Estrogen Negative Feedback Research Articles

Female obesity: profoundly reduced estrogen excretion after aromatase inhibition may underscore exaggerated sensitivity to estrogen negative feedback

Female obesity: profoundly reduced estrogen excretion after aromatase inhibition may underscore exaggerated sensitivity to estrogen negative feedback

The Role of cAMP Response Element-Binding Protein in Estrogen Negative Feedback Control of Gonadotropin-Releasing Hormone Neurons

The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.

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.

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight.

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.

The neuroendocrine regulatory mechanisms of mammalian seasonal reproduction

The seasonal reproduction of mammal means the reproduction experiences an annual period from quiescence to renaissance. Studies have shown that kisspeptin and RFRP play an important role in the reproductive seasonality. The non-breeding season is characterized by an increase in the negative feedback effect of estrogen on GnRH, and this effect is transmitted by kisspeptin neurons, which may be an important factor affecting the reproduction activities. The expression of RFRP depends on melatonin secretion, and shows an apparent inhibition on reproduction in non-breeding season. In addition, thyroid hormones influence termination of the breeding season. Dopaminergic neuron A14/A15 also contributes to the seasonal changes in estrogen negative feedback. These neural systems may synergistically modulate the seasonal changes of reproductive function with the photoperiod. This review makes a systematic expatiation on the relationship between seasonal reproduction and these neural systems.

Neurosteroids, trigger of the LH surge

Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The “astrocrine hypothesis” maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an “off-on-off” mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca2+]i response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gαq, and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca2+]i release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development.This article is part of a Special Issue entitled ‘Neurosteroids’.

Introduction to special topic—estrogenic control of hypothalamic GnRH neurons

In female mammals, sexual maturation is initiated by a change in the release pattern of pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and a consequential increase in the secretion of ovarian sex-steroids, estradiol and progesterone. Orchestrating these endocrine changes is a diffuse population of hypothalamic neurons that produce the neuropeptide, gonadotropin-releasing hormone (GnRH). Although the development and functional integration of GnRH neurons with the rest of the central nervous system is still poorly understood, recent progress has been made on several fronts. For example, there is now evidence that some mammalian species express more than one molecular form of GnRH (GnRH-I and GnRH-II), and that the two corresponding GnRH neuronal sub-populations may play different roles in the regulation of reproductive function and behavior. Moreover, through the use of transgenic animal models, neuronal fiber tracing, gene expression profiling, and electrophysiologcal recordings, new insights have been gained into the mechanisms that regulate GnRH release. This volume brings together 10 articles that reflect current thinking about the role of GnRH in mediating estrogenic feedback to the neuroendocrine reproductive axis. The main focus is on the negative and positive actions that estrogens exert within the hypothalamus of mammals, especially around the time of the preovulatory LH surge. However, the articles also present findings from other vertebrate classes, and so provide an intersting comparative perspective. Taken together, the knowledge presented in this volume represents a foundation upon which to develop more effective therapies for pubertal disorders, infertility, and menopause. The first article (Sower and Baron, 2011) demonstrates the expression of estrogen receptors within the lamprey hypothalamus, suggesting potential feedback interactions between esatradiol and GnRH neurons. Phylogenetically, lampreys are positioned as basal vertebrates, and like many other non-mammalian vertebrates they express multiple forms of GnRH. Consequently, studies in lampreys provide us with important insights into the molecular evolution of estrogenic-GnRH interactions. A cluster of three articles then focus on hypothalamic neuropeptides that exert a major influences on the secretion of GnRH, and which are thought to mediate the feedback effects of sex-steroids on the reproductive axis. For example, studies in rodents have clearly shown the importance of kisspeptin to both pulsatile and surge modes of GnRH release (Navarro, 2012), while human studies have observed sexual dimorphism in kisspeptin neurons during aging (Hrabovszky et al., 2011). Again, an interesting evolutionary perspective is provided by comparing the steroid sensitivity of kisspeptin neurons in various non-mammalian vertebrates (Kanda and Oka, 2012). Although the exact mechanism by which estrogens interact with the reproductive neuroendocrine axis is unclear, our general understanding of this issue is neatly summarized by a series of three reviews (Micevych and Sinchak, 2011; Kenealy and Terasawa, 2012; Radovick et al., 2012). Of particular interest is the finding that sex-steroids exert their feedback not only through classic estrogen receptors located in the nucleus, but also more rapidly through receptors located in the cell membrane. Various physiological functions show adaptations for life in an environment that undergoes marked changes across the 24-h day. For example, in many mammals the timing of the preovulatory LH surge occurs at a very specific time of day. Two articles discuss the interactions between the circadian timing system and estrogen-sensitive neural circuits that regulate GnRH secretion and the preovulatory surge (Sun et al., 2012; Williams and Kriegsfeld, 2012). Finally, a novel hypothesis is proposed to explain the conundrum of how estrogens appear to exert both negative and positive feedback actions on the reproductive axis (Urbanski, 2012). Data is presented from non-human primate studies to suggest that negative and positive estrogen feedback are two completely separate mechanisms, which are mediated, respectively, by two distinct GnRH neuronal populations. This hypothesis challenges the common assumption that has dominated the field for the past four decades, namely that individual GnRH neurons are relatively homogenous and can mediate both negative and positive estrogen feedback.

