Design, Implementation, and Evaluation of an Intensive Course on Issues in Women's Health and Gender-Based Medicine.

OBJECTIVES: Sex and gender have profound effects on disease prevalence, presentation, and outcome, but these issues are not covered in depth in standard medical school curricula. To improve understanding of women's health, an intensive 1-month class was offered to fourth-year medical students. METHODS: The class combined background lectures on the biological and social determinants of women's health with presentations on specific medical conditions by practicing clinicians and students. Students' anonymous responses to end-of-class evaluation used by Stony Brook University School of Medicine as well as pre- and post-class answers to the question "why are women twice as likely to go to the doctor" were analyzed using quantitative, descriptive, and qualitative approaches. RESULTS: The class was given between 2017 and 2022 to a total of 154 students. Course evaluations were submitted by 133 students. Over 80% of responders ranked the class as good or excellent and many expressed surprise about how much sex and gender influence health. Furthermore, before taking the class responders favored gender stereotypes (82%) and OB/GYN visits (56%) as the main reasons why women utilize healthcare more often than men, whereas only 31% of post-class answers included these factors (p < .0001), which were replaced by others including misdiagnosis, high rate of adverse effects of medications, implicit bias, and longevity. CONCLUSION: A dedicated class given to students at the end of their undergraduate medical training increased awareness and knowledge of the effects of sex and gender on women's health.

Hypothalamic-Pituitary-Gonadal Axis Disorders Impacting Fertility in Both Sexes and the Potential of Kisspeptin-Based Therapies to Treat Them.

Impaired function of the hypothalamic-pituitary-gonadal (HPG) axis can lead to a vast array of reproductive disorders some of which are inherited or acquired, but many are of unknown etiology. Among the clinical consequences of HPG impairment, infertility is quite common. According to the latest report from the World Health Organization, the global prevalence of infertility during a person's lifetime is a staggering 17.5% which translate into 1 out of every 6 people experiencing it. In both sexes, infertility is associated with adverse health events, and if unresolved, infertility can cause substantial psychological stress, social stigmatization, and economic strain. Even though significant advances have been made in the management and treatment of infertility, low or variable efficacy of treatments and medication adverse effects still pose a significant problem. However, the discovery that in humans inactivating mutations in the gene encoding the kisspeptin receptor (Kiss1R) results in pubertal failure and infertility has expanded our understanding of the mechanisms underlying the neuroendocrine control of reproduction, opening up potential new therapies for the treatment of infertility disorders. In this chapter we provide an overview of common infertility disorders affecting men and women, their recommended treatments, and the potential of kisspeptin-based pharmacotherapies to treat them.

The PI3K/Akt Pathway in Meta-Inflammation.

Obesity is a global epidemic representing a serious public health burden as it is a major risk factor for the development of cardiovascular disease, stroke and all-cause mortality. Chronic low-grade systemic inflammation, also known as meta-inflammation, is thought to underly obesity's negative health consequences, which include insulin resistance and the development of type 2 diabetes. Meta-inflammation is characterized by the accumulation of immune cells in adipose tissue, a deregulation in the synthesis and release of adipokines and a pronounced increase in the production of proinflammatory factors. In this state, the infiltration of macrophages and their metabolic activation contributes to complex paracrine and autocrine signaling, which sustains a proinflammatory microenvironment. A key signaling pathway mediating the response of macrophages and adipocytes to a microenvironment of excessive nutrients is the phosphoinositide 3-kinase (PI3K)/Akt pathway. This multifaceted network not only transduces metabolic information but also regulates macrophages' intracellular changes, which are responsible for their phenotypic switch towards a more proinflammatory state. In the present review, we discuss how the crosstalk between macrophages and adipocytes contributes to meta-inflammation and provide an overview on the involvement of the PI3K/Akt signaling pathway, and how its impairment contributes to the development of insulin resistance.

