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Embryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n = 5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n = 9) or contralateral to the CL (n = 8) or into both horns (i.e., bilateral, n = 9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P < 0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean ± SE 2.85 ± 0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14.Summary SentenceDifferences in endometrial gene expression exist between the ipsilateral and contralateral uterine horns in cattle, but conceptus growth to day 14 is not different between the horns.

Reproductive performance of female pigs that do not receive sufficient colostrum from birth is permanently impaired. Whether lactocrine deficiency, reflected by low serum immunoglobulin immunocrit (iCrit), affects patterns of endometrial gene expression during the periattachment period of early pregnancy is unknown. Here, objectives were to determine effects of low iCrit at birth on the adult endometrial transcriptome on pregnancy day (PxD) 13. On the first day of postnatal life, gilts were assigned to high or low iCrit groups. Adult high (n = 8) and low (n = 7) iCrit gilts were bred (PxD 0), and humanely slaughtered on PxD 13 when tissues and fluids were collected. The endometrial transcriptome was defined for each group using mRNAseq and microRNAseq. Reads were mapped to the Sus scrofa 11.1 genome build. Mature microRNAs were annotated using miRBase 21. Differential expression was defined based on fold change (≥ ±1.5). Lactocrine deficiency did not affect corpora lutea number, uterine horn length, uterine wet weight, conceptus recovery, or uterine luminal fluid estrogen content on PxD 13. However, mRNAseq revealed 1157 differentially expressed endometrial mRNAs in high versus low iCrit gilts. Differentially expressed genes had functions related to solute transport, endometrial receptivity, and immune response. Six differentially expressed endometrial microRNAs included five predicted to target 62 differentially expressed mRNAs, affecting similar biological processes. Thus, lactocrine deficiency on the first day of postnatal life can alter uterine developmental trajectory with lasting effects on endometrial responses to pregnancy as reflected at the level of the transcriptome on PxD 13.Summary SentenceLactocrine deficiency on the first day of postnatal life alters the uterine developmental program with long-term effects on patterns of porcine endometrial gene expression during the periattachment period of early pregnancy.

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition. endometrium, female reproductive tract, uterine gland, uterus

Because sphingosine 1-phosphate (S1P) is a potent stimulator of angiogenesis, we hypothesized that the S1P pathway is activated to stimulate endometrial/placental angiogenesis during pregnancy. We initially localized S1P signaling pathway members in the gravid and nongravid uterine horns of unilaterally pregnant ewes. Sphingosine kinase-1 expression was greater in gravid compared to nongravid horns. In situ hybridization revealed elevated expression of sphingosine 1-phosphate phosphatase (SGPP1) in gravid interplacentomal endometrial stroma on Days 20 and 40 compared to the nongravid uterine horn, but expression increased in endometrium of the nongravid uterine horn between Days 40 and 120. SGPP1 expression increased in placentomes late in gestation. Sphingosine 1-phosphate lyase mRNA was modestly expressed at Day 20 and then decreased. In contrast, sphingosine 1-phosphate receptor 1 (S1PR1) mRNA increased in endometrium and caruncular stroma of the gravid uterine horn. Treatment with FTY720 and VPC23019, S1P receptor antagonists, blocked human and ovine endothelial cell invasion using an in vitro model of sprouting angiogenesis. Knockdown of S1PR1 with siRNA reduced invasion responses as well. We previously reported that delta-like 4 (DLL4) and A disintegrin and metalloproteinase with thrombospondin-like repeats 1 (ADAMTS1) participate in endothelial cell invasion stimulated by S1P and growth factors in vitro, and thus investigated whether their expression correlated with areas undergoing angiogenesis in vivo. DLL4 expression was similar to S1PR1, while ADAMTS1 mRNA was expressed by endometria of both nongravid and gravid horns, as well as conceptus and placentomes. These results establish that S1P signaling pathway members and S1P- and growth factor-regulated genes are prominent in uterine and placental tissue and in some cases are correlated with areas undergoing angiogenesis. Thus, S1P signaling may be crucial for proper fetal-placental development.

An asymmetric distribution of the sexes within the left and right uterine horns has been described in multiple species. A series of experiments were conducted to evaluate the sex ratio (% male) of calves gestated in the left and right uterine horns, as well as the sex ratio of embryos originating from the left and right ovaries of cattle. The sex ratio of calves gestated in the right uterine horn of naturally mated cows was significantly higher compared with the sex ratio of calves gestated in the left uterine horn. In addition, the sex ratio of the left and right uterine horns differed significantly from parity. The sex ratio of embryo transfer calves born following transfer to the left and right uterine horns was not significantly different. Additionally, the proportion of male embryos collected from the right uterine horns was significantly greater than from the left uterine horns of superovulated cows. The sex ratio of embryos collected from the left and right uterine horns of unilaterally ovariectomized cows was not significantly different. However, more female than male embryos were produced when left ovary oocytes fertilized in vitro. In conclusion, the results of these experiments demonstrate that a significantly greater proportion of males are gestated in the right uterine horn of cattle and a greater proportion of females in the left. Additionally, the data indicate that sex-specific selection pressure may be applied to embryos by ovarian factors rather than by the uterine environment.

The luteolytic effects of exogenous prostaglandin F2alpha (PGF) that did and did not simulate natural 13,14-dihydro-15-keto-PGF (PGFM) pulses were studied during mid-diestrus in 42 Holstein heifers. Plasma concentrations of PGF were assessed by assay of PGFM. In experiment 1, a single intrauterine injection of 4.0 mg of PGF into the uterine horn ipsilateral to the corpus luteum resulted in a precipitous progesterone decline, whereas sequential injections of 0.25 or 1.0 mg every 12 h resulted in a stepwise decrease (P < 0.05) following each injection. A progesterone increase occurred during the first 5 min before the luteolytic decrease but only for the 4.0-mg dose. From the results of experiment 2, a 2-h intrauterine infusion of a total of 0.5 mg of PGF was judged to best simulate a natural PGFM pulse. In experiment 3, simulation of sequential pulses at 12-h intervals resulted in a continuous precipitous decrease in progesterone to

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.

In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.

The spherical equine embryonic vesicle is mobile throughout the uterine lumen for several days before becoming fixed in the caudal segment of a uterine horn on Day 16 (ovulation = Day 0). Orientation refers to the position of the embryo proper at the periphery of the vesicle relative to the position of the mesometrial attachment. In mares, the embryonic pole of the vesicle is antimesometrial after completion of orientation. Day of vesicle fixation, differential thickening of the endometrium near the mesometrial attachment, and orientation of the embryonic vesicle were studied in 30 ponies, using B-mode and color-Doppler transrectal ultrasonography. The thickness of the endometrium at the mesometrial aspect of the vesicle divided by the thickness at the antimesometrial aspect was termed the encroachment ratio. At the future site of fixation, the first increase (P < 0.05) in the encroachment ratio occurred between 4 and 1 days before fixation. An early vascular indicator of the future position of the embryo proper was discovered by color-Doppler imaging and consisted of a colored spot in the image of the endometrium close to the wall of the embryonic pole. The early indicator was detected in each mare 0.5 ± 0.1 days after fixation and 2.5 ± 0.2 days before first detection of the embryo proper. The position of the early indicator when first detected at the periphery of the embryonic vesicle was not significantly different from the position of the embryo proper when first detected. Results supported the hypothesis that differential thickening of the endometrium precedes orientation and indicated that orientation occurs immediately after fixation.