63 EVALUATION OF HYPOTHALAMIC–PITUITARY RESPONSIVENESS DURING THE POSTPARTUM NELLORE COWS

The presence of calf, body condition score, energy balance, number of births (multiparous vs primiparous) and breed are factors that influence the duration of postpartum anoestrus in beef cows. The objective of the present study was to evaluate, during early postpartum, the time of re-establishment of pituitary LH stocks, measured by the hypothalamic-pituitary-axis responsiveness to exogenous administration of gonadotropin-releasing hormone (GnRH) or oestradiol benzoate (EB). Multiparous lactating Nellore cows (Bos indicus, n = 65) were randomly allocated into 6 groups, according to the hormonal treatment: EB group (1.0 mg of EB, IM, n = 7), GnRH group (50 μg of lecireline, IM, n = 16). The EB-supplemented (SUP; n = 9) and GnRH-SUP (n = 16) groups received the same treatments specified above and were SUP with a balanced diet, based on cotton meal and ground corn (3.5 kg cow–1 per day). Additionally, animals from EB-calf removed (CR; n = 4) and GnRH-CR (n = 13) groups received the same treatments of EB and GnRH group, respectively and had their CR shortly after parturition. The hormones were administered weekly, from 7 days postpartum (±5 days) until the occurrence of the first ovulation, which was determined by the presence of corpus luteum during ovarian ultrasonography performed weekly. Blood samples were collected just before and 2 h (GnRH groups) or 18 h (EB groups) after hormone administration, in order to determine LH concentration by radioimmunoassay. Data were analysed by ANOVA. Mean values in days (± standard error of the means) for the first postpartum LH surge were EB (73.0 ± 5.2); EB-CR (16.7 ± 5.8); EB-SUP (41.7 ± 6.7); GnRH (32.3 ± 3.0); GnRH-CR (11.0 ± 3.5); GnRH-SUP (15.6 ± 2.8). There were significant differences (P &lt; 0.05) between groups EB vs EB-CR; EB vs EB-SUP; BE vs GnRH; GnRH vs GnRH-CR; GnRH vs GnRH-SUP and a tendency between EB-SUP vs EB-CR (P &lt; 0.10). Results indicate that from the second week postpartum, there is sufficient LH in the pituitary to induce ovulation after GnRH or EB administration. However, the cows from the EB group ovulated later than animals from the other groups, possibly due to the sensitivity of the hypothalamus to negative feedback of estrogens, inhibiting the pre-ovulatory LH surge. Additionally, CR and food SUP reduced in 2 to 4 weeks the time of the first postpartum LH surge induced by GnRH or EB in Nellore cows. A fellowship was received from FAPESP. Support was received from FAPESP.