Shaping Microglial Phenotypes Through Estrogen Receptors: Relevance to Sex-Specific Neuroinflammatory Responses to Brain Injury and Disease.

In mammals, 17ß-estradiol (E2), the primary endogenous estrogen, maintains normal central nervous system (CNS) function throughout life and influences brain responses to injury and disease. Estradiol-induced cellular changes are mediated through the activation of nuclear and extranuclear estrogen receptors (ERs), which include ERα, ERß, and the G-protein coupled receptor, GPER1. ERs are widely expressed throughout the brain, acting as transcriptional effectors or rapidly initiating membrane and cytoplasmic signaling cascades in practically all brain cells including microglia, the resident immune cells of the CNS. Activation of ERs by E2 exerts potent anti-inflammatory effects through mechanisms involving the modification of microglial cell responses to acute or chronic brain injury. Recent studies suggest that microglial maturation is influenced by the internal gonadal hormone milieu and that their functions in the normal and diseased brain are sex specific. However, the role that each ER subtype plays in microglial development or in determining microglial function as the primary cellular defense mechanism against pathogens and injury remains unclear. This is partly due to the fact that most studies investigating the mechanisms by which E2-ER signaling modifies microglial cellular phenotypes have been restricted to one sex or age. This review examines the different in vivo and in vitro models used to study the direct and indirect regulation of microglial cell function by E2 through ERs. Ischemic stroke will be used as an example of a neurologic disease in which activation of ERs shapes microglial phenotypes in response to injury in a sex- and age-specific fashion. SIGNIFICANCE STATEMENT: As the primary immune sensors of central nervous system damage, microglia are important potential therapeutic targets. Estrogen receptor signaling modulates microglial responses to brain injury and disease in a sex- and age-specific fashion. Hence, investigating the molecular mechanisms by which estrogen receptors regulate and shape microglial functions throughout life may result in novel and effective therapeutic opportunities that are tailored for each sex and age.

Targeted Deletion of PTEN in Kisspeptin Cells Results in Brain Region- and Sex-Specific Effects on Kisspeptin Expression and Gonadotropin Release.

Kisspeptin-expressing neurons in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) of the hypothalamus relay hormonal and metabolic information to gonadotropin-releasing hormone neurons, which in turn regulate pituitary and gonadal function. Phosphatase and tensin homolog (PTEN) blocks phosphatidylinositol 3-kinase (PI3K), a signaling pathway utilized by peripheral factors to transmit their signals. However, whether PTEN signaling in kisspeptin neurons helps to integrate peripheral hormonal cues to regulate gonadotropin release is unknown. To address this question, we generated mice with a kisspeptin cell-specific deletion of Pten (Kiss-PTEN KO), and first assessed kisspeptin protein expression and gonadotropin release in these animals. Kiss-PTEN KO mice displayed a profound sex and region-specific kisspeptin neuron hyperthrophy. We detected both kisspeptin neuron hyperthrophy as well as increased kisspeptin fiber densities in the AVPV and ARC of Kiss-PTEN KO females and in the ARC of Kiss-PTEN KO males. Moreover, Kiss-PTEN KO mice showed a reduced gonadotropin release in response to gonadectomy. We also found a hyperactivation of mTOR, a downstream PI3K target and central regulator of cell metabolism, in the AVPV and ARC of Kiss-PTEN KO females but not males. Fasting, known to inhibit hypothalamic kisspeptin expression and luteinizing hormone levels, failed to induce these changes in Kiss-PTEN KO females. We conclude that PTEN signaling regulates kisspeptin protein synthesis in both sexes and that its role as a metabolic signaling molecule in kisspeptin neurons is sex-specific.

Loss of PI3K p110α in the Adipose Tissue Results in Infertility and Delayed Puberty Onset in Male Mice.

