Retinol-binding protein: a major secretory product of the pig conceptus

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.     

Characterization of proteins produced in vitro by periattachment bovine conceptuses

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(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.     

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.     

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F_2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.     

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.     

Onset of secretion of proteins with antiviral activity by pig conceptuses

In Exp. 1, antiviral activity was detected in Day-15 pregnant uterine flushings (6222 +/- 2167 units/ml) and in conceptus culture medium collected at 0, 1, 2, 4, 8, 16, 24, 32, 40, and 48 h (95, 375, 650, 1216, 1600, 2100, 2017, 2083, 3500 and 5000 units/ml, respectively; R2 = 0.81, P less than 0.01; y = 190.0 + 252.7x - 11.2x2 + 0.2x3. In Exp. 2, antiviral activity of Day-15 conceptus culture medium was reduced 99% after boiling for 20 min (P less than 0.01) and, after 18 h dialysis (6000-8000 Mr cut-off), 100% of the activity was in the retentate. In Exp. 3, antiviral activity was not detected in cultures of conceptuses from Days 10 and 11 and activity was maximal for Day 14 and Day 15 conceptuses (2100 and 2083 units/ml, respectively). Effects of day were best described by a quadratic regression equation (y = 17,652 - 3263x + 150x2; R2 = 0.55, P less than 0.01). In Exp. 4, changes in antiviral activity detected in uterine flushings from pregnant gilts on Days 8, 10, 11, 12, 14 and 15 (1.3, 0, 6.7, 63.3, 580 and 1663 units/ml, respectively) were described by the equation y = -20,743 + 6189x - 606x2 + 20x3 (R2 = 0.85, P less than 0.01). In Exp. 5, low antiviral activities (5-30 units/ml) were detected in all plasma samples collected from the uterine artery and uterine vein of pregnant and cyclic gilts, but values were not significantly influenced by pregnancy status, day or site of collection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postcopulatory effects of two antifertility agents on ova transport and implantation

The daily doses which prevented implantation in 50 percent of treated animals (ED50) of 2,3 - bis (4-hydroxyphenol) valeronitrile (SC-3402) and 2,3 - bis (4-methoxyphenyl) pent-2-enenitrile (SC-3296) injected in rats s on Days 1 to 3, or Days 4 to 7, or Days 1 to 7 (Day 1 = pregnancy) were 100, 200, and 40 mcg and 50, 100, and 12 mcg respectively, ED50 doses of estrone were 4,8 and 3.5 mcg. Control animals showed ova in the oviduct only on Days 1, 2 and 3, also in the uterus on Day 4, and only in the uterus on Day 5. Very few ova were found in rats treated with 10 mcg estrone daily Day 1-2 and autopsied on Day 3. The same treatment period with 200 mcg SC-3402 caused similar results. 64 mcg SC-3402 resulted in a smaller reduction of ova. Acceleratory potency of 200 mcg SC-3402 is greater than can be due to its estrogenic activity equivalent, 0.5 mcg estrone; that of 64 mcg SC-3296 (4.8 equivalents estrone) can be so ascribed. Rats receiving daily 4-8 mg 17 alpha-acetoxy-6 alpha-methylprogesterone (MAP) from Day 1 to 9 to delay nidation, and 200 mcg SC-3402, autopsied on Day 10 showed no free blastocysts and a few implantation sites in the process of resorption (Free blastocysts were found in rats similarly treated but with ligation of the uterus at the cervix on Day 5 to prevent expulsion of blastocysts). Control rats on Day 10 showed a few implantation sites and free blastocysts. The normal number of implantations were present in SC-3296 treated rats. The average weight of cornu traumatized by threading one cornu in psuedopregnant rats with a silk thread on Day 5 (Day 1=cervical stimulus) in rats treated with 200 mcg SC-3402 on Days 5-8, 404 plus or minus 50 mg was significantly (P less than .05) lower than mean control weight, 794 plus or minus 48 mg. The difference between the mean weight of non-traumatized cornu of rats given 100 mcg, 284 plus or minus 36 and 232 plus or minus 12 mg respectively was significantly (P less than .05) greater than in controls, 159 plus or minus 5.8 mg. The deciduoma-inhibiting activity of SC-3402 is further evidence that it initiates nidation but impedes early implantation stages.     

Effect of recombinant alpha interferons on fertility and interestrous interval in sheep

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 mug of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 mug recombinant human interferon alpha1 (rhlFN); or 3) 1500 ug of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 mug/conceptus) than from rblFN-treated (56 +/- 12 mug/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 mug/conceptus) and rblFN-treated (147 +/- 17 mug/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.     

Steroid regulation of retinol-binding protein in the ovine oviduct

Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17beta (E2), progesterone (P4), E2+P4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P4 alone or in combination with E2 significantly reduced steady-state RBP mRNA levels compared to those in E2-treated animals. Oviductal explants from E2- and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E2 and E2+P4 treatments. This study demonstrates that P4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.     

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.     

Oxytocin-stimulated inositol phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins

Three experiments (Exp) assessed the influence of stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins (oCSP) on turnover of inositol trisphosphate (the putative second-messenger for oxytocin-stimulated secretion of prostaglandin F2 alpha) in ovine endometrium during luteolysis and maternal recognition of pregnancy. In Exp 1, endometrium was collected from 5 cyclic (Cy) and 6 pregnant (P) ewes on Day 16 after onset of estrus. In Exp 2, endometrium was collected from Day 12 Cy (n = 5), Day 12 P (n = 3), Day 16 Cy (n = 4), and Day 16 P (n = 3) ewes. In Exp 3, 12 Cy ewes were allotted randomly, in a 2 x 2 factorial arrangement, to receive serum protein (SP), or oCSP and estradiol-17 beta (E2), or vehicle treatments. Ewes were injected i.v. with 0.5 mg E2 or vehicle on Day 12 and received twice-daily infusions of 1.5 mg SP or oCSP (containing 25 micrograms ovine trophoblast protein-1 by radioimmunoassay [RIA]) + SP (1.5 mg total protein) into each uterine horn on Days 12, 13, and 14. Blood samples for RIA of plasma progesterone were collected on Days 10-15 (before treatment on each day) and endometrium was collected on Day 15. For each Exp, 100 mg endometrium was incubated, in duplicate, for 2 h with 10 microCi [3H] inositol and treated with 0 or 100 nM oxytocin (OT) for 20 min, then [3H]inositol mono-, bis-, and trisphosphates (IP1, IP2, and IP3, respectively) were quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of medium proteins radiolabelled after culture of Day 13 to 16 pig conceptus tissue with [3H]leucine and absence of chorionic gonadotrophin-like activity

Day 13-16 pig conceptus tissue was cultured for 24 h in medium containing [3H]leucine. The patterns of radioactively labelled proteins that were released into the medium during culture were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. Day-13 conceptuses released two major radiolabelled proteins of Mr 23 000 and 26 000 and Day 14-16 conceptuses released these as well as proteins of Mr 14 000, 19 000, 44 000, 50 000 and 88 000. Various immunological and biological tests for a human chorionic gonadotrophin-like activity were performed on tissue extracts and on culture medium, but there was no evidence for its presence in the pig conceptus at Day 13-16 of gestation.     

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.     

Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.     

Pregnancy and interferon tau regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.     

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.