Fredrick Stormshak

Decreased Progesterone Levels and Progesterone Receptor Antagonists Promote Apoptotic Cell Death in Bovine Luteal Cells

Abstract We tested the hypothesis that progesterone (P4) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) ± P4 (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P4 levels by RIA. Aminoglutethimide inhibited P4 synthesis by > 95% and increased the level of apoptosis as evidenced by 32P-labeled oligonucleosomal DNA fragmentation (> 40%). P4 supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P4 has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P4 was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in 32P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P ≥ 0.05) in P4 levels after treatment with PR antagonists. These observations support the concept that P4 represses the onset of apoptosis in the CL by a PR-dependent mechanism.

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.

Apparent role of melatonin and prolactin in initiating winter fur growth in mink.

A study was conducted to determine the effects of exogenous melatonin and bromocriptine (CB-154), an inhibitor of prolactin synthesis and secretion, on the induction of winter fur growth in mink. Melatonin (10 and 120 mg) was administered to mink (N = 5/group) via silastic implants inserted sc over the scapular area during the last week of June 1985. Treatment of mink (N = 5) with CB-154 alone or in combination with 10 mg melatonin (N = 5) consisted of daily sc injections of 2 mg of the drug in sterile saline from June 25 through July 30. Control animals (N = 5) did not receive injections of vehicle or sham implants. Administration of CB-154 alone or in combination with 10 mg melatonin, as well as 120 mg melatonin alone, initiated growth of the winter fur significantly earlier than that of controls or mink treated with 10 mg melatonin (P less than 0.05). These data suggest that inhibition of prolactin secretion by melatonin is requisite for induction of molt of summer fur and growth of winter fur of mink.

Nongenomic Inhibition of Oxytocin Binding by Progesterone in the Ovine Uterus

Abstract 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-17β (2 days) and P4 (5 days) before being treated with estradiol-17β 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 × 10−9 and 1.74 × 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-17β, 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.

Distribution and regulation of aromatase activity in the ram hypothalamus and amygdala

Conversion of androgen to estrogen within the brain by cytochrome P450 aromatase is a component of the system controlling the display of normal male reproductive behavior and negative feedback inhibition of LH secretion in rams. In the present study, we used the highly sensitive 3H2O assay to measure aromatase activity in microdissected regions of the basal diencephalon and amygdala of intact and castrated rams. We found that aromatase activity was heterogeneously distributed. The highest activity was found in the medial amygdala, cortical amygdala, and bed nucleus of the stria terminalis. Intermediate levels of aromatase activity were found within the medial preoptic area/anterior hypothalamus, periventricular preoptic area, lateral preoptic area/anterior hypothalamus, ventromedial hypothalamus, and lateral hypothalamus. The lowest activity was present in the septum, infundibulum/median eminence, and dorsal medial hypothalamus. After castration, aromatase was significantly reduced in the medial preoptic area/anterior hypothalamus, periventricular preoptic area, lateral preoptic area/anterior hypothalamus, and infundibular nucleus/median eminence. In contrast, levels in castrate rams were unchanged in several other regions, most notably the bed nucleus of the stria terminalis and medial and cortical amygdala. These results provide a quantitative profile of aromatase activity in discrete regions of the ram hypothalamus and limbic system. They also demonstrate that aromatase activity is reduced in some, but not all brain regions after castration which suggests that different regulatory mechanisms control aromatase within different neuronal populations of the ram.

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.

The effects of photoperiod and melatonin on serum prolactin levels of mink during the autumn molt

An experiment was conducted to determine the effects of a reduced daily photoperiod and exogenous melatonin on serum prolactin levels of mink during the autumn molt and growth of the winter pelage. During the last week of June, adult standard dark female mink (Mustela vison) were exposed to natural changes in daylength (controls), a reduced photoperiod of 6 h light: 18 h dark (6L: 18D) or exposure to natural changes in daylength and treated with melatonin (10 mg) in a Silastic implant inserted subcutaneously over the scapular area. Beginning July 2, and continuing through October 22, blood samples were collected at nine biweekly intervals, and serum prolactin concentrations were quantified by a heterologous double antibody radioimmunoassay. Both reduced photoperiod and exogenous melatonin caused serum prolactin levels to decline rapidly after mid‐July, resulting in concentrations that were significantly lower than those of controls 6 to 8 wk earlier. These data suggest that growth of the winter pelage of mink is strongly associated with declining prolactin levels. It appears that part of the photoperiodic‐induced effects on fur growth of the mink are mediated through melatonin and its effects on prolactin synthesis and/or secretion.

Estrogen Synthesis in Fetal Sheep Brain: Effect of Maternal Treatment with an Aromatase Inhibitor

Abstract 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 (Km) 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.

Comparative aspects of the regulation of corpus luteum function in various species.

Central to understanding the pivotal role of the corpus luteum in governing reproductive cycles of mammals has been the study of those factors that control the function of this gland. Research on control of luteal function has encompassed a broad spectrum of mammalian taxa and has evolved from early studies to identify the source and nature of controlling factors to present day attempts to resolve their action at the level of the luteal cell. From this research data have emerged leading to the realization that among mammals a diversity of factors regulate the function of the corpus luteum. The fact that function of the corpus luteum, in most mammals, is influenced by hormones from several sources, namely the pituitary gland, uterus and placenta, makes this organ truly unique among endocrine glands. As the student of the corpus luteum is well aware, some of these hormones are involved in promoting steroidogenesis and(or) prolonging the life span of the corpus luteum while others serve to provoke its demise. In recent years several excellent reviews have appeared that discuss differences among mammals in regard to the hormonal regulation of luteal function (Rothchild, 1981; Keyes et al., 1983; Niswender et al., 1985; Khan-Dawood and Dawood, 1986). Consequently, certain portions of the present treatise on luteal function may appear to be redundant. Although old ground may be trod upon once again, an effort will be made to present new information.

Prolactin binding sites in the uterus of the mink

The present study was conducted to determine if specific binding sites for prolactin (PRL) are present in the uterus of the mink. Uteri of anestrous mink were homogenized and subjected to differential centrifugation into three particulate fractions, 1500, 15 000 and 50 000 X g. Binding of [125I]oPRL to membranes in an aliquot (200-400 micrograms protein) of the 50 000 X g particulate fraction was quantified. Time and temperature for optimal binding were 18 h at 25 degrees C. Scatchard plot analysis revealed a single set of binding sites for PRL with a Kd of 8.25 X 10(-11) +/- 0.68 M. The maximum amount of [125I]oPRL bound was 28 fmoles/mg protein. Prolactin binding sites were detected in both the uterus and kidney of mink, but not in skeletal muscle, spleen, diaphragm or lung. These data indicate that uterine cell membranes of the mink contain sites that bind prolactin with high affinity.