Negative Feedback Effect Of Estradiol Research Articles

Differential regulation of Kiss1 gene expression by oestradiol in the hypothalamus of the female Damaraland mole-rat, an induced ovulator

Kisspeptin, a product of the Kiss1 gene is considered a potent stimulator of gonadotropin release, by interacting with its receptor, the G protein-coupled receptor 54. Kiss1 neurons are known to mediate the positive and negative feedback effects of oestradiol on GnRH neurons that control the pulsatile and surge secretion of GnRH. While in spontaneously ovulating mammals the GnRH/LH surge is initiated by a rise in ovarian oestradiol secreted from maturing follicles, in induced ovulators, the primary trigger is the mating stimulus. Damaraland mole rats (Fukomys damarensis) are cooperatively breeding, subterranean rodents that exhibit induced ovulation. We have previously described in this species the distribution and differential expression pattern of Kiss1-expressing neurons in the hypothalamus of males and females. Here we examine whether oestradiol (E2) regulates the hypothalamic Kiss1 expression in a similar way as described for spontaneously ovulating rodent species. By means of in situ hybridisation, we measured Kiss1 mRNA among groups of ovary-intact, ovariectomized (OVX) and OVX females treated with E2 (OVX + E2). In the arcuate nucleus (ARC), Kiss1 expression increased after ovariectomy and decreased with E2 treatment. In the preoptic region, Kiss1 expression after gonadectomy was similar to the level of wild-caught gonad-intact controls, but was dramatically upregulated with E2 treatment. The data suggest that, similar to other species, Kiss1 neurons in the ARC, which are inhibited by E2, play a role in the negative feedback control on GnRH release. The exact role of the Kiss1 neuron population in the preoptic region, which is stimulated by E2, remains to be determined.

The differential secretion of FSH and LH: regulation through genes, feedback and packaging

While the role of oestradiol and progesterone in the control of GnRH pulsatile secretion and generation of the preovulatory GnRH surge to induce release of the LH surge has been fully investigated, less attention has been given to changes in the pituitary gland that may sensitize gonadotrophs to switch from pulsatile release to surge release of LH, in particular. Furthermore, in the follicular phase while pulsatile secretion of LH is maximal, FSH secretion is reduced, yet both hormones are produced by the same gonadotrophs. The mechanisms whereby this differential release can occur are still unclear. The main regulator of FSH secretion is through the negative feedback effects of oestradiol and inhibin, which directly affect FSHbeta mRNA content and subsequent synthesis of FSH. FSH is then released predominantly via a constitutive pathway and the amount released is closely related to the rate of synthesis. In contrast, while basal LH secretion occurs via a constitutive pathway, the principal release of LH through pulsatile secretion is through the regulated pathway with GnRH stimulating the release of pre-synthesized LH contained in storage granules without significant changes in LHbeta mRNA. Secretogranin II (SgII) is associated with LH in these electron-dense storage granules and LH-SgII granules appear to be the principal form of granule released in response to GnRH through the regulated pathway. At the time of the preovulatory LH surge, granule movement to the gonadotrope cell membrane abutting a capillary, polarization, appears to play an important part in the priming mechanism for release of LH during the preovulatory LH surge in response to the GnRH surge. As there appears to be limited or no gonadotroph cell division in the adult pituitary gland, each gonadotroph passes through this synthesis and secretion pathway repeatedly through successive oestrous cycles. Packaging of LH and FSH into different secretory granules within the same cell is thus pivotal for the differential secretion of these gonadotrophins.

Physiology of reproductive seasonality in sheep – an update

ABSTRACTThe seasonal breeding in sheep poses a challenge in improving productivity, owing to the incomplete knowledge of the mechanics of seasonal breeding, effective strategies for increasing the lamb crop are restricted. A major mechanism responsible for seasonal reproduction is a striking increase in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol that peaks during anestrous and decreases in the breeding season. Melatonin does not directly drive seasonal changes in reproduction; rather it appears to synchronize an endogenous circannual rhythm. The Pars Tuberalis (PT) is a single common region across species that shows strongest MT1 expression. Melatonin acts through high affinity, G-protein coupled receptors located in thyrotroph cells in PT of the pituitary, controlling the production of the hormone, thyrotropin (TSH). Probably, the thyroid hormones serve as a connecting link between environmental cues and reproductive activity. Two very important peptides viz. kisspeptins and RFamide relative peptide appear to be key targets of thyroid hormones. Kisspeptin has been demonstrated as a potent GnRH stimulator and RFRP as an inhibitor which gets modulated with the season and controls reproductive activity in sheep.