Deletion of PI3K catalytic subunit p110α in adipose tissue (aP2-Cre/p110αflx/flx, α-/- hereafter) results in increased adiposity, glucose intolerance, and liver steatosis. Because this endocrine organ releases hormones like leptin, which are important in reproductive physiology, we investigated the reproductive phenotype of α-/- males. Compared to controls, α-/- males displayed delayed onset of puberty accompanied by a reduction in plasma LH levels and testicular weight. At postnatal day 30, α-/- mice exhibited normal body weight but elevated fasted plasma leptin levels. Testicular leptin gene expression was increased, whereas expression of the cholesterol transporter StAR and of P450 cholesterol side chain cleavage enzyme was decreased. Adult α-/- males were infertile and exhibited hyperandrogenemia with normal basal LH, FSH, and estradiol levels. However, neither sperm counts nor sperm motility was different between genotypes. The mRNA levels of leptin and of 17-beta-dehydrogenase 3, and enzyme important for testosterone production, were significantly higher in the testis of adult α-/- males. The mRNA levels of ERα, an important regulator of intratesticular steroidogenesis, were lower in the testis of adult and peripubertal α-/- males. We propose that chronic hyperleptinemia contributes to the negative impact that disrupting PI3K signaling in adipocytes has on puberty onset, steroidogenesis, and fertility in males.

Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons.

Hypothalamic kisspeptin (Kiss1) neurons express estrogen receptor α (ERα) and exert control over GnRH/LH secretion in female rodents. It has been proposed that estradiol (E2) activation of ERα in kisspeptin neurons in the arcuate nucleus (ARC) suppresses GnRH/LH secretion (negative feedback), whereas E2 activation of ERα in kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) mediates the release of preovulatory GnRH/LH surges (positive feedback). To test these hypotheses, we generated mice bearing kisspeptin cell-specific deletion of ERα (KERαKO) and treated them with E2 regimens that evoke either negative or positive feedback actions on GnRH/LH secretion. Using negative feedback regimens, as expected, E2 effectively suppressed LH levels in ovariectomized (OVX) wild-type (WT) mice to the levels seen in ovary-intact mice. Surprisingly, however, despite the fact that E2 regulation of Kiss1 mRNA expression was abrogated in both the ARC and AVPV of KERαKO mice, E2 also effectively decreased LH levels in OVX KERαKO mice to the levels seen in ovary-intact mice. Conversely, using a positive feedback regimen, E2 stimulated LH surges in WT mice, but had no effect in KERαKO mice. These experiments clearly demonstrate that ERα in kisspeptin neurons is required for the positive, but not negative feedback actions of E2 on GnRH/LH secretion in adult female mice. It remains to be determined whether the failure of KERαKO mice to exhibit GnRH/LH surges reflects the role of ERα in the development of kisspeptin neurons, in the active signaling processes leading to the release of GnRH/LH surges, or both.

Kisspeptin cell-specific PI3K signaling regulates hypothalamic kisspeptin expression and participates in the regulation of female fertility.

Hypothalamic kisspeptin neurons integrate and translate cues from the internal and external environments that regulate gonadotropin-releasing hormone (GnRH) secretion and maintain fertility in mammals. However, the intracellular signaling pathways utilized to translate such information into changes in kisspeptin expression, release, and ultimately activation of the kisspeptin-receptive GnRH network have not yet been identified. PI3K is an important signaling node common to many peripheral factors known to regulate kisspeptin expression and GnRH release. We investigated whether PI3K signaling regulates hypothalamic kisspeptin expression, pubertal development, and adult fertility in mice. We generated mice with a kisspeptin cell-specific deletion of the PI3K catalytic subunits p110α and p110ß (kiss-p110α/ß-KO). Using in situ hybridization, we examined Kiss1 mRNA expression in gonad-intact, gonadectomized (Gdx), and Gdx + steroid-replaced mice. Kiss1 cell number in the anteroventral periventricular hypothalamus (AVPV) was significantly reduced in intact females but not in males. In contrast, compared with WT and regardless of steroid hormone status, Kiss1 cell number was lower in the arcuate (ARC) of kiss-p110α/ß-KO males, but it was unaffected in females. Both intact Kiss-p110α/ß-KO males and females had reduced ARC kisspeptin-immunoreactive (IR) fibers compared with WT animals. Adult kiss-p110α/ß-KO males had significantly lower circulating luteinizing hormone (LH) levels, whereas pubertal development and fertility were unaffected in males. Kiss-p110α/ß-KO females exhibited a reduction in fertility despite normal pubertal development, LH levels, and estrous cyclicity. Our data show that PI3K signaling is important for the regulation of hypothalamic kisspeptin expression and contributes to normal fertility in females.

