Progesterone inhibits uterine gland development in the neonatal mouse uterus

Uterine glands and their secretions are required for conceptus (embryo/fetus and associated placenta) survival and development. In most mammals, uterine gland morphogenesis or adenogenesis is a uniquely postnatal event; however, little is known about the mechanisms governing the developmental event. In sheep, progestin treatment of neonatal ewes permanently ablated differentiation of the endometrial glands. Similarly, progesterone (P4) inhibits adenogenesis in neonatal mouse uterus. Thus, P4 can be used as a tool to discover mechanisms regulating endometrial adenogenesis. Female pups were treated with sesame vehicle alone as a control or P4 from Postnatal Day 2 (PD 2) to PD 10, and reproductive tracts were examined on PD 5, 10, or 20. Endometrial glands were fully developed in control mice by PD 20 but not in P4-treated mice. All other uterine cell types appeared normal. Treatment with P4 stimulated proliferation of the stroma but suppressed proliferation of the luminal epithelium. Microarray analysis revealed that expression of genes were reduced (Car2, Fgf7, Fgfr2, Foxa2, Fzd10, Met, Mmp7, Msx1, Msx2, Wnt4, Wnt7a, Wnt16) and increased (Hgf, Ihh, Wnt11) by P4 in the neonatal uterus. These results support the idea that P4 inhibits endometrial adenogenesis in the developing neonatal uterus by altering expression of morphoregulatory genes and consequently disrupting normal patterns of cell proliferation and development.     

Developmental biology of uterine glands.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.     

Ovarian regulation of endometrial gland morphogenesis and activin-follistatin system in the neonatal ovine uterus

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. Previous results indicated that ovariectomy of ewes at birth did not affect uterine growth or initial stages of endometrial gland genesis on PND 14 but did affect uterine growth after PND 28. Available evidence from a number of species supports the hypothesis that the ovary does not affect endometrial gland morphogenesis in the postnatal uterus. To test this hypothesis in our sheep model, ewes were assigned at birth to a sham surgery as a control or bilateral ovariectomy (OVX) on PND 7. Uteri were removed and weighed on PND 56. Ovariectomy did not affect circulating levels of estradiol-17beta. Uterine weight was 52% lower in OVX ewes. Histomorphological analyses indicated that the thickness of the endometrium and myometrium, total number of endometrial glands, and endometrial gland density in the stratum spongiosum stroma was reduced in uteri of OVX ewes. In contrast, the number of superficial ductal gland invaginations and gland density in the stratum compactum stroma was not affected by ovariectomy. The uteri of OVX ewes contained lower levels of betaA subunit, activin receptor (ActR) type IA, ActRIB, and follistatin protein expression but higher levels of betaB subunit. In the neonatal ovary, follistatin, inhibin alpha subunit, betaA subunit, and betaB subunit were expressed in antral follicles between PNDs 0 and 56. These results led to rejection of the hypothesis that the ovary does not influence endometrial adenogenesis. Rather, the ovary and, thus, an ovarian-derived factor regulates, in part, the coiling and branching morphogenetic stage of endometrial gland development after PND 14 and expression of specific components of the activin-follistatin system in the neonatal ovine uterus that appear to be important for that critical process.     

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.     

Expression of wingless type (WNT) genes and their antagonists at mRNA levels in equine endometrium during the estrous cycle and early pregnancy

WNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4) and on days of 14 (P14, n=4), 18 (P18, n=4), 22 (P22, n=4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n=4) and after luteolysis in estrus phase (AL, on day of 17.5-18, n=4) of the cycle. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Eleven WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, WNT8A, WNT9B, WNT10B, WNT11 and WNT16) and their antagonists (SFRP1, SFRP2, SFRP5, DKK1, DKK2 and WIF-1) were detected in equine endometrium. Compared to d0, WNT2, WNT5B, WNT7A and SFRP1 expressions were downregulated by the pregnancy while DKK1 was upregulated. WNT5A, WNT11 and WIF-1 were upregulated on P14 and P18, but WNT2B increased only on P14. When LD and P14 were compared, level of WNT8A decreased on P14 while increase in WNT4 level on P14 was slightly significant (P<0.06). Levels of WNT7A and SFRP1 decreased while DKK1 and WIF-1 increased by the pregnancy on P18 compared to AL. Moreover, WNT2B, WNT5A, WNT9B, WNT10B, WNT11, WNT16 DKK1 and WIF-1 were upregulated on LD compared to AL whereas WNT4, WNT7A, SFRP1 were downregulated. In conclusion, the results demonstrate that WNT genes and their antagonists appear to be regulated during early pregnancy in equine endometrium possibly due to embryonic factors and/or maternal progesterone.     

Endometrial HSD11B1 and cortisol regeneration in the ovine uterus: effects of pregnancy, interferon tau, and prostaglandins

In ruminants, the elongating conceptus secretes interferon tau (IFNT), the pregnancy recognition signal, and prostaglandins (PGs). Progesterone from the ovary induces prostaglandin synthase two (PTGS2) and hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) in the endometrial epithelia, and PTGS2-derived PGs regulate endometrial functions and conceptus elongation. The enzyme HSD11B1 interconverts inactive cortisone and active cortisol. These studies determined the effects of pregnancy, IFNT, and PGs on endometrial HSD11B1 expression and activity in the ovine uterus. Study one found that HSD11B1 activity was present in both the endometrium and conceptus during early pregnancy. In study two, ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PTGS2 inhibitor, from Days 8 to 14 of pregnancy. Endometrial HSD11B1 activity and cortisol in the uterine lumen were substantially lower in MEL-infused ewes. In study three, cyclic ewes received intrauterine infusions of vehicle as a CX, MEL, recombinant ovine IFNT, or IFNT and MEL. Infusion of IFNT increased endometrial HSD11B1 expression and activity and cortisol in the uterine lumen, and this effect was diminished by coinfusion of MEL. In study four, cyclic ewes were infused with vehicle as a CX, IFNT, PGE2, PGF2 alpha, or PGI2. Infusion of all the PGs and IFNT increased endometrial HSD11B1 expression and activity, and IFNT and PGI2 infusion increased cortisol in the uterine lumen. These studies support the idea that IFNT and PGs from the conceptus regulate endometrial HSD11B1 expression and activity that regenerates bioactive cortisol in the ovine uterus during early pregnancy to influence endometrial functions and conceptus elongation.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe

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 conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.     

The activin-follistatin system in the neonatal ovine uterus

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.     

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.     

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.     

Uterine gland development begins postnatally and is accompanied by estrogen and progesterone receptor expression in the dog

During neonatal and juvenile life, mammalian uteri undergo extensive structural and functional changes, including uterine gland differentiation and development. In sheep and mice, inhibition of neonatal uterine gland development induced by progestin treatment led to a permanent aglandular uterine phenotype and adult infertility, suggesting that this strategy might be useful for sterilizing dogs and other companion animals. The goal of this study was to define temporal patterns of adenogenesis (gland development), cell proliferation, and progesterone and estrogen receptor expression in uteri of neonatal and juvenile dogs as a first step toward determining whether neonatal progestin treatments might be a feasible contraceptive approach in this species. Uteri obtained from puppies at postnatal wk 1, 2, 4, 6, or 8 were evaluated histologically and immunostained for MKI67, a marker of cell proliferation, estrogen receptor-1, and progesterone receptor. Adenogenesis was under way at 1 wk of age, as indicated by the presence of nascent glands beginning to bud from the luminal epithelium, and rapid proliferation of both luminal epithelial and stromal cells. By Week 2, glands were clearly identifiable and proliferation of luminal, glandular, and stromal cells was pronounced. At Week 4, increased numbers of endometrial glands were evident penetrating uterine stroma, even as proliferative activity decreased in all cell compartments as compared with Week 2. Whereas gland development was most advanced at Weeks 6 to 8, luminal, glandular, and stromal proliferation was minimal, indicating that the uterus was nearly mitotically quiescent at this age. Both estrogen receptor-1 and progesterone receptor were expressed consistently in uterine stromal and epithelial cells at all ages examined. In summary, canine uterine adenogenesis was underway by 1 wk of age and prepubertal glandular proliferation was essentially complete by Week 6. These results provided information necessary to facilitate development of canine sterilization strategies based on neonatal progestin treatments designed to permanently inhibit uterine gland development and adult fertility.     

Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy

Conceptus (embryo and associated extraembryonic membranes) implantation and development require a reciprocal biochemical and physical interactions between the extraembryonic membranes and the endometrium. However, the enzymatic antioxidative pathways controlling reactive oxygen species production at the endometrial-extraembryonic membrane interface early in pregnancy are not known. We aimed therefore to determine the content of malondialdehyde, as biomarkers of lipid peroxidation, and the activities of the major antioxidant enzymes, copper-zinc containing and manganese containing superoxide dismutases, catalase and glutathione peroxidase, in sheep extraembryonic membranes, caruncular and intercaruncular endometrium zones sampled at specific stages of pregnancy corresponding to the conceptus implantation (day 16) and the early post-implantation period (day 21). Malondialdehyde content in caruncular, intercaruncular and extraembryonic tissues was not different between stages of the pregnancy. Extraembryonic membranes demonstrated increased manganese containing superoxide dismutase and glutathione peroxidase activities, whereas catalase activity in these tissues decreased from day 16 to day 21. Caruncular tissues demonstrated increased manganese containing superoxide dismutase activity from day 16 to day 21. Intercaruncular tissues demonstrated increased copper-zinc containing superoxide dismutase, manganese containing superoxide dismutase and catalase activities from day 16 to day 21. The ovine extraembryonic membranes exhibit dynamic changes in enzymatic antioxidative pathways different from those of endometrial tissues during the transition from implantation to post-implantation period. This biochemical data provides novel insights into the developmental changes in antioxidative pathways of extraembryonic membranes and endometrium during early conceptus development.     

Oxytocin-induced prostaglandin release from perifused bovine caruncular and intercaruncular endometrial tissue on days 20, 30 and at first estrus postpartum

Twenty-two multiparous Brahman x Hereford F1 cows were utilized to determine the effect of oxytocin (OT) on prostaglandin F2 alpha (PGF) release from caruncular and intercaruncular endometrial tissues and prostaglandin E2 (PGE) release from intercaruncular tissue. The previously gravid uterine horn was removed on d 20 postpartum (n = 7), on d 30 postpartum (n = 7) or the uterine horn ipsilateral to the dominant follicle was removed 12-18 h after onset of first behavioral estrus postpartum (ES; n = 8). Tissues (200 mg wet wt) were cultured in Nutrient Mixture F-10 medium in a perifusion system. The medium and tissues were aerated with 95% O2: 5% CO2 and temperatures were maintained at 39 degrees C. The flow rate was 100 microliters/min and fractions were collected at 20 min intervals for 400 min. After a 2 h settling phase, the tissues were challenged with 1, 2 or 4 micrograms [Asu1,6]-OT/ml of media for 1 h. Basal release of PGE and PGF on d 20 was greater than on d 30 and at ES (P less than .02) which were similar. All doses of OT increased PGE and PGF with both remaining elevated throughout the duration of the perifusion (P less than .008). However, there were no differences among doses. Release of PGE in response to OT on d 20 and 30, was higher than at ES (P less than .008). More PGF was released in response to OT from intercaruncular than caruncular tissue on d 20 (P less than .0001) and at ES (P less than .003). Release of PGF in response to OT on d 20 was higher (P less than .0001) than on d 30 and d 30 was higher than at ES (P less than .007). Basal and OT-induced release of PGE and PGF declined as day postpartum increased. We conclude that intercaruncular tissue released more PGF than caruncular tissue and both intercaruncular and caruncular tissue responded to OT with a sustained release of prostaglandins in a non-dose-dependent manner on d 20, 30 and at ES postpartum.     

Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes.

The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.