Reproductive status-dependent kisspeptin and RFamide-related peptide (Rfrp) gene expression in female Damaraland mole-rats.

Damaraland mole rats (Fukomys damarensis) are cooperatively breeding, subterranean mammals that exhibit a high reproductive skew. Reproduction is monopolised by the dominant female of the group, whereas subordinates are physiologically suppressed to the extent that they are anovulatory. In these latter animals, it is assumed that normal gonadotropin-releasing hormone secretion from the hypothalamus is disrupted. The RFamide peptides kisspeptin (Kiss1) and RFamide-related peptide-3 (RFRP-3) are considered as potent regulators of gonadotropin release. To assess whether these neuropeptides are involved in the mechanism of reproductive suppression, we investigated the distribution and gene expression of Kiss1 and Rfrp by means of in situ hybridisation in wild-caught female Damaraland mole-rats with different reproductive status. In both reproductive phenotypes, substantial Kiss1 expression was found in the arcuate nucleus and only few Kiss1-expressing cells were detected in the anteroventral periventricular nucleus (AVPV), potentially as a result of low circulating oestradiol concentrations in breeding and nonbreeding females. Rfrp gene expression occurred in the dorsomedial nucleus, the paraventricular nucleus and the periventricular nucleus. While in female breeders and nonbreeders, plasma oestradiol levels were low and not significantly different, quantification of the hybridisation signal for both genes revealed significant differences in relation to reproductive status. Reproductively active females had more Kiss1-expressing cells and a higher number of silver grains per cell in the arcuate nucleus compared to nonreproductive females. This difference was most pronounced in the caudal part of the nucleus. No such differences were found in the AVPV. Furthermore, breeding status was associated with a reduced number of Rfrp-expressing cells in the anterior hypothalamus. This reproductive status-dependent expression pattern of Kiss1 and Rfrp suggests that both neuropeptides play a role in the regulation of reproduction in Damaraland mole-rats. Enhanced long-term negative feedback effects of oestradiol could be responsible for the lower Kiss1 expression in the arcuate nucleus of reproductively suppressed females.

Hypothalamic effects of progesterone on regulation of the pulsatile and surge release of luteinising hormone in female rats

Progesterone can block the oestradiol-induced GnRH/LH surge and inhibit LH pulse frequency. Recent studies reported that progesterone prevented premature LH surges during ovarian hyperstimulation in women. As the most potent stimulator of GnRH/LH release, kisspeptin is believed to mediate the positive and negative feedback effects of oestradiol in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei, while the region-specific role of progesterone receptors in these nuclei remains unknown. This study examined the hypothesis that progesterone inhibits LH surge and pulsatile secretion via its receptor in the ARC and/or AVPV nuclei. Adult female rats received a single injection of pregnant mare serum gonadotropin followed by progesterone or vehicle. Progesterone administration resulted in a significant prolongation of the oestrous cycle and blockade of LH surge. However, microinjection of the progesterone receptor antagonist, RU486, into the AVPV reversed the prolonged cycle length and rescued the progesterone blockade LH surge, while RU486 into the ARC shortened LH pulse interval in the progesterone treated rats. These results demonstrated that progesterone’s inhibitory effect on the GnRH/LH surge and pulsatile secretion is mediated by its receptor in the kisspeptin enriched hypothalamic AVPV and ARC respectively, which are essential for progesterone regulation of oestrous cyclicity in rats.

Curiouser and Curiouser: The Evolving Story of the Mechanisms Involved in Puberty.