PI3K: An Attractive Candidate for the Central Integration of Metabolism and Reproduction.

In neurons, as in a variety of other cell types, the enzyme phosphatidylinositol-3-kinase (PI3K) is a key intermediate that is common to the signaling pathways of a number of peripheral metabolic cues, including insulin and leptin, which are well known to regulate both metabolic and reproductive functions. This review article will explore the possibility that PI3K is a key integrator of metabolic and neural signals regulating gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release and explore the hypothesis that this enzyme is pivotal in many disorders where gonadotropin release is at risk. Although the mechanisms mediating the influence of metabolism and nutrition on fertility are currently unclear, the strong association between metabolic disorders and infertility is undeniable. For example, women suffering from anorectic disorders experience amenorrhea as a consequence of malnutrition-induced impairment of LH release, and at the other extreme, obesity is also commonly co-morbid with menstrual dysfunction and infertility. Impaired hypothalamic insulin and leptin receptor signaling is thought to be at the core of reproductive disorders associated with metabolic dysfunction. While low levels of leptin and insulin characterize states of negative energy balance, prolonged nutrient excess is associated with insulin and leptin resistance. Metabolic models known to alter GnRH/LH release such as diabetes, diet-induced obesity, and caloric restriction are also accompanied by impairment of PI3K signaling in insulin and leptin sensitive tissues including the hypothalamus. However, a clear link between this signaling pathway and the control of GnRH release by peripheral metabolic cues has not been established. Investigating the role of the signaling pathways shared by metabolic cues that are critical for a normal reproductive state can help identify possible targets in the treatment of metabolic and reproductive disorders such as polycystic ovarian syndrome.

Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons.

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms--a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor α (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus.

Male-biased effects of gonadotropin-releasing hormone neuron-specific deletion of the phosphoinositide 3-kinase regulatory subunit p85alpha on the reproductive axis.

GnRH neurosecretion is subject to regulation by insulin, IGF-I, leptin, and other neuroendocrine modulators whose effects may be conveyed by activation of phosphoinositide 3-kinase (PI3K)-mediated pathways. It is not known, however, whether any of these regulatory actions are exerted directly, via activation of PI3K in GnRH neurons, or whether they are primarily conveyed via effects on afferent circuitries governing GnRH neurosecretion. To investigate the role of PI3K signaling in GnRH neurons, we used conditional gene targeting to ablate expression of the major PI3K regulatory subunit, p85alpha, in GnRH neurons. Combined in situ hybridization and immunohistochemistry confirmed reduction of p85alpha mRNA expression in GnRH neurons of GnRH-p85alpha knockout (KO) animals. Females of both genotypes exhibited estrous cyclicity and had comparable serum LH, estradiol-17beta, and FSH levels. In male GnRH-p85alphaKO mice, serum LH, testosterone, and sperm counts were significantly reduced compared with wild type. To investigate the role of the other major regulatory subunit, p85beta, on the direct control of GnRH neuronal function, we generated mice with a GnRH-neuron-specific p85alpha deletion on a global betaKO background. No additional reproductive effects in male or female mice were found, suggesting that p85beta does not substitute p85 activity toward PI3K function in GnRH neurons. Our results suggest that p85alpha, and thus PI3K activity, participates in the control of GnRH neuronal activity in male mice. The sex-specific phenotype in these mice raises the possibility that PI3K activation during early development may establish sex differences in GnRH neuronal function.

Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels.

Reproductive hormone secretions are inhibited by fasting and restored by feeding. Metabolic signals mediating these effects include fluctuations in serum glucose, insulin, and leptin. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose sensing and many actions of insulin and leptin in neurons, we assessed their role in suppressing LH secretion during food restriction. Vehicle or a K(ATP) channel blocker, tolbutamide, was infused into the lateral cerebroventricle in ovariectomized mice that were either fed or fasted for 48 h. Tolbutamide infusion resulted in a twofold increase in LH concentrations in both fed and fasted mice compared with both fed and fasted vehicle-treated mice. However, tolbutamide did not reverse the suppression of LH in the majority of fasted animals. In sulfonylurea (SUR)1-null mutant (SUR1(-/-)) mice, which are deficient in K(ATP) channels, and their wild-type (WT) littermates, a 48-h fast was found to reduce serum LH concentrations in both WT and SUR(-/-) mice. The present study demonstrates that 1) blockade of K(ATP) channels elevates LH secretion regardless of energy balance and 2) acute fasting suppresses LH secretion in both SUR1(-/-) and WT mice. These findings support the hypothesis that K(ATP) channels are linked to the regulation of gonadotropin-releasing hormone (GnRH) release but are not obligatory for mediating the effects of fasting on GnRH/LH secretion. Thus it is unlikely that the modulation of K(ATP) channels either as part of the classical glucose-sensing mechanism or as a component of insulin or leptin signaling plays a major role in the suppression of GnRH and LH secretion during food restriction.

Neuroendocrine consequences of androgen excess in female rodents.

Androgens exert significant organizational and activational effects on the nervous system and behavior. Despite the fact that female mammals generally produce low levels of androgens, relative to the male of the same species, increasing evidence suggests that androgens can exert profound effects on the normal physiology and behavior of females during fetal, neonatal, and adult stages of life. This review examines the effects of exposure to androgens at three stages of development--as an adult, during early postnatal life and as a fetus, on reproductive hormone secretions in female rats. We examine the effects of androgen exposure both as a model of neuroendocrine sexual differentiation and with respect to the role androgens play in the normal female. We then discuss the hypothesis that androgens may cause epigenetic modification of estrogen target genes in the brain. Finally we consider the clinical consequences of excess androgen exposure in women.

Ovarian steroids stimulate adenosine triphosphate-sensitive potassium (KATP) channel subunit gene expression and confer responsiveness of the gonadotropin-releasing hormone pulse generator to KATP channel modulation.

The ATP-sensitive potassium (K(ATP)) channels couple intracellular metabolism to membrane potential. They are composed of Kir6.x and sulfonylurea receptor (SUR) subunits and are expressed in hypothalamic neurons that project to GnRH neurons. However, their roles in regulating GnRH secretion have not been determined. The present study first tested whether K(ATP) channels regulate pulsatile GnRH secretion, as indirectly reflected by pulsatile LH secretion. Ovariectomized rats received sc capsules containing oil, 17beta-estradiol (E(2)), progesterone (P), or E(2)+P at 24 h before blood sampling. Infusion of the K(ATP) channel blocker tolbutamide into the third ventricle resulted in increased LH pulse frequency in animals treated with E(2)+P but was without effect in all other groups. Coinfusion of tulbutamide and the K(ATP) channel opener diazoxide blocked this effect, whereas diazoxide alone suppressed LH. Effects of steroids on Kir6.2 and SUR1 mRNA expression were then evaluated. After 24hr treatment, E(2)+P produced a modest but significant increase in Kir6.2 expression in the preoptic area (POA), which was reversed by P receptor antagonism with RU486. Neither SUR1 in the POA nor both subunits in the mediobasal hypothalamus were altered by any steroid treatment. After 8 d treatment, Kir6.2 mRNA levels were again enhanced by E(2)+P but to a greater extent in the POA. Our findings demonstrate that 1) blockade of preoptic/hypothalamic K(ATP) channels produces an acceleration of the GnRH pulse generator in a steroid-dependent manner and 2) E(2)+P stimulate Kir6.2 gene expression in the POA. These observations are consistent with the hypothesis that the negative feedback actions of ovarian steroids on the GnRH pulse generator are mediated, in part, by their ability to up-regulate K(ATP) channel subunit expression in the POA.

