Terry M. Nett

Pituitary effects of steroid hormones on secretion of follicle-stimulating hormone and luteinizing hormone

Steroid hormones have a profound influence on the secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These effects can occur as a result of steroid hormones modifying the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, or a direct effect of steroid hormones on gonadotropin secreting cells in the anterior pituitary gland. With respect to the latter, we have shown that estradiol increases pituitary sensitivity to GnRH by stimulating an increase in expression of the gene encoding the GnRH receptor. Since an estrogen response element (ERE) has not been identified in the GnRH receptor gene, this effect appears to be mediated by estradiol stimulating production of a yet to be identified factor that in turn enhances expression of the GnRH receptor gene. However, the importance of estradiol for enhancing pituitary sensitivity to GnRH during the periovulatory period is questioned because an increase in mRNA for the GnRH receptor precedes the pre-ovulatory rise in circulating concentrations of estradiol. In fact, it appears that the enhanced pituitary sensitivity during the periovulatory period may occur as a result of a decrease in concentrations of progesterone rather than due to an increase in concentrations of estradiol. Estradiol also is capable of altering secretion of FSH and LH in the absence of GnRH. In a recent study utilizing cultured pituitary cells from anestrous ewes, we demonstrated that estradiol induced a dose-dependent increase in secretion of LH, but resulted in a dose-dependent decrease in the secretion of FSH. We hypothesized that the discordant effects on secretion of LH and FSH might arise from estradiol altering the production of some of the intrapituitary factors involved in synthesis and secretion of FSH. To examine this hypothesis, we measured amounts of mRNA for activin B (a factor known to stimulate synthesis of FSH) and follistatin (an activin-binding protein). We found no change in the mRNA for follistatin after treatment of pituitary cells with estradiol, but noted a decrease in the amount of mRNA for activin B. Thus, the inhibitory effect of estradiol on secretion of FSH appears to be mediated by its ability to suppress the expression of the gene encoding activin.

Use of deslorelin short-term implants to induce ovulation in cycling mares during three consecutive estrous cycles

Abstract Alternatives to human chorionic gonadotropin (hCG) for inducing ovulation in cycling mares over several consecutive cycles were explored. Placebo, one, three or five short-term implants each containing 2.2 mg of gonadotropin-releasing hormone (GnRH) analogue (deslorelin) were administered to cycling mares after identification of a follicle over 30 mm. Mares were treated over three consecutive cycles, and artificially inseminated during the third cycle only. Serum was assayed for concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone during each cycle. All deslorelin doses decreased the interval to ovulation (4.0 days, 2.6 days, 2.4 days and 2.0 days), increased the proportion of mares ovulating within 48 h (20.0%, 83.3%, 73.3% and 85.7%), and decreased the diameter of the largest follicle at ovulation (45.8 mm, 38.0 mm, 41.0 mm and 37.2 mm) for the placebo, 2.2 mg, 6.6 mg and 11.0 mg groups, respectively ( P ). The interovulatory interval was lengthened in the 11.0 mg group compared with all other groups (21.2 days, 21.8 days, 26.4 days and 32.7 days for the placebo, 2.2 mg, 6.6 mg and 11.0 mg groups, respectively, P P > 0.05) were detected in serum concentrations of progesterone or pregnancy rate among the groups. Cycle number had no effect ( P > 0.05) on the reproductive parameters or serum concentrations of progesterone. Concentrations of LH were higher in Cycle 3 in the 6.6 mg and 11.0 mg groups, and FSH concentrations were decreased in Cycles 2 and 3 in treated mares. Deslorelin was efficacious for inducing ovulation in cycling mares with no diminished activity over three consecutive cycles. However, ovarian suppression and changes in serum concentrations of gonadotropins were noted at higher doses.

Binding characteristics of the ovine membrane progesterone receptor alpha and expression of the receptor during the estrous cycle.