Differential effects of aging on estrogen negative and positive feedback

Recent studies have demonstrated an age-related decline in gonadotropins and a decrease in pituitary responsiveness to GnRH, indicating that aging influences the neuroendocrine components of the female reproductive axis independently of changes in ovarian function. To determine whether aging might also affect the luteinizing hormone (LH) negative and positive feedback responses to gonadal steroids, we administered a controlled, graded sex steroid infusion to 11 younger (45-56 yr) and nine older (70-80 yr) postmenopausal women (PMW) in whom endogenous ovarian steroids and peptides are uniformly low. The doses of estradiol (E(2)) and progesterone (P) were chosen to mimic levels across the normal follicular phase and have been shown previously to induce negative followed by positive feedback on LH. Similar E(2) and P levels were achieved in younger and older PMW (P = 0.4 and 0.3, respectively) and produced a biphasic LH response in all subjects. The early decline in LH to 53% of baseline was not different in older vs. younger PMW. However, the positive feedback effect was attenuated in older compared with younger PMW (peak LH 144.4 ± 19.5 vs. 226.8 ± 22.3 IU/l, respectively, P = 0.01). In conclusion, these studies in PMW demonstrate preservation of short-term steroid negative and positive feedback in response to exogenous E(2) and P with aging. Attenuation of positive feedback in older compared with younger PMW is consistent with previous reports of declining GnRH responsiveness with aging.

Elevated estrogen receptor expression in hypothalamic preoptic area decreased by electroacupuncture in ovariectomized rats

In the present study, effects of electroacupuncture (EA) on estrogen receptor alpha (ERα) and beta (ERβ) mRNA and protein expression in the hypothalamus of ovariectomized (OVX) rats were detected by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot analysis. Gonadotropin-releasing hormone (GnRH) release and GnRH mRNA level in hypothalamic preoptic area (POA) were evaluated by push–pull perfusion and qRT-PCR. Our results showed that elevated mRNA and protein expression of ERα and ERβ in hypothalamus were restrained following EA treatment in OVX rats. EA treatment also inhibited GnRH release and GnRH mRNA levels in OVX rats. These results provide novel evidence that EA treatment down regulates the expression of hypothalamic estrogen receptors (ERs), thus restores the response of GnRH neurons to estrogen depression, and partially resets the negative feedback of estrogen to hypothalamus–pituitary–ovary axis (HPOA) in OVX rats, which may be a critical mechanism for EA on female reproductive disorders.

Jak2 Is Necessary for Neuroendocrine Control of Female Reproduction

Gonadotropin-releasing hormone (GnRH) neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity. In vitro, GnRH neuronal cell lines respond to a variety of ligands that activate the Jak (Janus-activated kinase)/STAT (signal transducers and activators of transcription) intracellular signaling pathway. To determine its biological function in reproduction, we used Cre (cAMP response element)/LoxP technology to generate GnRH neuron-specific Jak2 conditional knock-out (Jak2 G(-/-)) mice. GnRH mRNA levels were reduced in Jak2 G(-/-) mice when compared with controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum luteinizing hormone (LH) levels were significantly lower in female Jak2 G(-/-) mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G(-/-) mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However, the activation of GnRH neurons following release from estrogen-negative feedback is retained. Female Jak2 G(-/-) mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity, and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice.

Sexual dimorphism of kisspeptin and neurokinin B immunoreactive neurons in the infundibular nucleus of aged men and women

