Conceptus interferons and maternal recognition of pregnancy

Ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1) are secreted by ovine and bovine conceptuses for a restricted period when the conceptus acts to block regression of the corpus luteum. Both are considered to be antiluteolytic, acting locally on the endometrium to prevent the production of the uterine luteolysin prostaglandin F2 alpha. Molecular cloning of cDNAs for oTP-1 and bTP-1 has shown that both are structurally related to alpha interferons (IFN-alpha s). In particular, they resemble members of the 172-residue long IFN-alpha II (or IFN-omega) subfamily. Both proteins possess the antiviral and antiproliferative properties of IFN-alpha s and appear to bind IFN-alpha receptors. A novel and previously unsuspected role for IFNs in early pregnancy is apparent.     

Ovine trophoblast protein-1 and bovine trophoblast protein-1 are present as specific components of uterine flushings of pregnant ewes and cows

A rabbit antiserum raised against ovine trophoblast protein-1 (oTP-1) was used to stain Western blots of the protein components from the uterine flushings of pregnant ewes (n = 61), non-bred cyclic ewes (n = 22), bred-but-nonpregnant ewes (n = 36), pregnant cows (n = 34), and bred-but-nonpregnant cows (n = 15). Nonpregnant animals were defined as ones from which no embryo was recovered. Uterine flushings of pregnant ewes contained oTP-1 between Days 14 and 24 of pregnancy, but not at Day 12. All of the cyclic ewes and 34 of 36 bred ewes, judged as nonpregnant, tested negatively for the presence of oTP-1. With one exception, oTP-1 was not detected in the nongravid uterine horns of pregnant ewes in which the conceptus had been confined to one uterine horn. Bovine trophoblast protein-1 (bTP-1), which cross-reacts immunologically with oTP-1, was also detectable specifically in the uterine flushings of pregnant cows when anti-oTP-1 antiserum was used. The urine (n = 14) and certival mucus (n = 20) samples of all the pregnant ewes tested were free of any detectable oTP-1. Thus, a useful pregnancy test for ewes based on oTP-1 release into these fluids seems unlikely. Results of this study show that oTP-1 and bTP-1 are pregnancy-specific proteins that are secreted into the uterine lumen where they possibly exert a local response.     

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.     

Ovine trophoblast protein-1 and human interferon alpha reduce prostaglandin synthesis by ovine endometrial cells

Ovine trophoblast protein-1 (oTP-1), a protein secreted by the sheep conceptus immediately prior to implantation has sequence homology with alpha interferon. We have previously shown that, in parallel with human alpha interferon (IFN), oTP-1 reduces the release of prostaglandins (PG) E and F2 alpha from cultured ovine endometrial cells. Here we have examined the time and dose dependence of these actions and the possible site of action of the peptides. The concentrations of oTP-1 and IFN required for 50% inhibition of PGE release were 92 pg/ml and 0.88 pg/ml and for PGF2 alpha release, 165 pg/ml and 1.12 pg/ml respectively. Significant effects on PG release were not measured before 12 h after addition of peptide to culture dishes. Following removal of the peptides, the cells released less PGs for a further 18 h but then recovered. A large increase in PG synthesis and release occurred from cells cultured with added serum or arachidonic acid (AA) and an interactive effect was demonstrated between them, AA having a greater stimulatory effect on PG released in the presence of serum. However, in all cases oTP-1 and IFN continued to attenuate prostaglandin release. We conclude that the IFNs act directly or indirectly on the prostaglandin synthase enzyme.     

Expression of trophoblastic interferon genes in sheep and cattle.