Regulation of KATP channel subunit gene expression by hyperglycemia in the mediobasal hypothalamus of female rats.

The ATP-sensitive potassium (K(ATP)) channels are gated by intracellular adenine nucleotides coupling cell metabolism to membrane potential. Channels comprised of Kir6.2 and SUR1 subunits function in subpopulations of mediobasal hypothalamic (MBH) neurons as an essential component of a glucose-sensing mechanism in these cells, wherein uptake and metabolism of glucose leads to increase in intracellular ATP/ADP, closure of the channels, and increase in neuronal excitability. However, it is unknown whether glucose and/or insulin may also regulate the gene expression of the channel subunits in the brain. The present study investigated whether regulation of K(ATP) channel subunit gene expression might be a mechanism by which neuronal populations adapt to prolonged changes in glucose and/or insulin levels in the periphery. Ovariectomized, steroid-replaced rats were fitted with indwelling jugular catheters and infused for 48 h with saline, glucose (hyperglycemia-hyperinsulinemia), insulin and glucose (hyperinsulinemia), diazoxide (control), or glucose and diazoxide (hyperglycemia). At the end of infusions, the MBH, preoptic area, and pituitary were dissected for RNA isolation and RT-PCR. Hyperglycemia decreased Kir6.2 mRNA levels in the MBH in both the presence and absence of hyperinsulinemia. These same conditions also produced a trend toward decreased SUR1 mRNA levels in the MBH; however, it did not exceed statistical significance. Hyperglycemia increased whereas hyperinsulinemia reduced neuropeptide Y mRNA levels when these groups were compared with each other. However, neither was significantly different from values observed in saline-infused controls. In conclusion, hyperglycemia per se may alter expression of K(ATP) channels and thereby induce changes in the excitability of some MBH neurons.

Estrogen receptors in neuropeptide Y neurons: at the crossroads of feeding and reproduction.

Hypothalamic neuropeptide Y (NPY) neurons function as physiological integrators in at least two different neuroendocrine systems - one governing feeding and the other controlling reproduction. Estrogen might modulate both systems by regulating NPY gene expression; it might reduce food intake by suppressing NPY expression, and evoke reproductive hormone surges by stimulating it. How can estrogen exert opposing effects in an ostensibly homogeneous NPY neuronal population? Recent work with immortalized NPY-producing cells suggests that the ratio of estrogen receptor alpha:estrogen receptor beta can determine the direction and temporal pattern of transcriptional responses to estrogen. Because this ratio might itself be physiologically regulated, these findings provide one explanation for multiple neuropeptidergic responses to a single steroid hormone.

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors.

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the delta opioid receptor agonist [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because delta opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by delta opioid receptors.

Participation of growth factor signal transduction pathways in estradiol facilitation of female reproductive behavior.

