Prepubertal Gonadotropin-Releasing Hormone Neuron Activity and the Effects of Prenatal Androgen Exposure

Abstract

Reproductive function is governed by the hypothalamic-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) neurons are central mediators of the reproductive axis and release GnRH, which controls pituitary and gonadal function. Polycystic-ovary syndrome (PCOS) is the leading cause of infertility in reproductive-aged women. PCOS is characterized by an upregulation in luteinizing hormone (LH), and thus GnRH release. Endocrine abnormalities of PCOS, including increased androgens, high-frequency LH pulses, and disrupted reproductive cycles, are present in prenatally androgenized (PNA) animal models. GnRH neuron activity in adult female PNA mice is also elevated. Clinical studies in girls with hyperandrogenemia revealed disrupted LH pulsatility prior to adulthood, suggesting that aspects PCOS may manifest before puberty. In this dissertation we demonstrate that GnRH neurons are prepubertally active and that this activity is disrupted in PNA mice. To examine this activity we used electrophysiological extracellular recordings conducted from GFP-identified GnRH neurons in brain slices from control and PNA mice at postnatal weeks 1-4. We found that GnRH neuron activity was present in both males and females at all time points tested. In control female mice prepubertal activity of GnRH neurons increased during early life, with firing being highest at 3 weeks. In contrast, firing activity in PNA mice did not increase across the postnatal period. To investigate if the downstream reproductive axis plays a role in GnRH neuron firing rate of PNA mice, we tested hypotheses that 1) increased GnRH neuron firing in adult PNA mice is due to ovarian factors, specifically androgens, and 2) that gonadal factors have minimal influence on GnRH neuron firing in 3-week old mice. Interestingly, removing ovarian inputs in adult PNA mice decreased firing rate and addition of androgen implants in ovariectomized mice restored firing to that of PNA sham-operated mice, suggesting that ovarian androgens are a primary contributor driving increased GnRH neuron firing in adult PNA mice. In 3-week old control and PNA mice however no differences were found between sham-operated and ovariectomized groups, suggesting a minimal role for ovarian factors on GnRH neuron firing in 3-week old PNA mice. Because ovarian inputs did not affect GnRH activity in 3-week old PNA mice, we next hypothesized that prepubertal GnRH neuron activity plays a role in establishing adult reproductive function and in setting up adult activity of the GnRH neuronal network. Either activating (hM3Dq) or inhibitory (hM4Di) designer receptors exclusively activated by designer drugs (DREADDs) were targeted to GnRH neurons to allow for remote control of neuronal activity. Injection of clozapine n-oxide (CNO) produced the expected changes in luteinizing hormone (LH) release from the pituitary in control studies. CNO was administered between two and three weeks of age, the period when GnRH neuron activity was reduced in PNA mice. Reducing GnRH neuron activity during development decreased adult GnRH neuron firing rate. Interestingly, neither manipulation altered the timing of pubertal makers. Thus GnRH neuron activity programs changes in the adult neuronal network governing reproductive function but altering prepubertal GnRH neuron activity cannot fully explain the phenotypes found in PNA mice. Overall this body of work indicates that the reproduce axis is active prior to puberty. These results also support a model in which prepubertal alteration of GnRH neuronal activity programs changes in the adult GnRH neuron network and indicate that androgens play a role in the manifestation of reproductive abnormalities observed in PNA mice.