Stress elicits activation of the hypothalamic-pituitary-adrenal axis, which leads to enhanced circulating glucocorticoids, as well as impaired gonadotropin secretion and ovarian cyclicity. Here, we tested the hypothesis that elevated, stress-levels of glucocorticoids disrupt ovarian cyclicity by interfering with the preovulatory sequence of endocrine events necessary for the LH surge. Ovarian cyclicity was monitored in female mice implanted with a cholesterol or corticosterone (Cort) pellet. Cort, but not cholesterol, arrested cyclicity in diestrus. Subsequent studies focused on the mechanism whereby Cort stalled the preovulatory sequence by assessing responsiveness to the positive feedback estradiol signal. Ovariectomized mice were treated with an LH surge-inducing estradiol implant, as well as Cort or cholesterol, and assessed several days later for LH levels on the evening of the anticipated surge. All cholesterol females showed a clear LH surge. At the time of the anticipated surge, LH levels were undetectable in Cort-treated females. In situ hybridization analyses the anteroventral periventricular nucleus revealed that Cort robustly suppressed the percentage of Kiss1 cells coexpressing cfos, as well as reduced the number of Kiss1 cells and amount of Kiss1 mRNA per cell, compared with expression in control brains. In addition, Cort blunted pituitary expression of the genes encoding the GnRH receptor and LHβ, indicating inhibition of gonadotropes during the blockage of the LH surge. Collectively, our findings support the hypothesis that physiological stress-levels of Cort disrupts ovarian cyclicity, in part, through disruption of positive feedback mechanisms at both the hypothalamic and pituitary levels which are necessary for generation of the preovulatory LH surge.

Background: The neuropeptide RFamide-related peptide-3 (RFRP-3; mammalian ortholog to gonadotropin-inhibiting hormone) can inhibit luteinizing hormone (LH) release and increases feeding, but the regulation and development of RFRP-3 neurons remains poorly characterized, especially in mice. Methods and Results: We first confirmed that peripheral injections of murine RFRP-3 peptide could markedly suppress LH secretion in adult mice, as in other species. Second, given RFRP-3's reported orexigenic properties, we performed double-label in situ hybridization for metabolic genes in Rfrp neurons of mice. While Rfrp neurons did not readily coexpress neuropeptide Y, thyrotropin-releasing hormone, or MC4R, a small subset of Rfrp neurons did express the leptin receptor in both sexes. Surprisingly, we identified no changes in Rfrp expression or neuronal activation in adult mice after acute fasting. However, we determined that Rfrp mRNA levels in the dorsal-medial nucleus were significantly reduced in adult obese (Ob) mice of both sexes. Given the lower Rfrp levels observed in adult Ob mice, we asked whether leptin might also regulate RFRP-3 neuron development. Rfrp gene expression changed markedly over juvenile development, correlating with the timing of the juvenile ‘leptin surge' known to govern hypothalamic feeding circuit development. However, the dramatic developmental changes in juvenile Rfrp expression did not appear to be leptin driven, as the pattern and timing of Rfrp neuron development were unaltered in Ob juveniles. Conclusion: Leptin status modulates RFRP-3 expression in adulthood, but is not required for normal development of the RFRP-3 system. Leptin's regulation of adult RFRP-3 neurons likely occurs primarily via indirect signaling, and may be secondary to obesity, as only a small subset of RFRP-3 neurons express the long form of the leptin receptor (LepRb).

In rodents, the ovulation-inducing luteinizing hormone (LH) surge is sexually dimorphic, occurring only in females, but the reasons for this sex difference are unclear. Two neuropeptides, kisspeptin and RFamide-related peptide 3 (RFRP-3), are hypothesized to regulate the gonadotropin-releasing hormone (GnRH)/LH surge. In females, both of these systems show circadian changes coincident with the LH surge, but whether males show similar temporal changes under comparable hormonal conditions is unknown. Here, we evaluated circadian time (CT)-dependent changes in gene expression and neuronal activation of Kiss1 and Rfrp neurons of female and male mice given identical LH surge-inducing estrogen regimens. As expected, females, but not males, displayed a late afternoon LH surge and GnRH neuronal activation. Kiss1 expression in the anteroventral periventricular nucleus (AVPV) was temporally increased in females in the late afternoon, whereas males demonstrated no temporal changes in AVPV Kiss1 expression. Likewise, neuronal activation of AVPV Kiss1 neurons was dramatically elevated in the late afternoon in females but was low at all circadian times in males. Estrogen receptor α levels in AVPV Kiss1 neurons were sexually dimorphic, being higher in females than males. AVPV progesterone receptor levels were also higher in females than males. Hypothalamic Rfrp messenger RNA levels showed no CT-dependent changes in either sex. However, Rfrp neuronal activation was temporally diminished in the afternoon/evening in females but not males. Collectively, the identified sex differences in absolute and CT-dependent AVPV Kiss1 levels, AVPV sex steroid receptor levels, and circadian-timed changes in neuronal activation of both Kiss1 and Rfrp neurons suggest that multiple sexually dimorphic processes in the brain may underlie proper LH surge generation.

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