Early Pregnancy In Pigs Research Articles

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) β1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72 h later. Gilts in the control group (n = 23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy ( P < 0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 ( P < 0.05) and 15 ( P < 0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFβ, LIF, and PGHS-2 on day 12 of pregnancy ( P < 0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

Leptin Gene and Protein Expression in the Ovary During the Oestrous Cycle and Early Pregnancy in Pigs

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT-PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid- and late-luteal phase of the oestrous cycle as well as during days 14-16 and 30-32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14-16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late-luteal phase of the cycle than on days 30-32 after conception. However, comparison of leptin protein expression in OS between days 14-16 of the cycle and days 30-32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14-16 of pregnancy in comparison to days 30-32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30-32 compared with days 14-16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.

Local and Systemic Effects of Embryos on Uterine Tissues During Early Pregnancy in Pigs

In the pig, the periimplantation period is critical for successful establishment of pregnancy. We studied the influence of embryos on oxytocin (OT) and progesterone (P(4)) regulated endometrial and myometrial secretion of 1) luteotrophic prostaglandin E(2) (PGE(2)) and 2) luteolytic prostaglandin F(2alpha) and its metabolite (PGFM) on days 12-14 of pregnancy in pigs. We used unilaterally pregnant pigs created by a surgical procedure in which one uterine horn remained intact and the second horn was cut transversely so that part of the horn was detached from the uterine body. The animals were divided into two groups, inseminated gilts (days 12-14 of pregnancy, n=6) and uninseminated cyclic gilts, which were used as controls (days 12-14 of estrous cycle, n=5). Embryos developed only in the patent part of the uterus and not in the occluded horn. The abundance of OTR mRNA was increased in the endometrium and decreased in the myometrium of the gravid uterine horn in the pregnant pigs compared with the non-gravid uterine horn or either uterine horn in the cyclic pigs, indicative of a local effect of the conceptus. The presence of embryos in the uterine horn during the periimplantation period determines endometrial metabolism of PGF(2alpha) and the local response of the endometrium to OT and P(4). OT stimulates PGF(2alpha) secretion and PGFM accumulation in endometrial cultures only from the non-gravid uterine horn and controls PGE(2) secretion from the endometrium and myometrium in the pregnant gilts. The results indicate a more systemic affect of pregnancy on the uterine response to OT and a possibly the local effect of the conceptus in promoting progesterone's inhibition of OT-stimulated PGE(2) secretion and uterine metabolism of PGF(2alpha).

Endometrial and myometrial secretion of androgens and estrone during early pregnancy and luteolysis in pigs

Previously, we found that in addition to embryos, the uterine tissues may be a source of estradiol-17beta (E(2)) during early pregnancy in the pig. The aim of the present study was to determine whether porcine endometrium and myometrium secrete androgens - androstenedione (A(4)), testosterone (T) and estrone (E(1)) during early pregnancy and luteolysis (Days 14-16) in pigs. Individual endometrial and myometrial slices (200 mg) were first pre-incubated (24 h) and then incubated (6 h, 37 degrees C in an atmosphere of 95% O(2) and 5% CO(2)) in the presence or absence of progesterone (P(4); 10(-5) M), oxytocin (OT; 10(-7) M) or OT plus P(4). Basal endometrial and myometrial secretion of A(4) and T did not differ between pregnant and cyclic gilts. Endometrial secretion of E(1) was higher in pregnant than cyclic gilts (p<0.05) while myometrial secretion of E(1) did not differ between the two groups of the examined pigs (p>0.05). Progesterone significantly increased A(4) and T secretion (p<0.001) by uterine tissues regardless of the reproductive status. In the presence of P(4), endometrial and myometrial secretion of E(1) was increased only during luteolysis (p<0.001). In both tissues, OT did not affect the examined steroid secretion and did not change the effect of P(4). 1) porcine endometrium and myometrium was found to produce A(4), T and E(1) in vitro; 2) basal endometrial and myometrial production of A(4) and T did not differ between the examined reproductive periods; 3) the endometrium released more E(1) during early pregnancy than luteolysis; 4) in the presence of substrate (P(4)), uterine tissues increased secretion of A(4) and T during early pregnancy and luteolysis; and 5) P(4) increased uterine production of E(1) only during luteolysis. These data demonstrated the presence of the active steroid pathway in porcine endometrium and myometrium which may serve as an alternative source of androgens and estrogens in pigs.

