Influence of the endometrium, protease inhibitors and freezing on antiviral activity of proteins secreted by pig conceptuses

In Exp. 1, only medium from cultures containing conceptus tissue had antiviral activity (P less than 0.05). Addition of Day-15 pregnant endometrium or Day-14 cyclic uterine flush proteins to cultures containing 200 mg conceptus tissue decreased antiviral activity (conceptus x endometrial protein interaction, P less than 0.06). Effects of endometrium (-54%) and uterine flush proteins (-40%) on antiviral activity of conceptus cultures did not differ from each other (P greater than 0.10). In Exp. 2, antiviral activity was only detected in cultures containing conceptus tissue (P less than 0.06). The amount of antiviral activity in cultures of Day-15 conceptus tissue was not influenced differently (P greater than 0.10) by culture in medium conditioned by endometrium from Day 10 or Day 12 of pregnancy. However, antiviral activity was undetectable in medium conditioned by endometrium from one of the Day-12 gilts. In Exp. 3, antiviral activity was present in medium from only 1 of 3 cultures from Day-12 gilts when assayed unfrozen. Antiviral activity was lower (P less than 0.01) in cultures of conceptuses from Day 12 than Day 14 of pregnancy; however, antiviral activity increased quadratically (P less than 0.05) when cultures contained 0, 0.01, 0.1 and 1.0 units/ml aprotinin, respectively. Freezing and thawing culture medium did not reduce (P greater than 0.10) antiviral activity compared to medium assayed unfrozen (1438 vs 1354 units/ml, respectively). These results suggest a regulatory influence of the endometrium on secretion of antiviral proteins by pig conceptuses in vitro.     

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.     

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 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.     

Proteins secreted by the sheep conceptus suppress induction of uterine prostaglandin F-2 alpha release by oestradiol and oxytocin

In Exp. I, 0.5 mg oestradiol or vehicle (0.5 ml absolute ethanol + 0.5 ml 0.9% NaCl) was injected i.v. at 08:00 h on Day 14 (onset of oestrus = Day 0). Blood samples were obtained via a jugular catheter at 30 and 1 min before oestradiol and every 30 min for 10 h afterwards. Plasma was obtained and assayed for 15-keto-13,14-dihydro-PGF-2 alpha (PGFM) by radioimmunoassay. Before oestradiol, PGFM basal values were higher (P less than 0.01) in pregnant (N = 10) than nonpregnant (N = 6) ewes (193 +/- 30 vs 67 +/- 8 pg/ml). However, at 4-10 h after oestradiol, pregnant ewes (N = 5) had less variable (P less than 0.01) PGFM values than did nonpregnant ewes (N = 5). In Exp II, conceptus secretory proteins (CSP) were obtained by pooling medium from cultures of Day-16 sheep conceptuses (N = 40). Ewes received 750 micrograms CSP + 750 micrograms plasma protein (N = 6) or 1500 micrograms plasma protein (N = 6) per uterine horn at 08:00 h and 18:00 h on Days 12-14. All ewes received 0.5 mg oestradiol at 08:00 h on Day 14 and blood samples were collected as in Exp. I and assayed for PGFM. On Day 15, 3 ewes in each group received 10 i.u. oxytocin and 3 received saline i.v. at 08:00 h and blood samples were taken continuously from 10 min before to 60 min after treatment. Mean PGFM response to oestradiol was suppressed (P = 0.05) in CSP- vs plasma protein-treated ewes (371 +/- 129 vs 1188 +/- 139 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of prostaglandin F2α as the luteolysin in swine: V comparison of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts

Uterine flushings were collected from 38 gilts representing Days 6,8,10,12,14,15,16 and 18 of the estrous cycle and pregnancy. The same group of gilts were represented within each of the respective days of the estrous cycle and pregnancy, i.e., three to six gilts per day per status. Uterine flushings (about 40ml) were assayed for prostaglandin F (PGF), estrone (E₁), estradiol (E₂), progestins (P) and protein. Nonpregnant gilts had higher (P<.01) concentrations of P in uterine flushings than pregnant gilts, but pregnant gilts had higher (P<.01) E₁ and E₂ concentrations. Significant day by status interactions were detected for E₁ (P<.05), but not for E₂ concentrations in uterine flushings. Total recoverable PGF and PGF concentrations in uterine flushings were greater (P<.01) in pregnant than nonpregnant gilts and significant (P<.01) day by status interactions were detected. In nonpregnant gilts, PGF increased between Days 12 and 16, i.e., during the period of corpora lutea (CL) regression. In pregnant gilts, PGF in uterine flushings increased markedly between Days 10 and 18. Total recoverable PGF on Day 18 of the estrous cycle was only 464.5 ± 37.6 ng as compared to 22,688.1 ± 1772.4 ng on Day 18 of pregnancy. Total recoverable protein was also higher (P<.01) in pregnant gilts. These data indicate that PGF synthesis and secretion by the uterine endometrium and/or conceptuses is not inhibited during pregnancy and suggest that PGF is sequestered within the uterine lumen of pregnant gilts, as is the total protein component of endometrial secretions referred to as histotroph.     

