N-glycodiversity of the Pregnancy-Associated Glycoprotein family (PAG) produced in vitro by trophoblast and trophectoderm explants during implantation, placentation and advanced pregnancy in the pig

The PAG family is encoded by distinct genes expressed in extra-embryonic chorionic membranes (TR--trophoblast, TRD--trophectoderm) of various pregnant mammals. The objective of our study was to determine N-glycodiversity of porcine PAG protein family (pPAG) produced in vitro by TR or TRD explants of gilts (n=26) throughout pregnancy (16-77 dpc). Explants were cultured for over 1200 h (TR, 16 dpc) or for 8 h (TRD, 17-77 dpc). Released proteins were isolated from media by separating ultra-filtration (>10 kDa). A deglycosylation (removal of N-linked carbohydrate side chains) of proteins was performed by glycopeptidase F, and compared to non-deglycosylated forms by PAGE-Western blotting with anti-pPAG sera and additionally to polypeptide pPAG precursors, coded by ORF of their cloned cDNAs. We demonstrated gestation-stage dependent diversity of deglycosylated/glycosylated forms of the pPAG proteins produced in vitro in the pig. TR explants harvested on 16 dpc during long term culture released 43 kDa pPAG proteins. These proteins were deglycosylated to approximately 36.9 and approximately 39.6 kDa (16 dpc). Tissue harvested on 17 dpc in vitro secreted 65-68 kDa pPAG proteins which were reduced to three forms, 50.6, 58.7 and 63.5 kDa. In addition, approximately 73.3 kDa major pPAG proteins (77 dpc) were reduced to at least three forms: approximately 39.6, approximately 36.9 and approximately 33.4 kDa. Such N-deglycosylation was not detected on days 25-61. N-deglycosylation of native pPAG proteins clearly corresponded to three N-glycosylation sites of asparagines (N-x-S/T) found in ORF of the pPAG2-like precursors, identified by their in silico translated cDNAs. Thus, the pregnancy-stage dependent N-glycodiversity of the pPAG protein family, containing an average 9.66% of N-linked oligosaccharides, may play some role(s) in porcine conceptus attachment, successful implantation and during advanced pregnancy.     

Radiocompetition of secretory pregnancy-associated glycoproteins as chorionic ligands with luteal and uterine gonadotrophin receptors of pregnant pigs

Porcine pregnancy-associated glycoprotein (pPAG) family is very promiscuous and its role(s) remains unknown. The objective of this study was to identify whether secretory placental proteins (including pPAGs), produced in vitro by porcine chorionic explants, may interact with other proteins/targets, i.e. luteal and uterine binding sites of pregnant pigs. Trophoblast (TRF) and trophectoderm (TRD) were harvested during peri-implantation and placentation periods (14-61 dpc-day post coitum). In vitro-produced TRF/TRD proteins were isolated from media by ultrafractionation (>10 kDa MWCO) or precipitation with 20-75% saturation of (NH(4))(2)SO(4) and pPAG proteins were monitored by Western blotting. Secretory TRF/TRD ligands (including PAGs) were serially diluted (0.78-25 microg/ligand) and examined by radioreceptor assay (RRA). Luteal and uterine membrane receptors of pregnant pigs (pRc) were isolated from corpora lutea (pCLRc), myometrium (pMYORc) and endometrium (pENDRc). The three pRc types were harvested during three periods of pregnancy: 14 dpc (14 Rc), 21-26 dpc (21-26 Rc) and 31 dpc (31 Rc). The RRA competitions of individual TRF or TRD ligands were performed with (125)I-hCG as tracer and different pRc types. The RRA results of TRF/TRD were compared to hCG/pLH ligands--as positive controls (0.39-50 ng/ml), and endometrial (END) proteins (0.78-25 microg/ml) produced in vitro by END explants of cyclic, pseudopregnant and pregnant gilts (cEND, PsEND and pEND, respectively)--as negative control ligands. Results indicated that secretory TRF/TRD proteins (+pPAGs) were able to compete with (125)I-hCG for binding with other proteins/targets, i.e. luteal and uterine receptors of pregnant pigs (pCLRc, pMYORc and pENDRc) in a concentration- and pregnancy stage-dependent manner. This study indicated that porcine secretory 14-15 dpc TRF (pPAG; 30-73 kDa) ligands, effectively displaced (125)I-hCG tracer from pCL14Rc (up to P< or =0.01), corresponding to displacement by hCG and porcine LH. During the early stage of pregnancy, some competition tendency (P< or =0.01) was also detected for TRF ligands (14-15 dpc) with pEND14Rc. As pregnancy advanced, significant (125)I-hCG competition (at least P< or =0.05) with secretory semi-purified TRD ligands (30-42 dpc) was determined for all types of examined receptors pCL31Rc, pMYO31Rc and pEND31Rc, mainly with TRD fractions precipitated by 20% saturation of (NH(4))(2)SO(4). It seems that chorionic pPAG family can be involved in luteoprotective mechanism during implantation and placentation, according to the binding-interaction with luteal and uterine gonadotropin receptors of pregnant pigs.     

Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.     

Localization of chorionic pregnancy-associated glycoprotein family in the pig

The objective of this study was to localize the immuno-positive porcine PAG (pPAG) proteins within chorionic cells throughout the intensive placenta development as pregnancy advances (16-61 days post coitum - dpc). Placental sections were used for double fluorescent histochemistry with selected primary rabbit anti-pPAG sera. The polyclonals were created against recombinant pPAG2 antigen or various secretory porcine native chorionic antigens produced in vitro. Among placental cells stained with fluorescent propidium iodine, the positive pPAG immuno-complexes were visualized by Alexa 488 fluorochrom - conjugated to secondary anti-rabbit goat immunoglobulins. This is the first report concerning cellular localization of the pPAG protein family within diffuse epitheliochorial placenta development throughout the first half of pregnancy in the pig. Fluorescent immuno-positive pPAG signals have been restricted to chorionic cell layers (branched mushroom-like and finger-like structures) that generate a epitheliochorial feto-maternal surface augmented by maternal endometrium interdigitations with the gestation progress in the pig. These results suggest that the pPAG proteins robustly expressed in chorionic cells are involved in the regulation of intensive development of diffuse porcine placenta during the first half of pregnancy.     

Pregnancy-associated glycoprotein family (PAG)--as chorionic signaling ligands for gonadotropin receptors of cyclic animals

The chorionic pregnancy-associated glycoprotein (PAG) family was identified in pigs, cattle and other eutherian mammals. The objective of this study was to examine whether secretory chorionic proteins (including PAGs), produced in vitro by explants of porcine and bovine placental membranes, may interact with other proteins, i.e. gonadal and extragonadal binding sites. Trophoblast (TRF) and trophectoderm (TRD) explants of pigs (n=38; 14-61 dpc-day post coitum) or cotyledons (CT) of cows (n=5; 40-110 dpc) were long-term cultured. Released chorionic proteins were ultra-fractionated from media (>10 kDa) or precipitated [20-75% of (NH(4))(2)SO(4)]. The PAGs were monitored by Western/PAGE (30-73 kDa). Secretory TRF/TRD/CT (+PAG) proteins (0.78-25 microg/ligand) were examined by radioreceptor assay (RRA) with iodinated hCG ((125)I-hCG) for binding-effectiveness by gonadotropin receptors of cyclic pigs and cows (cRc). Gonadal and extragonadal cRc isolated from luteal-phase corpora lutea and uteri (cCLRc, cMYORc and cENDRc) were tested with positive control ligands: porcine LH and hCG (0.39-50 ng/ml). Control proteins produced in vitro by endometrial (END) explants of cyclic (cEND), pseudopregnant (PsEND) and pregnant (pEND) gilts were utilised as negative ligands (0.78-25 microg/ligand). Positive control ligands competed with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc (18-61%/B(0) for hCG and 27-57%/B(0) for LH). Negative ligands (cEND, PsEND and pEND) did not show cRc bindings. This is the first RRA report indicating that in vitro produced porcine TRF/TRD proteins (+PAG) competed (P< or =0.05) with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc in a concentration- and pregnancy stage-dependent manner. The highest competition with (125)I-hCG (up to P< or =0.001) was found for ultra-fractionated TRF/TRD proteins (>10 kDa) during early pregnancy (<22 dpc). The greatest competition (P< or =0.05) of precipitated porcine TRD proteins (>30 dpc) was detected for fractions obtained by saturation with use of 20% of (NH(4))(2)SO(4). Bovine CT proteins revealed lower competition of (125)I-hCG for bovine cCLRc (during 45 dpc only) that was more efficient with CT (up to 71%) than with non-labelled hCG (82%). The PAG proteins may play a role as potential "signal molecules", because they were able to interact with gonadotropin receptors of luteal-phase animals. It seems that the pPAG proteins may be luteoprotective chorionic-origin signals during implantation and placentation, according to binding-effectiveness of the chorionic ligands that was comparable to LH/hCG ligands with gonadal and extragonadal receptors of cyclic animals.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

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.     