The effect of insulin-like growth factor-I, relaxin and luteinizing hormone on vascular endothelial growth factor secretion by cultured endometrial stromal cells on different days of early pregnancy in pigs

The effect of insulin-like growth factor-I (IGF-I), relaxin (RLX) and luteinizing hormone (LH) on vascular endothelial growth factor (VEGF) in vitro secretion by endometrial stromal cells in pigs was investigated on days 10-12 and 20-22 of gestation. LH-stimulated stromal cell secretion of VEGF did not differ among tested days of early pregnancy. However, IGF-I- and RLX-mediated release of VEGF depended on the day of pregnancy. It seems that IGF-I and RLX may be considered as potent activators of VEGF-mediated angiogenesis in porcine endometrium, and their action may be more pronounced during maternal recognition of pregnancy.

Expression of the vascular endothelial growth factor‐receptor system in the porcine endometrium throughout the estrous cycle and early pregnancy

The objective of this study was to investigate the protein and mRNA expression of vascular endothelial growth factor (VEGF), VEGFR-1 (fms-like tyrosine kinase, Flt-1) and VEGFR-2 (fetal liver kinase-1/kinase insert domain-containing receptor, Flk-1/KDR) in the endometrium during the estrous cycle and early pregnancy in pigs. The VEGF-receptor system was localized in epithelial and stromal cells, blood vessels, and myometrium. Western blot analysis showed higher levels of VEGF protein during the periovulatory and periimplantation periods (P < 0.001, and P < 0.05, respectively). Constant expression of VEGF mRNA during the cycle and significant upregulation on Days 22-25 of gestation (vs. Days 9-17; P < 0.001) was observed. Stable levels of VEGFR-1 mRNA and protein were detected in the endometrium of cyclic animals. However, higher VEGFR-1 protein expression was found on Days 16-17 of the estrous cycle (P < 0.01) and Days 13-15 of gestation (P < 0.05). Protein expression of VEGFR-2 was elevated on Days 2-4 of the estrous cycle (P < 0.001), but mRNA levels were constant during the cycle. In pregnancy, VEGFR-2 protein expression started to increase after Day 15 (vs. Days 9-12; P < 0.05), but induction of VEGFR-2 mRNA expression occurred earlier on Days 13-15. It appears from the present study that the VEGF-receptor system is regulated in a temporal and spatial manner during the estrous cycle and early pregnancy in pigs. The results suggest that VEGF-A family members are probably involved in appropriate preparation of endometrium for implantation and in vascular events during implantation in pigs.

Omega-3 fatty acid supplementation and the insulin-like growth factor (IGF) system in early pregnancy in pigs (2007)

The IGF system of growth factors, receptors and binding proteins functions from early in pregnancy. Recent evidence indicates improved embryo survival in gilts fed supplemental omega-3 fatty acids beginning before conception. Here we report effects of supplementing a corn-soybean meal diet (control) with a marine source of protected omega-3 fatty acids (PFA, 1.5% of diet) on mRNA expression for IGF-I, IGF-II, IGF Binding Protein-3 (IGFBP-3) and IGFBP-5 in the porcine gravid uterus. The PFA (Gromageâ„¢) contained equal amounts of eicosapentanoic (EPA) and docosahexanoic (DHA) acids and replaced corn in the diet beginning when gilts were approximately 170 d old (n = 13/treat-ment). Gilts were artificially inseminated at approximately 205 d of age. Conceptus and endometrial samples were collected on d 11, 15, and 19 of gestation. All gilts were pregnant. In the conceptus, message for IGF-II and IGFBP-3 increased (P<0.001) from d 15 to d 19, while there was an increase (P < 0.001) in IGF-I and IGFBP-5 from d 11 to 15 and a decrease (P<0.001) to d 19. In the endometrium, message for IGF-I was stable over the interval, but message for IGF-II and IGFBP-5 were increased by d 15 and IGFBP-3 by d 19 (P<0.01). There were trends for omega-3 fatty acid supplementation to increase endometrial IGF-II (P = 0.09) and IGFBP-5 (P = 0.12) on d 15. In the d-19 conceptus, embryonic but not extraembryonic IGF-I mRNA tended to be greater (P = 0.13) for PFA compared to control gilts. During d 11 to 19 the conceptus is elongating, attaching to the uterus, and the embryonic disc is differentiating from a homogenous tissue to form the tissues and organs of the adult. One mechanism for omega-3 fatty acid effects in early pregnancy could involve epigenetic effects on mRNA expression for the IGF and IGFBP proteins.; Swine Day, 2007, Kansas State University, Manhattan, KS, 2007

