Afternoon Of Proestrus Research Articles

Early-Life Resource Scarcity in Mice Does Not Alter Adult Corticosterone or Preovulatory Luteinizing Hormone Surge Responses to Acute Psychosocial Stress.

Early-life stressors can affect reproductive development and change responses to adult stress. We tested if resource scarcity in the form of limited bedding and nesting (LBN) from postnatal days (PND) 4 to 11 delayed sexual maturation in male and female mice and/or altered the response to an acute, layered, psychosocial stress (ALPS) in adulthood. Contrary to the hypotheses, age and mass at puberty were unaffected by the present application of LBN. Under basal conditions and after ALPS, corticosterone concentrations in males, diestrous females, and proestrous females reared in standard (STD) or LBN environments were similar. ALPS disrupts the luteinizing hormone (LH) surge in most mice when applied on the morning of proestrus; this effect was not changed by resource scarcity. In this study, the paucity of effects in the offspring may relate to a milder response of CBA dams to the paradigm. While LBN dams exited the nest more often and their offspring were smaller than STD-reared offspring on PND11, dam corticosterone concentrations were similar on PND11. To test if ALPS disrupts the LH surge by blunting the increase in excitatory GABAergic input to gonadotropin-releasing hormone (GnRH) neurons on the afternoon of proestrus, we conducted whole-cell voltage-clamp recordings. The frequency of GABAergic postsynaptic currents in GnRH neurons was not altered by LBN, ALPS, or their interaction. It remains possible that ALPS acts at afferents of GnRH neurons, changes response of GnRH neurons to input, and/or alters pituitary responsiveness to GnRH and that a more pronounced resource scarcity would affect the parameters studied.

Thyrotropin-releasing hormone stimulates NR4A3 expression in the pituitary thyrotrophs of proestrus rats.

The secretion of several hypothalamic peptide hormones is activated during the preovulatory period. Hypothalamic thyrotropin-releasing hormone (TRH) is one such hormone with reproductive and/or metabolic significance. However, it remains unclear whether thyroid-stimulating hormone (TSH)-producing thyrotrophs are produced during the preovulatory period. We previously found a transient increase in the expression of the nuclear receptor NR4A3, a well-known immediate early gene, in the proestrus afternoon in the anterior pituitary glands of rats. To investigate the relationship between TRH secretion and pituitary NR4A3 expression during proestrus, we used proestrus and thyroidectomized rats to identify NR4A3-expressing cells and examined the regulation of Nr4a3 gene expression via the hypothalamus-pituitary-thyroid (HPT) axis. The percentage of NR4A3-expressing cells increased in thyrotrophs at 14:00 h of proestrus. Incubation of rat primary pituitary cells with TRH transiently stimulated Nr4a3 expression. Thyroidectomy to attenuate the negative feedback effects led to increased serum TSH levels and Nr4a3 gene expression in the anterior pituitary, whereas thyroxine (T4) administration conversely suppressed Nr4a3 expression. Additionally, the administration of T4 or TRH antibodies significantly suppressed the increase in Nr4a3 expression at 14:00 h of proestrus. These results demonstrate that pituitary NR4A3 expression is regulated by the HPT axis, and that TRH stimulates thyrotrophs and induces NR4A3 expression during the proestrus afternoon. This suggests the potential involvement of NR4A3 in the regulation of the HPT axis during pre- and post-ovulatory periods.

Reciprocal Changes in Voltage-Gated Potassium and Subthreshold Inward Currents Help Maintain Firing Dynamics of AVPV Kisspeptin Neurons during the Estrous Cycle.

Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared with diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K+ current: fast-transient slow-transient, and residual. During proestrus, the V50 of inactivation of the fast-transient current was depolarized and the amplitude of the slow-transient component was reduced compared with diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K+ currents are suppressive compared with diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling postinhibitory rebound bursting. These findings suggest modulation of voltage-gated K+ and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge.

Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice.

Female reproductive success relies on proper integration of circadian- and ovarian- signals to the hypothalamic-pituitary-gonadal axis in order to synchronize the preovulatory LH surge at the end of the ovarian follicular stage with the onset of the main active period. In this study, we used a combination of neuroanatomical and electrophysiological approaches to assess whether the hypothalamic neurons expressing Arg-Phe amide-related peptide (RFRP-3), a gonadotropin inhibitory peptide, exhibit daily and estrous stage dependent variations in female mice. Furthermore, we investigated whether arginine vasopressin (AVP), a circadian peptide produced by the suprachiamatic nucleus regulates RFRP-3 neurons. The number of c-Fos–positive RFRP-3 immunoreactive neurons is significantly reduced at the day-to-night transition with no difference between diestrus and proestrus. Contrastingly, RFRP neuron firing rate is higher in proestrus as compared to diestrus, independently of the time of the day. AVP immunoreactive fibers contact RFRP neurons with the highest density observed during the late afternoon of diestrus and proestrus. Application of AVP increases RFRP neurons firing in the afternoon (ZT6-10) of diestrus, but not at the same time point of proestrus, indicating that AVP signaling on RFRP neurons may depend on circulating ovarian steroids. Together, these studies show that RFRP neurons integrate both daily and estrogenic signals, which downstream may help to properly time the preovulatory LH surge.

Neonatal overfeeding reduces estradiol plasma levels and disrupts noradrenergic-kisspeptin-GnRH pathway and fertility in adult female rats

This work evaluated the effects of neonatal overfeeding, induced by litter size reduction, on fertility and the noradrenaline-kisspeptin-gonadotrophin releasing hormone (GnRH) pathway in adult female rats. The litter size was adjusted to 3 pups with each mother in the small litters (SL) and 10 pups with each mother in the normal litters (NL). SL females exhibited metabolic changes associated with reproductive dysfunctions, shown by earlier vaginal opening and first estrus, later regular cyclicity onset, and lower and higher occurrences of estrus and diestrus phases, respectively, as well as reduced fertility, estradiol plasma levels, and mRNA expressions of tyrosine hydroxylase in the locus coeruleus, kisspeptin, and GnRH in the preoptic area in adult females in the afternoon of proestrus. These results suggest that neonatal overfeeding in female rats promotes reproductive dysfunctions in adulthood, such as lower estradiol plasma levels associated with impairments in fertility and noradrenaline-kisspeptin-GnRH pathway during positive feedback.

Impaired LH surge amplitude in gonadotrope-specific progesterone receptor knockout mice

The progesterone receptor (PR, encoded by Pgr) plays essential roles in reproduction. Female mice lacking the PR are infertile, due to the loss of the protein's functions in the brain, ovary, and uterus. PR is also expressed in pituitary gonadotrope cells, but its specific role therein has not been assessed in vivo. We therefore generated gonadotrope-specific Pgr conditional knockout mice (cKO) using the Cre-LoxP system. Overall, both female and male cKO mice appeared phenotypically normal. cKO females displayed regular estrous cycles (vaginal cytology) and normal fertility (litter size and frequency). Reproductive organ weights were comparable between wild-type and cKO mice of both sexes, as were production and secretion of the gonadotropins, LH and FSH, with one exception. On the afternoon of proestrus, the amplitude of the LH surge was blunted in cKO females relative to controls. Contrary to predictions of earlier models, this did not appear to derive from impaired GnRH self-priming. Collectively, these data indicate that PR function in gonadotropes may be limited to regulation of LH surge amplitude in female mice via a currently unknown mechanism.

Changes in GABAergic Transmission to and Intrinsic Excitability of Gonadotropin-Releasing Hormone (GnRH) Neurons during the Estrous Cycle in Mice.

