Mifepristone and PGF2α activate phosphatidylinositol hydrolysis in the ovine corpus luteum.

Two experiments were conducted to determine whether mifepristone (RU486) and PGF2α activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the ovine estrous cycle. In experiment 1, ewes on day 8 of the cycle were given 10 µg RU486 or vehicle into the ovarian artery with removal of the corpus luteum (CL) after 10 min. Blood collected prior to and after treatment was analyzed for progesterone. Aliquots of CL were incubated with 10 µCi of 3H-inositol and in the presence and absence of PGF2α (10 nM) for 15 min. Exposure of CL to RU486 and PGF2α increased phosphatidylinositol hydrolysis (p < 0.05). Serum progesterone was reduced in both control and RU486-treated ewes (p < 0.05) compared to concentrations before treatments. In experiment 2, aliquots of CL collected from ewes on day 8 of the cycle were incubated with 3H-inositol and exposed to RU486 (2 µM) in the presence and absence of PGF2α (1 µM) for 15 min. Treatments stimulated phosphatidylinositol hydrolysis as in Exp 1 (p < 0.05). Progesterone concentrations in incubation medium were increased in response to RU486 and PGF2α (p < 0.05). Collectively, these data suggest that RU486 and PGF2α act to stimulate phosphatidylinositol hydrolysis in the mature ovine CL.

Optimal dose of PG600 when given to progestogen-synchronized ewes during anestrus as affected by day of the year and temperature.

The objective of this long-term study was to improve the out-of-season breeding rates for ewes by examining effectiveness of dose levels of PG600, a combination of 80-IU/mL pregnant mare serum gonadotropin and 40-IU/mL human chorionic gonadotropin. Each week, during the anestrous season from 1998 through 2016, mature ewes were inserted with 1 of 2 progestogen devices. After 9 to 13 d, at removal of the device, ewes were injected intramuscularly with a specific dose of PG600. A total of 1,402 ewes were treated in this study, with weekly treatment groups ranging from 3 to 12 ewes. Ewes were assigned randomly in equal numbers to 1 of 2 ram pens with one fertile ram in each pen. Rams were fitted with a marking harness. Between 36 and 48 h after removal of the device, rams were switched. Ewes were checked for crayon marks (indicative of estrus) at 36, 48, and 96 h after removal of synchronizing devices. Rams were removed after 1 wk. Pregnancy status was diagnosed at 90 d of gestation using radiography and verified at 120 d or at lambing. To explore the impact of dose level, daylight, and temperature on pregnancy rate, we formulated an ordered multinomial probit model. The model controls statistically for dose level, synchronization device type, device reuse, and natural variation in minimum daily temperature and day of the year (DOY). Predicted pregnancy rate and type of birth at 90 d of gestation were significantly affected by DOY and temperature as well as dose level. Both observed and predicted pregnancy rates were higher for PG600 dose levels between 2.00 and 3.50 mL than for those below 2.00 mL or above 3.99 mL. By giving optimal amounts of PG600 to anestrous ewes, producers will have a greater number of pregnant ewes at lambing during the out-of-season lambing time.

Progesterone suppresses an oxytocin-stimulated signal pathway in COS-7 cells transfected with the oxytocin receptor.

The present study was conducted to determine if progesterone (P4) would inhibit oxytocin-stimulated phosphoinositide hydrolysis in COS-7 cells expressing transfected ovine oxytocin receptor (OTR) with little or no nuclear P4 receptor (nPR) protein present. The relative absence of nPR in these cells was confirmed by immunocytochemistry and RT-PCR. To investigate the effects of P4 on oxytocin (OT) signaling, cells were transiently transfected with the ovine OTR. Radioreceptor assay for [(3)H]-OT binding confirmed the presence of a high affinity binding site for OT in transfected cells, while treatment with P4 and GTPgammaS (which uncouples the OTR from the heterotrimeric G-protein) increased the K(d) for OT binding slightly. Cells were then assayed for inositol phosphate hydrolysis 48 h post-transfection. Pre-treatment of cells with P4 for 10 min significantly interfered with rapid (20 min) OT-stimulated inositol trisphosphate (IP(3)) production. This inhibition was specific to P4, because pre-treatment of cells with promegestone (R5020), testosterone, mifepristone (RU 486), or cortisol did not decrease OT-stimulated IP(3) levels. By radioreceptor assay for PR, no measurable specific binding of R5020 was observed for either transfected or non-transfected cells. We conclude that P4 can inhibit OTR-mediated phosphoinositide hydrolysis in COS-7 cells that express little or no nPR protein. These data support a role for a non-genomic action for P4 in OTR signaling via some mechanism other than by binding to a membrane progestin receptor in an immortalized, transfected cell.