Sometimes, like a giant jigsaw puzzle, concepts in endocrinology take time to develop, to form a coherent picture of how endocrine systems are integrated to support physiological responses. Such, it can be argued, has been the case with respect to our understanding of the mechanisms underlying puberty. For more than 50 years, it has been known that mammals experience a period of agonadal restraint during development, in which reproductive function is held in check (1). As puberty approaches, this restraint is switched off and pulsatile GnRH secretion is activated. What caused the juvenile period to end remained a matter of conjecture, although a shift in steroid feedback was suggested by observations showing that immature rats had enhanced sensitivity to the negative feedback effects of estradiol (2), whereas the onset of ovarian cyclicity could be advanced by prepubertal injections of estradiol (3). In the past 5 decades, several other important pieces of the puzzle have been filled in. The brain was itself found to be a site of gonadal steroid synthesis (4, 5). Data emerged to indicate that neurosteroid synthesis might contribute to reproductive function (6). Dramatic advances were made in understanding the neurotransmitter and neuropeptide circuitry responsible for regulating the onset of reproductive function, in particular the kisspeptin system (7, 8). However, what actually triggered the change in feedback control of gonadotrophin secretion at puberty remained obscure. A shift from a predominantly inhibitoryhypothalamic environment toone inwhichexcitatory neurotransmitters activated the GnRH neurons was believed to occur (9, 10), but what caused this shift remained unknown. In the current issue of Endocrinology, Kenealy et al (11) develop the novel idea that in female primates, the control of GnRH secretion before and during puberty may actually operate in a relatively similar hypothalamic steroidal milieu: only the source of the estradiol is different. To determine hypothalamic steroid concentrations, perfusates were collected in vivo from the stalk-median eminence (S-ME) region of 32 female rhesus monkeys at different stages of prepubertal and pubertal development. The samples were assayed for estradiol, estrone, testosterone, androstenedione, and progesterone, using a sensitive and specific liquid chromatography-mass spectrometry-based method. Concomitantly, GnRH in the perfusates, as well as LH and steroid levels in circulating serum samples from the same animals, were also assayed. As expected, GnRH and LH levels followed a similar developmental pattern, levels at midpuberty being higher than either prepuberty or early puberty. Circulating estradiol levels also were higher in midpuberty. The steroid measurements in the S-ME perfusates, however, exhibited a strikingly different pattern. Although androstenedione, testosterone, and progesterone did not change significantly in the perfusates across the different stages of puberty, estrone and estradiol were found to be high in the S-ME perfusates from prepubertal animals, declining in midpuberty and early puberty, respectively. Because serum estradiol levels increased over the same period, the S-ME to serum ratio fell dramatically in the midpubertal animals, more than 5-fold in the case of estradiol and more than 60-fold in the case of estrone. These results suggest that although the prepubertal period is indeed characterized by a period of GnRH inhibition that does not depend on ovarian hormone secretion, it is not in fact steroid independent. Rather, it may be mediated at least in part by estradiol produced in situ,

KNDy Neurons Modulate the Magnitude of the Steroid-Induced Luteinizing Hormone Surges in Ovariectomized Rats.

Kisspeptin is the most potent stimulator of LH release. There are two kisspeptin neuronal populations in the rodent brain: in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus. The arcuate neurons coexpress kisspeptin, neurokinin B, and dynorphin and are called KNDy neurons. Because estradiol increases kisspeptin expression in the AVPV whereas it inhibits KNDy neurons, AVPV and KNDy neurons have been postulated to mediate the positive and negative feedback effects of estradiol on LH secretion, respectively. Yet the role of KNDy neurons during the positive feedback is not clear. In this study, ovariectomized rats were microinjected bilaterally into the arcuate nucleus with a saporin-conjugated neurokinin B receptor agonist for targeted ablation of approximately 70% of KNDy neurons. In oil-treated animals, ablation of KNDy neurons impaired the rise in LH after ovariectomy and kisspeptin content in both populations. In estradiol-treated animals, KNDy ablation did not influence the negative feedback of steroids during the morning. Surprisingly, KNDy ablation increased the steroid-induced LH surges, accompanied by an increase of kisspeptin content in the AVPV. This increase seems to be due to lack of dynorphin input from KNDy neurons to the AVPV as the following: 1) microinjections of a dynorphin antagonist into the AVPV significantly increased the LH surge in estradiol-treated rats, similar to KNDy ablation, and 2) intra-AVPV microinjections of dynorphin in KNDy-ablated rats restored LH surge levels. Our results suggest that KNDy neurons provide inhibition to AVPV kisspeptin neurons through dynorphin and thus regulate the amplitude of the steroid-induced LH surges.

