Mistimed feeding disrupts circadian rhythms of male mating behavior and female preovulatory LH surges in mice

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 hours. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to subfertility.

Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction

Estrogen produced by ovarian follicles plays a key role in the central mechanisms controlling reproduction via regulation of gonadotropin-releasing hormone (GnRH) release by its negative and positive feedback actions in female mammals. It has been well accepted that estrogen receptor α (ERα) mediates both estrogen feedback actions, but precise targets had remained as a mystery for decades. Ever since the discovery of kisspeptin neurons as afferent ERα-expressing neurons to govern GnRH neurons, the mechanisms mediating estrogen feedback are gradually being unraveled. The present article overviews the role of kisspeptin neurons in the arcuate nucleus (ARC), which are considered to drive pulsatile GnRH/gonadotropin release and folliculogenesis, in mediating the estrogen negative feedback action, and the role of kisspeptin neurons located in the anteroventral periventricular nucleus-periventricular nucleus (AVPV-PeN), which are thought to drive GnRH/luteinizing hormone (LH) surge and consequent ovulation, in mediating the estrogen positive feedback action. This implication has been confirmed by the studies showing that estrogen-bound ERα down- and up-regulates kisspeptin gene (Kiss1) expression in the ARC and AVPV-PeN kisspeptin neurons, respectively. The article also provides the molecular and epigenetic mechanisms regulating Kiss1 expression in kisspeptin neurons by estrogen. Further, afferent ERα-expressing neurons that may regulate kisspeptin release are discussed.

Neuroprogesterone synthesis in hypothalamic astrocytes is associated with a pubertal increase in membrane ERα levels

ABSTRACTThe development of estrogen positive feedback is a hallmark of female puberty. Both estrogen and progesterone signaling are required for the functioning of this neuroendocrine feedback loop but the physiological changes that underlie the emergence of estrogen positive feedback remain unknown. Only after puberty does estradiol (E2) facilitate progesterone synthesis in female hypothalamic astrocytes (neuroP) (Mohr et al. 2018), an event critical for estrogen positive feedback and the LH surge. We hypothesize that prior to puberty, these astrocytes have low levels of membrane estrogen receptor alpha (ERα), making them unable to respond to E2 with increased neuroP synthesis prior to puberty. To test this hypothesis, pure populations of primary astrocyte cultures were derived from female mice at three different stages of development: pre-puberty (postnatal week 3), pubertal onset (week 5), and post-puberty (week 8). Hypothalamic astrocyte responses were measured after treatment with E2. Hypothalamic astrocytes increased progesterone synthesis across pubertal development. Prior to puberty, mERα expression was low in hypothalamic astrocytes, but expression increased across puberty. The increase in mERα expression in hypothalamic astrocytes also corresponded with an increase in caveolin-1 protein, PKA phosphorylation, and a more rapid [Ca2+]i flux in response to E2. Together, these results indicate that increased mERα in hypothalamic astrocytes contributed to the post-pubertal response to E2 that results in neuroP synthesis, critical for ovulation.SIGNIFICANCE STATEMENTHypothalamic astrocytes, when exposed to estradiol, make progesterone that is necessary for estrogen positive feedback during adulthood in the female rodent. However, little is known about what cellular changes occur during puberty that allow for the estradiol facilitation of progesterone synthesis. In this study, we compared cell excitability, progesterone synthesis, and levels of membrane estrogen receptor in hypothalamic astrocytes derived from pre-, mid-, and post-pubertal female mice to characterize their maturation that allows them to increase progesterone synthesis facilitating estrogen positive feedback. This study suggests that hypothalamic astrocytes acquire the necessary cellular machinery during puberty to enable E2-facilitated progesterone synthesis.

Classical and Membrane-Initiated Estrogen Signaling in an In Vitro Model of Anterior Hypothalamic Kisspeptin Neurons

The neuropeptide kisspeptin is essential for sexual maturation and reproductive function. In particular, kisspeptin-expressing neurons in the anterior rostral periventricular area of the third ventricle are generally recognized as mediators of estrogen positive feedback for the surge release of LH, which stimulates ovulation. Estradiol induces kisspeptin expression in the neurons of the rostral periventricular area of the third ventricle but suppresses kisspeptin expression in neurons of the arcuate nucleus that regulate estrogen-negative feedback. To focus on the intracellular signaling and response to estradiol underlying positive feedback, we used mHypoA51 cells, an immortalized line of kisspeptin neurons derived from adult female mouse hypothalamus. mHypoA51 neurons express estrogen receptor (ER)-α, classical progesterone receptor (PR), and kisspeptin, all key elements of estrogen-positive feedback. As with kisspeptin neurons in vivo, 17β-estradiol (E2) induced kisspeptin and PR in mHypoA51s. The ERα agonist, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, produced similar increases in expression, indicating that these events were mediated by ERα. However, E2-induced PR up-regulation required an intracellular ER, whereas kisspeptin expression was stimulated through a membrane ER activated by E2 coupled to BSA. These data suggest that anterior hypothalamic kisspeptin neurons integrate both membrane-initiated and classical nuclear estrogen signaling to up-regulate kisspeptin and PR, which are essential for the LH surge.

Hypothalamic IGF-I Gene Therapy Prolongs Estrous Cyclicity and Protects Ovarian Structure in Middle-Aged Female Rats

There is substantial evidence that age-related ovarian failure in rats is preceded by abnormal responsiveness of the neuroendocrine axis to estrogen positive feedback. Because IGF-I seems to act as a permissive factor for proper GnRH neuronal response to estrogen positive feedback and considering that the hypothalamic content of IGF-I declines in middle-aged (M-A) rats, we assessed the effectiveness of long-term IGF-I gene therapy in the mediobasal hypothalamus (MBH) of M-A female rats to extend regular cyclicity and preserve ovarian structure. We used 3 groups of M-A rats: 1 group of intact animals and 2 groups injected, at 36.2 weeks of age, in the MBH with either a bicistronic recombinant adeno-associated virus (rAAV) harboring the genes for IGF-I and the red fluorescent protein DsRed2, or a control rAAV expressing only DsRed2. Daily vaginal smears were taken throughout the study, which ended at 49.5 weeks of age. We measured serum levels of reproductive hormones and assessed ovarian histology at the end of the study. Although most of the rats injected with the IGF-I rAAV had, on the average, well-preserved estrous cyclicity as well as a generally normal ovarian histology, the intact and control rAAV groups showed a high percentage of acyclic rats at the end of the study and ovaries with numerous enlarged cysts and scarce corpora lutea. Serum LH was higher and hyperprolactinemia lower in the treated animals. These results suggest that overexpression of IGF-I in the MBH prolongs normal ovarian function in M-A female rats.