Gonadotropin-releasing hormone (GnRH) neurons form the final common central output pathway controlling fertility and are regulated by steroid feedback. In females, estradiol feedback action varies between negative and positive; negative feedback typically regulates episodic GnRH release whereas positive feedback initiates a surge of GnRH, and subsequently luteinizing hormone (LH) release ultimately triggering ovulation. During the estrous cycle, changes between estradiol negative and positive feedback occur with cycle stage and time of day, with positive feedback in the late afternoon of proestrus in nocturnal species. To test the hypotheses that synaptic and intrinsic properties of GnRH neurons are regulated by cycle stage and time of day, we performed whole-cell patch-clamp studies of GnRH neurons in brain slices from mice at two times considered negative feedback (diestrous PM and proestrous AM) and during positive feedback (proestrous PM). GABAergic transmission can excite GnRH neurons and was higher in cells from proestrous PM mice than cells from proestrous AM mice and approached traditional significance levels relative to cells from diestrous PM mice. Action potential response to current injection was also greater in cells from proestrous PM mice than the other two groups. Interestingly, the hormonal milieu of proestrous AM provided stronger negative feedback on both GnRH neuron excitability and GABAergic postsynaptic current (PSC) amplitude than diestrous PM. These observations demonstrate elements of both synaptic and intrinsic properties of GnRH neurons are regulated in a cycle-dependent manner and provide insight into the neurobiological mechanisms underlying cyclic changes in neuroendocrine function among states of estradiol negative and positive feedback.

Estradiol Increases Glutamate and GABA Neurotransmission into GnRH Neurons via Retrograde NO-Signaling in Proestrous Mice during the Positive Estradiol Feedback Period.

Surge release of gonadotropin-releasing hormone (GnRH) is essential in the activation of pituitary gonadal unit at proestrus afternoon preceded by the rise of serum 17β-estradiol (E2) level during positive feedback period. Here, we describe a mechanism of positive estradiol feedback regulation acting directly on GnRH-green fluorescent protein (GFP) neurons of mice. Whole-cell clamp and loose patch recordings revealed that a high physiological dose of estradiol (200 pM), significantly increased firing rate at proestrus afternoon. The mPSC frequency at proestrus afternoon also increased, whereas it decreased at metestrus afternoon and had no effect at proestrus morning. Inhibition of the estrogen receptor β (ERβ), intracellular blockade of the Src kinase and phosphatidylinositol 3 kinase (PI3K) and scavenge of nitric oxide (NO) inside GnRH neurons prevented the facilitatory estradiol effect indicating involvement of the ERβ/Src/PI3K/Akt/nNOS pathway in this fast, direct stimulatory effect. Immunohistochemistry localized soluble guanylate cyclase, the main NO receptor, in both glutamatergic and GABAergic terminals innervating GnRH neurons. Accordingly, estradiol facilitated neurotransmissions to GnRH neurons via both GABAA-R and glutamate/AMPA/kainate-R. These results indicate that estradiol acts directly on GnRH neurons via the ERβ/Akt/nNOS pathway at proestrus afternoon generating NO that retrogradely accelerates GABA and glutamate release from the presynaptic terminals contacting GnRH neurons. The newly explored mechanism might contribute to the regulation of the GnRH surge, a fundamental prerequisite of the ovulation.

Estradiol-induced enhancement of fear extinction in female rats: The role of NMDA receptor activation

