Kevin Sinchak

Direct regulation of adult brain function by the male-specific factor SRY.

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Membrane Estrogen Receptor-α Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats

In rats, female sexual behavior is regulated by a well defined limbic–hypothalamic circuit that integrates sensory and hormonal information. Estradiol activation of this circuit results in μ-opioid receptor (MOR) internalization in the medial preoptic nucleus, an important step for full expression of sexual receptivity. Estradiol acts through both membrane and intracellular receptors to influence neuronal activity and behavior, yet the mechanism(s) and physiological significance of estradiol-mediated membrane responses in vivo have remained elusive. Recent in vitro evidence found that stimulation of membrane-associated estrogen receptor-α (ERα) led to activation of metabotropic glutamate receptor 1a (mGluR1a). Furthermore, mGluR1a signaling was responsible for the observed downstream effects of estradiol. Here we present data that show that ERα and mGluR1a directly interact to mediate a rapid estradiol-induced activation of MOR in the medial preoptic nucleus, leading to female sexual receptivity. In addition, blockade of mGluR1a in the arcuate nucleus of the hypothalamus resulted in a significant attenuation of estradiol-induced MOR internalization, leading to diminished female sexual behavior. These results link membrane-initiated estradiol actions to neural events modulating behavior, demonstrating the physiological importance of ERα-to-mGluR1a signaling.

The Luteinizing Hormone Surge Is Preceded by an Estrogen-Induced Increase of Hypothalamic Progesterone in Ovariectomized and Adrenalectomized Rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17β-estradiol (50 µg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.

Neuroprogesterone: Key to estrogen positive feedback?

In the cycling female rat, estradiol and progesterone induce reproductive behavior and the surge of luteinizing hormone (LH) needed for ovulation. Circulating estradiol of ovarian origin induces progesterone receptors in the preoptic area and hypothalamus. Sequential activation of estrogen receptors (ER) and progesterone receptors coordinates reproductive physiology and behavior. In ovariectomized and adrenalectomized (ovx/adx) rats, administration of estradiol alone is sufficient to initiate an LH surge, and central infusion of aminoglutethimide (AGT), a blocker of the P450 side chain cleavage enzyme, disrupted the estrous cycle of intact rats without affecting peripheral estradiol levels, suggesting that an endogenous source of progesterone remains in these animals. In ovx/adx rats, progesterone levels in the hypothalamus increase prior to the LH surge, and inhibition of progesterone synthesis prevents the LH surge, suggesting that hypothalamic neuroprogesterone is necessary for estrogen positive feedback. In support of the idea that estradiol induces neuroprogesterone, estradiol increased expression of the progesterone-synthesizing enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the hypothalamus before the LH surge. Further, in vitro experiments demonstrate that estradiol stimulates progesterone synthesis in astrocytes, considered to be the most active steroidogenic cells in the CNS. To stimulate neurosteroidogenesis, estradiol acts through membrane ER and type 1a metabotropic glutamate receptors (mGluR1a) to increase free cytoplasmic calcium ([Ca(2+)](i)) via activation of the PLC-IP(3) pathway. Estradiol-induced progesterone synthesis is mimicked by thapsigargin-induced release of IP(3) receptor-sensitive Ca(2+) stores in astrocyte cultures. Thus, estradiol-induced progesterone synthesis is dependent on membrane ERs that act through mGluR1a to activate the PLC-IP(3) pathway. This neuroprogesterone also facilitated proceptive behavior. Blocking either progesterone synthesis or progesterone receptor in estrogen-primed ovx/adx prevented proceptive but not receptive behaviors.

Estrogen Induces de novo Progesterone Synthesis in Astrocytes

The brain is an established target for peripheral steroids, but also expresses steroidogenic enzymes and is capable of de novo ‘sex’ steroid synthesis (neurosteroidogenesis) independent of peripheral steroidogenic organs. In adrenalectomized and ovariectomized rats that do not have peripheral sources of steroids, estrogen treatment increased progesterone levels specifically in the hypothalamus, indicating that estrogen stimulates progesterone neurosteroidogenesis. Recent studies have demonstrated that specific cell types preferentially secrete specific steroids, and that astrocytes are the primary progesterone synthesizing cells in the nervous system. We hypothesized that estrogen could directly induce de novo synthesis of progesterone in astrocytes. To determine whether estrogen stimulates progesterone synthesis in astrocytes, astrocyte-enriched cultures were grown to confluence, then grown for an additional 48 h in an estrogen- and phenol-free Dulbecco’s Modified Eagle Medium (DMEM) and then treated with either 17β-estradiol or steroid-free media. After culturing for 48 h in steroid-free, phenol red-free DMEM, low levels of progesterone were detected in the media, whereas progesterone levels were significantly increased in the media of astrocytes cultured in DMEM with 17β-estradiol (10–7–10–4M). To determine whether estrogen regulated the mRNA expression of progesterone synthetic enzymes, P-450 side-chain cleavage and 3β-hydroxysteroid dehydrogenase, control and 17β-estradiol-treated astrocytes were harvested and prepared for Northern and slot blot analysis. Expression levels of enzyme mRNAs were very low and 17β-estradiol did not significantly increase mRNA levels of either steroidogenic enzyme. These results suggest that estrogen directly stimulated the de novo synthesis of neuroprogesterone in astrocytes, and demonstrate the potential for estrogen to regulate reproductive physiology and behavior through the paracrine actions of astrocyte-derived progesterone.