Effects of a preovulatory administered depot gonadotrophin-releasing hormone agonist on reproductive hormone levels and pregnancy outcome in gilts

The present study aimed to explore the influence of a preovulatory administered depot gonadotrophin-releasing hormone (GnRH) agonist (GnRHa; Decapeptyl Depot) on the endocrine parameters and pregnancy outcome of gilts (n = 6). A GnRHa-supported preovulatory luteinising hormone (LH) surge was detected in all treated gilts. LH pulses were abolished completely by depot GnRHa on Day 7 and partly on Day 21 of pregnancy. In this treatment group (n = 6) four gilts were pregnant at slaughter on Day 28. In the control group receiving Gonavet, a non-formulated GnRHa (n = 6), all pigs showed LH pulses and were pregnant at slaughter on Day 28 of gestation. Mean progesterone concentrations were elevated in controls during the early luteal phase and were similar for both groups during the implantation period. Mean concentration of unoccupied progesterone receptor was significantly higher in uterine myometrium than in endometrium, but without treatment effects. Peripheral estrone sulfate concentrations showed a similar increase in all pregnant gilts on Days 17 and 18, and remained elevated. In summary, treatment with a depot GnRHa for synchronisation of ovulation alters pulsatile LH secretion during early pregnancy in pigs. In general, this alteration seems not to exert an injurious influence on luteal function and, therefore, on embryo and early fetal development.

Expression of inter-alpha-trypsin inhibitor heavy chains in endometrium of cyclic and pregnant gilts

Attachment of the placenta to the uterus in pigs involves extracellular interaction between the expanding trophoblastic membrane and the thick glycocalyx present on the uterine epithelial microvilli. Formation of complexes between members of inter-alpha-trypsin inhibitor family may function in the maintenance of the extracellular matrix. This study investigated the change in the inter-alpha-trypsin inhibitor heavy chains (ITIH1, ITIH2, ITIH3 and ITIH4) during the oestrous cycle and early pregnancy in pigs. Gene expression of ITIH1, ITIH2, ITIH3 and ITIH4 was detected in the endometrium of cyclic and pregnant gilts; however, gene expression of ITIH was not altered throughout the oestrous cycle or early pregnancy. Western blot analysis with an ITIH antiserum identified the possible linkage forms of ITIH with the serine protease inhibitor, bikunin. Pregnancy altered the release of the various inter-alpha-inhibitor forms from the endometrium during the period of trophoblastic attachment. The results from this study indicate that the inter-alpha-trypsin inhibitor family plays an important role in maintenance of the uterine surface glycocalyx during placental attachment in pigs.

Effect of folic acid plus glycine supplement on uterine prostaglandin and endometrial granulocyte-macrophage colony-stimulating factor expression during early pregnancy in pigs

The objective was to determine the effects of folic acid+glycine supplement on uterine metabolism of prostaglandin and mRNA expression of endometrial granulocyte-macrophage colony-stimulating factor (GM-CSF) in nulliparous (NYL) and multiparous Yorkshire–Landrace (YL) sows, and in multiparous Meishan–Landrace sows (ML). In each of these three groups, sows were randomly assigned to two treatments: 15 ppm folic acid+0.6% glycine or no supplement. The dietary supplement was given from the estrus before mating to slaughter on Day 25 of pregnancy. At slaughter, endometrial tissue was collected to determine endometrial expression levels of GM-CSF mRNA, cyclooxygenase-1 (COX1) and -2 (COX2) and to evaluate in vitro endometrial secretion of prostaglandin E 2 (PGE 2) secretion. Allantoic fluid samples were also collected to determine the concentration of PGE 2, prostaglandin F 2α (PGF 2α), estradiol-17β (E 2), progesterone (P 4), and transforming-growth factor-β 2 (TGF-β 2). The allantoic contents of PGF 2α, E 2 and P 4, and endometrial in vitro secretion of PGE 2 were not significantly influenced by the folic acid+glycine supplement. The folic acid+glycine supplement tended ( P<0.07) to increase allantoic content of PGE 2 and TGF-β 2 in all sows and increased ( P<0.05) endometrial expression of COX2, especially in NYL sows. The endometrial expression of COX1 was decreased ( P<0.05) by folic acid+glycine supplement, especially in multiparous YL sows. The allantoic contents of PGE 2 and PGF 2α were not significantly affected by sow type. However, NYL sows had higher ( P<0.05) endometrial in vitro secretion of PGE 2 and allantoic content of P 4 than multiparous YL and ML sows. The allantoic content of E 2 was also higher ( P<0.05) in NYL sows than in multiparous ML sows only. The allantoic content of TGF-β 2 was lower ( P<0.05) in multiparous ML than in multiparous YL only sows. Finally, in YL and NYL sows, folic acid+glycine supplement decreased ( P<0.05) the endometrial expression of GM-CSF but not in ML sows. In summary, folic acid+glycine supplement altered endometrial expression of GM-CSF and uterine metabolism of prostaglandins during the post-attachment period of porcine embryos but some of these effects were manifest only in Meishan and nulliparous sows.