The myometrium undergoes dramatic changes as pregnancy progresses through phases of proliferation, hypertrophy, contractile state and labor. In this study, we showed that the composition of the muscle actin isoforms, a major component of the myometrial contractile apparatus and cytoskeleton, was modified during pregnancy. The expression of smooth muscle alpha-actin (Acta2, which we abbreviate as alpha-SM-actin) and gamma-actin mRNAs and proteins was examined by real-time polymerase chain reaction and Western immunoblot, and was localized with immunohistochemistry, in the nonpregnant, pregnant, and postpartum rat uterus. Both alpha-SM-actin (vascular specific actin isoform) and gamma-actin (predominant in visceral smooth muscle) were detected in the rat myometrium. Myometrial expression of alpha-SM-actin mRNA and protein was high throughout pregnancy. The transcript and protein levels of gamma-actin were increased significantly in the second part of gestation (31.8-fold increase for mRNA and 16.7-fold increase for protein relative to nonpregnant). The localization of gamma-actin was markedly altered during pregnancy. In early gestation, myometria from empty and gravid uterine horns of the unilaterally pregnant rats showed abundant gamma-actin immunostaining in the longitudinal layer but weak staining in the circular layer. Gamma-actin immunostaining increased in only the circular layer of the gravid horn after midgestation and remained low in the empty one. Gamma-actin protein translocated to the membranous region of uterine myocytes at late gestation. The temporal alteration in gamma-actin expression and localization at late gestation suggested that this change in myometrial composition of contractile proteins is important to adequately prepare the myometrium for the development of optimal contractions during labor.

The equine embryonic vesicle is mobile on Days 12‐14 (Day 0 5 ovulation), when it is approximately 9‐15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15‐17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n 5 16) or not detected (n 5 8) by Day 12. Endometrial vascularity (scored 1‐4, for none to maximal, respectively) did not differ on Days 1‐8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P , 0.05) on Days 12‐16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P , 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P , 0.05) than scores in the opposite horn on Days 12‐15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P , 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility. conceptus, embryo, endometrial vascular perfusion, endometrium, mares, pregnancy, uterine contractions, uterus

We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays. Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity. Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP. To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT). Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP. These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.

Androgens are known to attenuate some effects of estradiol-17beta (E) in the uterus. The objectives of the present experiment were to determine effects of 5alpha-dihydrotestosterone (DHT) on estrogenic actions in the pig uterus and its associations with changes in expression of the estrogen receptor (ER) alpha and ERbeta. Postpubertal gilts (120-130 kg of body weight; n = 16) were ovariectomized, and 3-4 weeks later received once-a-day injections (i.m.) of one of the following treatments during four consecutive days: 1) vehicle (corn oil), 2) E (250 microg), 3) E (250 microg) plus 1 mg DHT, or 4) E (250 microg) plus 10 mg DHT. Uterine tissues were collected 24 h after the last treatment. Gilts receiving E or E plus 1 mg DHT had greater uterine wet weight, uterine horn diameter, luminal epithelium thickness, and endometrial gland diameter compared with gilts treated with vehicle or E plus 10 mg DHT. Gilts receiving E or E plus 1 mg DHT were not different in these characteristics. Relative amounts of mRNAs in the endometrium for the cell proliferation marker histone H2a and the E-inducible protein complement component C3 increased in gilts treated with E compared with gilts treated with vehicle. E-induced increases in histone H2a and C3 mRNAs were not altered by cotreatment with E plus 1 mg DHT but were inhibited by E plus 10 mg DHT. Androgen receptor (AR) mRNA in the endometrium increased by treatment with E. Cotreatment of gilts with E and DHT did not alter the E-induced AR mRNA increase. Gilts treated with E plus 10 mg DHT had lesser amounts of immunoreactive ERalpha in cell nuclei of the myometrium and endometrial stroma and a tendency for a decrease in luminal epithelium compared with gilts treated with E. Amounts of immunoreactive ERalpha in glandular epithelium were not influenced by the treatments. Relative amounts of ERalpha and ERbeta mRNAs decreased in the endometrium of gilts treated with E plus 10 mg DHT compared with gilts treated with E. Downregulation of the ERs, particularly ERalpha in the myometrium and endometrial stroma, might be a relevant mechanism in the antagonism of estrogenic effects by DHT in the pig uterus.

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell, and morula stages were vitrified in EFS40 by a one-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Days 2 1t o22 of synchrony (i.e., at a point in pseudopregnancy 1‐2 days earlier than the embryos). Approximately half the vitrified embryos transferred into oviducts on Day 21 developed to term, but only a minority of embryos, whether vitrified (10%‐34%) or fresh (24%‐33%), transferred at later times did so, suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to 20.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos (;63%) developed to term in reasonably synchronous recipients (Day 0 to 20.5) but not in more asynchronous ones (6%; Day 21). A majority of vitrified morulae also developed to term (52%‐68%) in a wider range of recipients (Days 0 to 21), the greatest success occurring in recipients on Day 20.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo. early development, embryo, oviduct, pregnancy, uterus

Previous studies have documented that ubiquitin-related proteins are present in human, baboon, rhesus monkey, cow, sheep, and mouse pregnant uteri, indicating that the ubiquitin-proteasome pathway (UPP) may be involved in the extensive uterine remodeling during mammalian early pregnancy, but there is still no direct evidence. A mouse intrauterine injection model was employed to study the direct effect of the UPP on mouse embryo implantation and its possible mechanisms. On Day 3 of pregnancy in each mouse, one of the uterine horns in each mouse was injected with different concentrations of lactacystin, a specific proteasome inhibitor, or anti-ubiquitin antibody, and the other side was used as a control. On Days 5, 6, and 7, the number of implanted embryos was counted and the expression and gelatinolytic activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 were studied. Results presented here illustrate that injection of lactacystin and anti-ubiquitin antibody significantly inhibited mouse embryo implantation. Further investigations by reverse transcription-polymerase chain reaction and gelatin zymography showed that MMP-2 and MMP-9 mRNA expression, as well as the gelatinolytic activity of MMP-9 in the lactacystin-treated uterine horn, significantly decreased, whereas the activity of MMP-2 was not significantly affected. The results obtained from this study, together with previous reports, suggest that the UPP is involved in mouse embryo implantation, and UPP's effect on embryo implantation is achieved at least in part by regulating MMP-2 and MMP-9 mRNA expression and the gelatinolytic activity of MMP-9.