Converging cross-species evidence indicates that fear extinction (the laboratory basis of exposure therapy for anxiety disorders) in females is modulated by endogenous and exogenous estradiol. The mechanisms underlying estradiol's influences on fear extinction are largely undefined. However, one likely candidate is the NMDA-receptor (NMDAr), activation of which is necessary for estradiol-mediated enhancements in structural and functional neural plasticity, as well as extinction consolidation in males. Here, we demonstrate that systemic co-administration of the non-competitive NMDAr antagonist, MK801, blocked the enhancement of fear extinction by systemic estradiol in ovariectomized rats. In intact rats, MK801 during diestrus (rising estradiol) prevented the enhancement in extinction recall in rats that received extinction training during proestrus (peak estradiol). Systemic administration of the partial NMDAr agonist D-cycloserine (DCS) prior to extinction training facilitated extinction in ovariectomized rats, mimicking the effects of estradiol. In intact rats, DCS administered on the afternoon of proestrus and the morning of estrus (declining estradiol) facilitated extinction in rats that received extinction training during metestrus (low estradiol). Finally, DCS also facilitated extinction in ovariectomized rats when administered immediately after extinction training. Combined, these findings suggest that endogenous and exogenous estradiol enhance fear extinction via NMDAr-dependent mechanisms. Moreover, these findings raise the possibility that fear extinction deficits during periods of low endogenous estradiol levels can be reversed by increasing NMDAr activation via DCS administration, either well prior to, or immediately after, extinction training.

Roles of RFRP-3 in the Daily and Seasonal Regulation of Reproductive Activity in Female Syrian Hamsters.

In females, reproductive activity relies on proper integration of daily and environmental changes as well as cyclic sex-steroid feedback. This study sought to investigate the role of the hypothalamic Arg-Phe amide-related peptide (RFRP)-3 in the daily and seasonal control of reproductive activity in female Syrian hamsters by analyzing the RFRP system and investigating the effects of central administration of RFRP-3 at different reproductive stages. In long day-adapted sexually active female hamsters, the number of c-Fos-activated RFRP immunoreactive neurons was reduced in the afternoon of diestrus and proestrus; the latter was correlated with increased kisspeptin activity and the luteinizing hormone (LH) surge. Moreover, acute RFRP-3 administration decreased LH secretion when given midafternoon, before the LH surge, and had no effect at other time points of proestrus or diestrus. These data indicate that RFRP-3 exerts a tonic inhibition on LH secretion, which is lifted at the time of the preovulatory surge on the afternoon of proestrus. In short day-adapted sexually inactive female hamsters, Rfrp expression is strongly inhibited in a sex steroid-independent manner, and prolonged central infusion of RFRP-3 completely reactivated the reproductive axis through increased kisspeptin expression, gonadotropin and estradiol secretion, and gonadal weight. These findings reveal a critical role of RFRP-3 in the control of reproductive activity in female rodents and suggest that RFRP neurons, acting alongside kisspeptin neurons, are essential for proper synchronization of reproductive activity with the time of the day, the stage of the estrous cycle, and the seasonal changes in photoperiod.

The participation of the muscarinic receptors in the preoptic-anterior hypothalamic areas in the regulation of ovulation depends on the ovary.

BackgroundMuscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation.MethodsCyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 μl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB).ResultsAtropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection.ConclusionsThese results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.

Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.

Vasoactive Intestinal Peptide Excites GnRH Neurons in Male and Female Mice.

A variety of external and internal factors modulate the activity of GnRH neurons to control fertility in mammals. A direct, vasoactive intestinal peptide (VIP)-mediated input to GnRH neurons originating from the suprachiasmatic nucleus is thought to relay circadian information within this network. In the present study, we examined the effects of VIP on GnRH neuron activity in male and female mice at different stages of the estrous cycle. We carried out cell-attached recordings in slices from GnRH-green fluorescent protein mice and calcium imaging in slices from a mouse line expressing the genetically encoded calcium indicator GCaMP3 selectively in GnRH neurons. We show that 50%-80% of GnRH neurons increase their firing rate in response to bath-applied VIP (1nM-1000nM) in both male and female mice and that this is accompanied by a robust increase in intracellular calcium concentrations. This effect is mediated directly at the GnRH neuron likely through activation of high-affinity VIP receptors. Because suprachiasmatic nucleus-derived timing cues trigger the preovulatory surge only on the afternoon of proestrus in female mice, we examined the effects of VIP during the estrous cycle at different times of day. VIP responsiveness in GnRH neurons did not vary significantly in diestrous and proestrous mice before or around the time of the expected preovulatory surge. These results indicate that the majority of GnRH neurons in male and female mice express functional VIP receptors and that the effects of VIP on GnRH neurons do not alter across the estrous cycle.

