Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.     

Regulation of c-fos expression by EGF and GnRH in specific anterior pituitary cells from proestrous female rats

C-fos is an early expression oncogene that can be stimulated by a variety of regulators. It is expressed by subsets of all pituitary cells, with increased expression seen in proestrous rats. However, in freshly dispersed pituitary cells studied during different stages of the cycle, there is limited expression of fos by luteinizing hormone (LH) cells and little basal expression by cells with follicle-stimulating hormone (FSH) antigens. Proestrus is a time during which pituitary gonadotropes express peak levels of receptors for gonadotropin-releasing hormone (GnRH) and epidermal growth factor (EGF). We hypothesized that if GnRH or EGF stimulated fos activity in gonadotropes they would be most effective during the peak expression of their receptors. Anterior pituitaries were removed, cut into small pieces, and stimulated for 30 min. Total RNA was then collected and analyzed by Northern analysis. Both EGF and GnRH caused an increase in c-fos mRNA levels in the anterior pituitary gland compared with unstimulated pituitary glands assayed immediately after removal from the pituitary. However, the stimulatory effects were no greater than those seen with medium alone. This suggested that fos expression could be stimulated by local factors either in the pituitary or the medium itself. The second phase of the study focused on pituitary cells plated for 1 hr and then stimulated with EGF and GnRH for 15 min. Dual immunocytochemistry was done to learn which cell types expressed the fos proteins. After 15 min, EGF and GnRH both increased the percentages of fos-bearing cells above levels seen in medium alone. EGF stimulated fos proteins in subsets of FSH, adrenocorticotropin (ACTH), and growth hormone (GH) cells. GnRH increased fos proteins in subsets of ACTH and GH cells. These results suggest that EGF and GnRH may regulate fos expression, but not necessarily in gonadotropes. They also highlight the need for carefully timed experiments because endogenous factors in the pituitary itself may stimulate immediate early gene expression. (J Histochem Cytochem 46:935-943, 1998)     

Identification of three putative GnRH receptor subtypes in vertebrates

The majority of vertebrates have two or three forms of gonadotropin-releasing hormone (GnRH), which appear to have arisen by successive gene duplication events. This suggests the possibility of concordant gene duplications of the GnRH receptor to produce two or more cognate receptors. Since the extracellular loop 3 (EC3) domain of mammalian GnRH receptors plays a role in distinguishing the different forms of GnRH, we have contemplated that the sequence of this domain will differ significantly in the putative cognate receptors. Degenerate oliognucleotides encoding the sequences of the transmembrane domains preceding and following EC3 were used for PCR amplification of genomic DNA from zebrafish (Brachydanio rerio), goldfish (Carassius auratus), African clawed frog (Xenopus laevis), chicken (Gallus domesticus), and lizard (Agama atra). Isolation and sequencing of specific clones revealed that they fell into three groups. Two of these were most similar to the mammalian pituitary GnRH receptor and were therefore designated Type IA and Type IB. The third form (designated Type II) was most different from the others and was identified in Xenopus, lizard, and human DNA. These findings support the concept of the existence of three distinct GnRH receptors, which have evolved in conjunction with three distinct GnRH ligand classes present in many vertebrates.     

Differential signal transduction by five splice variants of the PACAP receptor

The two forms of pituitary adenylyl cyclase-activating polypeptide (PACAP-27 and -38) are neuropeptides of the secretin/glucagon/vasoactive intestinal polypeptide/growth-hormone-releasing hormone family and regulate hormone release from the pituitary and adrenal gland. They may also be involved in spermatogenesis, and PACAP-38 potently stimulates neuritogenesis and survival of cultured rat sympathetic neuroblast and promotes neurite outgrowth of PC-12 cells. The PACAP type-I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland and testes), specific for PACAP, is positively coupled to adenylyl cyclase and phospholipase C. The recently cloned type II receptor does not discriminate between PACAP and vasoactive intestinal polypeptide and is coupled to only adenylyl cyclase. Here we have used a new expression cloning strategy, based on the induction of a reporter gene by cyclic AMP, to isolate a complementary DNA encoding the type-I PACAP receptor. On transfection of this cDNA, both PACAP-27 and -38 stimulate adenylyl cyclase with similar EC50 values (50% effective concentration, 0.1-0.4 nM), whereas only PACAP-38 stimulates phospholipase C with high potency (EC50 = 15 nM). Four other splice variants were isolated with insertions at the C-terminal end of the third intracellular loop. Expression of these cDNAs revealed altered patterns of adenylyl cyclase and phospholipase C stimulation, suggesting a novel mechanism for fine tuning of signal transduction.     