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.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor α (ERα) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERα regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17β (E2) valerate (EV), an ERα agonist, 3) EM-800, an ERα antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterin...

Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.

The objective of the present study was to identify and characterize transcripts whose levels are increased in the mouse uterus during decidualization. Using the method of suppression subtractive hybridization, we identified a novel transcript. This transcript contained a potential open reading frame that coded for a 196-amino-acid protein that shows homologies to the heat shock protein 20 family of genes. This transcript was expressed in several adult tissues and in the embryo. Its steady-state level was significantly greater in implantation segments of the uterus compared to nonimplantation segments. Furthermore, the steady-state levels of this novel transcript were significantly greater in uterine horns undergoing artificially induced decidualization compared to control contralateral horns. Using in situ hybridization methods, signals for the transcript were localized to the endometrial stromal cells that were undergoing decidualization. Finally, we found that progesterone caused a significant ...

Reversible phosphorylation is essential in regulating uterine contractions. Identification, characterization, and functional understanding of myometrium protein phosphatase(s) are lacking. Okadaic acid (OA), which inhibits protein phosphatase-1 (PP1) and PP2A, has been shown to alter uterine contractions. Experiments were conducted to determine the 1) identity of the myometrial OA-sensitive PP, 2) influence of OA on spontaneous and oxytocin (OT)-stimulated myometrial contractions, and 3) expression of uterine PPs during sexual development. Western blot analysis indicated the presence of PP1(alpha) and PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1(alpha) in longitudinal and circular myometrial layers and endometrial epithelium. Conversely, PP2A was localized to the endometrial stroma. Cumulative addition of OA (n = 9; 10, 100, 250, 500, 1000 nM) did not significantly alter spontaneous contractions of mouse uterine horns in comparison to vehicle-treated controls (n = 9). By the end of the test period OA- and vehicle-treated uteri displayed a comparable decline in uterine contractions to 79.2% and 63.7%, respectively, of basal contractile activity. Pretreatment of uterine tissue with OA (1 microM; n = 7) significantly reduced contractile response to increasing concentrations of OT (8, 16, 32, 64 nM) in comparison to vehicle pretreatment (dimethyl sulfoxide; n = 7). At the end of the OT-administration period, contractile activity was 160.4% and 67.3% of basal contractile activity for vehicle (no OA) and OA-pretreated groups, respectively. During the early prepubertal period PP1(alpha) was expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1(alpha) was observed in both the longitudinal and circular myometrium. In summary, these studies have indicated 1) that PP1 is the primary myometrial OA-sensitive PP; 2) that inhibition of PP1 had no effect on spontaneous contractions, whereas it markedly inhibited OT-stimulated uterine contractions; and 3) that PP1 is differentially expressed in the circular and longitudinal myometrium in relation to sexual development.

The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) enzyme catalyses the conversion of the biologically inert glucocorticoid 11-dehydrocorticosterone to active corticosterone (11-oxoreductase activity) in vivo, and it is dramatically up-regulated in uterine myometrium in the days leading up to parturition. 11beta-HSD-1 is likely to enhance local concentrations of glucocorticoid within the myometrium and thus facilitate uterine contractility, but the stimulus for the increase in myometrial 11beta-HSD-1 is unknown. The objective of the present study was to test whether the induction of myometrial 11beta-HSD-1 is dependent on uterine occupancy or systemic hormonal signals of late pregnancy. This involved use of a unilateral pregnancy (ULP) model in which the gravid and nongravid uterine horns are both exposed to the normal systemic hormonal milieu of pregnancy. Western blot analysis showed that the 11beta-HSD-1 signal was only partially induced in the nongravid horn of ULP rats on Day 22 of pregnancy (term: Day 23). Moreover, artificial distension of this nongravid horn had no effect on myometrial 11beta-HSD-1 immunoreactivity or bioactivity at either Day 16 or Day 22 of pregnancy. Removal of fetuses and placentas on Day 18 reduced myometrial 11beta-HSD-1 bioactivity 4 days later, and this effect was not overcome by artificial maintenance of uterine distension. In contrast, after fetectomy at Day 18 (i.e., removal of the fetus but not placenta), myometrial 11beta-HSD-1 bioactivity was largely maintained on Day 22, indicative of placental support for myometrial 11beta-HSD-1 over this period. In conclusion, our data show that full induction of myometrial 11beta-HSD-1 expression and associated 11-oxoreductase bioactivity late in rat pregnancy is dependent upon intrauterine occupancy. Although the hormonal milieu of late pregnancy appears to stimulate myometrial 11beta-HSD-1 marginally, full induction clearly requires an additional stimulus. Manipulations involving fetectomy and artificial uterine distension indicate that the placenta provides at least part of this stimulus, but uterine stretch does not appear to play a role.

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.

Porcine conceptuses rapidly elongate within the uterine horns prior to the period of placental attachment. During the time of elongation, secretion of estrogen by the developing conceptuses occurs for the establishment of pregnancy through maintenance of corpora lutea and facilitation of placental attachment. Factors associated with the uterine luminal epithelium accentuate embryo attachment by allowing close contact between the conceptus and the uterine epithelium. Kallikrein, a serine protease, may be involved with the timing of conceptus expansion and placental attachment to the uterine surface. The objective of this study was to evaluate kallikrein enzymatic activity, protein, and gene expression in the pig during the estrous cycle and early pregnancy. Enzymatic activity was first detected in uterine flushings (UTF) on Day 12 of the estrous cycle and pregnancy. Activity was enhanced on Day 12 of pregnancy compared to that in cyclic gilts, with a reversal of increased kallikrein activity in cyclic compared to pregnant flushings on Day 15. Western blot analysis with antiserum to human plasma kallikrein detected a 50-kDa product similar to human plasma kallikrein from Day 10 to Day 15 of the estrous cycle and pregnancy. Kallikrein enzymatic activity in UTF was associated with the presence of a 23-kDa reactive product. Gene expression of kallikrein as determined by reverse transcription-polymerase chain reaction indicated the presence of kallikrein mRNA in the porcine endometrium and conceptuses. Results indicate that an increase in uterine luminal kallikrein activity occurs during the estrous cycle at a period that corresponds to rapid conceptus elongation during pregnancy of the pig. The present information suggests that kallikrein may play a role in opening the window for establishment of pregnancy in the pig.

