Analysis of STAT1 expression and biological activity reveals interferon-tau-dependent STAT1-regulated SOCS genes in the bovine endometrium.

Signal transducer and activator of transcription (STAT) proteins are critical for the regulation of numerous biological processes. In cattle, microarray analyses identified STAT1 as a differentially expressed gene in the endometrium during the peri-implantation period. To gain new insights about STAT1 during the oestrous cycle and early pregnancy, we investigated STAT1 transcript and protein expression, as well as its biological activity in bovine tissue and cells of endometrial origin. Pregnancy increased STAT1 expression on Day 16, and protein and phosphorylation levels on Day 20. In cyclic and pregnant females, STAT1 was located in endometrial cells but not in the luminal epithelium at Day 20 of pregnancy. The expression of STAT1 during the oestrous cycle was not affected by progesterone supplementation. In vivo and in vitro, interferon-tau (IFNT) stimulated STAT1 mRNA expression, protein tyrosine phosphorylation and nuclear translocation. Using chromatin immunoprecipitation in IFNT-stimulated endometrial cells, we demonstrated an increase of STAT1 binding on interferon regulatory factor 1 (IRF1), cytokine-inducible SH2-containing protein (CISH), suppressor of cytokine signaling 1 and 3 (SOCS1, SOCS3) gene promoters consistent with the induction of their transcripts. Our data provide novel molecular insights into the biological functions of STAT1 in the various cells composing the endometrium during maternal pregnancy recognition and implantation.

SOCS genes expression during physiological and perturbed implantation in bovine endometrium.

In mammals, suppressor of cytokine signalling (CISH, SOCS1 to SOCS7) factors control signalling pathways involved in the regulation of numerous physiological processes including pregnancy. In order to gain new insights into the biological functions of SOCS in the endometrium, a comprehensive analysis of SOCS gene expression was carried out in bovine caruncular (CAR) and intercaruncular (ICAR) tissues collected i) during the oestrous cycle, ii) at the time of maternal recognition of pregnancy and at implantation in inseminated females, iii) following uterine interferon-tau (IFNT) infusion at day 14 post-oestrus, iv) following a period of controlled intravaginal progesterone release and v) following transfer of embryos by somatic-cell nuclear transfer (SCNT). The regulatory effects of IFNT on in vitro cultured epithelial and stromal cells were also examined. Altogether, our data showed that CISH, SOCS4, SOCS5 and SOCS7 mRNA levels were poorly affected during luteolysis and pregnancy. In contrast, SOCS1, SOCS2, SOCS3 and SOCS6 mRNA levels were strongly up-regulated at implantation (day 20 of pregnancy). Experimental in vitro and in vivo models demonstrated that only CISH, SOCS1, SOCS2 and SOCS3 were IFNT-induced genes. Immunohistochemistry showed an intense SOCS3 and SOCS6 staining in the nucleus of luminal and glandular epithelium and of stromal cells of pregnant endometrium. Finally, SOCS3 expression was significantly increased in SCNT pregnancies in keeping with the altered immune function previously reported in this model of compromised implantation. Collectively, our data suggest that spatio-temporal changes in endometrial SOCS gene expression reflect the acquisition of receptivity, maternal recognition of pregnancy and implantation.

Lysophosphatidic acid receptors in ovine uterus during estrous cycle and early pregnancy and their regulation by progesterone.

In the present study, we examined the lysophosphatidic acid (LPA) pathway in the ovine uterus during the estrous cycle and early pregnancy. With the use of quantitative reverse transcription PCR, expression of LPAR1 and LPAR3 was analyzed. Both receptors were present in the ovine uterus. Immunolocalization showed that LPAR1 was mainly present in the stroma of the ovine endometrium, whereas LPAR3 was mostly restricted to epithelial compartments. In luminal and glandular epithelia, LPAR1 and LPAR3 levels were affected by pregnancy status, day, or the day-by-status interaction, whereas in stroma the receptors were not modified. Analysis of the whole endometrium from ovariectomized ewes showed that the expression of LPAR3 but not LPAR1 was regulated by the administration of progesterone. However, the examination of receptors at cellular levels showed that progesterone increases LPAR1 and LPAR3 in glandular epithelium and, in a minor extent, in endometrial stroma. Emerging evidence suggests that LPA is an essential component in the estrous cycle and early pregnancy regulation. We demonstrated that LPA induced stress fiber formation in ovine uterine epithelial cells, suggesting that LPA may be involved in cytoskeleton reorganization occurring cyclically in ovine uterus.

Developmental regulation of prostacyclin synthase and prostacyclin receptors in the ovine uterus and conceptus during the peri-implantation period.

