Sex Differences in Steroid Receptor Coexpression and Circadian-Timed Activation of Kisspeptin and RFRP-3 Neurons May Contribute to the Sexually Dimorphic Basis of the LH Surge

Abstract

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.

Gonadotrophin‐inhibitory hormone and its mammalian orthologue RFamide‐related peptide‐3: Discovery and functional implications for reproduction and stress

“…These injections led to a rapid, dose‐dependent suppression of LH, indicating a likely role in oestradiol negative‐feedback regulation. Additionally, our studies found that the removal of oestradiol negative‐feedback at the time of the LH surge is mediated, at least in part, via circadian‐controlled inhibition of the RFRP‐3 system, with analogous findings recently reported in mice . These studies showing coordinated suppression of RFRP‐3 cellular activity with pro‐oestrus were conducted in an oestrogen‐clamped LH surge model.…”

“…Additionally, our studies found that the removal of oestradiol negative-feedback at the time of the LH surge is mediated, at least in part, via circadian-controlled inhibition of the RFRP-3 system, 27 with analogous findings recently reported in mice. 69 These studies showing coordinated suppression of RFRP-3 cellular activity with pro-oestrus were conducted in an oestrogen-clamped LH surge model. However, RFRP-3 cellular activity is suppressed during pro-oestrus in naturally cycling hamsters as well.…”

Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction

“…Earlier work in female Syrian hamster reported a monosynaptic connection between the SCN and RFRP-3 neurons, suggesting that RFRP-3 neurons are regulated by the SCN clock (47). In accordance with this hypothesis, a daily rhythm in RFRP-3 neuronal activity has been reported, with a lower number of RFRP-3 neurons expressing c-FOS coincident with the timing of the LH surge in female Syrian hamsters (44, 47) and mice (101). Equivocal findings are reported regarding the association between RFRP-3 cell activation state and the number of Rfrp expressing neurons, with daily variation in RFRP-3 neuronal activity being associated (47) or not (44, 101), with corresponding changes in the number of Rfrp expressing cells.…”

Stress rapidly suppresses in vivo LH pulses and increases activation of RFRP-3 neurons in male mice

“…Double-label ISH for cfos+Kiss1 and cfos+Rfrp were performed on an additional set of brain sections in order to assess neuronal activation. Both single- and double-label ISH assays were performed using well-published protocols and validated P33 or DIG-labeled riboprobes, as previously reported (Poling, et al 2017; Semaan and Kauffman 2015; Stephens, et al 2016; Stephens, et al 2017). After ISH slides were developed and coverslipped, a custom automated image processing and quantification software program (Dr. Don Clifton, University of Washington) was used to analyze ISH slides under computer-assisted microscopy, by an investigator blinded to treatment groups.…”