Effect of pregnancy and exogenous ovarian steroids on endometrial prolactin receptor ontogeny and uterine secretory response in pigs

During gestation, pigs have constant circulating levels of prolactin (PRL), and lack decidual PRL and placental lactogens. Effects of PRL on uterine physiology in pigs may be due to changes in endometrial PRL receptors. In this study, effects of the conceptus and cyclic hormonal environment on endometrial PRL receptors were investigated. Endometrial PRL receptor numbers were similar between Days 8 and 15 for cyclic gilts. In contrast, endometrial PRL receptor numbers for pregnant gilts were similar between Days 8 and 11, then increased (p less than 0.05) on Day 12 and remained elevated between Days 14 and 30. This day-by-status interaction approached significance (p less than 0.06) and overall receptor numbers were higher (p less than 0.01) for pregnant than for cyclic gilts. Pig conceptuses secrete estrogen between Days 11 and 12; therefore, regulation of endometrial PRL receptors by acute administration of estradiol was investigated. Uterine flushings and endometrium were collected from one uterine horn of cyclic gilts on Day 11; then, at 1, 6, 12, and 24 h following a single injection of estradiol valerate (EV; 5 mg, into adipose tissue), uterine flushings and endometrium were collected from the second uterine horn. Endometrial PRL receptor numbers were higher (p less than 0.05) at both 1 h and 6 h after treatment with EV and then decreased (p less than 0.02) by 12 h to below pretreatment values. In uterine flushings, total recoverable protein (p less than 0.05), uteroferrin (p less than 0.01), leucine aminopeptidase (p less than 0.05), calcium (p less than 0.03), sodium (p less than 0.01), and potassium (p less than 0.05) increased between 12 and 24 h following EV treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Conceptus development, uterine response, blood gases and endocrine function of gilts exposed to increased ambient temperature during early pregnancy

Fifteen crossbred gilts were used to determine the influence of heat stress during Days 8 to 16 after onset of estrus on the development of conceptuses and uterine and endocrine functions. Ten gilts were bred 12 and 24 h after the onset of estrus (Day 0), and five gilts were nonbred controls. On Day 5, catheters were inserted into the uterine-ovarian vein (UV), saphenous artery (SA) and saphenous vein (SV) of each gilt. An electromagnetic blood flow transducer was implanted around the main uterine artery. Pregnant (n=5) and nonbred (n=5) control gilts were exposed to 21 +/- 1 degrees C, and pregnant heat-stressed gilts (n=5) were exposed to 37 +/- 1 degrees C for 12 h and 32 +/- 1 degrees C for 12 h daily during Days 8 through 16 after estrus. Treatment did not influence the partial pressure of oxygen (PO(2)) and of carbon dioxide (PCO(2)) in the UV, SA and SV blood. Uterine blood flow was not altered by heat stress. On Day 16, total wet weight of conceptuses was reduced in the gilts that were heat-stressed compared with conceptuses from control gilts. Incorporation of (3)H-leucine into macromolecules in vitro by conceptuses from the heat-stressed gilts was reduced compared with control gilts. Concentrations of 15-keto-13, 14-dihydro prostaglandin F(2alpha) (PGFM) in peripheral blood were greater than 1 ng/ml between Days 13 to 16 after estrus in 20% of the pregnant control gilts, 60% of the heat-stressed pregnant gilts, and 100% of the nonbred gilts. Concentrations of estradiol in the SA were affected by treatment. These results indicate that heat stress of gilts between Days 8 to 16 after estrus reduced the amount of conceptus tissue and altered concentrations of estradiol in the peripheral circulation, but uterine blood flow and PO(2) and PCO(2) in blood were not affected.     

