The anteroventral periventricular (AVPV) nucleus of the hypothalamus integrates estradiol (E2) and progesterone (P4) feedback signals from the ovaries to stimulate gonadotropin releasing hormone (GnRH) neurons and trigger an ovulatory surge in luteinizing hormone (LH). E2 maintains the daily cyclic LH surge and P4 quickly amplifies the surge and limits it to one day. P4 amplification of the surge and rapid signaling in the AVPV may occur through its non-classical progestin receptors. Previous in vitro studies using a microarray analysis with N42 mouse embryonic hypothalamic neurons suggest that progesterone membrane component 1 (Pgrmc1) regulates genes linked to the janus kinase (Jak)/signal transducer and activator of transcription (Stat) signaling pathway. I hypothesized that P4 alters Jak/Stats through Pgrmc1 regulation of one or more Jak or Stat molecules and then performed a set of in vitro and in vivo studies to test this. I transfected N42 cells with either scramble or Pgrmc1 siRNA followed by treatment with either ethanol vehicle control or 10 nM P4 and measured Jak1, Jak2, Stat3, Stat5a, Stat5b, and Stat6 mRNA levels via quantitative polymerase chain reaction (QPCR). Jak1 and Jak2 mRNAs increased with P4 treatments, and this upregulation required Pgrmc1. Silencing Pgrmc1 in the cells also produced an increase in Jak1 and Jak2 mRNA, suggesting that Pgrmc1 constitutively suppressed jak1 and jak2 in the absence of P4. None of the Stats were significantly regulated by P4 or Pgrmc1 silencing. To determine how Pgrmc1 regulates Jak/Stat in vivo, I took AVPV microdissections from Pgrmc1 and Pgrmc2 double conditional knockout (DCKO) mice and looked at gene expression of jak/stat. Transcript levels of Jak2, but not Jak1, were severely downregulated in the DCKO animals and Stat mRNAs were not significantly changed. Discrepancies from in vitro and in vivo data prompted me to analyze the role of the class II progestin and adipoQ (Paqr) receptors in Jak/Stat signaling. P4 treatments and siRNA experiments in N42 cells showed that Paqr8, but not Paqr7, was required for P4 upregulation of Jak1 and Jak2 mRNAs. Overall, these findings show that Pgrmc1 regulates Jak1 and Jak2 synthesis in a P4-dependent and -independent manner that requires interaction with Paqr8.

The anteroventral periventricular nucleus (AVPV) is a sexually dimorphic preoptic structure that is nearly three times larger in the female rat. Estradiol (E2) exposure during development decreases AVPV volume through apoptosis, a process which normally occurs preferentially in male. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupter that interferes with E2-dependent sexual differentiation of the AVPV. Whole-genome microarrays were used to identify sex-specific genes regulated by E2 and TCDD in postnatal day 2 (P2) AVPV punches from untreated males and females, males treated with TCDD or vehicle, and females treated with E2 or vehicle. Trio emerged as a gene target regulated by E2 and TCDD, and this gene is essential for neurite outgrowth which is sexually dimorphic in the AVPV. My goal was to verify microarray data that Trio was expressed in the P2 AVPV and to test whether expression was affected by sex, E2 and TCDD. The microarray did not discriminate among Trio splice variants, 9S, 9L and Duet, so I mapped each of these in the P2 AVPV using in situ hybridization. I used quantitative real-time PCR to examine the effects of sex, E2 and TCDD on mRNA levels encoding each splice variant. I found that the AVPV expressed 9S and 9L at moderate levels and Duet at low levels. The expression of 9S and 9L mRNA was increased by E2 in females. However, only 9S expression differed between sexes and it was lower in males. TCDD had no effect on expression of any of the splice variants. This work provides the first evidence that Trio splice variants are independently regulated and that Trio may mediate effects of E2 in the developing AVPV.

Female reproductive senescence is widespread among mammalian species, but menopause is limited to species with menstrual cycles. While hormonal changes at menopause have profound impacts in the lives of women at middle age, the complex mechanisms underlying this process remain obscure. All three levels of the hypothalamic-pituitary-gonadal (HPG) axis are involved in reproductive aging, and evidence highlights a critical role for the dysregulation of gonadotropin-releasing hormone (GnRH) neurons, the hypothalamic cells that drive reproductive function. To investigate neuroendocrine mechanisms that may initiate and perpetuate reproductive decline at each step in the transition to acyclicity, I utilized an ovarian-intact middle-aged female rat model of natural reproductive senescence. These studies focused on three hypothalamic nuclei that are known to control GnRH activity: the anteroventral periventricular nucleus (AVPV), the site of positive hormone feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback; and the median eminence (ME), the site of GnRH release, with the following specific aims: 1) Characterize neuroendocrine gene and protein expression in female rats throughout the natural transition to acyclicity; 2) Determine the effects of chronic N-methyl-D-asparate receptor subunit 2b (NMDAR-NR2b) inhibition in acyclic females; and 3) Examine neuroendocrine gene expression during premature reproductive senescence after perturbation of the HPG axis. The results of these studies identified novel molecular and cellular changes with age and reproductive cycle status in the ARC and ME, two regions that are underappreciated for their roles in reproductive senescence. Surprisingly, few molecular targets were identified in the AVPV, a region that is much better-studied in this context. In the ME and ARC, I found changes in transcription factors and evidence of altered hormone feedback via changes in sex steroid hormone receptors and enzyme expression with reproductive aging. I also discovered decreased expression of genes for the excitatory neuropeptides, kisspeptin and neurokinin B, as well as decreased percentage of kisspeptin immunoreactive cells and their co-expression with estrogen receptor alpha in the ARC. And finally, in the ME, neurotrophic factor expression was changed with age, and the presence and phosphorylation state of the NR2b subunit of the NMDA receptor contributes to a greater inhibitory tone with acyclicity. Together these studies have identified novel pathways, especially in the ARC and ME, that are related to reproductive decline. Furthermore, changes in the hypothalamic neural and glial network of neurotransmitters, neuropeptides, hormone receptors and other transcription factors are likely contributing to altered responses to hormonal feedback and decreased excitatory drive for GnRH release. / text