Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus

Progesterone (P4) is unequivocally required to maintain a uterine environment conducive to pregnancy. This study investigated the effects of P4 treatment on expression of selected growth factors (fibroblast growth factor 7 [FGF7], FGF10, hepatocyte growth factor [HGF], and insulin-like growth factors [IGF1 and IGF2]), their receptors (MET, FGFR2(IIIB), and IGF1R), and IGF binding proteins (IGFBPs) in the ovine uterus. Ewes received daily injections of corn oil vehicle (CO) or 25 mg of P4 in vehicle from 36 h after mating (Day 0) to hysterectomy on Day 9 or Day 12. Another group received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Day 8 through Day 12. Endometrial FGF10 mRNA levels increased between Day 9 and Day 12 and in response to P4 on Day 9 in CO-treated ewes, which had larger blastocysts, and were substantially reduced in P4+RU486-treated ewes, which had no blastocysts on Day 12. Endometrial FGF7 or HGF mRNA levels were not affected by day or reduced by RU486 treatment, but MET mRNA levels were higher in P4-treated ewes on Day 9 and Day 12. Levels of IGF1, IGF2, and IGF1R mRNA in the endometria were not affected by early P4 treatment. Although stromal IGFBPs were unaffected by P4, levels of IGFBP1 and IGFBP3 mRNA in uterine luminal epithelia were increased substantially between Day 9 and Day 12 of pregnancy in CO-treated ewes and on Day 9 in early P4-treated ewes. Therefore, FGF10, MET, IGFBP1, and IGFBP3 are P4-regulated factors within the endometrium of the ovine uterus that have potential effects on endometrial function and peri-implantation blastocyst growth and development.     

Intrauterine infusion of ovine conceptus secretory proteins reduces prostaglandin F2α release without affecting endometrial oxytocin receptor concentrations

Chronically ovariectomized ewes were pretreated with progesterone and oestradiol to induce oestrus and randomly allocated into four treatment groups. Progesterone injections were given to Groups 1 and 2 on Days 1–12 and Groups 3 and 4 on Days 1–15. Ewes in Groups 2 and 4 were infused with conceptus secretory proteins (oCSP), via an intrauterine catheter, twice daily on Days 13–15. Ewes in Groups 1 and 3 were similarly infused, but with serum proteins (oSP). Endometrial oxytocin receptor (OTr) concentrations and oxytocin-induced 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) release were measured on Day 16.Progesterone concentrations in ewes receiving 12 days of progesterone treatment declined after Day 12, reaching a nadir on Day 14. In contrast, plasma progesterone concentrations remained elevated until Day 16 in ewes receiving the extended progesterone treatment. On Day 16, endometrial OTr concentrations were significantly higher in ewes given 12 days of progesterone treatment than in ewes given 15 days of progesterone irrespective of the presence of oCSP or oSP. Treatment with oCSP significantly decreased oxytocin-induced PGFM release in ewes given 12 days of progesterone treatment compared with those ewes receiving oSP infusions. The extended 15 day progesterone treatment resulted in a further decrease in oxytocin-induced PGFM release in both oCSP and oSP infused ewes.These data indicate that, in steroid treated ovariectomized ewes, intrauterine infusion of oCSP will reduce oxytocin-induced PGFM response but not OTr concentrations. Progesterone appears to play a dominant role in the regulation of OTr as well as oxytocin-induced PGFM release.     

Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium

In ruminants, both the endometrium and the conceptus (embryo and associated extraembryonic membranes) trophectoderm synthesizes and secretes prostaglandins (PG) during early pregnancy. In mice and humans, PGs regulate endometrial function and conceptus implantation. In Study One, bred ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PG synthase (PTGS) inhibitor from Days 8-14 postmating, and the uterine lumen was flushed on Day 14 to recover conceptuses and assess their morphology. Elongating and filamentous conceptuses (12 cm to >14 cm in length) were recovered from all CX-treated ewes. In contrast, MEL-treated ewes contained mostly ovoid or tubular conceptuses. PTGS activity in the uterine endometrium and amounts of PGs were substantially lower in uterine flushings from MEL-treated ewes. Receptors for PGE2 and PGF2 alpha were present in both the conceptus and the endometrium, particularly the epithelia. In Study Two, cyclic ewes received intrauterine infusions of CX, MEL, recombinant ovine interferon tau (IFNT), or IFNT and MEL from Days 10-14 postestrus. Infusion of MEL decreased PGs in the uterine lumen and expression of a number of progesterone-induced endometrial genes, particularly IGFBP1 and HSD11B1. IFNT increased endometrial PTGS activity and the amount of PGs in the uterine lumen. Interestingly, IFNT stimulation of many genes (FGF2, ISG15, RSAD2, CST3, CTSL, GRP, LGALS15, IGFBP1, SLC2A1, SLC5A1, SLC7A2) was reduced by co-infusion with MEL. Thus, PGs are important regulators of conceptus elongation and mediators of endometrial responses to progesterone and IFNT in the ovine uterus.     