Effects of prolactin on conceptus survival and uterine secretory activity in pigs

Hypoprolactinaemia was induced by bromocriptine (CB154; 100 mg/day) which decreased circulating prolactin by 40% (P less than 0.06), but did not affect conceptus survival at Day 25 when administered on Days 10-16 when compared to saline:ethanol-treated control gilts. Bromocriptine or vehicle was administered to cyclic gilts on Days 10-11, oestradiol valerate was injected on Day 11 and uterine flushings were collected on Day 12. Total recoverable protein and uteroferrin in uterine flushings were not affected by treatment. However, leucine aminopeptidase activity (P less than 0.02) and total recoverable Ca2+, Na+, K+ and Cl- (P less than 0.05) were decreased in uterine flushings of gilts that received bromocriptine, suggesting that hypoprolactinaemia decreased general secretory activity of the endometrial epithelium and modulated ionic changes, respectively, in the uterine environment of pigs. Subcutaneous administration of pig prolactin (1 mg/12 h) increased (P less than 0.001) serum prolactin 4.5-fold. The interaction between hyperprolactinaemia and progesterone, without oestrogen, on components of uterine flushings were determined using gilts that received progesterone (200 mg/day) and prolactin or saline on Days 4-14 after ovariectomy on Day 4. On Day 15, there were no differences (P greater than 0.05) in any of the uterine secretory components measured. Hyperprolactinaemia (1 mg pig prolactin on Days 6-11) enhanced overall uterine secretory response on Day 12 to oestradiol (5 mg) administered on Day 11 compared to gilts that received 1 ml saline on Days 6-11 of the oestrous cycle. Total recoverable protein and leucine aminopeptidase activity were greater (P less than 0.05) for oestradiol-treated gilts, but effects of prolactin were not significant. Total recoverable glucose (P less than 0.01), PGF-2 alpha (P less than 0.02), uteroferrin (P less than 0.01) and specific activity of uteroferrin (P less than 0.001) were increased by prolactin and oestradiol, but not oestradiol alone. Calcium (P less than 0.05), chloride (P less than 0.05) and potassium (P less than 0.01) were increased in response to oestradiol. These results indicate an interaction between oestradiol and prolactin, but not progesterone and prolactin, which enhances secretion of some products of the pig uterine endometrium.     

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.     

Characterization of the interleukin-1beta system during porcine trophoblastic elongation and early placental attachment

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.     

Effect of porcine conceptus secretory proteins on interestrous interval and uterine secretion of prostaglandins

Porcine conceptus secretory proteins (pCSP) were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialyzed, and concentrated by Amicon ultrafiltration for intrauterine infusion. Serum proteins (SP) were obtained from blood collected from a Day 15 pregnant gilt and diluted for intrauterine infusion. Catheters were placed into both uterine horns and the inferior vena cava of cyclic (Day 8) gilts. Single blood samples were collected at 0800 h on Days 9, 10, and 11. On Day 11, all gilts received 1 mg estradiol-17 beta (E2) i.m. at 0800 h. Protein infusions commenced on Day 12 and continued through Day 15, twice daily at 0800 h and 2000 h. Protein infusions per uterine horn were (1) 4.0 mg pCSP + 4.0 mg SP (pCSP, 4 gilts) and (2) 8.0 mg SP (SP, 4 gilts). Blood samples were collected every 15 min on Days 12 through 17 between 0805 h and 1100 h. Single blood samples were collected at 0800 h after Day 17 until gilts exhibited estrus. Concentrations of prostaglandin (PG) E, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and progesterone (P4) were measured by specific radioimmunoassays. Interestrous intervals for pCSP-treated (18.2 days) and SP-treated (18.0 days) gilts were not different (SEM = 0.8 days) and temporal changes in concentrations of P4 in plasma did not differ between pCSP-treated (29.2 ng/ml) and SP-treated (31.2 ng/ml) gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid phosphatase and leucine aminopeptidase activity in the uterine flushings of non-pregnant and pregnant gilts

The activities of uteroferrin, measured as acid phosphatase (AP), and an aminoacylpeptidase (AA) were measured in uterine flushings collected from gilts on Days 6, 8, 10, 12, 14, 15, 16 and 18 of the oestrous cycle and pregnancy (N = 37). Changes in AP (P less than 0.05) were associated with day for both specific and total AP in non-pregnant and pregnant gilts. For pregnant and non-pregnant gilts, AP activity was greatest between Days 14 and 16 and then decreased to Day 18. The AA specific activity increased (P less than 0.01) between Days 10 and 12 of the oestrous cycle and pregnancy, but neither effects of pregnancy nor day by pregnancy status interaction were detected. The AA total activity was greater for pregnant gilts (P less than 0.01). These data suggest an inhibitory effect of oestrogens of blastocyst origin on synthesis and/or secretion of uteroferrin, but not AA.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Transcervical collection of equine conceptuses between 10 and 16 days after ovulation

