmed the above results
(not shown) but additionally indicated that
expression was highest in cells of the trophectoderm and was low or absent in the extraembryonic endoderm that lies immediately below
the trophectoderm, in the yolk sac and in
tissues of the embryonic disc. A series of
micrographs illustrating the tissue-specific expression of bTP-1 mRNA in trophectoderm is
provided in Figure 5. The technique of in situ
hybridization also allowed younger embryos
that could not provide sufficient RNA for dot
blot or Northern hybridization studies to be
examined for bTP-1 mRNA expression. Low,
but detectable, hybridization signals were
evident as early as d 12 (data not shown),
though maximal expression per cell occurred
three or more days later at the time of m a t 4
recognition of pregnancy.
These studies correlated well with results on
protein biosynthesis that indicated that bTP-1
production was high from d 15 to d 25 (Bart01
et al.. 1985) and possibly beyond (Godkin et
al., 1988). They contrasted with similar experiments carried out on OW-1whose synthesis
The existence of IFN was first inferred by
Isaacs and Lindemann (1957), but it required
almost 20 yr before the proteins were purified,
in part because of the astonishingly high
biological potency of these substances (see
Knight, 1987). For example, IFN-a rece tors
bind ligand with average Kd around 1WE M
(Rubenstein and Orchansky, 1986). Even now
“natural” IFN are in short supply. As far as we
are aware, for example, bTP-1 is the first
bovine IFN to be purified to homogeneity from
a natural source, and oTP-1 is certainly the
first ovine IFN of any kind either to be
purified or to have its mRNA cloned
However, as a result of recombinant DNA
procedures it is now possible to produce many
biologically active IFN in large quantities from
the bacterium E. coli (Pestka et al., 1987).
These recombinant IFN have been used for a
number of purposes, including treatment of
cancers (see Fauci et al., 1987) and viral
disease (Bielefeldt O h m et al., 1987). The
availability of a recombinant bovine I F N - ~ I
(rboIFN-ol11) has recently allowed this protein
to be tested for its effects on various reproductive parameters and, in particular, to be
compared with bTP-1 and OW-1with regard
to its dose effectiveness. The studies have
clearly demonstrated that some of the biological responses to the embryonic E N can be
mimicked by using the recombinant bovine
product. For example, infusion of milligram
quantities of rboIFN-a~l into the uteri of
cyclic ewes (Stewart et al., 1989) or intrauterine infusion (Plante et al., 1989) or intramuscular injection (Plante et al., 1990) of the
protein into cows during the period of maternal
recognition of pregnancy extended luteal
lifespan in the treated animals compared with
controls given placebo. Nevertheless, it has
become evident that considerably more
rboIFN-a~l had to be introduced into the
uterus to achieve an extension of interestrous
interval than if “natural” oTP-1 or bTP-1 were
used. There have also been indications that
rbolFNu11 and bTP-1 may have somewhat
different effects on prostaglandin metabolism
by endometrium (Helmer et al., 1989).
36
ROBERTS ET AL.
F55. In situ lodimtion of bTf-1 mRNA m d-19 bovine embryo trophectoderm. (A) Brightfield micrograph of
portions of trophectoderm(closed arrow) and yolk sac membranes (open mow) from a d-19 bovine embryo. Scale bar = 30
p.(B) Darkfield micrographof the same sectionUwtrated in (A)showing localizationof silver &as (white dots), which
hdicatepositive hybridbationof a [35S]-lfTP-1cDNA probe to bTF-1mRNA present in the trophectoderm.Note the lack of
hybridhationsignal in the yolk sac tissue. Q Darkfidd micrograph of an adjacent section hybridized with ["Sl-y-aain
cDNA showing localizationof actin mRNA in both trophectodermand yolk sac membranes.
It therefore remains an open question as to the standard range of biological activities of an
whether bTP-1 functions in maternal recogni- IFN-a. Clearly the genes for bTP-1 must
tion of pregnancy simply because it possesses possess regulatory control elements that permit
BOVINE CONCJPTUS INTERFWONS
37
them to be activated in trophoblast for a Godkin, J. D., F. W.Bazer, W. W. Thatcher and R. M.
relatively restricted period of development.
Roberts. 1984b. Proteins released by cultnred Day
1 5 1 6 conceptuses prolong luteal maintenance when
There is no evidence that other IFN-a genes
introduced into the uterine lumen of cyclic ewes. J.
are activated simultaneously and little indicaReprod. Fertil. 7157.
tion that bTp-1 is induced as a major product Godkin, J. D., B. J. Lifsey and B. A. GiUespie. 1988.
in leukocytes exposed to virus. Thus, what
Characterization of bovine conceptus proteins produced during the peri- and post-atwhaent periods of
may distinguish OW-1and bTp-1 from other
early pngnaecy. Bio,. Reprod. 38:703.
m4X not be
much their
Gross.T.S.,C.Plante,W.W.Thatcher.P.J.Hrmsen,S.D.
activities, but the tissue-specific and temporal
Helmer and D. I . Putney. 1988. Secretory proteins of
manner in which the two are expressed.
the bovine conceptus altes prostaglandin and protein
secretion in vitro. Biol. Reprod. 39:977.
Hansel, W. and E. M. Convey. 1983. Physiology of the
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