BackgroundClassically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors and subsequent modulation of gene expression. However, there is increasing evidence for rapid, non-genomic effects of progesterone in a variety of mammalian tissues and it is possible that a membrane PR (mPR) is causing these events. We recently isolated and characterized an ovine mPR referred to as mPR-alpha, distinct from the nuclear PR. Based on predicted structural analysis, the ovine mPR-alpha possesses seven transmembrane domains typical of G protein-coupled receptors. Despite the homology to other reported mPRs, information pertaining to the steroid binding characteristics of the ovine mPR-alpha was lacking. Additionally, the ovine mPR-alpha transcript has been identified in the hypothalamus, pituitary, uterus, ovary and corpus luteum, yet changes in expression of the ovine mPR-alpha in these tissues were not known. Consequently, the purpose of this work was to determine the steroid binding characteristics of the ovine mPR-alpha and to investigate possible changes in expression of the ovine mPR-alpha in reproductive tissues throughout the estrous cycle.MethodsBinding studies were performed using crude membrane fractions from CHO cells expressing the mPR-alpha. Using quantitative Real-time PCR we determined the expression pattern of mRNA for the ovine mPR-alpha during the ovine estrous cycle in tissues known to express the mPR-alpha. Jugular blood samples were also collected and analyzed for serum concentrations of P4 to ensure ewes were at the appropriate stage of their cycle.ResultsOnly progesterone, 20alpha-hydroxyprogesterone and 17alpha-hydroxyprogesterone were able to displace binding of 3H-P4 (P < 0.001) to membrane fractions from CHO cells expressing ovine mPR-alpha. The average B-max and Kd values for three separate experiments were 624 +/- 119 fmol/micro gram protein and 122 +/- 50 nM, respectively. Significant changes in expression of mRNA for the mPR-alpha during the estrous cycle were noted in the corpus luteum and uterus.ConclusionThe mPR-alpha specifically binds progestins and its expression was correlated to progesterone secretion during the ovine estrous cycle. Results from the present studies suggest that mPR-alpha may have an important physiological role during the ovine estrous cycle.

Binding and Cytotoxicity of Conjugated and Recombinant Fusion Proteins Targeted to the Gonadotropin-Releasing Hormone Receptor

Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We and others have delivered this protein to various cell types, including cancer cells, using hormones to specifically target cells bearing the hormone receptor. Here, we compare binding and cytotoxicity of GnRH-PAP hormonotoxins prepared either by protein conjugation (GnRH-PAP conjugate) or through recombinant DNA technology (GnRH-PAP fusion). Although GnRH-PAP conjugate protein bound specifically to and caused cell death in cells bearing the gonadotropin-releasing hormone (GnRH) receptor, we could not detect binding or cytotoxicity using two different versions of the fusion protein in receptor-positive cells. We conclude that generation of an active GnRH-PAP fusion protein may not be feasible either because both ends of the GnRH molecule are required for receptor binding, but only the NH2 terminus is free in the fusion protein and/or that more potent analogues of GnRH (inclusion of which is not feasible in the fusion protein) are needed for efficient targeting. In contrast, the GnRH-PAP conjugate shows promise as a novel anticancer agent, capable of targeting cancer cells expressing the GnRH receptor such as prostate, breast, ovarian, endometrial, and pancreatic cells. It may also be useful as a therapeutic agent to eliminate pituitary gonadotrophs, eliminating the need for chronic GnRH analogue administration to treat hormone-sensitive diseases.

Measurement of equine prolactin with an equine-canine radioimmunoassay: seasonal effects on the prolactin response to thyrotropin releasing hormone

Abstract A radioimmunoassay (RIA) based on anti-equine prolactin antiserum and radioiodinated canine prolactin was used to assess the dose response of plasma prolactin to thyrotropin releasing hormone (TRH) in mares in the nonbreeding season (winter) and in mares in estrus in the breeding season (summer). Mares were administered TRH intravenously and blood samples were collected via jugular catheters at −15, 0, 15, 30, 45, 60, 90, 120, 180 and 240 min relative to injection. Doses of TRH were 0, .08, .40, 2.0 and 10.0 mg per mare (n = 3 per dose within each season). The prolactin response was assessed by absolute hormonal concentrations before and after TRH injection and by net area under the curve. Prolactin concentrations in plasma before injection of TRH were higher (P

A Nongenomic Action of Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone in Ovariectomized Ewes

Abstract The objective of the present study was to determine how rapidly estradiol (E2) was able to suppress the secretion of LH in ovariectomized (OVX) ewes and to evaluate the ability of conjugated forms of E2 (E2 conjugated to BSA [1,3,5(10)-estratrien-3,17β-diol-6-one-6-carboxymethyloxime:BSA [E2-BSA] and a novel conjugate, E2 conjugated to a small peptide [E2-PEP]) to mimic the actions of E2 on secretion of LH and FSH. Animals (n = 5–6 per group) were given infusions for 4 h of 50 μg of E2 or equimolar concentrations of E2-BSA or E2-PEP. Treatments with E2, E2-BSA, and E2-PEP each induced an acute suppression of LH secretion (<20 min, P < 0.01). In contrast, E2, but not E2-BSA or E2-PEP, induced the characteristic preovulatory-like surge of LH (at 10 h after priming treatment) and decreased secretion of FSH (at 4 h after priming treatment). In conclusion, the acute inhibition of LH secretion induced by E2 in OVX ewes supports the concept of a nongenomic action as the mechanism underlying the sudden suppression in secretion of LH. In addition, the fact that conjugated forms of E2 mimicked only the acute suppression of secretion of LH, without inducing the putative genomic actions on secretion of LH or FSH (i.e., a preovulatory-like surge), suggests that the acute effect of E2 may be mediated via the plasma membrane.

