Regulatory effect of steroid hormones and fetal tissues on expression of oxytocin receptor in the endometrium of late pregnant ewes.

Oxytocin receptors play an important role in the establishment of pregnancy and parturition in ruminants. Previous studies in cyclic and early pregnant ewes have indicated that receptor concentrations are regulated by steroid hormones and fetal secretory products. This study investigated the effect of oestradiol and progesterone, or co-culture with placenta or corpus luteum on oxytocin receptor expression. Endometrial explants from late pregnant ewes were cultured for up to 96 h in various treatment combinations. After culture, tissues were subjected to in situ hybridization and autoradiography with 125I-labelled oxytocin receptor antagonist to localize and measure the expression of oxytocin receptor mRNA and protein. Results were quantified as absorbance units from autoradiographs. Oxytocin receptors were confined to the endometrial luminal epithelium and both mRNA and 125I-labelled oxytocin receptor antagonist binding were upregulated spontaneously in basic serum-free medium. Upregulation occurred earlier in the presence of oestradiol (0.1 mumol l-1) but the final receptor concentration was similar to that found in the basic medium. Continuous progesterone treatment (1 mumol l-1) and co-culture with corpus luteum both delayed the increase in oxytocin receptor mRNA, but a short initial (4 h) period in progesterone-free basic medium resulted in loss of the inhibitory effect. Co-culture with placental tissues had no effect. In conclusion, oxytocin receptor expression in the luminal epithelium increased immediately on removal from the maternal environment. This occurred regardless of treatment and did not require the presence of steroid hormones, but could be accelerated or delayed by oestradiol and progesterone, respectively. There may be an additional inhibitory factor present in the corpus luteum.     

Ovarian oxytocin and the maternal recognition of pregnancy

The secretion of oxytocin by the corpus luteum is thought to stimulate the episodic release of PGF-2 alpha from the uterus, thereby contributing to luteolysis. In pregnancy corpus luteum function is maintained, and secretion of oxytocin, or its actions on the uterus, appear to be inconsistent with the successful establishment of gestation. Protection against the effects of oxytocin is ensured by a number of mechanisms, including the cessation of luteal oxytocin secretion, which is evident by Day 20 after mating in sheep, and the maintenance of low levels of the oxytocin receptor in the uterus.     

Biochemical and endocrine aspects of oxytocin production by the mammalian corpus luteum

A review of the current state of knowledge of oxytocin production by the preovulatory follicle and corpus luteum is presented. Corpora lutea of a number of mammalian species have been found to synthesize oxytocin. However, the synthesis and secretion of this nanopeptide by the corpus luteum of the ruminant has been most extensively studied because of the potential role of this peptide in facilitating luteal regression. While much information exists relative to various biochemical and endocrine factors that impact on oxytocin gene expression, this aspect about luteal synthesis of this peptide hormone remains enigmatic. Prostaglandin F-2α (PGF-2α) has been shown to be a primary endogenous hormone responsible for triggering luteal secretion of oxytocin. Details are provided regarding the PGF-2α-induced intracellular signal transduction pathway that ultimately results in exocytosis of luteal oxytocin. Evidence is also presented for potential autocrine/paracrine actions of oxytocin in regulating progesterone production by luteal and granulosa cells. Concluding remarks highlight aspects about luteal oxytocin production that require further research.     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.     

Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy

Concentrations of oxytocin in corpora lutea were reduced from 1706 to less than 15 ng/g wet wt after hysterectomy in sheep during the oestrous cycle. Hysterectomy also blocked the appearance of raised levels of oxytocin in ovarian and jugular venous plasma caused by cloprostenol. Administration of cloprostenol to hysterectomized ewes resulted in luteal regression, which occurred as rapidly as in intact animals. Therefore oxytocin in the corpus luteum during the oestrous cycle is unlikely to be involved in intraluteal events mediating prostaglandin-induced luteolysis.     

Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid-pseudopregnant rats. These data indicate that P4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.     

Progesterone production by dispersed luteal cells of non-pregnant cows: effects of oxytocin and oestradiol

The effects of oxytocin and oestradiol on progesterone production by dispersed luteal cells of non-pregnant cows were studied. In acute incubation (3 h), oxytocin, at a concentration of 800 mIU/ml, significantly inhibited the production of progesterone induced by HCG (10 IU/ml). Suppression of basal progesterone production was evident in some corpora lutea. Lower oxytocin concentrations (4 and 40 mIU/ml) had no effect. At a concentration of 400 mIU/ml, oxytocin may be inhibitory to basal and HCG-induced progesterone production. Oestradiol (1 μkg/ml) had no effect on basal progesterone production but may suppress the production of progesterone induced by HCG. However, incubation with oxytocin (400 mIU/ml) plus oestradiol (1 μg/ml) resulted in a significant inhibition of HCG-induced progesterone production. These data provide evidence for an inhibitory effect of oxytocin on the corpus luteum of non-pregnant cows. Oestradiol may interact with oxytocin to inhibit the bovine corpus luteum function.     

