No abstract

Puberty is a complex developmental stage that elicits a cascade of neuroendocrine changes leading to reproductive maturation. In females, the development of reproductive circuits during puberty culminates in the brain responding positively to ovarian oestradiol (E2), triggering the luteinizing hormone (LH) surge that underlies ovulation. There is mounting evidence that alongside E2, brainderived progesterone (neuroP) is needed for full activation of oestrogen positive feedback: (1) astrocyte-derived neuroP is crucial for fully stimulating kisspeptin release in an in vitro model of adult anterior hypothalamic kisspeptin neurons, 1,2 (2) blocking neuroP synthesis arrests oestrous cyclicity and decreases the LH surge, 3,4 and(3) nuclear progesterone receptor (PGR) signalling in anteroventral

33The development of estrogen positive feedback is a hallmark of female puberty. Both estrogen 34 and progesterone signaling are required for the functioning of this neuroendocrine feedback 35 loop but the physiological changes that underlie the emergence of estrogen positive feedback 36 remain unknown. Only after puberty does estradiol (E2) facilitate progesterone synthesis in 37 female hypothalamic astrocytes (neuroP) (Mohr et al. 2018), an event critical for estrogen 38 positive feedback and the LH surge. We hypothesize that prior to puberty, these astrocytes 39 have low levels of membrane estrogen receptor alpha (ERα), making them unable to respond to 40 E2 with increased neuroP synthesis prior to puberty. To test this hypothesis, pure populations of 41 primary astrocyte cultures were derived from female mice at three different stages of 42 development: pre-puberty (postnatal week 3), pubertal onset (week 5), and post-puberty (week 43 8). Hypothalamic astrocyte responses were measured after treatment with E2. Hypothalamic 44 astrocytes increased progesterone synthesis across pubertal development. Prior to puberty, 45 mERα expression was low in hypothalamic astrocytes, but expression increased across 46 puberty. The increase in mERα expression in hypothalamic astrocytes also corresponded with 47 an increase in caveolin-1 protein, PKA phosphorylation, and a more rapid [Ca 2+ ] i flux in 48 response to E2. Together, these results indicate that increased mERα in hypothalamic 49 astrocytes contributed to the post-pubertal response to E2 that results in neuroP synthesis, 50 critical for ovulation. 51 52 SIGNIFICANCE STATEMENT 53Hypothalamic astrocytes, when exposed to estradiol, make progesterone that is necessary for 54 estrogen positive feedback during adulthood in the female rodent. However, little is known about 55 what cellular changes occur during puberty that allow for the estradiol facilitation of 56 progesterone synthesis. In this study, we compared cell excitability, progesterone synthesis, 57 and levels of membrane estrogen receptor in hypothalamic astrocytes derived from pre-, mid-, 58 and post-pubertal female mice to characterize their maturation that allows them to increase 59 progesterone synthesis facilitating estrogen positive feedback. This study suggests that 60 hypothalamic astrocytes acquire the necessary cellular machinery during puberty to enable E2-61 facilitated progesterone synthesis. 62 *

No abstract

Puberty involves the maturation of female reproductive circuits to the point of supporting estrogen positive feedback that elicits a luteinizing hormone (LH) surge, triggering ovulation. We have shown that in post-pubertal female rodents, peripheral estradiol (E2) increases progesterone synthesis in hypothalamic astrocytes (neuroP), initiating an LH surge. Interestingly, E2-facilitated neuroP synthesis occurs in adult females but not in males or prepubertal females. The focus of the current study was to determine changes in female astrocytes during puberty that allow E2 to facilitate neuroP synthesis. Of particular interest are responses to estrogen indicated by intracellular calcium flux, levels of estrogen receptor-alpha (ERα)-- trafficked to the cell surface by interaction with caveolin 1 (Cav1) proteins—that couple with metabotropic glutamate receptors (mGluRs) and mediate E2’s induction of neuroP synthesis by activating protein kinase A (PKA). Hypothalamic astrocyte cultures were established from female mice at postnatal day (PND) 23 (prepubertal), PND 35 (pubertal), and PND 60 (adult) and treated with 1nM E2 or vehicle. Calcium imaging revealed that PND 60 astrocytes had a more rapid response to E2 compared to PND 23 astrocytes that had a slower and more sustained response to E2. Surface biotinylation and western immunoblotting determined changes in membrane ERα, Cav1, phosphoPKA. We observed a developmental increase in phosphoPKA and Cav1 proteins (ANOVAs, main effects of age, p values < 0.005; n = 3-11/treatment/age. Surface biotinylation and western blotting revealed an age-related increase in full-length (66 kDa) mERα (ANOVA, main effect of age, p < 0.05, n = 7-11/treatment/age), while there is an age-related decrease in a 36 kDa mERα (ANOVA, main effect of age, p < 0.05, n = 2-6/treatment/age). E2 signaling through the 36 kDa mERα splice variant, which is abundant prior to puberty, could initiate downstream inhibitory signaling cascades attenuating E2-facilitated P4 synthesis. Current studies will determine if the response to E2 in hypothalamic astrocytes undergoes a transition from inhibitory to excitatory signaling due to differential coupling of mERs and mGluRs. These experiments highlight how cellular signaling changes during puberty in hypothalamic astrocytes to allow for neuroP biosynthesis that contributes to estrogen positive feedback. Supported by HD042635 and UL1TR001881.