A Nongenomic Action of 17β-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone

Abstract The objective of the present study was to determine the ability of 17β-estradiol (E2) and conjugated forms of E2 (E2 conjugated to BSA [E2-BSA] and a novel conjugate, E2 conjugated to a small peptide [E2-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERα, also known as ESR1, and ERβ, also known as ESR2) in the mediation of the acute action of E2 in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E2, E2-BSA, or E2-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E2-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E2 and the E2-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERα agonist (propylpyrazole-triol), but not the ERβ agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E2 prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E2. The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERα. The fact that E2-BSA or E2-PEP mimicked the action of E2 suggests that this effect was mediated by an ER associated with the plasma membrane.

SDZ 200–110 Induces Leydig Cell Tumors By Increasing Gonadotropins in Rats

The administration of 62.5 mg/kg/day of SDZ 200–110, a calcium channel blocker, for 2 years increased the incidence of Leydig cell tumors while decreasing pituitary tumors in Sprague-Dawley rats. Lower doses did not change the incidence of these tumors. No other endocrine tumors were seen in rats or mice of either sex. A single gavage dose of 62.5 mg/kg/day decreased serum testosterone levels by 90% 4 hr after dosing. In vitro testosterone production by Leydig cells from these animals was minimally decreased, which suggests that a direct inhibition of steroid synthesis was removed during cell isolation. Dietary administration of the drug for 10 weeks did not significantly alter levels of serum hormones or testicular luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) receptors, although a significant elevation of testicular testosterone levels was seen. Increased serum levels of LH and follicle-stimulating hormone (FSH) were seen after 52 and 66 weeks, respectively, of dietary feeding of 62.5 mg/kg/day. The increase in serum LH was observed to week 104, while FSH levels returned to control levels by week 94. No effect on gonadotropin receptors was seen at the 6.25 mg/kg/day dosage. The age-related increase in serum prolactin was markedly reduced by 62.5 mg/kg/day of SDZ 200–110 in weeks 66 to 104 and to a lesser extent at the 6.25 mg/kg/day dosage. Testicular LH receptors were decreased by the high dose in animals sacrificed after 90–104 weeks. In conclusion, SDZ 200–110 increases the incidence of Leydig cell tumors by elevating levels of serum gonadotropins. The suggested mechanism for this increase in gonadotropins is a result of the effects of SDZ 200–110 on serum hormones and testicular LH receptors. The drug was judged not to pose a risk to humans since no change in gonadotropin levels was observed after chronic treatment.

GONADOTROPIN-RELEASING HORMONE AGONIST: A NEW APPROACH TO REVERSIBLE CONTRACEPTION IN FEMALE DEER

Fertility control offers a potential alternative for controlling an abundance of wild ungulate populations where lethal methods are infeasible or unacceptable. A promising nonsteroidal, nonimmunologic approach to reversible contraception consists of agonist of gonadotropinreleasing hormone (GnRH). We evaluated the effects of the GnRH agonist, leuprolide, on reproduction, the suppression of luteinizing hormone (LH) and progesterone, blood parameters, and reproductive behavior in captive female mule deer (Odocoileus hemionus) during December 1999 through June 2001. Leuprolide, administered as a controlled release formulation (ATRI-GEL®), was 100% effective in preventing pregnancy for one breeding season. Infertility was achieved by suppressing LH levels, which prevented ovulation and the formation of corpus luteum. Treated females regained normal ovarian function and conceived the following breeding season. Leuprolide had no adverse effects on blood chemistry and hematology, body weight dynamics, or the general health of treated females. In contrast to our predictions, leuprolide did not suppress estrous behavior in female deer during the “normal” breeding period, nor did treated females return to normal ovarian function and exhibit reproductive behaviors during the postbreeding period. This prolonged-release leuprolide formulation offers an alternative approach to reversible contraception in female deer that overcomes some of the problems associated with existing technology.

Thyroid stimulating hormone and prolactin secretion after thyrotropin releasing hormone administration to mares: Dose response during anestrus in winter and during estrus in summer

Abstract The response of thyroid stimulating hormone (TSH) and prolactin (PRL) concentrations to administration of thyrotropin releasing hormone (TRH) was determined in light-horse mares during the anestrous season (winter) and during estrus (standing heat) in the summer. Within each season, mares (4/group) were treated with either saline (controls) or one of four doses of TRH (80, 400, 2,000 or 10,000 ug) intravenously. Samples of blood were drawn at −15, −.5, 15, 30, 45, 60, 90, 120, 180 and 240 min relative to TRH injection. Concentrations of TSH and PRL in pre-TRH samples were greater (P .10) between reproductive state and TRH dose for TSH concentrations. Concentrations of PRL were not significantly affected by any TRH dose during either season. It appears that mares differ from many mammalian species in that they do not respond to an injection of TRH with increases in both TSH and PRL.