The secretory output of gonadotropin-releasing hormone (GnRH) neurons is critically influenced by peptidergic neurons synthesizing kisspeptins (KP) and neurokinin B (NKB) in the hypothalamic infundibular nucleus (Inf). These cells mediate negative feedback effects of sex steroids on the reproductive axis. While negative feedback is lost in postmenopausal women, it is partly preserved by the sustained testosterone secretion in aged men. We hypothesized that the different reproductive physiology of aged men and women is reflected in morphological differences of KP and NKB neurons. This sexual dimorphism was studied with immunohistochemistry in hypothalamic sections of aged human male (≥50 years) and female (>55 years) subjects. KP and NKB cell bodies of the Inf were larger in females. The number of KP cell bodies, the density of KP fibers, and the incidence of their contacts on GnRH neurons were much higher in aged women compared with men. The number of NKB cell bodies was only slightly higher in women and there was no sexual dimorphism in the regional density of NKB fibers and the incidence of their appositions onto GnRH cells. The incidences of NKB cell bodies, fibers, and appositions onto GnRH neurons exceeded several-fold those of KP-IR elements in men. More NKB than KP inputs to GnRH cells were also present in women. Immunofluorescent studies identified only partial overlap between KP and NKB axons. KP and NKB were colocalized in higher percentages of afferents to GnRH neurons in women compared with men. Most of these sex differences might be explained with the lack of estrogen negative feedback in aged women, whereas testosterone can continue to suppress KP, and to a lesser extent, NKB synthesis in men. Overall, sex differences in reproductive physiology of aged humans were reflected in the dramatic sexual dimorphism of the KP system, with significantly higher incidences of KP-IR neurons, fibers and inputs to GnRH neurons in aged females vs. males.

Estrogen Negative Feedback on Gonadotropin Secretion: Evidence for a Direct Pituitary Effect in Women

Previous animal and human studies using low-dose estrogen have demonstrated estrogen negative feedback on gonadotropin secretion through gonadotropin-releasing hormone (GnRH) release at the level of the hypothalamus in women. Whether low-dose estrogen also exerts a direct negative feedback at the level of the pituitary is unclear due to insufficient data. In a recent study, a decrease was found in pituitary responsiveness to GnRH with aging in women. Studies in human beings aimed to establish a direct pituitary site of estrogen negative feedback and investigate the interaction of estrogen, and aging in the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) feedback loop have been hampered by the inability thus far to isolate the pituitary response from endogenous hypothalamic input. This study tested the hypothesis that estrogen has a direct negative feedback effect at the pituitary and also investigated whether this effect would vary with aging. Isolation of pituitary responsiveness from hypothalamic and ovarian input was achieved through blockade of the GnRH receptor using the NAL-GLU GnRH antagonist, followed by graded doses of GnRH before and after 1 month of estrogen administration. The participants were healthy postmenopausal women aged 48 to 56 years (n = 8) or 70 to 75 years (n = 8). After estrogen administration, there was a significant decrease in the LH (P = 0.01) and FSH (P = 0.0001) responses to GnRH in all study subjects. There was also a decrease in the ratio of FSH to LH amplitudes in response to GnRH (P = 0.04). The inhibitory effect of estrogen on FSH responses was significantly diminished with aging (P = 0.02). In contrast to FSH, LH responses were not significantly affected by aging (P = 0.4). These findings demonstrate that estrogen has a direct inhibitory effect on pituitary responsiveness to GnRH in women. This estrogen negative feedback diminishes with aging. FSH but not LH responses are inhibited by estrogen.

Knockdown of GABAA Receptor Signaling in GnRH Neurons Has Minimal Effects upon Fertility

The amino acid gamma-aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 +/- 1%) GnRH neurons had the gamma2-subunit of the GABA(A) receptor deleted. Electrophysiological recordings of GABA(A)-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the gamma2-subunit knocked out (GnRH gamma2 KO) showed that the amplitude and frequency of GABA(A) postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH gamma2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH gamma2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-beta-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH gamma2 KO male mice. Together, these findings show that 70-90% reductions in the normal levels of GABA(A) receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

Estrogen Negative Feedback on Gonadotropin Secretion: Evidence for a Direct Pituitary Effect in Women

Estrogen Negative Feedback on Gonadotropin Secretion: Evidence for a Direct Pituitary Effect in Women

Estrogen Negative Feedback on Gonadotropin Secretion: Evidence for a Direct Pituitary Effect in Women