Neural mechanisms controlling seasonal reproduction: Principles derived from the sheep model and its comparison with hamsters

Seasonal reproduction is a common adaptive strategy among mammals that allows for breeding to occur at times of the year when it is most advantageous for the subsequent survival and growth of offspring. A major mechanism responsible for seasonal reproduction is a striking increase in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol. The neural and neuroendocrine circuitry responsible for mammalian seasonal reproduction has been primarily studied in three animal models: the sheep, and two species of hamsters. In this review, we first describe the afferent signals, neural circuitry and transmitters/peptides responsible for seasonal reproductive transitions in sheep, and then compare these mechanisms with those derived from studies in hamsters. The results suggest common principles as well as differences in the role of specific brain nuclei and neuropeptides, including that of kisspeptin cells of the hypothalamic arcuate nucleus, in regulating seasonal reproduction among mammals.

Developmental Programming: Impact of Prenatal Bisphenol-A on Estradiol Negative Feedback in Sheep.

Endocrine disruptors are hormonally active substances that impact the trajectory of the developing fetus/offspring. An NHANES study found that 99% of humans studied had measurable urinary levels of bisphenol-A (BPA), a chemical with steroidogenic potential used in the plastic industry. Developmental exposure to BPA leads to detrimental effects at several levels including the reproductive axis. In sheep, prenatal exposure to BPA dampens ovulatory LH surges and induces LH hypersecretion. This study tested the hypothesis that LH hypersecretion in prenatal BPA-treated females is the consequence of reduced sensitivity to estrogen negative feedback. A dose response study was conducted involving 4 groups: control (C; n=7), BPA 0.05 mg/kg/day (B1; n=8), 0.5 mg/kg/day (B2; n=8), and 5 mg/kg/day (B3; n=7). Pregnant sheep were treated daily with BPA (in corn oil, s.c.) from days 30 to 90 of gestation. Controls received corn oil. Sensitivity to estrogen negative feedback was tested in all female offspring at ~12 weeks of age. All animals were administered a GnRH antagonist (acycline, 10 μg/kg, s.c.) every 12 h for 72 h to arrest follicular growth and suppress endogenous estradiol production. Eighty hours after cessation of acyline treatment, animals were implanted with a 3 mm Silastic implant containing estradiol (produces prepubertal levels of estradiol). Blood samples were collected at 20 min intervals for 6 h before, at the end of, and 72 h after cessation of GnRH antagonist treatment, and 67 h after insertion of estradiol implant for measurement of LH. LH pulses were detected by cluster algorithm. To test negative feedback effects of estradiol on LH pulse dynamics (frequency, amplitude, and area under the pulse), a paired t-test was used for all LH pulse variables to compare both pre- and post- estradiol periods. To assess BPA dose response relationship, the delta change between pre- and post- estradiol periods was subjected to regression analyses. GnRH antagonist treatment ablated LH pulsatility in all females. Cessation of GnRH antagonist treatment induced high frequency LH pulses in all groups. In controls, estradiol treatment suppressed LH pulse frequency 61.7±10.3% (P<0.01) and increased LH pulse amplitude and area under the pulse 152.2±65.2% (P<0.01) and 440.0±153.8% (P<0.05), respectively. Estradiol treatment also suppressed LH pulse frequency (B1: 45.2±10.1, B2: 48.3±7.2, B3: 66.1±8.8%) and increased LH pulse amplitude (B1: 200.4±34.9, B2: 157.1±38.7, B3: 302.2±101.9%), and area under the pulse (B1: 470.3±60.1, B2: 280.4±64.9, B3: 808.9±232.7%) in all 3 BPA-treated groups (P<0.01 for all doses and variables). The high degree of variability (P<0.05, homogeneity test) found within each BPA dose group, in response parameters, resulted in lack of BPA effect on LH pulse frequency and amplitude. Regression analysis uncovered a significant, but subtle positive linear correlation between BPA dose and area under the pulse (adjusted r2=0.140, P<0.05). In summary, reduced sensitivity to estradiol negative feedback, manifested as increased LH released per pulse, may be a contributing factor to the LH hypersecretion seen in these animals. The high variability in response parameters indicate that the organizational program involved in establishing sensitivity to steroid feedback is the result of the interplay between individual genetic susceptibility and developmental insults. Supported by NIH ES016541.