Effect of dietary fat sources on systemic and intrauterine synthesis of prostaglandins during early pregnancy in gilts.

The present experiment was conducted to determine the influence of dietary fatty acids C18:2n-6 and C18:3n-3 on the modulation of intrauterine synthesis of prostaglandin E2 (PGE2) and F2alpha (PGF2alpha) during early pregnancy in pigs. Prostaglandin E2 in uterine fluid has been previously reported to be associated with embryo survival and development. Thirty-two Yorkshire-Landrace nulliparous gilts were randomly allocated to four diets containing 5% supplemental fat. The four dietary treatments were: HT, hydrogenated tallow (26.5% C16:0 and 54.8% C18:0); SO, sunflower oil (61.3% C18:2n-6); LO, linseed oil (50.4% C18:3n-3); and SO(CLA), a mixture of sunflower oil and conjugated linoleic acids to provide 20% CLA. Treatments started 2 d after the first pubertal estrus (d -21) and lasted for 36 d (slaughter), which was 15 d after the second estrus (d 0; insemination). Fatty acids and PGE2 were measured in the peripheral blood plasma on d -19, d -7, d 0, and d 14. Fatty acids in endometrial tissues and PGE2 and PGF2alpha in the uterine fluid collected on d 15 were also measured. Concentrations of fatty acids in the plasma reflected the content of fatty acids in the diet as early as d -7. From d -7, PGE2 concentrations in the plasma were higher in gilts fed SO compared with HT (P < 0.05). Plasma PGE2 concentrations were lower (P < 0.01) on d 14 in gilts fed LO compared with HT. Total PGF2alpha contents in the uterine fluid of gilts fed LO were more than 70% lower (P < 0.05) than for the HT group. A similar trend was observed for total PGE2 content and for the ratio PGF2alpha:PGE2, but the effect (LO vs HT) was less marked (P < 0.07 and P < 0.10, respectively). There was no effect of SO or SO(CLA) on total PGE2 contents in the uterine fluid. Dietary enrichment in C18:2n-6 and/or C18:3n-3 for early pregnant gilts can influence fatty acids in plasma and endometrial tissue and can modulate circulatory and intrauterine prostaglandins.

Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in pig

In 1977 Bazer and Thatcher proposed that maternal recognition of pregnancy in the pig involves the secretion of PGF 2α towards the uterine lumen (exocrine) rather than towards the uterine venous drainage (endocrine) as occurs in the non-pregnant pig during the mid to late stages of the estrous cycle. The retrograde transfer of PGF 2α from the venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF 2α could constitute a part of putative mechanism of corpus luteum protection during early pregnancy. A luteotropic/anti-luteolytic effect of PGE 2 in the pig also has been frequently demonstrated and it seems that the most effective agent in changing PGE 2:PGF 2α secretion is estradiol. The role for oxytocin during luteolysis and early pregnancy is controversial. It appears, however, that the main function of this hormone is autocrine and/or paracrine stimulation of PGF 2α secretion. Pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an anti-luteolytic effect in pigs. It is likely, that cytokines and angiogenic growth factors are involved in the initiation of luteolysis and/or maintenance of corpora lutea (CL). A discovery of functional LH receptors in porcine endometrium opened a new possibility for this hormone in luteolysis and perhaps in recognition of pregnancy in pigs. The endogenous LH pulses can provoke prostaglandin secretion from endometrium in pigs. On the other hand prolongation of up-regulation of LH receptors in endometrium of early pregnant gilts can additionally increase angiogenic factor production before the process of implantation is completed. Finally new integrated concepts of luteolysis and inhibition of luteolysis in pigs based on selectively reviewed information are presented.