Endocrine therapy of transsexualism and potential complications of long-term treatment

Physiological principles of the interrelationship of sex hormones and their regulation are the foundation of understanding appropriate treatment of the transsexual patient. While both genetic males and females have estrogens and androgens, the quantitative sex hormone production is genetically predetermined by sex hormone production both in the gonads and via peripheral conversion of hormone precursors to sex steroids. Sex hormones exert a negative feedback on the hypothalamus and pituitary gland whereby gonadotropin-releasing hormone (GnRH), pituitary luteinizing hormone (LH), and follicle-stimulating hormone (FSH) are regulated or suppressed by the endogenous levels of these hormones. Sex hormonal therapy induces attenuated GnRH stimulation of LH and FSH causing a reduction of serum sex hormone levels. It is clear that estrogen as well as androgen therapy have a dual role: (i) induction of feminization or virilization and (ii) suppression of the hypothalamic-pituitary-gonadal axis leading to a reduction of endogenous estradiol or testosterone secretion. Cross-sex hormonal treatment may have substantial medical side effects. The smallest dosage of hormonal therapy compatible with the above clinical aims should be used.     

Phenomenon of intrinsic rhythm of luteinizing hormone release from isolated anterior pituitary gland of female rat

The intrinsic nature of rthymic release of luteinizing hormone (LH) of isolated human and rat anterior pituitary gland reported independently by Macro Gambacciani and Xie in 1987 can be more directly demonstrated by a computer programme of Time Series-HSY Hidden Periodic Analytic Approach for continuous monitoring the LH output of the perfusate from a perfusion system with in vitro anterior pituitary of SD female rat. The results are as follows: (1) Under various reproductive conditions the average frequency (min/cycle) and amplitude (ng/ml) of the intrinsic rhythm of LH release were quite different: In proestrous group the frequency and amplitude were the highest, being intermediate in the ovariectomized group and lowest in the lactation group. (2) The intrinsic rhythm of LH release could be changed by either peptide or steroid hormones. In proestrous group with 30 min of gonadotropin-releasing hormone (GnRH), stimulation would reduce both frequency and amplitude. In case of lactation, the frequency was unchanged, but amplitude lowered, while in the ovariectomized rat pituitary, the 30 min GnRH stimulation decreased the frequency of release only. The intrinsic rhythm of the LH release could also be influenced by steriod hormones (Ru486 and Anordrin). With 120 min before removal of the anterior pituitary gland the rats receiving i.m. injection of Ru486 (2 mg/kg bw) or Anordrin (2 mg/kg), the results showed that Ru486 decreased frequency, while Anordrin decreased only the frequency to a less extent, both without amplitude affected. (3) Verapamil and EGTA added to the perfusion system did not abolish but only decreased the rhythmic phenomenon by using proestrous pitutary. This suggests that participation of Ca2+ may take place in the intrinsic release of LH. The above results indicated that the intrinsic rhythm of LH release of isolated anterior pituitary gland is different from various reproductive hormonal conditions and capable of being modified by exogenous hormones. The physiological function of the intrinsic rhythm of LH release of anterior pituitary gland remains to be elucidated.     

Normal reproductive neuroendocrinology in the female

The reproductive axis in women comprises a number of components that must function in a highly orchestrated manner for reproductive potential to be optimal. The neuroendocrine components of this axis, including the hypothalamus and the pituitary gland, are central to this system. Within the hypothalamus, the specialized neuronal system responsible for synthesizing and secreting gonadotropin-releasing hormone (GnRH) is itself modulated by a number of peptide and biogenic amine neurotransmitters that mediate feedback signals of ovarian origin. The luteinizing hormone and follicle-stimulating hormone secreting anterior pituitary gonadotropes perceive and transduce neural input in the form of GnRH, but are themselves also modulated by the ambient gonadal hormone concentrations. The authors review the physiologic relevance of the pulsatile nature of the GnRH signal, and some proposed mechanisms through which these signals are stimulated and modulated and subsequently perceived and transduced by gonadotropes.     