In order to examine whether sperm migration into and through the oviduct follows an invariable pattern or is subject to regulation, rats in proestrus, estrus, metestrus, or diestrus were inseminated in the upper third of each uterine horn with 10-20 million epididymal spermatozoa. Three or eight hours later, the numbers of spermatozoa free and adhering to the epithelium in the ampullary and isthmic segments were determined. A significantly higher number of spermatozoa were recovered in estrus than in other stages, at 3 h than at 8 h, and at all stages from the isthmus than from the ampulla. Spermatozoa adhering to the epithelium were observed only in proestrus and estrus and in the isthmus. The effect of exogenous estradiol-17beta (E2) and progesterone (P4) on sperm migration was investigated in rats in which the estrous cycle was inhibited pharmacologically. E2 facilitated sperm migration into the oviduct and P4 antagonized this effect, whereas P4 alone had no effect. Concomitant treatment with E2+P4 induced adhesion of spermatozoa to the oviductal epithelium. In conclusion, the pattern of sperm migration into and through the rat oviduct varies with the stage of the cycle, being dependent on E2 and P4. The adhesion of spermatozoa to the rat oviductal epithelium is stage- and segment-specific and requires the combined action of both hormones.

We investigated the expression of the mRNA encoding the oxytocin receptor (OTR) in rat myometrium throughout gestation and its regulation by progesterone and mechanical stretch. Using a semiquantitative reverse transcription-polymerase chain reaction approach, OTR mRNA was found to increase abruptly at the onset of spontaneous labor at term. Progesterone (4 mg/day) starting on Day 20 of gestation blocked this increase. Ovariectomy on Day 17 induced preterm labor 96 h after surgery and a significant increase in myometrial OTR mRNA levels 48 and 96 h after surgery. Both preterm labor and the rise in myometrial OTR expression were blocked by progesterone. To investigate the effects of stretch on myometrial OTR mRNA expression, unilaterally pregnant rats underwent either sham operation or placement of a tube in the nongravid uterine horn to distend the myometrium. On Day 20, stretch had no effect on OTR expression in the nongravid horns. During labor, OTR mRNA was highly expressed in the gravid horns as well as the nongravid stretched horns. In contrast, the level remained low in the nongravid unstretched horns. These results indicate that expression of rat myometrial OTR mRNA during pregnancy and labor is regulated by coordinated interactions between mechanical and endocrine signals.

Available evidence suggests that interferon tau (IFNτ), the signal for pregnancy recognition in ruminants, suppresses transcription of the estrogen receptor (ER) gene in the endometrial lumenal epithelium (LE) and superficial glandular epithelium (sGE) to prevent oxytocin receptor (OTR) expression and pulsatile release of luteolytic prostaglandin F 2α . The IFN regulatory factors one (IRF-1) and two (IRF-2) are transcription factors induced by type I IFNs that activate and silence gene expression, respectively. The objectives of these studies were to determine effects of pregnancy and IFNτ on expression of immunoreactive IRF-1 and IRF-2 proteins in the ovine endometrium. In study one, IRF-1 and IRF-2 were not detected in the LE or sGE of cyclic ewes. In pregnant ewes, IRF-1 expression was detected transiently in the LE and sGE only on Days 11 and 13, and IRF-2 was detected in these same epithelia on Days 13, 15, 17, and 20. In study two, 36 ewes were fitted with uterine catheters on Day 5 of the estrous cycle, and one uterine horn was double-ligated at the base. Uterine horns of each ewe received twice-daily injections of either recombinant ovine IFNτ or control proteins beginning on Day 11 until hysterectomy at 1, 3, 6, 12, 24, 48, 72, 96, or 120 h after initial injection. The IRF-1 was detected transiently in the endometrial LE and sGE only at 12 and 24 h in the uterine horn receiving IFNτ but not in those tissues receiving control proteins. The IRF-2 was expressed in the LE and sGE at 24 h and thereafter in the IFNτ-treated, but not control uterine horns. In control uterine horns, ER and OTR were first detected in the LE at 48 h and 72 h, respectively, and remained abundant thereafter. In horns receiving IFNτ, ER and OTR expression was not detected in the endometrial LE and sGE. Results suggest that IFNτ acts directly on the LE and sGE during pregnancy to sequentially induce IRF-1 and then IRF-2 gene expression, which is correlated temporally with an absence of ER and OTR. The ovine ER gene may contain an IFNτ-responsive element(s) that binds negative-acting, IFNτ-inducible transcription factors, such as IRF-2, which silences transcription of the ER gene in the endometrial epithelium during maternal recognition of pregnancy.

The modulatory effects of gestational age and circulating concentrations of progesterone, 5-pregnane-3,20-dione, and estradiol-17 on the uterine sex steroid hormone receptor levels of the African elephant were investigated. Uterine tissue biopsies and blood samples were obtained from animals culled in the Kruger National Park. Estrogen and progesterone receptor concentrations were determined in uterine biopsies from subadult, lactating, early-, mid-, and late-pregnant elephants, by equilibrium binding assays. Circulating estradiol-171 and progesterone concentrations were measured by means of RIAs, while plasma concentrations of 5-pregnane-3,20-dione were determined with an amplified ELISA. Significant inverse correlations of the concentrations of estrogen and progesterone receptors with the gestational stage of the elephants were observed. Pregnant uterine horns of individual animals contained lower levels of estrogen and progesterone receptors than the nonpregnant horns of the same animals. A strong positive correlation existed between uterine estrogen and progesterone receptors levels. Circulating concentrations of 5a-pregnane-3,20dione and progesterone decreased with an increase in the concentrations of progesterone receptors as well as with fetal age. We conclude that the progesterone receptor concentrations are down-regulated with progressing gestation in the African elephant. This down-regulation appears to be linked to an increase in circulatory 5-pregnane-3,20-dione concentration in the

Thirty-five crossbred and 22 Meishan contemporary gilts were necropsied on Day 1, 14, 28, 42, or 56 of age (birth = Day 0). At necropsy, a cross section of one uterine horn was fixed for histomorphometric study, and minced uterine tissue was cultured with 50 LCi 3H]leucine. Secreted proteins were identified by two-dimensional PAGE, fluorography, and incorporation of radioactivity. Body weights at necropsy were similar for the two breeds and increased (p < 0.01) between Days 1 and 56 of age. Ovarian and uterine weights, as well as histomorphometric areas, were similar for the two breeds on Day 1 but increased markedly (p < 0.01) in Meishan gilts on Day 56. In gilts of both breeds, secretion of uterine proteins 1 (Mr x 10- 3 /pl; 45/6.0), 2a and 2b (doublet, 25/6.2), and 3 (20/5.5) increased in association with endometrial gland development. A fourth protein (97/4.0) was observed in gilts of both breeds but was more abundant in Meishan; a fifth protein (13/6.0) was detected only in crossbred gilts on Day 56. Although specific regulatory roles for locally produced uterine proteins remain to be defined, the increase in specific uterine proteins and breed differences in uterine protein secretion suggest that uterine proteins may influence early uterine development.