Studies in humans and animals indicate that estrogen negative feedback occurs at the level of the hypothalamus, but it is unclear whether estrogen also exerts an inhibitory effect directly at the pituitary. The aim of the study was to determine whether estrogen has a direct negative feedback effect at the pituitary and whether this varies with aging. A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness before and after estrogen administration in Clinical Research Center studies at an academic medical center. Subjects were healthy postmenopausal women aged 48-56 yr (n = 8) or 70-75 yr (n= 8). A suppressive dose of the NAL-GLU GnRH antagonist was administered, followed by graded doses of GnRH before and after 1 month of estrogen administration. LH and FSH responses to GnRH decreased after estrogen administration (P = 0.01 and P = 0.0001, respectively). The ratio of FSH to LH amplitudes decreased in response to estrogen (P = 0.04) indicating a greater sensitivity of FSH than LH to inhibition by estrogen. The inhibitory effect of estrogen on FSH was attenuated with aging (P = 0.02), but was maintained for LH (P = 0.4). Studies that control for endogenous GnRH and estradiol demonstrate a direct pituitary site of estrogen negative feedback on LH and FSH responsiveness to GnRH in women. The effect of estrogen on FSH responsiveness is greater than on LH and is attenuated with aging. These studies indicate that estrogen negative feedback occurs directly at the pituitary and contributes to the differential regulation of FSH and LH secretion.

132 INDUCTION OF FIRST POSTPARTUM OVULATION AFTER ADMINISTRATION OF GnRH, ESTRADIOL BENZOATE, OR LH ASSOCIATED OR NOT WITH CALF REMOVAL IN NELORE COWS

There are reports in the literature indicating that the presence of calf, body condition score, energy balance, number of births (multiparous v. primiparous), and breed are factors that influence the duration of postpartum anoestrus in beef cows. The purpose of the present study was to evaluate, during early postpartum, the time necessary for reestablishment of pituitary LH stocks, determined by the hypothalamic-pituitary axis responsiveness to exogenous administration of GnRH and estradiol benzoate (EB). Multiparous lactating Nelore cows (n = 51) were kept in Brachiaria brizantha pasture. The animals were randomly allocated in 8 groups, according to the hormonal treatment: EB Group (1.0 mg of EB, i.m. Estrogin®, Farmavet, Sao Paulo, Brazil; n = 7), EB and calf removal (CR) group (EB/CR, calves were removed at the time of birth, n = 4), EB/SUP group (cows were supplemented with a balanced diet, based on cotton meal and ground corn, from the beginning of the experiment until ovulation), GnRH group (100 μg, licereline, i.m. Gestran Plus®, ARSA SLR, Buenos Aires, Argentina; n = 7), GnRH/CR group (n = 4), GnRH/SUP group (n = 8), LH group (12.5 mg, i.m. Lutropin® Bioniche, Ontario, Canada; n = 7), and control group (did not receive any hormone to induce ovulation or diet supplementation, n = 5).The hormones were administered weekly, from 7 days postpartum (±5 days) until the occurrence of the first ovulation, which was determined by the presence of CL during weekly ovarian ultrasonography (Aloka 900, Tokyo, Japan, 7.5 MHz probe). The data were analyzed by ANOVA. On average (±SEM) the first ovulation occurred 86.7 ± 0.5 (EB); 19.7 ± 0.6 (EB/CR); 57.8 ± 0.4 (EB/SUP); 44.7 ± 0.5 (GnRH); 25.9 ± 0.6 (GnRH/CR); 32.8 ± 0.4 (GnRB/SUP); 42.4 ± 0.5 (LH); and 59.3 ± 0.5 (control) days postpartum. There were significant differences between groups: EB v. EB/SUP (P &lt; 0.01), EB v. EB/CR (P &lt; 0.01), EB v. control (P &lt; 0.04), EB v. GnRH (P = 0.01), and there was a tendency between groups: GnRH v. GnRH-SUP (P = 0.10) and LH v. control (P = 0.10). The first ovulations were detected from the second week postpartum (animals from Groups GnRH and EB/CR) until the last ultrasound examination on Day 120 postpartum (Group EB). The results indicate that from the second week postpartum there is sufficient LH in the pituitary to induce ovulation after GnRH or EB administration. However, the cows from EB group ovulated later than animals from the other groups, possibly due to the sensitivity of hypothalamus to negative feedback of estrogens, inhibiting the pre-ovulatory LH surge. Additionally, calf removal and food supplementation reduced in 2 to 4 weeks the time of the first postpartum ovulation induced by GnRH or EB. Fellowship from FAPESP. Supported from FAPESP (Sao Paulo, Brazil).