Effect of folic acid and glycine supplementation on embryo development and folate metabolism during early pregnancy in pigs.

The present work aimed to determine if different levels of prolificacy either by parity or by genetic origin are linked to folate metabolism. Nulliparous Yorkshire-Landrace (YL) and multiparous YL, and multiparous Meishan-Landrace (ML) sows were randomly assigned to two treatments: 0 ppm or 15 ppm folic acid+0.6% glycine. Supplements were given from the estrus before mating until slaughter on d 25 of gestation. At slaughter, embryo and endometrial tissues were collected to determine concentrations of DNA, protein, and homocysteine. Allantoic fluid samples were also collected to determine concentrations of folates, vitamin B12 and amino acids. Blood samples were taken at first estrus, at mating, and on d 8, 16, and 25 of gestation to determine serum concentrations of folates, vitamin B12, and relative total folate binding capacity (TFBC). Over the entire experiment, multiparous YL sows had higher average serum concentrations of folates than nulliparous YL sows (P < 0.05) but had similar serum concentrations of relative TFBC. Concentrations of folates and relative TFBC averaged higher in ML measured over the entire experiment than in multiparous YL sows (P < 0.05). Concentrations of serum vitamin B12 were higher in multiparous YL than in ML sows or YL nulliparous sows (P < 0.05) over the entire experiment. In allantoic fluid, folates, vitamin B12, and essential amino acids contents were significantly lower in ML than in YL multiparous sows (P < 0.05). The folic acid+glycine supplement increased concentrations of serum folates, but the increase was more marked in nulliparous YL sows (nulliparous x folic acid+glycine, P < 0.05). The folic acid+glycine supplement had no effect on litter size and embryo survival, but it tended to increase embryo DNA in multiparous YL sows (P = 0.06) but not in ML and nulliparous YL sows. Homocysteine was decreased by folic acid+glycine supplement in embryos from all sows, but in endometrium, the folic acid+glycine effect was dependent on parity (nulliparous x folic acid+glycine, P < 0.05). The effects of folic acid+glycine on litter size and embryo development and survival and some aspects of folate metabolism suggest that the basal dietary content of folic acid+glycine was adequate for ML and nulliparous YL sows but not to optimize embryo development in YL multiparous sows.

Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs

Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs

Characterization of uterine epidermal growth factor during early pregnancy in pigs☆

Genomic research has identified a quantitative trait locus for uterine capacity, a component trait contributing to litter size, on porcine chromosome 8. The epidermal growth factor (EGF) gene, on porcine chromosome 8, may influence uterine capacity because of its growth-promoting activities. Using reverse transcription-polymerase chain reaction (PCR) and iterative screening of a porcine reproductive tissue cDNA library, 4932 bp cDNA sequence coding for porcine EGF precursor was obtained. The predicted protein sequence of the EGF precursor contained 1214 amino acids, similar to human EGF precursor (1207 amino acids, 81% identity). Curiously, the sequence of the mature peptide was less homologous between species than other regions of EGF precursor. The presence of conserved regions outside the mature peptide may suggest that these regions are functionally important. Expression of EGF mRNA in the endometrium of White crossbred gilts ( n = 3 to 5 each) was determined by Northern blotting using 20 μg of total RNA from endometrium of D 10, 13, and 15 cyclic, and D 10, 13, 15, 20, 30, and 40 of pregnant gilts. A 3342 bp probe from EGF precursor was used. The bands corresponding to EGF mRNA were quantified by densitometry and results were analyzed by ANOVA. EGF mRNA expression decreased significantly from D 13 to 15 of the cycle and pregnancy (P = 0.04), and from D 30 to 40 of pregnancy (P = 0.01). These findings show that EGF mRNA expression is temporally regulated during the cycle and early pregnancy, and this pattern of gene expression may be important during early conceptus development.

Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs

During early pregnancy, pig conceptuses initiate the synthesis of oestrogens and on day 12 their trophoblastic membranes undergo a rapid expansion throughout the uterine horns. The insulin-like growth factor (IGF) system may be involved with conceptus development and steroidogenesis in pigs. Changes in uterine luminal IGF, insulin-like growth factor binding proteins (IGFBPs) and enzymatic activity for cleavage of IGFBPs during the oestrous cycle and early pregnancy were investigated. Uterine luminal content of IGF-I and IGF-II in uterine flushings from pigs on day 12 of pregnancy were two and three times greater, respectively, compared with uterine flushings collected from gilts during the oestrous cycle. Both IGF-I and -II content decreased on day 15 of gestation but content of IGF-II in uterine flushings remained three times greater than that of cyclic gilts. IGFBP-2 and -3 were the predominant binding proteins present in uterine flushings during days 0-10 of the oestrous cycle or day 10 of pregnancy. No IGFBPs were detected in the uterine flushings of either cyclic or pregnant pigs after day 10 by ligand blotting. Incubation of [125I]-labelled IGFBPs with various protease inhibitors indicated that cleavage of [125I]-labelled IGFBP-2 and -3 in uterine flushings involved serine proteases such as tissue kallikrein and matrix metalloproteinases. The results of the present study indicate that an increase in tissue kallikrein activity on day 12 of the oestrous cycle and pregnancy in pigs can directly, or indirectly through activation of matrix metalloproteinases, cleave IGFBP-2 and -3, thus allowing uterine release of IGF-I and -II in the uterine lumen to stimulate conceptus development.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F2α secretion by luminal epithelial, glandular epithelial and stromal cells from pig endometrium. II. Responses of cyclic, pregnant and pseudopregnant pigs on Days 12 and 1

In pigs, the exact mechanism for the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine mode during pregnancy recognition is not known. The objective of this study was to examine whether this shift involved a change in the responsiveness of luminal epithelial, glandular epithelial and stromal cells to 0 or 100 nM oxytocin. Luminal epithelial cells, glandular epithelial cells and stromal cells were isolated from cyclic, pregnant or oestrogen-induced pseudopregnant gilts on Day 12 (Experiment 1) or Day 16 (Experiment 2) post oestrus (oestrus = Day 0). For cells obtained on Day 12, oxytocin stimulated PGF2alpha secretion by stromal cells (P<0.01) similarly for each reproductive status, whereas oxytocin stimulated PGF2alpha secretion from luminal and glandular epithelial cells (P<0.05) from pregnant and pseudopregnant gilts but not from cyclic gilts. For both concentrations of oxytocin, mean PGF2alpha secretion was less (P<0.05) from stromal cells of pregnant than cyclic gilts. For cells obtained on Day 16, oxytocin stimulated PGF2alpha release from stromal cells of cyclic gilts but not from stromal cells of pregnant gilts. Mean PGF2alpha secretion also was less (P<0.05) from stromal cells of pregnant gilts than cyclic gilts. Oxytocin tended to stimulate PGF2alpha release (P<0.07) from glandular epithelial cells of cyclic but not pregnant or pseudopregnant gilts. Luminal epithelial cells from all reproductive statuses were similarly unresponsive to oxytocin. In conclusion, the increased PGF2alpha secretory response to oxytocin of luminal and glandular epithelial cells from pregnant gilts on Day 12, combined with the decreased response of stromal cells from pregnant gilts on Days 12 and 16, may contribute, in part, to the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine direction during early pregnancy in pigs.

Effects of active and passive gonadotrophin-releasing hormone immunization on recognition and establishment of pregnancy in pigs.

This study investigated the effects of a reduction in gonadotrophins, by means of differently timed active and passive gonadotrophin-releasing hormone (GnRH) immunization at various stages, on the maintenance of early pregnancy in pigs. In the first experiment crossbred sows (n = 11) were immunized against GnRH using a commercial vaccine on the day of farrowing, mated at the first oestrus, and a booster immunization was administered 10 days (n = 7) or 20 days (n = 4) after mating. Plasma samples were collected every second day and assayed for GnRH antibodies and progesterone. Pregnancy testing was carried out by real time ultrasound. None of the sows receiving the booster immunization 10 days after mating were pregnant on Day 18 after mating. All sows receiving the booster on Day 20 after mating aborted, with a mean vaccination-to-abortion interval of 10.0 +/- 1.5 days. In the second experiment, crossbred gilts (n = 6) were passively immunized by infusing (i.v.) GnRH immune pig serum on Day 12 after mating. Luteinizing hormone profiles were determined on the day before immunization and one day afterwards. Daily plasma samples were assayed for GnRH antibodies and progesterone. None of the gilts were pregnant 18 days after mating, compared with 5 of 6 non-immunized controls. Booster immunization 10 days after mating resulted in failure of embryonic development and establishment of pregnancy before the corpora lutea (CL) regressed, according to progesterone profiles, whereas immunization 20 days after mating resulted in regression of CL followed by abortion. Passive immunization 12 days after mating had a similar effect to the active immunization 10 days after mating. These results demonstrate two different outcomes of active GnRH immunization depending on the timing of immunization, and indicate that loss of pregnancy between Days 12 and 18 may occur due a reduction in progesterone rather than complete failure of the CL, as occurs at later stages. The findings may provide an explanation for the reduced fertility of pigs in the summer-autumn period.