Uterine growth, cell proliferation, and endometrial expression of the c-fos proto-oncogene were evaluated for nonpregnant ewes (n = 6 ewes per day) on Days 0 (estrus), 2, 4, 8, 12, and 15 of the estrous cycle. Fresh and dry weights of uterine horns decreased (p < 0.01) linearly from Days 0 to 8 and then remained similar from Day 8 through Day 15. Tissue water content (1 - [dry weight/fresh weight]) was greater on Day 0 (p < 0.01) than on Days 2, 4, and 8, and the latter values were greater (p < 0.01) than on Days 12 and 15. DNA content was similar on Days 0, 2, 4, 8, and 15 but increased (p < 0.01) on Day 12. Although DNA content was greatest on Day 12, the ratios of RNA to DNA and of protein to DNA were least (p < 0.01) on that day. Thus, changes in uterine fresh weight were associated primarily with changes in tissue hypertrophy (RNA:DNA and protein:DNA ratios) and water content. In addition, changes in uterine fresh weight were associated with changes in the ratio of estradiol to progesterone in systemic blood, which was greatest (p < 0.01) on Days 0 and 2, decreased (p < 0.01) from Day 2 to Day 8, remained low through Day 12, and then was elevated again (p < 0.01) on Day 15. Moreover, compartmentalized changes in endometrial cell proliferation (labeling index [LI]; percentage of cells exhibiting nuclear incorporation of bromodeoxyuridine, a thymidine analogue) also were associated with the changes in fresh weight. The epithelium of the uterine lumen and luminal glands exhibited the greatest changes in rate of cell proliferation and accounted for most of the changes in LI seen across days of the estrous cycle. Endometrial expression of c-fos mRNA and protein also reflected changes in uterine weight and the systemic estradiol:progesterone ratio. The level of c-fos mRNA was greatest at estrus, low on Days 2-8, and elevated slightly on Days 12 and 15; Fos protein was greatest on Days 0 through 4, least on Day 8, and intermediate on Days 12 and 15. Characterization of uterine growth, cell proliferation, and c-fos expression throughout the estrous cycle will provide a foundation for future studies of gene expression regulating growth of the nonpregnant uterus.

In order to determine whether or not ovum transport acceleration induced by estradiol (E 2) requires RNA and protein synthesis in the oviduct, inhibitors of RNA and protein synthesis were injected locally in rats treated with E 2. We also tested whether administration of oviductal RNA from E,-treated rats could mimic the effect of E 2 on ovum transport. Rats on Day 2 of pregnancy were given a single s.c. injection of 10 ,ig E 2 and an intraoviductal (i.o.) injection of actinomycin D, ao-amanitin, or cycloheximide (Chx). In control groups, either the steroid or the inhibitor or both were replaced by the respective vehicle. RNA obtained from oviduct or ileum of E2-treated rats or from the oviduct of propylene glycol-treated rats was injected into the oviducts of recipient rats on Day 1 of pregnancy. Animals were autopsied 24 h later to determine the number and distribution of eggs in the genital tract. All three inhibitors partially blocked the E2-induced ovum transport acceleration, whereas administration of inhibitors alone did not affect oviductal egg recovery. Only oviductal RNA obtained from E 2-treated rats decreased the number of oviductal eggs (active extract). To interpret this finding, the active extract was preincubated with RNase or DNase before i.o. administration. Other groups of recipient rats also treated with active extract were injected s.c. with Chx, or their uterine horns were ligated to disclose the fate of the missing oviductal eggs. Active extract treated with RNase did not decrease the number of oviductal eggs; Chx blocked the effect of the active extract; and eggs missing from the oviduct were partially recovered in the uteri of ligated recipient rats. It is concluded that protein synthesis in the oviduct is required for the full effect of E 2 on ovum transport and that one or more RNA species induced by E 2 in the oviduct are by themselves able to mimic, and therefore mediate, the effect of E 2 on ovum transport.

Changes in the CD-1 mouse uterine transcriptome during proestrus and estrus were investigated to help elucidate mechanisms of uterine tissue remodeling during the estrus cycle and their regulation by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 weeks of age, and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis of whole uterine horn RNA identified 2428 genes differentially expressed in estrus compared to proestrus, indicating there is extensive remodeling of mouse uterus during the estrus cycle, affecting ~10% of all protein-encoding genes. Many (~50%) of these genes showed the same differential expression in independent analyses of isolated uterine lumenal epithelial cells. Changes in gene expression associated with structural alterations of the uterus included remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis and changes in major histocompatibility complex class II (MHCII) presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and transcription factors with poorly understood roles in reproductive tissues, included several genes and gene networks that have been implicated in pathological states. Many of the molecular pathways and biological functions represented by the genes differentially expressed from proestrus to estrus are also altered during the human menstrual cycle, although not necessarily at the corresponding phases of the cycle. These findings establish a baseline for further studies in the mouse model to dissect mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases.

In the mouse, estrogen and progesterone are required to prime the uterus for decidual cell reaction (DCR) in response to an intraluminal stimulus and, once DCR is induced, progesterone is required to maintain DCR. However, some evidence indicates that certain nonprogestational steroid hormones may also be involved in regulating DCR. The present study determined whether androgen plays any role in DCR. Adult CD1 mice were ovariectomized and treated with a regimen of estradiol and progesterone to prime the uterus for DCR and to maintain DCR. Sesame oil was injected into the uterine lumen to induce DCR on Day 5 of the treatment. DCR was determined by deciduomal weight-the difference between the wet weights of oil-injected and noninjected uterine horns. Testosterone, given at 1 mg/day during Days 3-5, could not replace progesterone in priming the uterus for DCR. However, the same dose of testosterone given during Days 6-8 maintained DCR. Alkaline phosphatase activity, a bio-marker for DCR, was present in the deciduoma maintained by either progesterone or testosterone, although the distribution of this enzyme activity was more intense in the antimesometrial pole in progesterone-maintained deciduoma. A nonaromatizable androgen, 5 alpha-dihydrotestosterone (DHT), was also effective in maintaining DCR, and this action of DHT was blocked by an androgen receptor antagonist, hydroxyflutamide. The relative potency of DHT in maintaining DCR was similar to that of progesterone. However, the regression of the deciduoma appeared to be advanced in DHT-treated mice. Ovariectomy on Day 6 of pregnancy resulted in resorption of the conceptus and regression of the decidua within 48 h. Treatment with DHT at the time of ovariectomy could not prevent fetal resorption, but it delayed the regression of decidua, as indicated, in part, by the presence of granulated metrial gland cells. In summary, androgen cannot prime the uterus for DCR, but it can maintain DCR once it is induced. The physiological significance of this finding remains to be determined.