The ventromedial hypothalamus oxytocin induces locomotor behavior regulated by estrogen

Our previous studies demonstrated that excitation of neurons in the rat ventromedial hypothalamus (VMH) induced locomotor activity. An oxytocin receptor (Oxtr) exists in the VMH and plays a role in regulating sexual behavior. However, the role of Oxtr in the VMH in locomotor activity is not clear. In this study we examined the roles of oxytocin in the VMH in running behavior, and also investigated the involvement of estrogen in this behavioral change. Microinjection of oxytocin into the VMH induced a dose-dependent increase in the running behavior in male rats. The oxytocin-induced running activity was inhibited by simultaneous injection of Oxtr-antagonist, (d(CH2)51, Try(Me)2, Orn8)-oxytocin. Oxytocin injection also induced running behavior in ovariectomized (OVX) female rats. Pretreatment of the OVX rats with estrogen augmented the oxytocin-induced running activity twofold, and increased the Oxtr mRNA in the VMH threefold. During the estrus cycle locomotor activity spontaneously increased in the dark period of proestrus. The Oxtr mRNA was up-regulated in the proestrus afternoon. Blockade of oxytocin neurotransmission by its antagonist before the onset of the dark period of proestrus decreased the following nocturnal locomotor activity. These findings demonstrate that Oxtr in the VMH is involved in the induction of running behavior and that estrogen facilitates this effect by means of Oxtr up-regulation, suggesting the involvement of oxytocin in the locomotor activity of proestrus female rats.

Excitability and Burst Generation of AVPV Kisspeptin Neurons Are Regulated by the Estrous Cycle Via Multiple Conductances Modulated by Estradiol Action.

The preovulatory secretory surge of gonadotropin-releasing hormone (GnRH) is crucial for fertility and is regulated by a switch of estradiol feedback action from negative to positive. GnRH neurons likely receive estradiol feedback signals via ERα-expressing afferents. Kisspeptin neurons in anteroventral periventricular nucleus (AVPV) are thought to be critical for estradiol-positive feedback induction of the GnRH surge. We examined the electrophysiological properties of GFP-identified AVPV kisspeptin neurons in brain slices from mice on the afternoon of diestrus (negative feedback) and proestrus (positive feedback, time of surge). Extracellular recordings revealed increased firing frequency and action potential bursts on proestrus versus diestrus. Whole-cell recordings were used to study the intrinsic mechanisms of bursting. Upon depolarization, AVPV kisspeptin neurons exhibited tonic firing or depolarization-induced bursts (DIB). Both tonic and DIB cells exhibited bursts induced by rebound from hyperpolarization. DIB occurred similarly on both cycle stages, but rebound bursts were observed more often on proestrus. DIB and rebound bursts were both sensitive to Ni2+, suggesting that T-type Ca2+ currents (ITs) are involved. IT current density was greater on proestrus versus diestrus. In addition to IT, persistent sodium current (INaP) facilitated rebound bursting. On diestrus, 4-aminopyridine-sensitive potassium currents contributed to reduced rebound bursts in both tonic and DIB cells. Manipulation of specific sex steroids suggests that estradiol induces the changes that enhance AVPV kisspeptin neuron excitability on proestrus. These observations indicate cycle-driven changes in circulating estradiol increased overall action potential generation and burst firing in AVPV kisspeptin neurons on proestrus versus diestrus by regulating multiple intrinsic currents.

Influence of dynorphin on estradiol- and cervical stimulation-induced prolactin surges in ovariectomized rats.