This study documents the expression of prostacyclin (PGI2) synthase (PTGIS) and PGI2 receptors in the trophoblast and uterus of the ewe at the time of maternal recognition of pregnancy (i.e. days 7, 9, 12, 14 and 17). The membrane receptor for PGI2 (PTGIR) and the nuclear receptors, i.e. peroxisome proliferator-activated receptors (PPAR) and their heterodimer partners the retinoid X receptors (RXR), were analysed. In the endometrium, PTGIS transcript and protein were expressed at day 9 of pregnancy and levels declined from days 12 to 17. Immunohistochemistry and in situ hybridization indicated that PTGIS was mainly located in the luminal epithelium of the endometrium. Endometrial PTGIR, PPARA, PPARG and RXRG expression was regulated during the peri-implantation period whereas PPARD, RXRA and RXRB were consistently expressed. In the trophoblast, PTGIS transcript levels rose as development progressed and peaked at day 17. PTGIR and PPARA transcripts peaked before day 12 and then declined and became nearly undetectable by day 17, whereas PPARD and PPARG transcript levels rose steadily from days 12 to 17. Because the PPARs and the RXRs display different expression profiles, we suggest that different heterodimers may form and support distinct functions as development proceeds. Our results also underline the importance of PTGIS and PPARD in the trophoblast and PTGIR in the uterus, suggesting that PGI2 is of both uterine and trophoblastic origin and is involved in a complex signalling pathway at around the time of implantation in the ewe.

Identification of differentially regulated genes during elongation and early implantation in the ovine trophoblast using complementary DNA array screening.

Following hatching, pre-elongated conceptuses undergo elongation by intense proliferation, until implantation. We investigated the changes in gene expression associated with these physiological events using human cDNA arrays containing 2370 known genes. Comparison of pre-elongated, elongated, and implanting trophoblasts allowed the determination of 313 expressed genes, 63 of which were differentially regulated. These were classified into four functional families. Pre-elongated trophoblasts were characterized by preferential expression of genes involved in protein trafficking, whereas only latter developmental stages expressed cell signaling genes and receptors. Among the 63 developmentally regulated genes, four exhibited the highest levels of expression (TMSB10, CTNNA1, NMP1, and CX3CL1). Each of these also represents a functional family and display a specific expression pattern. One of them, CX3CL1 (CX3C chemokine, also known as fractalkine), is a chemokine that seems to have potential importance in trophoblast development, and which deserves further clarification of its role in implantation.

Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells.

Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.

Effect of roscovitine, a cdk1 inhibitor, and of the presence of oocyte on bovine cumulus cell expansion and cyclooxygenase-2 expression.

Oocyte maturation is accompanied by differentiation of surrounding cumulus cells. These cells produce hyaluronic acid (HA) and its storage in intercellular spaces results in expansion of the cells. The cumulus cells also accumulate cyclooxygenase-2 (cox-2) during maturation. Both expansion and cox-2 storage are regulated by FSH and EGF. The aim of this study was to determine whether oocyte meiotic resumption is involved in the regulation of cumulus differentiation or not. We investigated the effects of roscovitine, a reversible inhibitor of meiosis resumption of cattle oocytes on EGF induced expansion and cox-2 expression at the transcript and protein levels respectively (RT-PCR and Western blot), in cumulus oocyte complexes (COCs) and cumulus complexes alone (CCs). EGF induced expansion and cox-2 expression in both COCs and CCs. These effects were prevented by roscovitine, whether in the presence or in the absence of oocyte. However, the oocyte was essential for the reversibility of inhibition by roscovitine. In conclusion, our results indicate that i) oocyte secreted-factors are not essential for cumulus expansion, and ii) roscovitine mediated inhibition of meiotic resumption also respects the functionality of the surrounding somatic cells.

Cyclooxygenase-2 is expressed by cumulus cells during oocyte maturation in cattle.

Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase.

Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes.

IFN-tau: a novel subtype I IFN1. Structural characteristics, non-ubiquitous expression, structure-function relationships, a pregnancy hormonal embryonic signal and cross-species therapeutic potentialities.

