Jon E. Levine

Circadian Clock Mutation Disrupts Estrous Cyclicity and Maintenance of Pregnancy

Classic experiments have shown that ovulation and estrous cyclicity are under circadian control and that surgical ablation of the suprachiasmatic nuclei (SCN) results in estrous acyclicity in rats. Here, we characterized reproductive function in the circadian Clock mutant mouse and found that the circadian Clock mutation both disrupts estrous cyclicity and interferes with the maintenance of pregnancy. Clock mutant females have extended, irregular estrous cycles, lack a coordinated luteinizing hormone (LH) surge on the day of proestrus, exhibit increased fetal reabsorption during pregnancy, and have a high rate of full-term pregnancy failure. Clock mutants also show an unexpected decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy, suggesting that maternal prolactin release may be abnormal. In a second set of experiments, we interrogated the function of each level of the hypothalamic-pituitary-gonadal (HPG) axis in order to determine how the Clock mutation disrupts estrous cyclicity. We report that Clock mutants fail to show an LH surge following estradiol priming in spite of the fact that hypothalamic levels of gonadotropin-releasing hormone (GnRH), pituitary release of LH, and serum levels of estradiol and progesterone are all normal in Clock/Clock females. These data suggest that Clock mutants lack an appropriate circadian daily-timing signal required to coordinate hypothalamic hormone secretion. Defining the mechanisms by which the Clock mutation disrupts reproductive function offers a model for understanding how circadian genes affect complex physiological systems.

Timing and completion of puberty in female mice depend on estrogen receptor α-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.

Endocrine Defects in Mice Carrying a Null Mutation for the Progesterone Receptor Gene

Mice carrying a null mutation of the progesterone receptor gene exhibit several reproductive abnormalities, including anovulation, attenuated lordotic behavior, uterine hyperplasia, and lack of mammary gland development. The hormonal correlates of these abnormalities are unknown, however, and were the focus of these studies. Serum samples from female wild-type (WT) and progesterone receptor knockout (PRKO) mice were obtained and analyzed by RIA for LH, FSH, PRL, estrogen (E2), and progesterone. Hypothalamic tissues were also processed for measurement of LHRH by RIA. Serum LH levels in PRKO mice were found to be elevated by approximately 2-fold over basal (metestrus) values in WT mice. By contrast, basal FSH levels were not different in PRKO and WT mice. Basal levels of E2 and progesterone in serum were likewise similar in the two groups, as were hypothalamic LHRH concentrations. Basal PRL levels were slightly higher in PRKO vs. WT mice. Ovariectomy of both groups of mice was accompanied by significant inc...

Nonclassical estrogen receptor α signaling mediates negative feedback in the female mouse reproductive axis

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) α but not ERβ knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERα. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERα signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERα (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERα-null (−/−) background, we examine the ability of ERE-independent ERα signaling pathways to convey estrogen feedback regulation of the female hypothalamic–pituitary axis in vivo. ERα−/AA exhibited 69.9% lower serum LH levels compared with ERα−/− mice. Additionally, like wild type, ERα−/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERα−/AA mice. However, unlike wild type, both ERα−/AA and ERα−/− mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERα signaling is sufficient to convey a major portion of estrogens negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERα signaling.

Regulation of Kiss1 and Dynorphin Gene Expression in the Murine Brain by Classical and Nonclassical Estrogen Receptor Pathways

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17β (E2), which acts via the estrogen receptor α isoform (ERα) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E2 inhibits the expression of Kiss1 mRNA. However, E2 induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E2 is unknown. ERα signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E2 on Kiss1 and Dyn expression, we studied the effects of E2 on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ERα signaling pathways. We found that stimulation of Kiss1 expression by E2 in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E2 in the Arc involved ERE-independent mechanisms. Thus, distinct ERα signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E2 can act within the same neuron through divergent ERα signaling pathways to regulate different neurotransmitter genes.

Neuroendocrine Consequences of Prenatal Androgen Exposure in the Female Rat: Absence of Luteinizing Hormone Surges, Suppression of Progesterone Receptor Gene Expression, and Acceleration of the Gonadotropin-Releasing Hormone Pulse Generator