Neuropeptide-Y stimulates growth hormone and gonadotropin-II secretion in the goldfish pituitary: involvement of both presynaptic and pituitary cell actions

We have previously reported that neuropeptide-Y (NPY) stimulates GH and gonadotropin-II (GtH-II) release from perifused pituitary fragments in the goldfish. Since the teleost pituitary is directly innervated by neurosecretory terminals from the brain, we further investigated the possible sites of action of NPY. Both synthetic human NPY and NPY-(18-36), an agonist selective for the NPY Y2-receptor, stimulated GH and GtH-II release from the pituitary fragments; the magnitude of the response to NPY (18-36) was smaller than that to the whole molecule of NPY. NPY also stimulated the release of GH and GtH-II from perifused dispersed pituitary cells. In contrast, NPY-(18-36) had no effect on either GH or GtH-II release from dispersed pituitary cells. These data suggest that Y2 action is not direct at the level of pituitary cells, but may be indirect through actions on nerve terminals in the pituitary. The hypothesis that the action of NPY on GH and GtH-II release is mediated in part by GnRH was then tested. Both NPY and NPY-(18-36) stimulated the GnRH release from preoptic-anterior hypothalamic slices and pituitary fragments with similar potency. Furthermore, a GnRH antagonist significantly reduced the effects of NPY on both GH and GtH-II release in perifused pituitary fragments. Similar to previous findings, NPY, when given at 55-min intervals, desensitized the hormone responses in pituitary fragments. Similarly, the same treatment with NPY in perifused dispersed pituitary cells induced desensitization of GH and GtH-II responses. Together, these results suggest that 1) more than one type of NPY receptors are present in the goldfish pituitary; and 2) NPY has at least two sites of action in the pituitary. One site of action is the pituitary cells, where NPY directly stimulates GH and GtH-II secretion; the second is the nerve terminals, where NPY presynaptically stimulates GnRH release via Y2-like receptors, and GnRH, in turn, stimulates GH and GtH-II release.     

Balloon expandable stents for systemic venous pathway stenosis late after Mustard''s operation

Gonadotropin-releasing hormone (GnRH) receptor expression is regulated by estradiol and GnRH itself. The objective of this experiment was to determine the extent to which low levels of estradiol, similar to those observed during the transition from the luteal to the follicular phase of the estrous cycle, and GnRH interact to regulate expression of GnRH receptors and GnRH receptor mRNA. Ewes were ovariectomized (OVX) at least 2 wk prior to initiation of the experiment, and the pituitary gland was surgically disconnected from the hypothalamus to remove ovarian and hypothalamic inputs to the pituitary. Within 24 h after hypothalamic-pituitary disconnection, ewes received pulses of GnRH (250 ng/pulse) every 2 h for 6 d. At the end of 6 d, ewes were randomly assigned to treatments in a 2 x 2 factorial arrangement as follows: half of the animals received a single estradiol implant and half received an empty implant (placebo). At the same time, animals also received one of the following treatments: (1) saline or (2) GnRH (100 ng/pulse/2 h). Additionally, one group of ewes was ovariectomized, but not subjected to hypothalamic-pituitary disconnection (OVX controls). Blood samples were collected 15 min prior to each pulse of GnRH or saline and at 15-min intervals for 1 h after each pulse until tissues were collected and concentrations of luteinizing hormone (LH) were determined. Anterior pituitaries were collected 24 h after implant insertion to quantitate steady-state amounts of GnRH receptor mRNA and numbers of GnRH receptors. Mean LH was greatest in ovariectomized control ewes compared to all other treatments (p < 0.05). Mean LH and LH pulse amplitude in the placebo and GnRH-treated group most closely mimicked LH secretion in ovariectomized control animals. Mean LH and LH pulse amplitude were similar between both GnRH-treated groups (p < 0.05). Mean LH and LH pulse amplitude were significantly lower in all animals treated with saline compared to OVX controls (p < 0.05). Treatment with an estradiol implant and pulsatile GnRH increased (p < 0.05) relative amounts of GnRH receptor mRNA and the number of GnRH receptors compared to all other treatments. There were no differences in GnRH receptor expression between the remaining treatment groups (p > 0.05). Therefore, in OVX ewes after hypothalamic-pituitary disconnection, low levels of estradiol and GnRH are required to increase GnRH receptor mRNA and GnRH receptor numbers. Since we only observed an increase in GnRH receptor expression in the presence of both estradiol and GnRH, we conclude that there is a synergistic interaction between these two hormones in the regulation of GnRH receptor expression.     