Pregnancy, spontaneous term labor (TL), and postpartum (PP) involution are associated with changes in the cellular and extracellular matrix composition of the uterus. Both the uterine smooth muscle (myometrium) and the infiltrating peripheral blood leukocytes involved in the activation of labor secrete extracellular matrix-degrading enzymes (matrix metalloproteinases, MMPs) that can modulate cellular behavior and barrier function. MMP expression is induced by mechanical stretch in several tissues. We hypothesized that the expression and activity of myometrial MMPs and their tissue inhibitors (TIMPs) are modulated in preparation for TL and PP involution and are regulated by mechanical stretch of uterine walls imposed by the growing fetus. Myometrial tissues were collected from bilaterally and unilaterally pregnant rats across gestation, TL, and PP. Total RNA and proteins were subjected to real-time PCR and immunoblotting, respectively, and tissue localization and activity was examined by immunohistochemistry and in situ zymography. We found that Mmp7, Mmp11, and Mmp12 mRNA levels were upregulated during TL and PP, while Mmp2, Mmp3, Mmp8, Mmp9, Mmp10, and Mmp13 mRNAs were only upregulated during PP. Timp1–Timp4 were stably expressed throughout gestation with some fluctuations PP. Active MMP2 was induced in the empty uterine horn during gestation and in the gravid PP uterus, suggesting negative regulation by biological mechanical stretch. We conclude that specific subsets of uterine MMPs are differentially regulated in the rat myometrium in preparation for two major events: TL and PP uterine involution.

We investigated the role of carbohydrates in blastocyst attachment to the uterine epithelium. Le(y) (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc) was localized by indirect immunofluorescence to the surface of the mouse blastocyst and uterine epithelium. Western blot analysis showed that Le(y) is carried on many uterine glycoproteins in both pregnant and nonpregnant females; however, new species were detected on Day 4 postcoitum (p.c.) coincident with the onset of uterine receptivity. The function of Le(y) in implantation was tested by injecting monoclonal antibody (mAb) directly into the uterine lumen on Days 3-5 p.c. The effects of intrauterine injections on implantation were scored by comparing the number of viable embryos to the number of CL on Day 10 p.c. Injection of purified anti-Le(y) IgM into the uterine lumen on the afternoon of Day 4 significantly inhibited implantation. This effect was dose-dependent and was obtained during a narrow time window, from 87 to 93 h p.c. Inhibition of implantation was not observed in contralateral uterine horns injected with saline, nor was it observed in uterine horns injected with other anti-carbohydrate mAbs. We conclude that binding of anti-Le(y) to the blastocyst or luminal epithelium masks a ligand involved in implantation. Although the mechanism of inhibition is unknown, we show that Le(y) can interact with another oligosaccharide (H) that has been described as a possible uterine ligand for blastocyst attachment. We hypothesize that Le(y) and H form carbohydrate-carbohydrate interactions that promote close apposition of cell surface membranes during an early step in implantation.

Since pharmacological evidence indicates that nitric oxide (NO) operates in the control of uterine motility, we have studied the distribution of NADPH diaphorase and NO synthases in the rat uterus using histochemical and immunohistochemical methods. Numerous nerve fibers displayed NADPH diaphorase activity and immunoreactivity to antisera raised against neuronal NO synthase. Nerve fibers appeared in all stages of the estrous cycle and also after ovariectomy. NADPH diaphorase activity was also present in endothelia and cells dispersed in the different uterine layers. Most NADPH diaphorase-positive (ND) cells had eosinophilic granules with occasional cells expressing the ED 1I macrophage-monocyte marker. Immunoreactivity for an inducible NO synthase was found in a small number of macrophage-like cells without NADPH diaphorase activity. Thus, ND cells may express another NO synthase isoform not detected by the available antisera. In normal cycling rats, ND cells were most abundant during proestrus, and their number further increased after estrogen treatment. ND cells were not observed after ovariectomy but were present after estrogen replacement therapy. ND cells could be involved in the estrogenic control of in vivo and in vitro uterine motility.

Fertility was evaluated at various times during the postpartum period in the llama. Fifty-six parous female llamas chosen at random were bred at 10, 20, and 30 days postpartum with six intact males. Half of the females copulated only once and the other half twice within an interval of 24 h. Ovarian activity was monitored by ultrasonography and analysis of urinary estrone sulfate and pregnanediol glucuronide (PdG). At the time of copulation, all females had developed ovulatory-size follicles. Ovulation was confirmed by the presence of a CL at 8 days after breeding and PdG concentrations > 1 ng/mg creatinine (Cr). Conception was defined as PdG concentrations > I ng/mg Cr at 15 days after breeding and the presence of a CL. Pregnancy was defined as the presence of a CL, PdG concentrations > 1 ng/mg Cr and an embryonic vesicle 20 days postbreeding. There were no significant differences in the proportion of females ovulating after breeding at different times postpartum; however, conception and pregnancy were significantly greater in females bred at Day 20 or 30. Ovarian follicle size was significantly larger at 30 days (9.1 mm) than at 10 and 20 days (7.9 and 8.8 mm, respectively) of breeding, with no difference in concentrations of estrone sulfate. The proportion of females conceiving as a result of breeding at 10 days postpartum (6 out of 10) was significantly less than for females bred at 20 (13 out of 15) or 30 days (16 out of 18). There were significant differences in the diameter of the uterine horns in females bred at 10 days (5.2-cm diameter) in contrast to females bred at 20 or 30 days (2.9 and 2.7 cm, respectively). These data demonstrate that the llama may ovulate as early as 10 days postpartum; however, pregnancy rates from breeding at 20 or 30 days were threefold higher (61%) than those in females bred at 10 days postpartum (21%).