Progesterone modulation of osteopontin gene expression in the ovine uterus

Osteopontin (OPN) is an acidic phosphorylated glycoprotein component of the extracellular matrix that binds to integrins at the cell surface to promote cell-cell attachment and cell spreading. This matrix constituent is a ligand that could potentially bind integrins on trophectoderm and endometrium to facilitate superficial implantation and placentation. OPN mRNA increases in the endometrial glandular epithelium (GE) of early-pregnant ewes, and OPN protein is secreted into the uterine lumen. Therefore, progesterone and/or interferon-tau (IFNtau) may regulate OPN expression in the uterine GE. Cyclic ewes were ovariectomized and fitted with intrauterine (i. u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 136.317 (ZK; progesterone receptor [PR] antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. Progesterone induced the expression of endometrial OPN mRNA in the GE and increased secretion of a 45-kDa OPN protein from endometrial explants maintained in culture for 24 h. Administration of ZK ablated progesterone effects. Intrauterine infusion of roIFNtau did not affect OPN gene expression or secretion in any of the steroid treatments. Interestingly, OPN mRNA-positive GE cells lacked detectable PR expression, although PR were detected in the stroma. Results indicate that progesterone regulates OPN expression in GE through a complex mechanism that includes PR down-regulation, and we suggest the possible involvement of a progesterone-induced stromal cell-derived growth factor(s) that acts as a progestamedin.     

Uterine luminal proteins and estrogens in gilts on a normal nutritional plane during the estrous cycle and on a normal or high nutritional plane during early pregnancy

In gilts, a high plane of nutrition during early pregnancy often results in increased embryo mortality, possibly related to changes in embryo-uterine asynchrony at a critical stage of pregnancy (around Day 11). Therefore, in the present study, uterine luminal proteins and estrogens were studied between Days 5 and 16 after the onset of estrus in gilts on either a normal (2.5 kg/d, cyclic and pregnant gilts) or a high (4.0 kg/d, pregnant gilts only) feeding level. Conceptus recovery rate between Days 5 and 12 was not affected by the feeding level during early pregnancy, neither were systemic progesterone levels. Between Days 9 and 11, dramatic changes took place in the protein composition of the uterine luminal 10kD+ proteins, shifting from most (90%) of the acidic proteins at Day 5 and 7 to approximately 50% at Day 11/12, especially due to an increase in basic proteins with an iso-electrical point of more than 8. This shift occurred most rapidly for the pregnant gilts at the high feeding level and least rapidly in the cyclic gilts, resulting in significant differences in the relative amount of acidic proteins at Day 10 and 11 after the onset of estrus (P < 0.05). Similarly, levels of estrogens in the uterine flushings at Days 10, 11 and 12 were always highest for the pregnant gilts on the high feeding level and were always lowest in the cyclic gilts (P < 0.05); pregnant gilts on the normal feeding level showed intermediate estrogen levels. The fact that gilts on a high feeding level during early pregnancy show more rapid changes in the uterine luminal protein composition and embryonic estrogen production seems to suggest that the rate of these changes may be related to embryo survival.     

Contractility of the ovarian vascular bed during the oestrous cycle and early pregnancy in gilts

The vasoconstrictor activity of the ovarian vascular bed in vitro was investigated during the oestrous cycle and early pregnancy. Gilts were killed during the follicular phase (Days 20 to +1; N = 5) or luteal phase (Days 11 to 13; N = 4) of the oestrous cycle, or on Day 13 of pregnancy (N = 5). Immediately before death, a sample of vena cava blood was obtained for determination of progesterone and oestrogen (oestrone and oestradiol-17 beta) concentrations. One ovary was removed, cannulated, perfused in vitro, and subjected to 10-min infusions of saline (vehicle control) and noradrenaline. Vasoconstriction was provoked by electrical stimulation at the end of each infusion. Ovaries from luteal-phase gilts exhibited greater (P less than 0.01) vasoconstriction than did ovaries from follicular-phase and pregnant gilts at the end of saline and noradrenaline infusions. The oestrogen to progesterone ratio was less (P less than 0.01) for luteal-phase and pregnant than for follicular-phase gilts. Vasoconstriction was negatively correlated (r = -0.99, P less than 0.01) with the oestrogen to progesterone ratio in systemic blood of gilts during the oestrous cycle but not during early pregnancy (r = +0.39, P greater than 0.10), possibly due to an effect of the conceptuses.     

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI₂) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF_1α (a PGI₂ metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF_1α concentration. Regulation of PGI₂ synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF_1α in the endometrium. The concentration of 6-keto PGF_1α in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI₂ metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI₂ secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI₂ synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI₂ release during the implantation period.     