IFN-tau (IFN-tau) constitutes a new class of type I IFN which is not virus-inducible, unlike IFN-alpha and IFN-beta, but is constitutively produced by the trophectoderm of the ruminant conceptus during a very short period in early pregnancy. It plays a pivotal role in the mechanisms of maternal recognition of pregnancy in ruminants and it displays high antiviral and antiproliferative activities across species with a prominent lack of cytotoxicity at high concentrations in vitro in cell culture and possibly in vivo. It exhibits high antiretroviral activity against HIV and exhibits immunosuppressive activity in a multiple sclerosis model and reduces embryo and fetal mortality by stimulation of IL-10 production. In this review all the biochemical and para-hormonal properties of this novel IFN-tau are described in detail: structural characteristics of proteins and genes, trophoblast expression, regulation of its expression, structure of its gene promoter, its absence in human species and in non-ruminant animals, the evolution of the IFN-tau genes, its structure-function relationships with its three-dimensional structure, structural localization of biological activities, its lack of cytotoxicity and its receptor. Surprisingly, for an IFN, IFN-tau is also a pregnancy-embryonic signal with paracrine antiluteolytic activity. In order to maintain luteal progesterone secretion, IFN-tau inhibits PGF-2alpha pulsatile secretion and oxytocin uterine receptivity in early pregnancy. It is believed to suppress pulsatile release of endometrial PGF-2alpha by preventing oxytocin and estrogen receptor expression. Additionally, it directly regulates prostaglandin metabolism and possibly the PGE:PGF-2alpha ratio.

Characterization of conceptus-produced goat interferon tau and analysis of its temporal and cellular distribution during early pregnancy.

Two proteins (17 and 22-24 kDa) produced by day 17 goat conceptuses were purified from in vitro culture media. Analysis of their N-terminal amino acid sequences and of their antiviral activity confirmed that both proteins belonged to the interferon tau family characteristic of ruminant conceptuses. The two molecules were glycosylated (22-24 kDa) or nonglycosylated (17 kDa) isoforms of the same protein. The time course of secretion was plotted and immunoblotting of the protein contents of uterine flushings from day 13 to day 21 of pregnancy was performed. The nonglycosylated isoform (17 kDa) was first detected on day 16; both isoforms were present at day 17 and, thereafter during pregnancy, the two proteins were not present in uterine flushings. Immunohistochemistry was used to show that the goat interferon tau was present in the trophoblastic cells as early as day 14 and until day 17. However, immunostaining was not uniform along the conceptus; labelling was greater at the abembryonic pole than at the embryonic pole. By day 18, as implantation proceeded, goat interferon tau was no longer detected. These results confirmed that the goat conceptus secretes interferon tau during the period of maternal recognition of pregnancy but its rapid decrease suggests that other factors need to be present by day 18 to take over its role in the maintenance of luteal function.

Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period.

In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.

Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy.

In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

Preferential esterification of arachidonic acid into ethanolamine phospholipids in epithelial cells from ovine endometrium.

In sheep, the pulsatile release of prostaglandin F2 alpha by the endometrium is necessary to achieve luteolysis which occurs at the end of the oestrous cycle. The production of prostaglandins is known to depend upon the availability of arachidonic acid, the fatty acid precursor of prostaglandin biosynthesis. Consequently, the mechanisms controlling intracellular amounts of arachidonate may be involved in the regulation of prostaglandin synthesis. Since arachidonic acid is mostly found in phospholipids and the endometrial epithelium is the primary source of prostaglandin F2 alpha during luteolysis, the fate of arachidonic acid when incorporated into epithelial cells from the ovine uterus was investigated. Endometrial epithelial cells isolated from cyclic ewes at day 15 after oestrus were cultured in the presence of [3H]arachidonic acid. Incorporation and distribution of the radiolabelled arachidonic acid into the various phospholipid classes were examined using HPLC. We observed that ethanolamine glycerophospholipids contained 61% of the total tritiated arachidonic acid incorporated into cellular lipids, whereas phosphatidylinositols, phosphatidylcholines and phosphatidylserines contained 17%, 13% and 4.7%, respectively. In addition, the radioactivity measured within phosphatidylethanolamines was preferentially detected in the 1-alkenyl-2-acyl (44%) forms of ethanolamine phospholipids, also called plasmalogens. The kinetic study of arachidonic acid uptake into ethanolamine phospholipids showed that arachidonic acid was rapidly esterified into the diacyl forms and then uptake decreased, whereas the incorporation increased continuously into the plasmalogen forms for at least 24 h. These results demonstrate that the primary pool of esterified arachidonic acid is found in ethanolamine plasmalogens of epithelial cells from the ovine endometrium. The high arachidonate content of ethanolamine plasmalogens suggests that these phospholipids play a crucial role in the control of arachidonic acid availability and ultimately in the regulation of prostaglandin synthesis.

Phospholipase A2 activity in endometrium from early pregnant and non-pregnant ewes.