The effect of the conceptus and of reduced uterine space on endometrial protein secretion was examined on Days 40, 60, and 80 of gestation in white crossbred gilts. Twenty-nine gilts were checked daily for estrus, and 15 were given 5 mg estradiol valerate daily from Days 11 to 15 (Day 0 = day of estrus) of the estrous cycle to induce pseudopregnancy. The remaining 14 pigs were mated during estrus. All pigs were laparotomized on Day 4, and one uterine horn was ligated to produce one crowded and one roomy uterine environment. Pigs were killed on Days 40, 60, and 80 of pregnancy or pseudopregnancy. The reproductive tracts were collected, and placental tissues from pregnant pigs and endometrial tissues from all pigs were cultured in the presence of 3H-leucine to evaluate protein secretion. Conditioned medium was dialyzed, measured for incorporation of radioactivity into nondialyzable macromolecules, and then subjected to two-dimensional (2D)-PAGE to determine the effect of uterine space and day of pregnancy or pseudopregnancy on overall protein secretion rate and secretion of specific proteins. Fetal survival, fetal weight, and placental weight were decreased (p < 0.01) in the crowded uterine environment compared to the roomy uterine environment. Incorporation of 3 H-leucine into nondialyzable macromolecules by endometrial tissue in culture was not affected by uterine space. Secretion of nondialyzable macromolecules by endometrium from pregnant pigs was not different from that by endometrium from pseudopregnant pigs on Day 40 but was greater (p < 0.01) on Days 60 and 80. Results of 2D-PAGE indicated that secretion of two endometrial proteins appeared to be increased (M, 13 000, pi 7 and M, 13 000, p 5.5), and secretion of one protein appeared to be decreased (M, 14 000, pi 6.2), in pregnant vs. pseudopregnant pig endometrial cultures. Decreasing uterine space did not affect incorporation of 3 H-leucine by placental tissue in culture. Examination of 2D-PAGE fluorographs of proteins secreted by placental tissue indicated that uterine space had no consistent effect on the array of proteins observed. However, secretion of a protein of Mr 35 000, pi 4 increased by Day 60. Another protein (M, 45 000, pi 4) increased by Day 80. Two other proteins (M, 12 500, pi 4.6 and Mr 13 000, pi 4.8) increased in some placental cultures by Day 60 of pregnancy and were consistently present in cultures from Day 80 placenta. These data indicate that uterine space does not influence endometrial or placental protein secretion on Days 40-80 of pregnancy. Secretion of specific proteins was greater by endometrium from pregnant compared to pseudopregnant pigs as early as Day 40 of pregnancy, and total endometrial protein secretion (measured as incorporation of 3'H-leucine into nondialyzable macromolecules) was greater for pregnant compared to pseudopregnant pigs by Day 60 of pregnancy; these findings suggest the possibility that the conceptus can alter endometrial protein secretion. The increase in endometrial protein secretion coincides with changes in secretion of specific placental proteins. These proteins or other compounds secreted by the conceptus may stimulate endometrial protein secretion during pregnancy.

The effect of altering uterine space available to developing conceptuses on placental and endometrial protein secretion and endometrial 3H-thymidine incorporation on Days 25 and 35 of gestation was tested. Gilts bred at estrus (Day 0) were laparotomized on Day 4, and the uterine horn ipsilateral to the ovary with the most ovulations was ligated midway between the uterine tip and uterine bifurcation to create a crowded and a roomy uterine environment. On Day 25 (n = 7) or 35 (n = 6), gilts were killed and the reproductive tract was dissected from each. Each placenta and fetus was weighed, and a sample of endometrium adjacent to each placenta was collected. Within each uterine environment, tissues (500 mg) from the heaviest and lightest (by weight) placentae and adjacent endometrial tissue were cultured for 24 h at 37 degrees C in 15 ml Minimal Essential Medium having 0.1 times the normal amount of leucine plus 50 microCi 3H-leucine. Aliquots (2 ml) of dialyzed medium were subjected to two-dimensional PAGE and fluorography to examine proteins with molecular weight ranges of approximately M(r) 20,000-130,000, and a further aliquot was subjected to SDS-PAGE to examine low molecular weight proteins (M(r) 2000-20,000). In addition, endometrium (200 mg in duplicate) adjacent to the heaviest and lightest placentae within each uterine environment was incubated for 4 h in 5 ml MEM plus 1 microCi 3H-thymidine to measure DNA synthesis. After culture, 3H-thymidine incorporation and total DNA were determined. Fetal survival in the crowded uterine environment decreased (p < 0.05) on Day 35 compared to Day 25. On both days, the heaviest and lightest placentae from the crowded uterine environment weighed less (p < 0.01) than the heaviest and lightest placentae from the roomy uterine environment. The fetuses associated with the heaviest and lightest placentae from the crowded uterine environment also weighed less (p < 0.05) than fetuses associated with the heaviest and lightest placentae from the roomy uterine environment. Nondialyzable radioactivity in medium per gram of tissue cultured from placental cultures decreased (p < 0.05) and from endometrial culture increased (p < 0.05) between Days 25 and 35. Placental secretion of a protein with M(r) 46,000, pI 5, decreased between Days 25 and 35. Endometrial secretion of three proteins (M(r) 35,000, pI 6.2; M(r) 25,000, pI 4.9; and M(r) 14,000, pI unknown) increased, and secretion of three other proteins (M(r) 24,000, pI 6.5 doublet; M(r) 22,000, pI 7.5, and M(r) 7000, pI unknown) decreased from Day 25 to Day 35 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth and microvascular development of the uterus were evaluated for ewes on Days 12, 18, 24, and 30 after mating (3-4 ewes/day; Day 0 = day of mating) in two experiments. In experiment 1, fresh weight and dry weight of gravid uterine horns were increased on Days 24 and 30 after mating, whereas those of nongravid uterine horns were elevated only on Day 30. The increased fresh and dry weights of gravid uterine horns on Day 24 were associated with uterine hyperplasia (increased DNA content). Increased fresh and dry weights of gravid uterine horns on Day 30, however, were associated with hypertrophy (increased RNA:DNA and protein:DNA ratios) of uterine tissues. In experiment 2, vascularity of endometrial tissues was elevated on Days 24 and 30 after mating. In addition, dramatic changes in uterine architecture (increased lumenal diameter and decreased endometrial thickness) and in uterine microvascular development (increased abundance of large microvessels and development of a subepithelial capillary plexus) were observed by Day 24 after mating. Characterization of the patterns of uterine growth and microvascular development will enable us to further define the role of previously reported uterine and conceptus-derived growth and angiogenic factors during early pregnancy.

This study was conducted to determine whether platelet-activating factor (PAF) (1) attenuated oxytocin-induced secretion of the prostaglandin (PG) F2 alpha metabolite, PGFM, by the ovine uterus in situ and (2) inhibited the generation of the inositol phosphate secondary messengers by endometrial tissue in response to oxytocin challenge in vitro. Ovariectomized ewes received steroid replacement to mimic the luteal phase. Six ewes received intrauterine injections of 200 micrograms PAF/uterine horn/day on Days 11-15, and 6 ewes were treated with vehicle. All ewes received 1 microgram oxytocin i.v. on Days 13-16. Pretreatment of ewes with PAF significantly suppressed PGFM release in response to oxytocin on Days 14 and 15 (p less than 0.005) compared to vehicle-treated ewes. PAF was not administered on Day 16, and the PGFM response to oxytocin was not different between groups. In a second experiment, ewes were given intrauterine injections of 200 micrograms PAF/uterine horn/day (n = 8) or vehicle (n = 7) on Days 11-15, and all ewes received 1 microgram oxytocin i.v. on Days 13 and 14. On Day 15 the uterus was removed, and the incorporation of 3H-inositol into inositol phosphates was determined in caruncular endometrium. Treatment of ewes with PAF in vivo reduced inositol monophosphate (IP1) generated by oxytocin (10(-6) M) by 56.4%, compared to that in endometrium from vehicle-treated controls, and also inhibited the incorporation of 3H-inositol into glycerophosphoinositol (GPI). If PAF was added to the endometrium during the incubation in vitro, the attenuation of inositol phosphate generation did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Controlled delivery of monoclonal antibodies to the mucus secretions of the vagina might provide women with passive immunoprotection against both sexually transmitted diseases and unwanted pregnancy. We have developed intravaginal devices composed of poly(ethylene-co-vinyl acetate) (EVAc) that continuously release IgG antibodies for over 30 days into buffered saline, and we have tested these devices in the vagina of mice. Polymeric devices containing either BSA (as a test reagent for proteins) or anti-hCG antibody, when inserted into the vaginas of mice, provided a continuous supply of either BSA or hCG-binding antibodies to the vaginal mucus for 30 days. Antibodies released by the devices achieved high concentration in the mucus within the lumen of the vagina, but did not significantly ascend into the uterine horns, as determined by epifluorescence microscopy of fluorescently labeled mouse IgG and by immunohistochemical localization of rabbit IgG. Our results suggest that long-term intravaginal delivery of functionally intact antibodies can be achieved with devices composed of EVAc.