227 EFFECTS OF A SINGLE DOSE OF A NONSTEROIDAL AROMATASE INHIBITOR ON OVARIAN FUNCTION IN CATTLE

Many countries have banned the use of estrogenic products in farm animals. Nonsteroidal aromatase inhibitors prevent the body from producing its own estrogen. The effects on the ovary are thought to be a result of suppression of estrogen-producing follicles and a rebound in endogenous levels of FSH through the removal of the negative feedback effect of estradiol. An experiment was designed to determine the effects of a nonsteroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest growth of the dominant follicle, resulting in emergence of a new follicular wave at a predictable interval post-treatment. Beef heifers were assigned randomly to 4 groups and given phosphate-buffered saline [control; (n = 10)] or letrozole at a dose of 500 (n = 9), 250 (n = 10), or 125 (n = 10) μgkg-1 i.v. 4 days after follicular ablation (≈ 2.5 days after wave emergence, at the time dominant follicle selection is manifest). Blood samples were collected and ovarian structures were monitored daily by transrectal ultrasonography. Analysis of variance for repeated measures, one-way ANOVA, paired t-test, and 2-sample t-test were applied to the analysis of the data. The diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P &lt; 0.05). The intervals from treatment to new wave emergence and from treatment to onset of regression of the extant dominant follicle were longer (P &lt; 0.05) in heifers treated with letrozole than in controls, although variances in the intervals were not different. A small but significant reduction in circulating estradiol concentration was observed, and plasma LH concentrations were higher (P &lt; 0.05) in letrozole-treated heifers than in controls. Lower plasma concentrations of FSH in letrozole-treated heifers than in controls (P &lt; 0.03) was interpreted as an indirect effect resulting from prolonged follicular dominance. In summary, a single dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a nonsteroidal method of controlling ovarian function in cattle but further studies are needed to clarify dosage and timing of treatment. Research supported by the Natural Sciences and Engineering Research Council of Canada and Bioniche Life Science Inc.

Noradrenaline Involvement in the Negative‐Feedback Effects of Ovarian Steroids on Luteinising Hormone Secretion

Noradrenaline has been shown to modulate the ovarian-steroid feedback on luteinising-hormone (LH) release. However, despite the high amount of evidence accumulated over many years, the role of noradrenaline in LH regulation is still not clearly understood. The present study aimed to further investigate the involvement of noradrenaline in the negative-feedback effect of oestradiol and progesterone on basal LH secretion. In experiment 1, ovariectomised (OVX) rats received a single injection of oil, oestradiol, or progesterone at 09.00-10.00 h and were decapitated 30 or 60 min later. Levels of noradrenaline and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined in microdissections of the preoptic area (POA) and medial basal hypothalamus-median eminence (MBH-ME) and correlated with LH secretion. Basal LH levels were decreased 30 and 60 min after oestradiol or progesterone injection, and this hormonal response was significantly correlated with a reduction in POA MHPG levels, which reflect noradrenaline release. In addition, noradrenaline levels in the POA were increased, whereas noradrenaline turnover (MHPG/noradrenaline ratio) was decreased 60 min after the injection of both hormones. No effect was found in the MBH-ME. In experiment 2, i.c.v. administration of noradrenaline (60 nmol), performed 15 min before oestradiol or progesterone injection in jugular vein-cannulated OVX rats, completely prevented the ovarian steroid-induced inhibition of LH secretion. The data obtained provide direct evidence that LH secretion in OVX rats is positively regulated by basal noradrenergic activity in the POA, and its reduction appears to play a role in the negative-feedback effect of ovarian steroids on LH secretion in vivo.

Regulation by Estradiol of Hypothalamic Somatostatin Gene Expression: Possible Involvement of Somatostatin in the Control of Luteinizing Hormone Secretion in the Ewe

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.

Physiological classification of anovulatory conditions in cattle

Evaluation of follicular growth patterns by ultrasound combined with measurement of circulating reproductive hormones has allowed designation of three functionally critical follicular sizes during the final stages of follicular growth: emergence (∼4 mm), deviation (∼9 mm), and ovulation (variable from 10 to 20 mm). Classification of anovulatory conditions on the basis of these three critical points is logical and provides for rational diagnosis and treatment of the underlying physiological condition. In extreme undernutrition, there is growth of follicles to emergence but not to deviation; however, the underlying pathophysiology is not defined because of relatively few scientific investigations of this condition. Anovulatory conditions with growth of follicles to deviation but not to ovulatory size have been extensively studied. Undernutrition and/or suckling can cause this anovulatory condition. It is characterized by a greater negative feedback effect of estradiol on GnRH/LH pulses than found in normally cycling cows. Another anovulatory condition that is common in high producing lactating dairy cows is characterized by growth of follicles to larger than ovulatory size, such as is observed in cows with follicular cysts. This condition is characterized by an insensitivity of the hypothalamus to the positive feedback effects of estradiol. Thus, these last two common anovulatory conditions appear to be primarily due to changes in the responsiveness of the hypothalamus to estradiol. Treatments that increase circulating progesterone concentrations can help in the treatment of these two conditions by potentially altering GnRH/LH pulses and allowing the final stages of follicular growth or resetting the hypothalamic responsiveness to the positive feedback effects of estradiol.