The trophoblastic interferons ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1, respectively) have been implicated as mediators of maternal recognition of pregnancy in sheep and cattle. The objective of this study was to describe the onset and duration of gene expression for oTP-1 and bTP-1 in preimplantation ovine and bovine conceptuses by in situ hybridization and Northern analysis. Sections from paraffin-embedded ovine conceptuses, collected on Days 10, 11, 12, 13, and 15 of gestation (n = 1, 3, 3, 2, 2), and bovine conceptuses, collected on Days 12/13, 15/16, and 19 (n = 2, 4, 5), were hybridized to specific [35S]-labeled cDNA probes. Two different probes, one encompassing bases 442-918 and representing both coding and 3'-untranslated regions, and a second 3'-specific probe (bases 650-912) were used to detect oTP-1 mRNA. At all stages examined, oTP-1 mRNA was confined to trophectoderm of ovine conceptuses. Consistent with earlier studies, expression increased markedly at Day 13. oTP-1 mRNA was detected at low levels in seven of seven ovine conceptuses prior to Day 13 when the longer probe was employed. With the 3'-specific probe, however, oTP-1 mRNA was detected in only one of the seven ovine conceptuses prior to Day 13. Thus, although low amounts of oTP-1 mRNA may be present in ovine conceptuses prior to Day 13, massive induction of this mRNA occurs on Day 13 coincident with the initiation of maternal recognition of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of antiviral activity and induction of 2',5'-oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1

Establishment of pregnancy in cattle has been proposed to depend on production of a conceptus protein, bovine trophoblast protein-1 (bTP-1), which has a high degree of sequence homology with bovine interferon-alpha (bIFN-alpha), especially the alpha II subfamily. A preparation of bovine conceptus secretory proteins enriched for bTP-1 has antiviral and physico-chemical properties similar to other bIFN-alpha. Antiviral activity is initially detectable in uterine flushings on Day 14 of pregnancy, when the conceptus measures 4-5 mm in length, and increases as the conceptus elongates through Day 18. Day 17 conceptuses produce more than 10(6) U antiviral activity during 24 h of culture. All IFNs induce the enzyme 2',5'-oligoadenylate synthetase, which catalyzes production of 2',5'-oligo(A), which in turn is involved in antiviral and growth inhibitory effects of IFNs. This enzyme activity is induced in Madin-Darby bovine kidney cells by the partially purified bTP-1 preparation similarly to IFN-alpha, -beta, and -gamma. Likewise, the partially purified bTP-1 and bIFN-alpha 1 induce 2',5'-oligo(A) synthetase activity in monolayers of endometrial epithelial and stromal cells. Compared to epithelial cells, stromal cells have higher baseline activity of 2'-5'-oligo(A) synthetase activity (p less than 0.01) and show a greater degree of induction in the presence of either the partially purified bTP-1 or bIFN-alpha 1 (p less than 0.01). Also, 2',5'-oligo(A) synthetase of endometrial stromal cells is induced to a greater degree by our enriched bTP-1 preparation than by bIFN-alpha 1 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.     

Intrauterine infusion of highly enriched bovine trophoblast protein-1 complex exerts an antiluteolytic effect to extend corpus luteum lifespan in cyclic cattle

Intrauterine infusion of enriched bovine trophoblast protein-1 complex (bTP-1) resulted in extension of interoestrous intervals and corpus luteum function in cyclic cattle. Conceptus proteins were obtained by culture of Day 17-18 conceptuses for 72 h. Media from the first (n = 28), second (n = 26) and third (n = 19) 24 h of conceptus incubations were utilized. A highly enriched preparation of bTP-1 was obtained by a combination of ammonium sulphate precipitation, ion-exchange chromatography, and h.p.l.c. gel filtration. Degree of purity of the final preparation was confirmed by gel electrophoresis and immunoblotting with antiserum to ovine trophoblast protein-1. Jersey cattle (3 per group) received intrauterine infusions, twice daily from Day 15.5 to 21.0, of bovine serum albumin, the entire array of bovine conceptus secretory proteins (bCSP) from the 3 days of conceptus culture, or bTP-1. Infusions were via a catheter into the uterine horn ipsilateral to the corpus luteum. Oestrous cycle length in bTP-1-treated cows (26.1 +/- 1.3 days) was greater than for cows given BSA (19.5 +/- 1.3 days) or bCSP (21.5 +/- 1.3 days). Similarly, progesterone concentrations in serum remained elevated for a longer period of time for bTP-1-treated cows than for cows treated with BSA or bCSP. Residual variance associated with vena cava concentrations of PGF-2 alpha at Days 19-21 after oestrus (which included the variance between 15-min periods within cows) was reduced in cows treated with bTP-1 as compared to other groups. Lack of a bCSP effect may have been due to low amounts of bTP-1 in conceptus-conditioned medium from cultures of greater than 24 h. None the less, purified bTP-1 was effective in extending luteal function and appears to be the antiluteolytic agent of early pregnancy.     