Prolactin is an anterior pituitary hormone necessary for fertility, pregnancy maintenance, lactation, and aspects of maternal behavior. In rodents, there is a surge of prolactin on the afternoon of proestrus, and a semi-circadian pattern of prolactin surges during early pregnancy, with a diurnal and nocturnal surge every day. Both of these patterns can be replicated in ovariectomized rats. A prior study demonstrated that central antagonism of κ-opioid receptors, the target of dynorphin, largely abolished the nocturnal prolactin surge in pregnant rats. We build on this to determine whether dynorphin, perhaps from the arcuate population that co-express kisspeptin, neurokinin B, and dynorphin (KNDy neurons), also contributes to the estradiol- or cervical stimulation-induced surges in ovariectomized rats. Ovariectomized rats were treated with either estradiol or cervical stimulation to induce prolactin surge(s). Blood samples were taken around the expected surge time to determine the effect of either acute κ-opioid receptor antagonism or previous chemical ablation of the KNDy population on prolactin levels. Dynorphin antagonism does significantly disrupt the nocturnal prolactin surge, but it does not contribute to the estradiol-induced surge. Chemical ablation of KNDy neurons had opposite effects; ablation of 40% of the KNDy neurons had no impact on the nocturnal prolactin surge, while a somewhat larger ablation significantly reduced the size of the estradiol-induced surge. We conclude that dynorphin is likely a controlling factor for the nocturnal surge induced by cervical stimulation, and that other KNDy neuron products must play a role in the estradiol-induced surge.

In vitro Autoradiographic Analysis of Regional Changes in Estrogen Receptor Alpha in the Brains of Cycling Female Rats

Background/Aims: The contributions of the three principal ovarian steroid hormones (estradiol, progesterone and testosterone) to the regulation of estrogen receptor alpha (ERα) levels in the rat brain were examined during the estrous cycle. Methods: Receptor concentrations were measured using an in vitro autoradiographic technique designed to separately quantify free, unoccupied receptors and receptors ‘occupied' by (bound to) endogenous hormone. Results: ERα occupation increased at proestrus and declined at estrus, reflecting changes in circulating estradiol and testosterone levels. Total ERα content followed a pattern that was the inverse of the occupation data, falling over the night of proestrus. Between 2.00 and 10.00 a.m. on the day of estrus, total ERα concentrations recovered in all brain regions except the ventromedial nucleus (VMN), in which ERα binding remained depressed at estrus. Administration of the progesterone antagonist mifepristone on the afternoon of proestrus resulted in recovery of ERα levels in the VMN by the morning of estrus, consistent with the hypothesis that the preovulatory progesterone surge selectively inhibits VMN ERα expression. Residual ERα occupation observed at estrus, when estradiol is not detectable in the serum, likely reflects intracranial aromatization of circulating androgens, since the pattern of receptor occupation observed at this stage of the cycle could be reproduced in ovariectomized rats by replacement with testosterone. Conclusion: These findings indicate that ERα binding in the brain fluctuates during the rat estrous cycle in a region-specific manner and suggest that local aromatization of testosterone may contribute significantly to ERα occupation when circulating estradiol levels are low.

Understanding the regulation of pituitary progesterone receptor expression and phosphorylation.

Administration of human FSH (hFSH) during the diestrus phase in cyclic rats is followed by a reduction in the preovulatory LH surge. This inhibitory action of FSH involves a decrease in the stimulatory effect of gonadotrope progesterone receptor (PR) activation, in a ligand-dependent (progesterone) and -independent (GNRH) manner. PR activation and action are mandatory for LH surge, and are dependent on the phosphorylation of serine (Ser) residues. Together with this post-translational modification, PR is marked for downregulation by proteasome machinery. These experiments used the western blotting technique to measure pituitary expression of PR-A and PR-B isoforms and phosphorylation levels of Ser294 and Ser400 PR-B in rats bearing i) hFSH treatment or ii) PR downregulation. Treatment with hFSH reduced LH secretion and increased that of estradiol in proestrus afternoon. hFSH injections, without altering PR-A and PR-B content or ratio, caused a reduction in phosphorylation of Ser294 and Ser400 but only when pituitaries were previously challenged with progesterone or GNRH for 2 h. However, while pSer294 levels increased after 2 h of pituitary incubation with progesterone or GNRH, those of pSer400 were not modified by these in vitro treatments. Finally, progesterone had a biphasic effect: in 2-h incubations increased pituitary PR-A and PR-B content, but after 8 h caused downregulation and altered PR-A:PR-B ratio. The results provide a potential mechanism through which LH levels are decreased by hFSH administration and better understanding of the control of PR expression and phosphorylation in rat pituitaries.

