Abstract
Mammalian reproduction and fertility are dependent upon gonadal steroid hormone feedback within the hypothalamic-pituitary-gonadal (HPG) axis. In polycystic ovary syndrome (PCOS), impaired reproductive function is associated with a reduced sensitivity to progesterone negative feedback and a resultant hyperactive pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). While the specific mechanisms of progesterone negative feedback to the HPG axis remain unknown, clinical and preclinical evidence suggests that progesterone acting through progesterone receptors (PR) in the arcuate nucleus of the hypothalamus (ARC) are involved. To identify the critical location of progesterone negative feedback, we tested whether disrupting progesterone sensitivity specifically in the ARC is sufficient to drive hyperactivity within the HPG axis. To do this we used adeno-associated virus (AAV)-mediated Cre-LoxP recombination in a transgenic PRfloxed mouse (PRfl/fl). Stereotaxically placed bilateral injections of AAV-Cre or AAV-control were performed in ARC of adult (3-4 months) female PRfl/fl mice, and immunohistochemistry was used to assess AAV targeting and AAV-Cre mediated loss of PR. For reproductive phenotyping, vaginal cytology was monitored for 14 days before and 4 weeks after Cre-mediated PR knockdown. LH cyclicity was assessed in serial blood samples by ELISA using Pulsar software. All data analysis was performed blinded to treatment group. PR expression in the ARC of AAV-Cre injected animals was significantly reduced in comparison to mice injected with AAV-control (p = 0.00037), with PR knockdown spanning the rostral, mid, and caudal extent of the ARC (p < 0.05). PRfl/fl mice injected with AAV-control (n = 5), or AAV-Cre outside of the ARC (off target) (n = 12) exhibited no changes in estrous cyclicity or LH release (p > 0.05). In contrast, PRfl/fl mice with AAV-Cre restricted to the ARC (n = 12) exhibited significantly decreased cycle frequency (p = 0.00036), increased cycle length (p = 0.0195), and a significant reduction in the percentage of time spent in proestrous (p = 0.0039). Similarly, PRfl/fl mice with AAV-Cre restricted to the ARC exhibited a significant increase in the number of LH peaks over a 2 hour time period in comparison to AAV-Cre off target (p < 0.05), with no differences in LH concentration or amplitude. Using a mouse model, we have demonstrated that knockdown of PR exclusively in the ARC in adulthood is sufficient to drive LH hypersecretion and mimic other reproductive impairments common to PCOS. Together, this work supports the ARC as the critical site for progesterone negative feedback regulation of the reproductive axis.
Oral Presentation.