Trace LC/MS/MS quantitation of 17β‐estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain

A sensitive LC/MS/MS method has been developed by derivatization of 17beta-estradiol (E2) with dansyl chloride to quantitate 17beta-E2 in female rat serum. The use of E2-d(5) minimized interferences from endogenous 17beta-E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 microl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17beta-E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic-pituitary-ovarian (HPO) axis. Variations in 17beta-E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing.

Molecular Characterization and Estrogen Regulation of Hypothalamic KISS1 Gene in the Pig1

Kisspeptin-GPR54 signaling plays an essential role in normal reproduction in mammals via stimulation of gonadotropin secretion. Here, we cloned the porcine KISS1 cDNA from the hypothalamic tissue and investigated the effect of estrogen on the distribution and numbers of KISS1 mRNA-expressing cells in the porcine hypothalamus. The full length of the cDNA was 857 bp encoding the kisspeptin of 54 amino acids, with the C-terminal active motif designated kisspeptin-10 being identical to that of mouse, rat, cattle, and sheep. In situ hybridization analysis revealed that KISS1-positive cell populations were mainly distributed in the hypothalamic periventricular nucleus (PeN) and arcuate nucleus (ARC). KISS1 expression in the PeN of ovariectomized (OVX) pigs was significantly upregulated by estradiol benzoate (EB) treatment. On the other hand, KISS1-expressing cells were abundantly distributed throughout the ARC in both OVX and OVX with EB animals. The number of KISS1-expressing neurons was significantly lowered by EB treatment only in the most caudal part of the ARC, but other ARC populations were not affected. The present study thus suggests that the PeN kisspeptin neurons could be responsible for the estrogen positive feedback regulation to induce gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) surge in the pig. In addition, the caudal ARC kisspeptin neurons could be involved in the estrogen negative feedback regulation of GnRH/LH release. This is the first report of identification of porcine KISS1 gene and of estrogen regulation of KISS1 expression in the porcine brain, which may be helpful for better understanding of the role of kisspeptin in reproduction of the pig.

Kisspeptin Neurons in the Ovine Arcuate Nucleus and Preoptic Area Are Involved in the Preovulatory Luteinizing Hormone Surge

Kisspeptin is the product of the Kiss1 gene that regulates GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the preoptic area (POA) and arcuate nucleus (ARC), and expression is up-regulated in the caudal ARC during the periovulatory period. We hypothesized that kisspeptin neurons in the ARC are activated by estradiol-17beta prior to the preovulatory LH surge. Ovariectomized ewes were treated as follows: 1) estradiol-17beta implants (sc 2 wk) to cause tonic negative feedback; 2) vehicle (no estrogen negative or positive feedback); or 3) positive feedback/GnRH surge-inducing injection of estradiol-17beta (50 microg iv). For groups 2 and 3, brains were collected 1 h after treatment and kisspeptin/Fos immunoreactivity was examined. In the caudal and mid-ARC, the percentage of kisspeptin cells that were Fos immunoreactive increased after acute estradiol treatment (group 3) over that seen in the other two groups. Kisspeptin/Fos colocalization was also quantified in ewes during the luteal and late-follicular phase of the estrous cycle, showing a trend toward an increase in colocalization in the late-follicular phase. Kisspeptin/Fos colocalization was similar in the POA across groups in both experiments. Analysis of Kiss1 mRNA by in situ hybridization revealed an increase in expression during the late-follicular phase in the caudal ARC and POA. These data suggest kisspeptin neurons located in the caudal extent of the ARC are involved in generating the positive feedback preovulatory GnRH/LH surge in the ewe, but there may also be a role for Kiss1-expressing cells in the POA.