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.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes

In experiment (Exp) 1, 12 cyclic ewes had catheters placed into each uterine horn on Day 7 (estrus = Day 0). On Days 11-15, 6 ewes received twice-daily intrauterine infusions of 1.5 mg serum protein (SP) into each uterine horn and 6 ewes received infusions of 1.08 mg SP + 0.42 mg ovine conceptus secretory proteins (oCSP) containing 25 micrograms ovine trophoblast protein-one (oTP-1) as determined by radioimmunoassay (25-35% bioactive by antiviral assay). SP-infused and oCSP-infused ewes had similar plasma 13,14-dihydro-15-keto prostaglandin F2 alpha (PGF2 alpha) profiles in response to oxytocin on Day 11, but SP ewes became more responsive (p less than 0.01) to oxytocin on Days 13 and 15 than oCSP ewes. SP ewes also had greater incorporation of [3H]inositol into inositol trisphosphate (IP3) (+3449%, p less than 0.01) and total inositol phosphate (IP) (+760%, p less than 0.08), in response to oxytocin, than did oCSP ewes (+553 and +168% for IP3 and total IP, respectively) in endometrium collected at ovariectomy/hysterectomy on Day 16. Mean CL weights on Day 16 and mean concentrations of progesterone in plasma collected at 12-h intervals on Days 6-16 were not different for SP and oCSP ewes, but concentrations of progesterone were lower (p less than 0.05) in SP ewes on Days 15-16 than for oCSP ewes. These results indicate that oTP-1 may prevent luteolysis by inhibiting development of endometrial responsiveness to oxytocin and, therefore, reduce oxytocin-induced synthesis of IP3 and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Galectin 15 (LGALS15): a gene uniquely expressed in the uteri of sheep and goats that functions in trophoblast attachment

Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.     

Differential effects of progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factors and their binding proteins define specific phases of myometrial differentiation during pregnancy in the rat

While the insulin-like growth factor (IGF) system is known to regulate uterine function during the estrous cycle, there are limited data on its role in myometrial growth and development during pregnancy. To address this issue, we defined the expression of the Igf hormones (1 and 2), their binding proteins (Igfbp 1-6), and Igf1r receptor genes in pregnant, laboring, and postpartum rat myometrium by real-time PCR. IGF family genes were differentially expressed throughout gestation. Igf1 and Igfbp1 mRNA levels were upregulated during proliferative phase (Days 6-12) of rat gestation. Igfbp3 gene expression also was elevated in proliferating smooth muscle cells (SMCs) and was highest at the time of transition between proliferative and synthetic phases (Days 12-15). Igfbp6 gene expression profile paralleled plasma progesterone (P4) concentrations, peaking during the synthetic phase (Days 17-19) and decreasing thereafter. Administration of P4 at late pregnancy (starting from Day 20) to maintain elevated plasma P4 concentrations blocked the onset of labor and prevented the fall in Igfbp6 mRNA levels. In contrast, the treatment of pregnant rats with the P4 receptor antagonist RU486 on Day 19 induced preterm labor and the premature decrease of Igfbp6 gene expression. Igfbp2 gene expression was transiently upregulated during the contractile phase of gestation (Days 21-23) solely in the gravid horn of unilaterally pregnant rats, but it was not affected in P4- or RU486-treated animals, supporting a role for mechanical stretch imposed by the growing fetuses. Igfbp5 gene was induced during postpartum involution. Our results suggest the importance of the IGF system in phenotypic and functional changes of myometrial SMCs throughout gestation in preparation for labor.     

Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone

The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe

The effects of administration of progesterone and oestradiol on ovine endometrial oxytocin receptor concentrations and plasma concentrations of 13,14-dihydro-15-keto prostaglandin F-2 alpha (PGFM) after oxytocin treatment were determined in ovariectomized ewes. Ewes received progestagen pre-treatment, progesterone and/or oestradiol in 11 different treatment schedules. Progestagen pre-treatment decreased oxytocin receptor concentrations in endometrium from ewes treated subsequently with either progesterone for 5 days or progesterone for 5 days plus oestradiol on Days 4 and 5 of progesterone treatment. Oestradiol increased endometrial oxytocin receptor concentrations when administered on Days 4 and 5 of 5 days progesterone treatment. Progestagen pre-treatment followed by progesterone treatment for 12 days caused a large increase in oxytocin receptors and no further increase occurred when ewes were given oestradiol on Days 11 and 12, or when progesterone was withdrawn on Days 11 and 12, or these two treatments were combined. Oxytocin administration caused an increase in plasma PGFM concentrations in ewes which did not receive progestagen pre-treatment, and subsequently received progesterone treatment for 5 days and oestradiol treatment on Days 4 and 5 of progesterone treatment. Similarly treated ewes which received progestagen pre-treatment did not respond to oxytocin. Oxytocin administration also increased plasma PGFM concentrations in ewes which received progestagen pre-treatment followed by progesterone treatment for 12 days, progesterone treatment for 12 days plus oestradiol on Day 11 and 12 of progesterone treatment, progesterone withdrawal on Day 11 and 12, or progesterone withdrawal and oestradiol treatment combined. The results indicate that (1) progesterone pre-treatment affects oxytocin receptor concentrations in the endometrium and uterine responsiveness to oxytocin and (2) progesterone treatment alone for 12 days after a treatment which mimics a previous luteal phase and oestrus is sufficient to induce oxytocin receptors and increase oxytocin-induced PGF release. These results emphasize the importance of progesterone and provide information which can be used to form an hypothesis for control of luteolysis and oestrous cycle length in the ewe.     

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.     

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.     

Differential suppression of endometrial prostaglandin F2alpha by the equine conceptus

Prostaglandin F2alpha secretion by the uterine endometrium between Days 13 and 14 postovulation causes luteal regression in mares. A mechanism involving interruption or suppression of this secretion causes pregnancy to be maintained. The present study was designed to determine the age of the conceptus when maximal suppression of PGF2alpha secretion occurs. Mares were examined daily during estrus with ultrasonography (day 0 = day of ovulation). Conceptus tissues were recovered nonsurgically on Days 9 (n = 7), 12 (n = 5), 13 (n = 5), and 16 (n = 7) and uterine biopsies on Day 14. Both uterine and conceptus tissues were washed in phosphate-buffered saline (PBS) with 100 units penicillin G/ml + 100 microg streptomycin/ml, pH 7.4. Endometrial tissue (approximately 200 mg) plus conceptus tissues were incubated in 15 ml of tissue culture medium 199 (M199) + 10% fetal calf serum and 10 units penicillin G/ml and 10 microg streptomycin/ml at 37 degrees C under 5% CO(2): 5% O(2) : 90% N(2). Samples were taken at 4, 8, and 24 h. Two plates that contained only endometrial tissue and two additional plates with 25 mg flunixin meglumine added along with endometrial tissue were also included in the incubations. Concentrations of PGF2alpha were measured in all samples using radioimmunoassay. There was a trend toward suppression of PGF2alpha secretion by conceptus tissues, regardless of age. However, Day 12 concepti significantly suppressed PGF2alpha secretion compared with that of endometrial tissue incubated alone (P = 0.03).     

Endometrial glands are required for preimplantation conceptus elongation and survival

Endometrial glands secrete molecules hypothesized to support conceptus growth and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by neonatal progestin exposure. The resulting stable adult uterine gland knockout (UGKO) phenotype was used here to test the hypothesis that endometrial glands are required for successful pregnancy. Mature UGKO ewes were bred repeatedly to fertile rams, but no pregnancies were detected by ultrasound on Day 25. Day 7 blastocysts from normal superovulated ewes were then transferred synchronously into Day 7 control or UGKO ewes. Ultrasonography on Days 25-65 postmating indicated that pregnancy was established in control, but not in UGKO ewes. To examine early uterine-embryo interactions, four control and eight UGKO ewes were bred to fertile rams. On Day 14, their uteri were flushed. The uterus of each control ewe contained two filamentous conceptuses of normal length. Uteri from four UGKO ewes contained no conceptus. Uteri of three UGKO ewes contained a single severely growth-retarded tubular conceptus, whereas the remaining ewe contained a single filamentous conceptus. Histological analyses of these uteri revealed that endometrial gland density was directly related to conceptus survival and developmental state. Day 14 UGKO uteri that were devoid of endometrial glands did not support normal conceptus development and contained either no conceptuses or growth-retarded tubular conceptuses. The Day 14 UGKO uterus with moderate gland development contained a filamentous conceptus. Collectively, these results demonstrate that endometrial glands and, by inference, their secretions are required for periimplantation conceptus survival and development.     

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.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).