The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon)

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine trophoblast protein-1 complex alters endometrial protein and prostaglandin secretion and induces an intracellular inhibitor of prostaglandin synthesis in vitro

Evidence exists that conceptuses alter endometrial protein secretion and modulate prostaglandin (PG) synthesis and secretion in cattle. The present study was designed to test the effect of bovine conceptus secretory proteins (bCSP) in general and the bovine trophoblast protein-1 complex (bTP-1) in particular on endometrial function. Endometrial explants from cyclic cows (N = 4) at Day 17 after oestrus were incubated for 24h with 0, 4.8, 24 or 120 micrograms BSA/ml, 1 microgram bTP-1/ml or 12.7 micrograms bCSP/ml. Both bCSP and bTP-1 decreased (P less than 0.005) release of radiolabelled macromolecules into medium and incorporation of radio-labelled precursors into tissue compared to BSA-treated tissues but not tissues treated with medium alone. Secretion of a protein of Mr = 14,900 was enhanced by bCSP treatment as compared to treatment with bTP-1 (P less than 0.025). Both bCSP and bTP-1 decreased PGF secretion of explants (P less than 0.01) compared to BSA. Overall, PGE-2 secretion by bCSP- and bTP-1-treated tissues did not differ from that of BSA cultures, but PGE-2 secretion was greater (P less than 0.025) for bTP-1 than bCSP-treated endometrium. Cotyledonary microsomes from parturient cattle were utilized as a PG-generating system for the detection of inhibitors of PG synthesis. PGF synthesis by the generating system was decreased (P less than 0.05) by 9% by cytosol from BSA-treated explants, whereas cytosol from bCSP- and bTP-1-treated explants decreased (P less than 0.01) PGF synthesis by 42% and 35%, respectively. In summary, both bCSP and bTP-1 inhibit PGF secretion, induce synthesis of an intracellular inhibitor of PGF synthesis, and decrease protein synthesis and secretion. The bTP-1 complex therefore alters PG dynamics by explants in a manner that would function to prevent luteolysis and support the establishment of pregnancy.     

Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast.

Yeast has been analysed for its potential to secrete an ovine member of the type-I interferon (IFN) family, trophoblastin (oTP-1). The processing potential of the yeast KEX2 gene product (KEX2p) was evaluated using gene oTP-1 fused to the pre-pro sequence encoding the pre-pro peptide of the yeast alpha-factor precursor. High-level accumulation of nonprocessed (unmatured) recombinant oTP-1 (re-oTP-1) was observed in the medium. In order to short-circuit the limiting activity of KEX2p and to obtain a fully matured re-oTP-1, secretion was directed using a pre::oTP-1 fusion, relying only on signal peptidase-dependent processing. However, secretion of oTP-1 was impaired. High-level secretion was restored when the gene product contained a peptide spacer between oTP-1 and the signal peptidase cleavage site. The oTP-1 variant was shown to have an extended N terminus. An N-extended form was examined further and shown to have the correct size. Surprisingly, the variant retained its in vitro and in vivo biological activities. This system is likely to represent a general method for high-level secretion of type-I IFNs.     

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.     

N-glycosylated and unglycosylated forms of caprine trophoblast protein-1 are secreted by preimplantation goat conceptuses

Goat conceptuses secrete caprine trophoblast protein-1 (cTP-1) which is related antigenically to the abundant embryonic interferon-alpha II of sheep and cattle. Antiserum to ovine and bovine TP-1s immunoprecipitated three molecular weight classes (23,000, 21,000 and 17,000, each with two isotypes) of cTP-1 from goat conceptus culture medium. Cultures which contained tunicamycin resulted in a shift in the Mr = 23,000 and Mr = 21,000 forms to a Mr of 17,000. The Mr = 23,000 and 21,000 forms, but not the Mr = 17,000 form, bound to Concanaval in A-Sepharose and were eluted under conditions selective for glycoproteins bearing complex-type oligosaccharide(s). Thus cTP-1 is a mixture of glycosylated and unglycosylated polypeptides.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)