Neurobehavioral actions of coumestrol and related isoflavonoids in rodents

Isoflavonoids are plant estrogens that are increasingly advocated as a natural alternative to estrogen replacement therapy (ERT) and are available as dietary supplements. As weak estrogen agonists/antagonists with a range of other enzymatic activities, the isoflavonoids provide a useful model for the actions of endocrine disruptors. This paper reviews the responses of rodents to diets containing coumestrol or an isoflavone supplement in comparison to animals fed the phytoestrogen-free AIN76A diet. Neural mechanisms were investigated by examining isoflavonoid effects on ERα-dependent (regulation of oxytocin receptor [OTR] binding in the ventromedial nucleus of the hypothalamus [VMN]) and ERβ-dependent (regulation of ERβ mRNA in the paraventricular nucleus [PVN]) endpoints. Activational as well as organizational effects on sexual behavior and gonadotropin secretion were observed for coumestrol. Treatment of rat dams with a 100-ppm coumestrol diet from birth to postnatal day (PND) 21 induced premature anovulation in female offspring, and treatment from birth to PND 10 suppressed sexual behavior in male offspring. One-week treatment of ovariectomized (OVX) female rats with the same coumestrol diet increased ERβ mRNA expression in the PVN, an effect opposite to that of estradiol. Ten days of treatment with a 200-ppm coumestrol diet increased LH secretion in OVX wild-type mice, an effect opposite to the normal negative feedback effects of estradiol. No effects were observed in ERα knockout (ERαKO)-OVX females, indicating that coumestrol's action on LH was mediated through ERα. Similar activational effects were observed for the isoflavone diet. The lordotic response to estrogen was significantly reduced by 2 days of treatment of OVX adult females with an isoflavone diet providing 13 ppm genistein and 33 ppm daidzein. One week of treatment with the same isoflavone diet produced an effect opposite to that of estradiol in the PVN, increasing ERβ mRNA expression above control levels. These investigations show that, in spite of their preferential affinity for ERβ, isoflavonoids act through both ERα and ERβ. Moreover, their neurobehavioral actions were antiestrogenic, either antagonizing or producing an action in opposition to that of estradiol. This work demonstrates that even small, physiologically relevant exposure levels can alter estrogen-dependent gene expression in the brain and complex behavior.

Suppression of fetal gonadotrophin concentrations by maternal passive immunization to GnRH in sheep.

The hypothalamo-pituitary-gonadal axis matures during fetal life and may be susceptible to adverse effects. Model systems can be used to understand its physiological role. The aim of this study was to determine whether antibodies to GnRH, administered to the mother, can cross the ovine fetal-placental barrier and suppress fetal gonadotrophin secretion. Maternal passive immunization to GnRH in pregnant Greyface ewes (day 103 of gestation) resulted in GnRH antibody titres of from 1:6000 to 1:9000 after 1 day and a suppression of the pulsatile secretion of fetal LH after 2 days. Fetal FSH concentrations declined gradually over the 11 days of the experiment and were only significantly different from control animals immunized against BSA in male fetuses. The slower decrease in fetal FSH concentrations than in LH concentrations shows that the secretion of FSH, unlike that of LH, is not dependent on short term changes in GnRH release. The lack of a suppressive effect of the maternal GnRH immunoneutralization on female fetal FSH secretion may be due to removal of the negative feedback effect of oestradiol and, possibly, inhibin. There was no sexual dimorphism in the effect of maternal GnRH immunoneutralization on fetal GnRH antibody titres or fetal LH secretion. These findings show that maternal passive immunization against GnRH results in GnRH antibodies crossing the fetal-placental barrier and suppressing fetal LH and FSH secretion in males, but only suppressing LH secretion in the females. Although the lack of effect on FSH secretion in the females needs to be investigated further, the present study provides evidence of a non-invasive procedure for blocking fetal gonadotrophin secretion which may be used to investigate hypothalamo-pituitary-gonadal function during early gestation in sheep.