Estrogen receptor‐α is required for estrogen‐induced μ‐opioid receptor internalization

Endogenous opioid circuits are pivotal for the regulation of sexual receptivity. Treatment of mice with morphine, a preferential μ‐opioid receptor (MOR) agonist, severely attenuates lordosis. Estrogen induces internalization of MOR in cell groups of the limbic‐hypothalamic lordosis‐regulating circuit. Because rapid MOR internalization is mediated by estrogen release of endogenous opioid peptides, internalization has been used as a neurochemical signature of estrogen action in the central nervous system. Together these results indicate that estrogen induces a MOR mediated inhibition of sexual receptivity. To determine which estrogen receptor, estrogen receptor‐α (ERα) or estrogen receptor‐β (ERβ), mediates MOR internalization, ERα knockout (ERαKO), ERβ knockout (ERβKO) and wild‐type (WT) mice were used in the present study. WT, ERαKO and ERβKO mice had similar MOR distributions in the limbic‐hypothalamic lordosis‐regulating circuit. Estrogen treatment internalized MOR in the medial preoptic nucleus of ovariectomized WT and ERβKO, but not ERαKO mice. Treatment of ERαKO mice with the selective endogenous MOR ligand, endomorphin‐1, induced levels of MOR internalization similar to WT mice suggesting that MOR in ERαKO mice could be activated and were probably functional. The results of the present experiments indicate that ERα is required for estrogen‐induced MOR internalization and suggest that ERα can mediate rapid actions of estrogen.

Estradiol regulation of progesterone synthesis in the brain

Steroidogenesis is now recognized as a global phenomenon in the brain, but how it is regulated and its relationship to circulating steroids of peripheral origin have remained more elusive issues. Neurosteroids, steroids synthesized de novo in nervous tissue, have a large range of actions in the brain, but it is only recently that the role of neuroprogesterone in the regulation of arguably the quintessential steroid-dependent neural activity, regulation of the reproduction has been appreciated. Circuits involved in controlling the LH surge and sexual behaviors were thought to be influenced by estradiol and progesterone synthesized in the ovary and perhaps the adrenal. It is now apparent that estradiol of ovarian origin regulates the synthesis of neuroprogesterone, and it is the locally produced neuroprogesterone that is involved in the initiation of the LH surge and subsequent ovulation. In this model, estradiol induces the transcription of progesterone receptors while stimulating synthesis of neuroprogesterone. Although the complete signaling cascade has not been elucidated, many of the features have been characterized. The synthesis of neuroprogesterone occurs primarily in astrocytes and requires the interaction of membrane-associated estrogen receptor-alpha with metabotropic glutamate receptor-1a. This G protein-coupled receptor activates a phospholipase C that in turn increases inositol trisphosphate (IP3) levels mediating the release of intracellular stores of Ca2+ via an IP3 receptor gated Ca2+ channel. The large increase in free cytoplasmic Ca2+ ([Ca2+]i) stimulates the synthesis of progesterone, which can then diffuse out of the astrocyte and activate estradiol-induced progesterone receptors in local neurons to trigger the neural cascade to produce the LH surge. Thus, it is a cooperative action of astrocytes and neurons that is needed for estrogen positive feedback and stimulation of the LH surge.

Orphanin FQ/nociceptin in the ventromedial nucleus facilitates lordosis in female rats.

THE localization of opioid receptor-like (ORL-1) orphan receptor in the ventromedial nucleus of the hypothalamus (VMH) suggested a role for this opioid system in the regulation of lordosis behavior. Recently, the ligand for ORL-1, orphanin FQ/nociceptin (OFQ/N), has been characterized and also demonstrated to be present in the VMH. The present experiments examined whether OFQ/N in the VMH facilitates lordosis behavior in estrogen-primed, sexually unreceptive female rats, and whether estrogen regulates ORL-1 levels in the VMH. Estrogen was shown to increase ORL-1 immunoreactivity in the VMH, and microinfusions of OFQ/N into the VMH facilitated lordosis behavior in a dose-dependent manner.

Visualizing activation of opioid circuits by internalization of G protein-coupled receptors

Mu-opioid receptor (MOR) and opioid receptor-like receptor (ORL-1) circuits in the limbic hypothalamic system are important for the regulation of sexual receptivity in the female rat. Sexual receptivity is tightly regulated by the sequential release of estrogen and progesterone from the ovary suggesting ovarian steroids regulate the activity of these neuropeptide systems. Both MOR and ORL-1 distributions overlap with the distribution of estrogen and progesterone receptors in the hypothalamus and limbic system providing a morphological substrate for interaction between steroids and the opioid circuits in the brain. Both MOR and ORL-1 are receptors that respond to activation by endogenous ligands with internalization into early endosomes. This internalization is part of the mechanism of receptor desensitization or down regulation. Although receptor activation and internalization are separate events, internalization can be used as a temporal measure of circuit activation by endogenous ligands. This review focuses on the estrogen and progesterone regulation of MOR and ORL-1 circuits in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus that are central to modulating sexual receptivity.

Synthesis and function of hypothalamic neuroprogesterone in reproduction.

The physiology and regulation of steroid synthesis in the brain have emerged as important for understanding brain function. Neurosteroids, those steroids synthesized de novo in nervous tissue, have been associated with numerous central nervous system functions, including myelination, mental retardation, and epilepsy. Central regulation of reproduction was thought to depend on steroids of peripheral origin. Only recently has the role of neurosteroids in reproduction been appreciated. This minireview describes our work trying to understand how circulating estradiol modulates the synthesis of neuroprogesterone. The synthesis of neuroprogesterone occurs primarily in astrocytes, and requires the interaction of membrane-associated estrogen receptor with metabotropic glutamate receptor and the release of intracellular calcium stores. The newly synthesized neuroprogesterone acts on estradiol-induced progesterone receptors in nearby neurons to initiate the LH surge.