Characteristics of preimplantation embryonic development to Day 3.5 of gestation were evaluated in lines of mice after 21 generations of selection for litter size or components of litter size. Selection criteria were direct selection for number born (LS), selection on an index of ovulation rate and the proportion of ova shed that resulted in fully formed pups (IX), selection for number born in unilaterally ovariectomized females as an indication of uterine capacity (UT), and an unselected control (LC). Comparison of the average distributions of embryonic stage of development on the left side of the uterus showed that selection (average effect of LS, IX, and UT vs. LC) tended to advance (p = 0.07) the average stage of embryonic development at Day 3.5 and shift the distribution (p = 0.10) by increasing the frequency of expanded blastocysts and decreasing the frequency of pre-morula embryos. A similar shift in the distribution on the right side of the uterus was not statistically significant. Selection decreased (p = 0.06) variability in developmental stage among embryos within the right uterine horn. These selection criteria evaluated in the mouse appear to have changed the frequencies of genes that affect some determinants of average stage of embryonic development and uniformity of development within a uterine horn at Day 3.5 of gestation.

In order to determine the effects of endothelin (ET) and relaxin on uterine contractility, immature female rats were treated with estrogen (E, 1 microgram s.c., Days 1-3) or estrogen and progesterone (2 mg s.c. [E + P], Days 2 and 3), and killed; the uterine horns were removed and suspended in muscle baths. Initially, we determined the contractile response to varying doses of ET and how this response was altered by pretreatment with progesterone. Uterine strips from animals treated with E + P (n = 10) were less sensitive to the stimulatory effects of ET than were strips from E-treated animals (n = 10). This difference was significant at ET doses above 2.5 nM. After completion of the dose-response studies, contractile patterns in response to ET and relaxin were then studied in animals treated with E (n = 10) or E + P (n = 9). ET (5 nM) significantly increased uterine contractility, mostly through an effect on the frequency of contractions (p less than 0.01). Relaxin (25 ng/ml) decreased contractility, affecting all contractile parameters measured (p less than 0.01). ET stimulated contractility in uterine horn segments previously inhibited by relaxin (p less than 0.01), and relaxin reduced the increased contractility produced by earlier exposure to ET (p less than 0.01). These data indicate that ET and relaxin can interact reversibly to control contractility in uterine horn segments in vitro, and that progesterone pretreatment can diminish the contractile response to the stimulatory effects of ET.

Mice homozygous for a mutant allele (an/an) causing a lifelong macrocytic anemia (Hertwig's anemia) also demonstrate an inability to deliver their offspring, despite normal ovulation, conception, implantation, and fetal development. We investigated the roles of estrogen and relaxin in the etiology of the reproductive defect in the Hertwig's anemia mice. Immunoreactive relaxin levels were undetectable in the nonpregnant controls, whereas levels in both timed-pregnant controls and timed-pregnant affected mice were significantly higher than in nonpregnant controls, but not significantly different from each other. Mean interpubic ligament length in the pregnant Hertwig's anemia mice was significantly greater than that in nonpregnant controls, but significantly less than that in the pregnant controls on Day 18 of pregnancy. Porcine relaxin was administered to nonpregnant affected and unaffected littermates and to nonpregnant controls. Whereas controls showed a significant response to porcine relaxin, neither the Hertwig's anemia mice nor their unaffected littermates responded to the porcine relaxin. Additional study was performed to determine estradiol effects in the affected and control animals utilizing detailed computerized morphometric analysis of uterine horns and cervices from immature, estradiol-injected controls and Hertwig's anemia mice. Results demonstrated a statistically significant trophic effect of estradiol upon uterine horn and cervical enlargement, as assessed by weight and volume, in controls. Only a slight, non-significant effect was seen in Hertwig's anemia mice. Additional histological effects of estradiol, including endometrial enfolding observed in controls, were not present in Hertwig's anemia mice. Lack of response to both estrogen and relaxin is responsible for the parturitional defect in Hertwig's anemia mice.

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.

A previous study indicated rabbit endomethal relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745 [1]). 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 insmunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesomethal surface occurs on Days 8-11. Relaxin was observed in antimesometrial endomethal 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 epitheial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epitheilal cells of the non-conceptusbearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epitheial 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, endomethal epitheial 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 epithdlial 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).

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)

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 PHI receptor numbers were similar between Days 8 and 15 for cyclic gilts. In contrast, endometrial PHI receptor numbers for pregnant gilts were similar between Days 8 and 11, then increased (p < 0.05) on Day 12 and remained elevated between Days 14 and 30. This day-bystatus Interaction approached significance (p < 0.06) and overall receptor numbers were higher (p

Thirty gilts were used to examine if the sequence in which oocytes were released at ovulation contributed to differences in embryonic development and uterine secretions by Day 12 (Day 0 = onset of estrus). Oocytes of follicles destined to ovulate last were recovered 42 h after injecting proestrous gilts with hCG, incubated with a fluorescent stain, and returned to the donor's oviduct. These later-maturing oocytes subsequently became the lesser-developed (p less than 0.01) embryos on Day 4. In a second experiment, lesser- vs. more-developed Day 4 embryos from additional gilts were transferred to ligated uterine horns of nonpregnant gilts. Subsequently, the lesser-developed Day 4 embryos became the smaller (p less than 0.01) blastocysts within a litter on Day 12. Uterine flushings associated with lesser-developed embryos on Day 12 contained less estradiol (p less than 0.01), less total protein (p less than 0.10), and less acid phosphatase activity (p less than 0.05), but total content of calcium was not different compared to flushings that contained more-developed embryos. Analysis of uterine flushings with two-dimensional PAGE procedures indicated advanced uteroferrin-associated glycoprotein secretion from the horn that contained more-developed embryos. Results of these experiments suggested that oocytes of later-ovulating follicles were progenitors of smaller embryos, which probably stimulated uterine secretion later than more advanced littermates on Day 12.