Osteopontin improves in vitro development of porcine embryos and decreases apoptosis

An optimal environment for fertilization and early embryonic development is provided by the mammalian oviduct and uterus. The secretory cells lining the lumen of the oviduct and uterus synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. Western blotting in this study demonstrated that a 50-kDa secreted phosphoprotein 1 (SPP1) form was present in the uterus on Days 0, 3, and 5 in pregnant and nonbred gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5 in pregnant gilts, but in nonbred gilts the concentration of SPP1 on Day 0 was higher than Day 3, but not Day 5. In addition, we show that addition of 0.1 microg/ml SPP1 to the culture medium after fertilization increased the percent cleaved (24 hr: 23.6 +/- 1.29(a) vs. 18.7 +/- 0.65(b) (2-cell %)), and the percent blastocyst (37.2 +/- 1.12(a) vs. 30.9 +/- 0.56(b)) derived from IVF (P < 0.05). In parthenogenetic-derived embryos the percent cleaved was increased due to SPP1 at 24 hr (24.0 +/- 1.59(a) vs. 19.7 +/- 1.59(b) (>2-cell %)), and at 48 hr (72.9+/- 2.99(a) vs. 63.3 +/- 2.99(b)), but not the percent blastocyst. By TUNEL assay, SPP1 decreased both apoptosis (7.9 +/- 0.04(a) vs. 13.1 +/- 0.02(b)) and the percent fragmentation (45.2 +/- 0.07(a) vs. 58.8 +/- 0.03(b)). We conclude that SPP1 can improve development in vitro possibly by reducing the rate of apoptosis.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Effect of in-vitro heat stress on prostaglandin and protein secretion by endometrium from pregnant and cyclic gilts at day 14 after oestrus

Endometrium from cyclic (N = 4) and pregnant (N = 4) gilts at Day 14 after oestrus was placed into three bilateral perifusion devices which allow separate perifusion of luminal and myometrial sides. Perifused endometrium was subjected to 39 or 42 degrees C. Incorporation of [3H]leucine into secreted and tissue proteins by endometrial explants following incubation at 39 or 42 degrees C was examined using trichloroacetic acid (TCA) precipitation and one-dimensional SDS polyacrylamide gel electrophoresis. Secretion of PGF was greater from the myometrial side for cyclic gilts (endocrine orientation), but greater from the luminal side for pregnant gilts (exocrine orientation). Regardless of reproductive status or endometrial side, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. However, PGF secretion in response to heat stress was greater (P less than 0.01) from the myometrial side and greater (P less than 0.01) for pregnant gilts. PGF secretion rates increased by 63% and 42% from the luminal side, and 40% and 156% from the myometrial side in response to heat stress for cyclic and pregnant gilts, respectively (status x treatment x side interaction; P less than 0.01). Heat stress did not alter incorporation of [3H]leucine into secreted proteins regardless of reproductive status, while incorporation into tissue proteins was decreased (P less than 0.05) by heat stress for pregnant gilts, but not altered for cyclic gilts. Heat stress, in vitro, redirects PGF secretion for endometria of pregnant gilts from an exocrine to an endocrine orientation where it would be available to effect luteolysis and compromise the establishment of pregnancy.     

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.     

Temporal patterns of secretion of porcine uterine suppressor and stimulatory macromolecules

Temporal secretory patterns of porcine uterine suppressor (>/=230 kD) and stimulatory (29 kD) macromolecules were evaluated within uterine luminal protein (ULP) secretions recovered during early pregnancy. The ULP was recovered by uterine flushing from four Landrace gilts each on Days 9, 12, 15 and 18 of pregnancy. Unfractionated and fractionated ULP (using Sephacryl S-200) was tested for suppression or stimulation of phytohemagglutinin-induced peripheral blood T-lymphocyte proliferation. For all days of pregnancy, unfractionated ULP suppressed (P<0.002 to 0.0001) lymphocyte proliferative responses, with the greatest (P<0.05) activity observed for ULP collected on Day 9 of pregnancy. Suppressor activity resulted from the >/=230 kD component, in which the activity was greater (P<0.05) for ULP from gilts on Day 9 than Days 12, 15 and 18 of pregnancy. The 29 kD component failed (P>0.05) to stimulate lymphocyte proliferation, although there was a nonsignificant stimulatory trend for 2 of 4 gilts each at Days 12 and 15 of pregnancy. These findings demonstrate a temporal secretory pattern for the >/=230 kD lymphocyte suppressor component, which may be requisite for the immunological survival of the conceptus during early pregnancy. The inconsistent appearance of the lymphocyte stimulatory factor (29 kD component) tends to minimize its biological significance relative to the immunology of pregnancy.     

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.     

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.