Abstract Preovulatory GnRH and LH surges depend on activation of estrogen (E2)-inducible progesterone receptors (PGRs) in the preoptic area (POA). Surges do not occur in males, or in perinatally androgenized females. We sought to determine whether prenatal androgen exposure suppresses basal or E2-induced Pgr mRNA expression or E2-induced LH surges (or both) in adulthood, and whether any such effects may be mediated by androgen receptor activation. We also assessed whether prenatal androgens alter subsequent GnRH pulsatility. Pregnant rats received testosterone or vehicle daily on Embryonic Days 16–19. POA-hypothalamic tissues were obtained in adulthood for PgrA and PgrB (PgrA+B) mRNA analysis. Females that had prenatal exposure to testosterone (pT) displayed reduced PgrA+B mRNA levels (P < 0.01) compared with those that had prenatal exposure to vehicle (pV). Additional pregnant animals were treated with vehicle or testosterone, or with 5α-dihydrotestosterone (DHT). In adult ovariectomized offspring, estradiol benzoate produced a 2-fold increase (P < 0.05) in PgrA+B expression in the POA of pV females, but not in pT females or those that had prenatal exposure to DHT (pDHT). Prenatal testosterone and DHT exposure also prevented estradiol benzoate-induced LH surges observed in pV rats. Blood sampling of ovariectomized rats revealed increased LH pulse frequency in pDHT versus pV females (P < 0.05). Our findings support the hypothesis that prenatal androgen receptor activation can contribute to the permanent defeminization of the GnRH neurosecretory system, rendering it incapable of initiating GnRH surges, while accelerating basal GnRH pulse generator activity in adulthood. We propose that the effects of prenatal androgen receptor activation on GnRH neurosecretion are mediated in part via permanent impairment of E2-induced PgrA+B gene expression in the POA.

Genetic rescue of nonclassical ERα signaling normalizes energy balance in obese Erα-null mutant mice

In addition to its role in reproduction, estradiol-17β is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα-/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα-/- mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17β on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women.

Neuroendocrine Regulation of the Luteinizing Hormone-Releasing Hormone Pulse Generator in the Rat

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.

Transient prenatal androgen exposure produces metabolic syndrome in adult female rats

Androgen exposure during intrauterine life in nonhuman primates and in sheep results in a phenocopy of the reproductive and metabolic features of polycystic ovary syndrome (PCOS). Such exposure also results in reproductive features of PCOS in rodents. We investigated whether transient prenatal androgen treatment produced metabolic abnormalities in adult female rats and the mechanisms of these changes. Pregnant dams received free testosterone or vehicle injections during late gestation, and their female offspring were fed regular or high-fat diet (HFD). At 60 days of age, prenatally androgenized (PA) rats exhibited significantly increased body weight; parametrial and subcutaneous fat; serum insulin, cholesterol and triglyceride levels; and hepatic triglyceride content (all P < 0.0125). There were no significant differences in insulin sensitivity by intraperitoneal insulin tolerance test or insulin signaling in liver or skeletal muscle. HFD had similar effects to PA on body weight and composition as well as on circulating triglyceride levels. HFD further increased hepatic triglyceride content to a similar extent in both PA and control rats. In PA rats, HFD did not further increase circulating insulin, triglyceride, or cholesterol levels. In control rats, HFD increased insulin levels, but to a lesser extent than PA alone ( approximately 2.5- vs. approximately 12-fold, respectively). We conclude that transient prenatal androgen exposure produces features of the metabolic syndrome in adult female rats. Dyslipidemia and hepatic steatosis appear to be mediated by PA-induced increases in adiposity, whereas hyperinsulinemia appears to be a direct result of PA.

Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner that is dependent on circulating 17β-estradiol (E2) and glucose concentrations. However, the intrinsic conductances responsible for the episodic firing pattern underlying pulsatile release and the effects of E2 and glucose on these conductances are primarily unknown. Whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons revealed that the KATP channel opener diazoxide induced an outward current that was antagonized by the sulfonylurea receptor 1 (SUR1) channel blocker tolbutamide. Single-cell reverse transcription (RT)-PCR revealed that the majority of GnRH neurons expressed Kir6.2 and SUR1 subunits, which correlated with the diazoxide/tolbutamide sensitivity. Also, a subpopulation of GnRH neurons expressed glucokinase mRNA, a marker for glucose sensitivity. Indeed, GnRH neurons decreased their firing in response to low glucose concentrations and metabolic inhibition. The maximum diazoxide-induced current was approximately twofold greater in E2-treated compared with oil-treated ovariectomized females. In current clamp, estrogen enhanced the diazoxide-induced hyperpolarization to a similar degree. However, based on quantitative RT-PCR, estrogen did not increase the expression of Kir6.2 or SUR1 transcripts in GnRH neurons. In the presence of ionotropic glutamate and GABAA receptor antagonists, tolbutamide depolarized and significantly increased the firing rate of GnRH neurons to a greater extent in E2-treated females. Finally, tolbutamide significantly increased GnRH secretion from the preoptic-mediobasal hypothalamus. Therefore, it appears that KATP channels and glucokinase are expressed in GnRH neurons, which renders them directly responsive to glucose. In addition, KATP channels are involved in modulating the excitability of GnRH neurons in an estrogen-sensitive manner that ultimately regulates peptide release.