Oxytocin in baboon (Papio anubis) corpus luteum

Recently, using a highly specific radioimmunoassay, we have demonstrated that the concentration of oxytocin in the corpus luteum of the human and cynomolgus monkey are several fold higher than in the peripheral circulation. In this study, we have examined the corpora lutea and ovarian stroma from the ovaries of normal adult cycling baboons (Papio anubis) for the presence of oxytocin through the use of immunocytochemical procedures. Tissues obtained at laparotomy were fixed in Bouin's solution and embedded in paraffin; immunoreactive oxytocin was localized with peroxidase-antiperoxidase and 3.3' diaminobenzidine. Six corpora lutea with stroma were obtained--two each from the early (Day 14-20), mid-(Day 21-24), and late (Day 25-30) stages of the luteal phase. Immunoreactive oxytocin was localized in all corpora lutea examined but was absent from all stroma samples. Larger areas of the corpus luteum from the mid-luteal phase showed staining for oxytocin, and the intensity of staining for this peptide was maximal in this phase of the cycle.     

Hormonal responses following treatment with different prostaglandin analogues for estrous cycle regulation in cattle

The effect of four different prostaglandin F(2)alpha analogues on secretion of the pituitary hormones prolactin, LH, FSH and oxytocin was evaluated in heifers. In addition, the luteolytic activity was measured by means of progesterone concentrations. For the sake of comparison, the corpus luteum of two heifers was enucleated and the effect on the above hormones determined. There seemed to be no differences among the various analogues concerning their luteolytic action on the corpus luteum. No effect on FSH secretion was observed. Basal concentrations of LH increased about 4 - 5 h after injection of the analogues, concurrently with the decrease of progesterone. Enucleation of the corpus luteum was followed by a similar pattern of hormone secretion. Therefore, the increase in LH concentrations did not appear to be a specific effect of the analogues, but was probably a result of the decrease in progesterone concentration. Within twenty minutes after administration of the analogues a clear increase in plasma prolactin (2-4 h) and oxytocin concentrations (1-2 h) could be demonstrated.     

Inhibition of luteal function by oxytocin antagonist in goats (Capra hircus).

Early corpus luteum development in nonpregnant and pregnant goats was characterized by a steady increase in peripheral plasma concentrations of progesterone and a high release of prostacyclin (PGI-2) but low release of prostaglandin F-2 alpha (PGF-2 alpha). Jugular administration of oxytocin antagonist (OXA) (0.2 microgram/kg/day) on the day of oestrus and for 3 days thereafter to cyclic and mated goats, significantly (P less than 0.001) inhibited progesterone and prostaglandin secretion and reduced conception rate. Co-administration of PGI-2 (200 micrograms/day) with OXA resulted in a steady increase of progesterone and establishment of pregnancy, but co-administration of PGF-2 alpha (175 micrograms/day) with OXA had no effect. It is suggested that oxytocin is required for early development of the corpus luteum and such effects may be mediated via PGI-2 production.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects

Recent analysis of mice deficient in both oxytocin (OT) and cyclooxygenase-1 has shown that OT exerts significant effects on both the ovarian corpus luteum and the uterine myometrium during pregnancy. To better define the roles of OT during pregnancy, we evaluated OT action and OT receptor regulation in wild-type and OT-deficient knockout (KO) mice. Continuous infusion of OT revealed that OT can either delay labor at low doses or initiate preterm labor at high doses. The infusion rates of OT necessary for these effects were reduced in OT KO mice. The dose of OT that delayed labor also delayed the normal decrease in plasma progesterone late in gestation, implicating a primary effect on the corpus luteum. Consistent with this hypothesis, luteal OT receptor expression exceeded that of the myometrium until luteolysis occurred. We propose that the downregulation of OT receptors in the corpus luteum and induction of OT receptors in the myometrium serve to shift the predominant consequence of OT action during murine pregnancy from labor inhibition to labor promotion.     

Suppression by luteectomy of the simultaneous increase in intramammary pressure and blood oxytocin levels induced by the injection of PGF2-alpha in ewes

Seven lactating Lacaune ewes underwent either a total luteectomy on day 19 of pregnancy (D19) (compensated from that stage by a daily progesterone supplementation of 25 mg to ensure embryonic survival; group 1:4 animals) or a control laparotomy (group 2: 3 animals). Intra-jugular injection of 200 micrograms of a synthetic PGF2 alpha analogue (Dinolytic, Upjohn) caused an increase in the intramammary pressure (IMP) and a concomitant rise in oxytocinaemia only in the presence of a corpus luteum, ie in all ewes of groups 1 and 2 before D19 and only in those of group 2 after that stage. These experiments confirm that the corpus luteum, and not the other ovarian compartments, releases oxytocin when prostaglandin F2 alpha is administered.     

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.     

Expression of mRNA for follicle-stimulating hormone suppressing protein in ovarian tissues of cows.

The expression of bovine follicle-stimulating hormone (FSH)-suppressing protein (FSP) mRNA was investigated in different ovarian tissues of cows. Northern blot analysis, using a cDNA probe to bovine FSP, demonstrated that the FSP gene in the bovine ovary is highly expressed in a pool of isolated granulosa cells. Two bands (2.8 and 1.8 kb) were observed in all tissues expressing the mRNA. FSP mRNA was low in small antral follicles and increased in growing follicles to reach a maximum in preovulatory follicles. Low amounts of mRNA of steady state FSP were observed in all stages of the corpus luteum as well as in the corpus luteum of pregnant cows, in the corpus albicans and theca tissue, whereas this mRNA could not be detected in the liver. These results are consistent with the hypothesis that, in cows, FSP functions as an autocrine regulator in developing follicles to facilitate luteinization of granulosa cells.