Estradiol (E(2)) regulates female reproductive behavior (lordosis) by acting on estrogen-sensitive neurons. We recently showed that E(2) facilitation of lordosis behavior requires concurrent activation of brain IGF-I receptors. The present study confirmed this finding and sought to identify the downstream signaling pathways involved in estrogen/IGF-I priming of lordosis. Intracerebroventricular infusions of a selective IGF-I receptor antagonist were administered to ovariectomized rats every 12 h beginning 1 h before the first of two daily E(2) injections. IGF-I receptor blockade partially inhibits lordosis if the antagonist is infused throughout the 2-d estrogen treatment period but not if it is administered only during the first or last 12 h of estrogen treatment. Because E(2) and IGF-I can activate phosphatidylinositol-3-kinase (PI3K) and MAPK, we infused agents that block PI3K and/or MAPK activity as described above. Both PI3K inhibitors (wortmannin and LY294002) and MAPK inhibitors (PD98059 and U0126) partially attenuate lordosis when administered during estrogen priming. None of these drugs modifies lordosis if they are infused only once, during the last 12 h of estrogen treatment. When both wortmannin and PD98059 are infused during E(2) priming, lordosis behavior is completely abolished. These data suggest that activation of both PI3K and MAPK by E(2) and IGF-I mediates hormonal facilitation of lordosis behavior.

Estrogen modulation of mu-opioid receptor-stimulated [35S]-GTP-gamma-S binding in female rat brain visualized by in vitro autoradiography.

The mu-opioid receptor (OR) is involved in several aspects of female reproductive neuroendocrinology, such as the control of gonadotropin release and the display of lordosis behavior. Even though the neuroendocrine events modulated by mu-ORs are steroid hormone-dependent, few studies have shown how steroid hormones such as estrogen and/or progesterone can affect mu-OR function. Therefore, the present study investigated if in vivo estrogen or estrogen plus progesterone treatment of ovariectomized (OVX) rats affects mu-OR coupling to its G proteins. We used autoradiographic analysis of agonist-stimulated [(35)S]-GTPgammaS binding, in which brain sections were incubated in the presence or absence of the mu-OR agonist [D-Ala(2), N-Me-Phe(4), Gly(2)ol]-enkephalin (DAMGO). Film images were quantified using calibrated [(14)C] standards. Analysis was performed in steroid-responsive hypothalamic regions such as the medial preoptic area (mPOA) and the ventromedial nucleus of the hypothalamus, as well as in non-hypothalamic brain regions. Treatment with estrogen, alone or with progesterone, significantly increased DAMGO-stimulated [(35)S]-GTPgammaS binding in the mPOA when compared to control OVX animals. In addition, estrogen increased mu-OR coupling in the caudate putamen. Steroid treatment had no effect on either basal or DAMGO-stimulated binding in the other brain regions examined. These findings suggest that estrogen modulates mu-OR function in a brain region-specific fashion. This could have important implications in terms of how these hormones synchronize reproductive behavior and gonadotropin release.

The role of delta-opioid receptors in estrogen facilitation of lordosis behavior.

The present study investigated the role of delta-opioid receptors (ORs) in estrogen facilitation of female rat reproductive behavior (lordosis). Infusion of 2 microg of the selective delta-OR agonist [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE), into the third ventricle facilitated lordosis behavior in ovariectomized (OVX) rats injected with estrogen (E) 48 and 24 h before behavioral testing. Pretreatment with the selective delta-OR antagonist naltrindole (NTDL) blocked DPDPE effects on lordosis behavior. Ventricular infusion of NTDL (40 microg) also suppressed lordosis behavior in fully receptive OVX rats primed with both E and progesterone (P). In addition, NTDL blocked lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMH) but not into the medial preoptic area (mPOA). Site-specific infusion of DPDPE into the VMH had dose-dependent, dual effects on lordosis behavior. While a very low dose of DPDPE (0.01 microg) facilitated lordosis behavior, a higher dose (1.0 microg) inhibited receptivity in OVX rats primed with E and a low dose (50 microg) of P. We used 3H-DPDPE to measure the density of delta-ORs in OVX rats treated with vehicle or with E by receptor autoradiography. E treatment did not have any effect on the density of DPDPE binding sites in the VMH, mPOA, medial amygdala, or caudate putamen. The behavioral effects of the ligands used in this study suggest that activation of delta-OR in the VMH by endogenous opioids facilitates estrogen-dependent lordosis behavior.