Breastfeeding and the Suppression of Fertility

Breastfeeding suppresses fertility for a variable length of time, but when certain criteria are applied, it can be a reliable contraceptive. The individual variation in duration of infertility is directly related to suckling activity. As suckling declines, follicle growth resumes and ovulation may occur. Infertility is maintained by suckling-induced disruption of the normal pattern of pulsatile luteinizing hormone release and is related to an increased sensitivity of the hypothalamus to the negative feedback effects of oestradiol. Even if ovulation does resume, the corpus luteum function is often insufficient to maintain pregnancy. Attempts to alter suckling activity to prolong lactational infertility are not successful, but maintenance of reasonable suckling frequencies of normal duration can protect women from pregnancy for a prolonged period postpartum.

Seasonal Changes in Gonadotropin-Releasing Hormone Secretion in the Ewe: Alteration in Response to the Negative Feedback Action of Estradiol1

Two experiments were performed to test the hypothesis that there is a seasonal change in the negative feedback effect of estradiol on episodic secretion of GnRH in the ewe. The first experiment identified a specific estradiol treatment (delivered by s.c. Silastic implant) that produced a 50% decrease in the frequency of pulsatile secretion of LH in ovariectomized ewes during the anestrous season. In the second experiment, this estradiol treatment was administered to ovariectomized ewes during the mid-breeding and anestrous seasons. Separate groups of ovariectomized ewes not treated with estradiol were included during each season to test for a seasonal difference in the effect of estradiol on episodic GnRH and LH secretion. Samples of hypophyseal portal blood (for GnRH) and jugular blood (for LH) were obtained at 5-min intervals approximately one month after placement of the estradiol implants. During the breeding season, no effect of estradiol was observed on either the frequency or size of GnRH and LH pulses. During anestrus, however, estradiol produced a profound suppression of the frequency of GnRH and LH pulses, and an increase in GnRH pulse size. No significant seasonal change was observed in the characteristics of GnRH and LH pulses in ovariectomized ewes in the absence of estradiol treatment. These findings lead to the conclusion that there is a marked seasonal change in the negative feedback effect of estradiol on episodic GnRH secretion in the ewe, with the steroid being maximally effective during anestrus.

Luteinizing hormone regulation by sex steroids in men with germinal and Leydig cell tumours

We examined the gonadotrophin secretion in patients with increased plasma concentrations of testosterone and oestradiol due to hCG-producing tumours. Comparison of plasma gonadotrophin concentrations before and after stimulation by GnRH, in eight men with hCG-producing tumours resulting in increased testosterone and oestradiol plasma levels, and in 29 men with Leydig cell tumours resulting in increased oestradiol and normal to low testosterone plasma levels. Eight men with hCG-producing tumours (six with testicular tumours, two with extratesticular tumours), 29 men with Leydig cell tumours and 15 normal men. The six men with germinal cell tumours of the testis were studied before and after unilateral orchidectomy. Plasma concentrations of hCG, testosterone and oestradiol were measured before and after intramuscular injection of hCG. LH and FSH were measured before and after intravenous injection of 100 micrograms GnRH. Plasma LH and FSH concentrations were low in patients with germ cell tumours, who exhibited increased plasma testosterone and oestradiol concentrations, and were normal in patients with Leydig cell tumours, in whom oestradiol only was increased. Plasma LH and FSH were normalized in the five patients with successful (e.g. normal hCG, testosterone and oestradiol) unilateral orchidectomy. Basal plasma testosterone concentrations correlated positively (P < 0.01) with plasma oestradiol concentrations in patients with germ cell tumours and negatively (P < 0.01) in patients with Leydig cell tumours. In patients with hCG-secreting germ cell tumours complete suppression of plasma LH and FSH with increased plasma concentrations of both testosterone and oestradiol are often discovered. No such gonadotrophin suppression is found in patients with Leydig cell tumours, but the negative correlation observed between plasma testosterone and oestradiol in these patients suggests a weak negative feedback effect of oestradiol on LH secretion, which cannot be demonstrated by basal LH measurements in plasma.