Modulation of the action of gonadotrophin surge-attenuating factor by gonadotrophin-releasing hormone

Gonadotrophin surge-attenuating factor (GnSAF) is a putative non-steroidal ovarian factor which attenuates the luteinizing hormone (LH) surge in superovulated women through the reduction of the pituitary response to gonadotrophin-releasing hormone (GnRH). The mechanism of action of GnSAF on gonadotrophin secretion was further studied by investigating six normally ovulating women in two cycles--a spontaneous and a follicle-stimulating hormone (FSH)-treated cycle. The response of the pituitary to five consecutive pulses of GnRH was investigated in late follicular phase (follicle size 15 mm) of both cycles. GnRH pulses, 10 micrograms each, were injected i.v. every 2 h and LH was measured in blood samples taken before and 30, 60 and 120 min after each pulse. FSH was injected daily at the fixed dose of 225 IU starting on cycle day 2. Peak values of LH increment occurred 30 min after each pulse. However, maximal LH increment occurred in both cycles after the second GnRH dose. In the FSH cycles the response of LH to the first three pulses was significantly attenuated compared with the spontaneous cycles, while the response to the fourth and fifth pulses was similar in the two cycles. In both cycles, LH increment 30 min post GnRH (net increase above the previous value) was similar after the fourth and fifth pulses. Serum concentrations of oestradiol and immunoreactive inhibin, although higher in the FSH cycles, remained stable throughout the GnRH experimental period in both cycles. These results demonstrate that multiple submaximal doses of GnRH can override the attenuating effect of GnSAF on LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systematic relationships among pocket gopher chewing lice (Phthiraptera: Trichodectidae) inferred from electrophoretic data

This study was designed to explore the efficacy of gonadotrophin-releasing hormone (GnRH) to antagonize the effect of gonadotrophin surge-inhibiting factor (GnSIF) on the timing of the induction by GnRH of the maximal self-priming effect on pituitary LH responsiveness. The GnSIF levels were increased by FSH treatment and reduced after gonadectomy. Female rats were injected s.c. with 10 IU FSH or saline (control) on three occasions during the 4-day cycle. Serial i.v. injections of GnRH (500 pmol/kg body weight) were administered to intact rats on the afternoon of pro-oestrus or 15-30 min after ovariectomy. Intact male rats were given 10 IU FSH and 500 or 2000 pmol GnRH/kg body weight on an equivalent time-schedule. Endogenous GnRH release was suppressed with phenobarbital. In intact female control rats, the timing of the maximally primed LH response was delayed as the GnRH pulse-interval increased. FSH treatment of female rats induced a suppression of the initial unprimed LH response and delayed the maximally primed LH response, which showed further delay as the GnRH pulse-interval was increased. When the pulsatile administration of GnRH was started 15-30 min after ovariectomy, the priming effect of GnRH did not change as the GnRH pulse-interval was increased in the saline-treated rats. However, FSH treatment caused a suppression of the unprimed LH response, a delay in the primed LH response and decreased the delay of the maximally primed LH response to GnRH when the GnRH pulse-interval was decreased. Increasing the interval between ovariectomy and the first GnRH pulse to 4 h diminished the efficacy of the FSH treatment: GnRH-induced priming was delayed by only one pulse instead of the two pulses in control rats. In intact males but not in orchidectomized rats, a self-priming effect was demonstrated during GnRH pulses which were 1 h apart. The effect of 2 nmol GnRH/kg body weight was the most pronounced. Compared with intact female rats, the timing of the maximally primed LH response was delayed by 1 h. FSH treatment did not affect the pituitary LH response to both dose levels of GnRH. It is concluded that FSH treatment increased the release of GnSIF by the ovary, then induced a state of low responsiveness of the pituitary gland to GnRH and subsequently delayed GnRH-induced maximal self-priming. The efficacy of GnRH to prime the pituitary gland was higher when GnSIF levels were decreasing after removal of the ovaries.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.     

PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland

Pituitary adenylate cyclase activating polypeptide (PACAP) and its close relative vasoactive intestinal polypeptide (VIP) were demonstrated in the anterior pituitary gland. The cells which exhibited PACAP immunoreactivity were oval or round shaped. Their distribution was similar to that of gonadotropes but the number of PACAP immunoreactive cells was less. Double labeling revealed that PACAP immunoreactivity partially colocalized with luteinizing and follicle-stimulating hormone; however, colocalization with other pituitary hormone immunoreactivities was not demonstrated. Our results suggest an autocrine or paracrine role of PACAP in the regulation of pituitary functions.     

The use of long- and short-acting forms of gonadotrophin-releasing hormone analogues in women undergoing oocyte donation

Evidence accumulated in in-vitro fertilization (IVF) cycles suggests that the use of long-acting forms of gonadotrophin-releasing hormone analogues (GnRHa) for pituitary desensitization may impair the outcome of IVF as compared to classical short-acting formulations. Whether the negative effects are directed against the corpus luteum, the endometrium, or both is unknown. However, the presence of high affinity binding sites for gonadotrophin-releasing hormone (GnRH) in the human endometrium suggests a possible role of these analogues on this target organ, affecting embryo implantation. In the present study, we tested direct effects of two different forms of GnRHa on implantation using the ovum donation model. Patients were prospectively allocated to one of the three study groups: the short-acting form of the analogue leuprolide acetate (group I; n=64), the long-acting form of the same analogue (group II; n=58), and the long-acting preparation of the analogue tryptorelin (group III; n=61). A total of 68 cycles of embryo transfer was carried out in group I, whereas 67 were performed in group II and 65 in group III. Cancellation rates were respectively 18.1, 17.3 and 18.8% because of bleeding while being on the waiting list for anonymous oocyte donation. The number of oocytes donated, fertilization rates and embryos replaced in each group were similar. As a result, pregnancy rate per transfer was 38.2, 49.3 and 44.6% respectively. Implantation rates per embryo replaced were respectively 13.4, 19.1 and 17.0%. These data suggest that the use of a long-acting form of GnRHa provides success rates similar to the short-acting preparations, resulting in more convenient medication for patients with ovarian function included in ovum donation programmes.     

Regulation of gonadotropin-releasing hormone (GnRH) receptor gene expression in sheep: interaction of GnRH and estradiol

GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 microg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 microg i.m.) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone. Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHbeta and FSHbeta subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHbeta subunit (P < 0.005) and FSHbeta subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHbeta subunit mRNA (P < 0.01) but did not affect amounts of LHbeta subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHbeta subunit (P < 0.01) and FSHbeta subunit (P < 0.005). From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell's ability to respond to estradiol.     

The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay

Human gonadotrophins undergo metabolic transformations which result in the presence of several smaller, structurally and immunologically related forms of gonadotrophins in the urine. For luteinizing hormone (LH), a beta core fragment (LHbeta cf) has been isolated from the pituitary and characterized. The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physicochemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of LHbeta cf have a different structure, probably in the carbohydrate moieties. This communication characterizes the expression of LHbeta cf in the urine of both reproductive and post-reproductive age women and in men, employing assays highly specific for the pituitary form of the fragment. It was found that LHbeta cf is the predominant LH associated molecular form in the urine during peri-ovulatory period, peaking 1-3 days later than intact LH and reaching a concentration of approximately 600 fmol/mg creatinine, 7-fold higher than either LH or LH free beta subunit. Corresponding concentrations of human chorionic gonadotrophin (HCG) beta cf were <1% that of LHbeta cf. LHbeta cf cross-reaction with some LH or LHbeta monoclonal antibodies may well interfere with the accurate estimation of the day of the LH surge when urinary tests are utilized.     