To recover intact Day-10.5 to Day-16.5 equine conceptuses (Day 0 = ovulation), a rigid catheter was used for 131 collections from donor mares diagnosed pregnant by ultrasonography. A total of 139 conceptuses were recovered, comprising 124 singletons, six pairs of twins and one set of triplets. Of these, 120 (86%) were intact after the collection, 14 (10%) had collapsed, and in five cases (4%), collapsed trophoblastic membranes were surrounded by an intact capsule. The recovery rate of intact conceptuses ranged from 99% on Days 10.5 to 12.5 to 40% on Day 16.5. More uterine flushes per recovery were needed to collect conceptuses on Day 14.5 than on Days 10.5 and 11.5 (x +/- SEM : 3.1 +/- 0.5 vs 1.4 +/- 0.1 and 1.3 +/- 0.2 flushes, respectively, P<0.05), and the total volume of flushing medium used was greater on Day 14.5 than on Days 10.5, 11.5 and 12.5 (1040 +/- 193 vs 406 +/- 49, 396 +/- 48 and 499 +/- 59 ml, respectively, P<0.01). Seventy of the 100 mares inseminated at the first estrus following embryo collection became pregnant, indicating that the technique used had no major effect on subsequent fertility.     

Porcine endometrial expression of kininogen,factor XII,and plasma kallikrein in cyclic and pregnant gilts

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.     

Secretion of a progesterone-induced inhibitor of plasminogen activator by the porcine uterus

The indirect ¹²⁵I-fibrin plate assay has been used to measure the levels of plasminogen activator (PA) in uterine flushings from pigs through the estrous cycle and during early pregnancy, and to measure the production of PA by pig conceptuses cultured in vitro. Activity in the flushings was high at the beginning and end of the estrous cycle, but only low levels were detected in mid cycle (the luteal phase). In pregnant animals, uterine PA levels became low around day 12 and did not show any further increase. Cultured day 12 blastocysts, however, released large amounts of PA into the medium in a time-dependent fashion over a 48 hr period, suggesting that this activity was inhibited in vivo. The presence of a protease inhibitor in uterine flushings has been demonstrated in cycling gilts, and follows a hormone-directed trend, with flushings taken during the luteal phase showing inhibitory activity against PA secreted early or late in the cycle. By assaying flushings from ovariectomized gilts given daily injections of progesterone, estrogen, both hormones together, or corn coil, it has been verified that the inhibitor is progesterone-induced and is also active against both PA produced by day 12 conceptuses and urokinase. It also inhibits PA, as determined using a direct fluorometric assay with glutaryl-glycyl-L-arginine-4-methyl-coumarinyl-7-amide as substrate. The PA inhibitor is acid-stable, and of low molecular weight (15,000 ± 5000), as determined by Sephacryl S-200 gel filtration. Unlike most animals, the trophoblast of the pig is not invasive in the uterus, but is invasive if transplanted to some ectopic site. The progesterone-induced inhibitor may possibly play a role in preventing invasive implantation.     

Effect of progesterone,mifepristone, and estrogen treatment during early pregnancy on conceptus development and uterine capacity in Swine

A series of experiments was performed to investigate the influence of progesterone at Days 2 and 3 of pregnancy on conceptus development and uterine capacity. In experiment 1, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts were given no treatment, estradiol valerate (5 mg given on Days 11 and 12), or progesterone (200 mg/day on Days 2 and 3 after mating). On Day 105 of pregnancy, each fetus and its associated placenta were weighed, and the number of live and dead fetuses was recorded for each litter. Early progesterone treatment reduced (P < 0.05) litter size (a measure of uterine capacity in UHO gilts). In experiment 2, intact white crossbred gilts were mated, given no treatment or progesterone treatment on Days 2 and 3 of pregnancy, and farrowed. Progesterone treatment decreased (P < 0.05) pregnancy rates. In pregnant gilts, progesterone had no effect on the number of live or stillborn piglets at birth, and gestation length was decreased (P < 0.05). Progesterone treatment did not affect the number of large or small piglets. In experiment 3, intact gilts were mated at estrus and then received 1). no treatment or treatment with 2). 100 mg, 3). 200 mg, or 4). 400 mg mifepristone (also known as RU486) on Day 2 of pregnancy. On Day 11 of pregnancy, both uterine horns were flushed, the number and diameter of each conceptus was recorded, and the flushed material was assayed for total protein and acid phosphatase. The 400 mg mifepristone treatment decreased conceptus diameter (P < 0.05) and total protein (P = 0.06) in the uterine flushings. In experiment 4, UHO gilts were mated at estrus, injected with either corn oil (control) or mifepristone (400 mg) on Day 2 of pregnancy, and killed on Day 105 of pregnancy, and the number and weight of live fetuses and placentas was recorded. In contrast to the effect of progesterone treatment, mifepristone decreased uterine capacity by decreasing the number of small conceptuses. These data suggest that progesterone concentrations on Days 2 and 3 of pregnancy in swine influence the rate of conceptus development during early pregnancy and uterine capacity during later pregnancy.