1‐ Exposure to ambient ozone affects circulating progesterone levels and ovulation in mice.

Ambient ozone reacts with biological molecules and causes lung inflammation, oxidative stress and other health effects. Our preliminary research has shown that oxidative stress can affect the expression of fertility in rodents. Here, we investigated the effects of ozone exposure on the female reproductive axis. For this, cycling female mice were exposed to ozone (2ppm) or filter air (FA) for 3 h in the morning of proestrous (n=18) and sacrificed 4 h post‐exposure (proestrous evening). The hypothalamus, pituitary, and ovaries were harvested and pulverized, and RNA was extracted to determine gene expression of GnRH1, GnRH receptor and LH receptor. Ovarian protein expression of StAR and HSD3B1 was also detected through Western Blot. Blood was collected to determine serum pituitary and ovarian hormonal levels. A second group of animals (n=12) was sacrificed 24 h post‐exposure (morning of estrus). Ovaries were collected and histologically analyzed to determine the number of pre‐ovulatory follicules and corpora lutea. Blood was collected to compare post‐ovulatory hormone levels. We found that ozone exposure in the afternoon of proestrus decreased both hypothalamic expression of GnRH and GnRH‐receptor and circulating progesterone levels. Histological analysis in the morning of estrus revealed that ovaries from females exposed to ozone had more preovulatory follicules and less corpora lutea than females exposed to FA (p<0.05). Together, these results indicate that ozone exposure impairs ovulation in mice, potentially via affecting steroidogenesis and/or folliculogenesis.

Estrogen-dependent post-translational change in the nitric oxide system may mediate the leptin action on LH and prolactin secretion

Gonadotrophin-releasing hormone (GnRH) neurons do not express the leptin receptor (OB-R) in the medial preoptic area (MPOA) and the medial basal hypothalamus (MBH). We assessed whether the effect of leptin on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in proestrus could be mediated by nitric oxide (NO) under estrogen modulation. Female rats were treated with an estrogen antagonist (tamoxifen s.c. 3mg/rat) or vehicle during metestrus and diestrus. At proestrus, they received leptin (3 or 10μg/μl) or intracerebroventricular saline at 11:00am and were decapitated at 5:00pm. The following were analyzed in this work: plasma LH, FSH and PRL levels (radioimmunoassay); neuronal NO-synthase (nNOS) and OB-R transcription (RT-PCR); nNOS and phosphorylated nNOS (pnNOS) translation levels (western blotting); and pSTAT3 immunoreactivity. Tamoxifen reduced the plasma LH and PRL levels and decreased the nNOS mRNA and pnNOS expression in the MPOA. Three micrograms of leptin increased the LH secretion and pnNOS protein levels in the MPOA and MBH. Ten micrograms of leptin decreased the transcription, translation and phosphorylation of nNOS in the MPOA. In the MBH, 10μg of leptin increased the protein expression of nNOS but not the mRNA expression neither pnNOS protein. Tamoxifen did not change either the mRNA or protein expression of nNOS or the phosphorylation of nNOS but decreased the number of cells that contained pSTAT3 immunoreactivity in both areas. In conclusion, the stimulatory effect of leptin on the secretion of LH and PRL on the afternoon of proestrus may be mediated by estrogen-dependent post-translational changes in the nNOS in the MPOA and MBH.