A transient effect of estrogen on calcium currents and electrophysiological responses to gonadotropin-releasing hormone in ovine gonadotropes

Episodic release of luteinizing hormone (LH) by the pituitary gland is controlled by hypothalamic gonadotropin-releasing hormone (GnRH). In the period leading up to the preovulatory surge of LH, estrogen increases the number of pituitary receptors for GnRH and sensitises the gonadotropes to GnRH. The postreceptor events that are responsible for the increase in responsiveness to GnRH are not clearly delineated, but LH release is known to be Ca2+ dependent. The present study addressed the question as to whether or not estrogen may act to modify voltage-dependent Ca2+ entry in normal gonadotropes. Primary cultures enriched in gonadotropes or somatotropes were produced from anestrous female sheep. Conventional whole-cell patch-clamp recording was used to measure inward membrane current in the absence of GnRH treatment, with and without 10 nM estradiol-17beta (E2) treatment for 0 to 36 h. Nystatin-perforated whole-cell patch-clamp recording was used to record membrane voltage responses to GnRH. Ca2+ current density (ICa, pA/pF) began to increase after 2 h exposure to E2, and reached peak values of about 200% of control by 16-20 h (p < 0.005), then declined. If E2 was withdrawn at 24 h, ICa returned towards control values by 36 h. If E2 treatment was continued beyond 24 h, however, ICa fell to about 75% of control by 36 h (p < 0.005). Actinomycin D prevented the enhancement of ICa. E2 was without effect on Na+ current density in gonadotropes, or on ICa in somatotropes. The proportion of ICa carried by L-type and N-type channels in gondadotropes was not changed by E2. Ovine gonadotropes respond to GnRH with membrane potential fluctuations driven by periodic activation of Ca2+-dependent K+ channels, and synchronised action potential generation. This response was found to be sensitive to E2. Responses were categorised according to the pattern of activity evoked by 10 nM GnRH. Without E2 treatment, 11/14 cells responded with oscillations and 3/14 cells responded with spiking (hyperpolarizations following single action potentials). After 20 h 10 nM E2, just 1/14 cells responded with hyperpolarizing oscillations while 13/14 cells showed spiking activity. The predominance of the spiking pattern in E2-treated cells is consistent with the increased Ca2+ flux, and with enhanced LH release. We conclude that E2 has a transient effect on gonadotropes to enhance voltage-gated Ca2+ channel function. The time-course and biphasic nature of the influence of E2 on ICa may be physiologically appropriate to the preovulatory LH surge. Enhanced Ca2+ influx may participate in increased Ca2+-dependent hormone release, while the delayed inhibitory action of E2 on ICa may serve to limit the duration of the surge.     

Short-term regulation of gonadotropin subunit mRNA levels by estrogen: studies in the hypothalamo-pituitary intact and hypothalamo-pituitary disconnected ewe

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone beta (LHbeta), follicle stimulating hormone beta (FSHbeta) and the common alpha-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 micrograms of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary alpha-subunit and FSHbeta mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHbeta mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injections and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHbeta subunit mRNA seen after estrogen injection of OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHbeta mRNA are reflected in changes in FSH secretion; the same is not true for LH.     

Age-dependent effects of hippocampal muscarinic receptor blockade on memory-based learning in the developing rat

In the male rat, testosterone has been shown to regulate gonadotrophin synthesis and secretion under experimental conditions such as castration or gonadotrophin-releasing hormone (GnRH) antagonist with or without testosterone. The present study aims at clarifying the effects of non-steroidal antiandrogens, Casodex and flutamide, and ethane dimethane sulphonate (EDS) on the regulation of gonadotropin synthesis and secretion. To enable a direct comparison within this study to expected effects of testosterone, a GnRH antagonist-treated group and a castrated group were included. The gene expression of the subunits was correlated with changes in the pituitary and plasma content of immunoreactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH), free subunits and pituitary content of in vitro bioactive LH and FSH. Groups of ten male rats each received the following treatments for 7 days: (1) vehicle; (2) castration; (3) EDS (75 mg/kg); (4) GnRH antagonist (Cetrorelix 250 micrograms/kg/day), (5) Casodex (20 mg/kg/day) or (6) flutamide (20 mg/kg/day). The effectiveness of testosterone deprivation was demonstrated by the reduction of weight in androgen-dependent organs such as epididymides and seminal vesicles in the treated groups. Treatment with flutamide, EDS or castration significantly increased (p < 0.05) serum levels of LH, FSH and alpha-subunit, whereas serum gonadotrophin levels were decreased in the GnRH antagonist-treated group. alpha-Subunit mRNA levels were elevated in the castrated, EDS and flutamide group and LH-beta mRNA levels were increased in the castrated and EDS group. FSH-beta mRNA levels were increased in the castrated group and decreased in the GnRH antagonist group, but remained unchanged in the flutamide and EDS group.(ABSTRACT TRUNCATED AT 250 WORDS)