Nongenomic action of progesterone inhibits oxytocin-induced phosphoinositide hydrolysis and prostaglandin F2alpha secretion in the ovine endometrium.

Experiments were conducted to characterize the nongenomic effects of progesterone (P4) on binding of oxytocin (OT) to its receptor and signal transduction in the ovine endometrium. The dose-response relationship of P4 to OT binding was examined. Membranes from endometrial tissue of ovariectomized hormone-treated ewes were preincubated in the presence of P4 for 1 h followed by OT receptor analysis. P4 interfered with the binding of OT in a dose-dependent manner. Endometrium was then recovered from cyclic ewes and divided into explants. Treatment consisted of two dosages of P4 and two dosages of OT. Explants were analyzed for total inositol monophosphate, bisphosphate (IP(2)), and trisphosphate (IP(3)) content. Preincubation with P4 for 10 min significantly interfered with OT stimulation of IP(2) and IP(3) synthesis. Oxytocin increased monophosphate production, but there was no detectable effect of P4. In the next experiment, endometrial explants were cultured in the absence or the presence of arachidonic acid. Explants were then exposed for 1 h to medium containing vehicle or P4. After incubation, explants were challenged with OT and the media were collected and analyzed for 13,14 dihydro-15-keto prostaglandin F(2alpha) by RIA. Treatment of explants with AA increased PGF(2alpha) content compared with that of controls. Brief exposure to P4 significantly decreased OT-induced PGF(2alpha) secretion from explants previously exposed to medium or AA. Collectively, these data are interpreted to indicate that the observed reduction in OT-induced IP(2) and IP(3) production and OT-induced PGF(2alpha) secretion was due to P4 inhibition of OT binding to its receptor.

Changes in bovine luteal progesterone metabolism in response to exogenous prostaglandin F(2alpha).

An experiment was conducted to determine the effect of prostaglandin F2alpha (PGF2alpha) on luteal synthesis of progesterone (P4) and related progestins. Sixteen beef heifers were assigned in equal numbers to four groups in a 2x2 factorial arrangement of treatments. The experiment consisted of two levels of PGF2alpha analog (0 and 500 microg) and two levels of time (4 and 24 h after injection) of corpus luteum collection. All heifers were injected intravenously with saline (2 ml) or PGF2alpha (cloprostenol) on day 8 of the estrous cycle (estrus=day 0). Jugular blood was collected at 0, 1, 2, 3, 4 and 20, 21, 22, 23, and 24 h after injection. Resulting sera were analyzed for P4 by use of radioimmunoassay. Luteal tissue was analyzed by gas chromatography/mass spectrometry for P4, 20beta-hydroxyprogesterone, pregnenolone, and allopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one). Treatment with PGF2alpha reduced serum concentrations of P4 as early as 1 h after injection (P<0.005) and steroid levels remained low over 24 h. Similarly, administration of PGF2alpha caused a decline in luteal P4 (P<0.005), 20beta-hydroxyprogesterone (P<0.10), and pregnenolone (P<0.05). In contrast, treatment with PGF(2alpha) caused an increase in luteal allopregnanolone over time (time x treatment interaction; P<0.05). These data are interpreted to suggest that PGF2alpha promotes conversion of P4 to the metabolite allopregnanolone.

Sexual partner preference, hypothalamic morphology and aromatase in rams.

The male-oriented ram is a unique and valuable animal model for the study of hormonal, developmental and genetic contributions to sexual partner preference. Unlike most other mammalian models that are in use currently, variations in sexual attraction occur spontaneously in domestic ram populations. It is estimated that as many as 8-10% of rams exhibit a sexual partner preference for other males, classifying them as male-oriented rams. Studies have failed to identify any compelling social factors that can predict or explain the variations in sexual partner preferences of rams. Nor is there consensus on the endocrine and sensory responsiveness of male-oriented rams to other rams. However, a number of studies have reported differences in brain structure and function between male-oriented and female-oriented rams, suggesting that sexual partner preferences are neurologically hard-wired. Recently, we identified a sexually dimorphic nucleus (oSDN) in the sheep preoptic area-anterior hypothalamus. The oSDN is larger in female-oriented rams than in male-oriented rams and similar in size in male-oriented rams and ewes. In addition, mRNA levels for aromatase in the oSDN were higher in males than in females and were higher in female-oriented rams than in male-oriented rams. These results suggest a relationship between steroid hormones, specifically estrogens and oSDN morphology. In this review, we provide an overview of sexual behavior in rams and discuss the multiple factors that may contribute to the development and adult expression of same-sex partner preferences in rams.

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus.

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.

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-2alpha (PGF-2alpha) has been shown to be a primary endogenous hormone responsible for triggering luteal secretion of oxytocin. Details are provided regarding the PGF-2alpha-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.

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.