In the ewe, synthesis of the luteolytic factor, prostaglandin F2 alpha, increases from day 13 to the end of the estrous cycle. Availability of free arachidonic acid is usually the rate-limiting step in prostaglandin biosynthesis. Phospholipase A2 (PLA2) may be the key enzyme for the hydrolysis of arachidonic acid from membrane-bound phospholipids. To investigate uterine PLA2 activity during the estrous cycle and early pregnancy, we monitored the release of [14C]oleic acid from the substrate 1-palmitoyl-2-[14C]oleoyl-phosphorylcholine by homogenates and cytosolic fractions of endometrium from ewes on days 12, 14 and 16 of the estrous cycle or pregnancy. We observed that PLA2 activity dropped by 58% (p < 0.02) in day-16 pregnant endometrium compared to day-16 non-pregnant endometrium. We then investigated whether the reduced PLA2 activity was due to induction of a specific inhibitor. The PLA2-inhibitor activity was determined by monitoring the inhibition of release of [14C]oleic acid from the radioactive substrate by porcine pancreatic PLA2. Inhibition by endometrial homogenates of pregnant animals of the control enzyme activity was 27% and only 14% by cyclic ones. Inhibition was dose-dependent and was as high as 53% (p < 0.01) with 1 mg protein from pregnant endometrial homogenates. Endometrial PLA2 behaved as a Michaëlian enzyme in the endometrium of day-16 cyclic ewes (Km = 79.4 mumol/l). Furthermore, the inhibitory activity from pregnant endometrium had characteristics of competitive inhibition. Our results suggest that inhibition of endometrial PLA2 activity could occur in early pregnant ewes.

Recombinant ovine trophoblastin (roTP) inhibits ovine, murine and human lymphocyte proliferation.

Ovine trophoblastic protein (oTP) is a 20-kDa embryonic secretory product constitutively secreted by ovine conceptus trophoblast from days 12-22 of pregnancy. Amino acid sequencing as well as molecular cloning revealed it to bear structural analogies with interferons of the class 2 alpha subfamily, defining the tau interferon group. It is endowed with classical interferon-like biological activities. Recombinant ovine trophoblastin (roTP), produced by genetic engineering, was purified by anion exchange HPLC to a high degree of homogeneity (98%). It behaved in immunodetection and antiviral activity assays like the natural form. We show here that when assayed on PHA-driven murine, human, and ovine (sheep) lymphocyte proliferation, roTP is immunosuppressive. It also inhibits unidirectional and bidirectional murine and human mixed lymphocyte reactions (MLRs). Since natural oTP possesses (at least) 5 isoforms, we also assayed these for immunosuppressive activities. All of them inhibited PHA-driven human and ovine lymphoblastogenesis. Finally, CD4+ and CD8+ ovine T cell selection was performed by panning. In contrast with earlier observations assaying roTP activity on human lymphocytes, both ovine CD4 and CD8 T cell subsets were sensitive to roTP in a PHA-driven proliferation assay. It is therefore suggested that trophoblast interferons might have a strategic function in preventing early embryonic demise by immunologic rejection, at least in ovine species.

Immunoregulatory effects of ovine trophoblastin protein (oTP): all five isoforms suppress PHA-induced lymphocyte proliferation.

The immunosuppressive properties of ovine trophoblastin protein (oTP) isoforms purified to homogeneity by DEAE HPLC have been studied within and across species barriers by in vitro assays. It has been demonstrated that not only the classical oTP 1, but in fact all 5 isoforms, are immunosuppressive in a PHA-induced proliferation assay, whilst being ineffective on IL-2 dependent CTL-L2 cell replication. The significance of these findings is discussed.

Evidence for extended maintenance of the corpus luteum by uterine infusion of a recombinant trophoblast alpha-interferon (trophoblastin) in sheep.

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

Is oxytocin of conceptus origin involved in inhibition of luteal regression in early pregnancy in ewes?

This study describes the presence in and production by the ovine conceptus of an oxytocin-like peptide during the early stages of development. Oxytocin was measured by radioimmunoassay in ovine conceptuses from days 14 to 30 of pregnancy. Tissue concentrations of oxytocin increased from day 14 (24.8 +/- 5 pg/100 mg) until day 19 (122.9 +/- 52 pg/100 mg) and then decreased (3 +/- 1 pg/100 mg). Oxytocin was released into culture medium by day-15 ovine conceptuses at a rate of 262 +/- 55 pg/24 h. Reverse-phase high-performance liquid chromatography (HPLC) analysis of peptides extracted from a pool of ovine conceptuses was conducted using chromatographic conditions developed to separate oxytocin from other nonapeptides. Radioimmunoassay of HPLC fractions for oxytocin revealed an immunoactive conceptus peptide in a single fraction at the same retention time as chromatographed authentic oxytocin. Radioimmunoassay and chromatographic data therefore suggest that this oxytocin-like peptide is similar, if not identical, to authentic oxytocin. Concentrations of oxytocin in conceptus tissue were maximal during the period of inhibition of luteal regression (days 14-19). It is proposed that conceptus oxytocin is involved in the maintenance of luteal function in early pregnancy.