Endometrial Responsiveness to Oxytocin during Diestrus and Early Pregnancy in Pigs Is Not Controlled Solely by Changes in Oxytocin Receptor Population Density1

These studies were performed to test the hypotheses that: 1) endometrial responsiveness to oxytocin (OT) in pig endometrium is associated with changes in OT receptor (OTr) population density resulting from corresponding regulation of OTr gene transcription, 2) endometrial responsiveness to OT is controlled solely through a mechanism involving changes in OTr population density, and 3) OTr population density and endometrial responsiveness to OT differ between diestrus and early pregnancy in pigs. In experiment 1, OTr population density and dissociation constant (Kd) in cyclic pigs were constant on Days 10-16 but increased (p < 0.05) between Days 10 and 12 of pregnancy before decreasing (p < 0.05) through Day 16. OT induced phosphoinositide (PI) hydrolysis and prostaglandin (PG) F2 alpha secretion in cyclic pigs only on Day 16 (p < 0.05), and during pregnancy only on Day 12 (p < 0.05). Activation of G protein by aluminum fluoride (AIF4-) treatment maximally stimulated (p < 0.05) PI hydrolysis and PGF2 alpha secretion in cyclic pigs on all days, indicating that downstream from the OTr, the PGF2 alpha secretory pathway was fully functional. During pregnancy, PI hydrolysis and PGF2 alpha secretion in response to AIF4- decreased (p < 0.01) on Days 14 compared to Days 10 and 12, and AIF4- did not stimulate PGF2 alpha release on Day 16. In experiment 2, abundance of OTr mRNA in cyclic pigs decreased between Days 0 and 5 before increasing between Days 5 and 12 (p < 0.05), but it was higher (p < 0.05) on Days 10-15 of pregnancy than on equivalent days in cyclic gilts. These results indicate that control of PGF2 alpha secretion in cyclic pigs appeared to occur primarily at the level of OTr coupling to G protein because changes in OTr number were not associated with increased sensitivity to OT or G-protein activation by AIF4-. During pregnancy, control was exerted at multiple levels, which included the OTr, G protein, phospholipase C, and subsequent aspects of the secretory pathway. The present study also indicated that endometrium was responsive to OT during luteolysis in cyclic pigs but not during corpus luteum maintenance in pregnant pigs.

Expression of the Estrogen and Progesterone Receptors and Insulin-like Growth Factor-I Genes in the Porcine Myometrium during Early Pregnancy.

After conception and during early pregnancy in pigs, uterine motility plays a keyrole in sperm transport and embryo spacing prior to placentation. This process is modulated by gonadal steroids. Early pig embryos secrete estradiol with a peak d. 12-13 and estradiol elicits part of its influence on target cells in the genital tract via the insulin-like growth factor-I (IGF-I). Therefore, in the present study, the levels of the estrogen receptor (ER) and progesterone receptor (PR), as well as their mRNAs and the IGF-I mRNA, were measured in the myometrium of early pregnant pigs (days 8-30 after onset of estrus) by EIA and solution hybridization, in order to establish relationships among the expressions of IGF-I, ER and PR during early pregnancy. The ER level was low on days 8-10 and decreased even further to very low levels on days 25-30 while the level of ER mRNA was unchanged during the same period. The PR level was decreasing during the days monitored, although PR mRNA showed a tendency to increase from days 12-13 to days 25-30. The level of myometrial IGF-I mRNA was higher on days 25-30 as compared to days 8-18 indicating that myometrial IGF-I mRNA expression is not affected by the production of conceptus estradiol on days 12-13. Significant correlations were found between the expressions of IGF-I mRNA and PR mRNA and between the levels of ER and PR. The results suggest that in the porcine myometrium the expressions of IGF-I and PR genes, respectively ER and PR, may have common regulating factors.