Evidence that Dopaminergic Neurons are not Involved in the Negative Feedback Effect of Testosterone on Luteinizing Hormone in Rams in the Non‐Breeding Season

In this study we tested the hypothesis that the negative feedback effects by testosterone on the secretion of luteinizing hormone (LH) in rams involves dopaminergic afferents to gonadotrophin-releasing hormone neurons operating via D(2) receptors in the non-breeding season. In the first experiment, three groups (n = 5) of rams were treated with an intravenous injection of vehicle or 10 or 20 mg of the dopaminergic D(2) antagonist pimozide and jugular venous samples were collected every 10 min for 3 h before and 3 h following treatment. The plasma was assayed for LH. Three groups of ewes (n = 4 to 5) were similarly treated. There were no significant effects of treatment of the rams with pimozide on the plasma concentrations of LH or LH pulse frequency or pulse amplitude and the response of individual rams in each group was inconsistent. In contrast, treatment of the ewes with 20 mg pimozide significantly (P<0.001) increased the mean (± SEM) plasma LH concentrations (pretreatment 0.37 ± 0.04; post-treatment 2.42±0.25 ng/ml) and decreased (P<0.001) the LH inter-pulse interval (pretreatment 180.0; post-treatment 88.0±11.1 min); the 10 mg dose of pimozide did not affect these parameters. In the second experiment, two groups of rams (n = 5) and ewes (n = 7) were treated with an intravenous injection of vehicle or 0.33 mg pimozide/kg liveweight and jugular venous samples were collected every 10 min for 2 h before and 6 h following treatment. As in the first experiment, the mean (± SEM) concentrations of plasma LH were not affected by treatment with pimozide in the rams (pretreatment 0.18 ± 0.25; post-treatment 0.43 ± 0.14 ng/ml) but were significantly (P<0.05) increased in the ewes (pretreatment 1.12±0.22; post-treatment 1.93 ± 0.23 ng/ml). In the third experiment, four adult rams were castrated and 3 weeks later each animal had two cannulae inserted to allow injection into the lateral cerebral ventricles. Vehicle or 100 μg pimozide was injected intracerebroventricularly and blood samples were collected as in the other experiments. A Latin Square design was used so that each animal received each treatment (n = 4). This procedure was repeated after the animals had been injected (intramuscularly) with 16 mg testosterone propionate twice daily for at least 7 days. Treatment with testosterone propionate significantly decreased (P < 0.001) the plasma concentrations of LH (pre-treatment 7.71±0.27; post-treatment 0.75 ± 0.27 ng/ml; mean ± SEM) and follicle-stimulating hormone (pre-treatment 79.61±8.47; post-treatment 42.53 ± 6.08 ng/ml; mean ± SEM) and increased the mean (± SEM) LH inter-pulse interval (53.14 ± 3.58 min pre-treatment and 292.5 ± 32.94 min post-treatment) but had no effect on the amplitude of LH pulses (pre-treatment 3.61 ± 0.36; post-treatment 1.86±1.76 ng; mean ± SEM). Pimozide had no effect on the plasma concentrations of gonadotrophins. These results suggest that, in the ram, dopaminergic neurons do not influence the gonadotrophin-releasing hormone neurons via D(2) receptors in the non-breeding season and are not involved in the negative feedback effect of testosterone on the secretion of gonadotrophins. Conversely, our data suggest that such a mechanism is integral to the negative feedback effects of oestradiol on LH in anoestrous ewes. Finally, it also appears that the steroid-independent suppression of the secretion of gonadotrophins during the non-breeding season in rams is not mediated via D(2) receptors.

Effects of intracerebroventricular administration of opiate receptor antagonists on the suppressed pulsatile LH release during acute fasting in ovariectomized estradiol-treated rats

The involvement of specific opiate receptors in the suppression of LH release during acute fasting in ovariectomized estradiol-treated rats was examined by intracerebroventricular (i.c.v.) administration of opiate receptor antagonists that exert a specificity directed mainly, although not absolutely, towards the δ-, κ- or μ-opiate receptors. Fasting for 48 h significantly decreased mean plasma LH levels in estradiol-treated animals by increasing sensitivity to the negative feedback effect of estradiol. Injecting i.c.v. the μ-opiate receptor antagonist naloxone (10 or 100 nmol in 2 μl of saline) blocked the inhibitory effect of fasting on pulsatile LH release and reinstated LH pulses. On the other hand, i.c.v. administration of the same dosages of a δ-opiate receptor antagonist ICI 174,864 or a κ-opiate receptor antagonist WIN 44441-3 did not have any effect. These results suggest that the increased sensitivity of the LH-releasing mechanism to the negative feedback effect of estradiol during fasting involves the endogenous opioids mainly through the selective activation of the μ-opiate receptors.