Project Summary/Abstract
Stress is considered a major factor in the development of menstrual cycle disorders, amenorrhea, and infertility,
affecting 25% of reproductive age women. To date, the neuroendocrine causes of stress-induced infertility are
not completely understood. Enhanced secretion of glucocorticoids (CORT) is a common and critical response to
all stressors. We demonstrated that a stress level of CORT disrupts the ovulatory cycle of female mice via
suppression of kisspeptin (Kiss1) neurons located in both arcuate (ARCKiss1) and anteroventral periventricular
(AVPVKiss1) nuclei, which are essential components of the neural control of reproduction. The effect of CORT
depends on ovarian steroid milieu. Specifically, low diestrus levels of estradiol enable CORT to impair ARCKiss1
control of luteinizing hormone (LH) pulses and high surge estradiol levels enable CORT to prevent AVPVKiss1
neuronal activation of the LH secretion. Receptors for estrogen and glucocorticoid receptors (GR) are both in
Kiss1 neurons; however, the interaction of GR and ER to modulate the transcriptomic and epigenomic landscape
within ARCKiss1 or AVPVKiss1 neurons to suppress LH secretion remains unclear. This proposal employs a cutting-
edge method to capture and isolate Kiss1 cells from heterogeneous ARC or AVPV cell populations. The Isolation
of Nuclei Tagged in specific Cell-Types (INTACT) protocol employs expression of a tagged nuclear envelope
protein (Sun1) only in Kiss1 cells (i.e. Cre-dependent) for rapid isolation of tagged nuclei for downstream
transcriptomic and epigenomic sequencing. Aim 1 will test the hypothesis that estradiol enables CORT to alter
the transcriptome of Kiss1 neurons, facilitating suppressed ARCKiss1 and AVPVKiss1 neuronal activity and lowered
pulsatile and surge-type LH secretion, respectively. Mice will be OVX and receive diestrus-like (ARCKiss1) or
surge-level replacement (AVPVKiss1) of estradiol and treated with CORT or cholesterol (control), and the
transcriptome of INTACT-isolated Kiss cells will be evaluated by RNA-sequencing. Aim 2 will test the hypothesis
that estradiol drives changes of the chromatin landscape of ARCKiss1 and AVPVKiss1 neurons allowing GR to bind
and alter the neuronal transcriptome. INTACT will be coupled with: (2.1) Assay for Transposase-Accessible
Chromatin (ATAC-seq) to evaluate the epigenome for open chromatin in mediated by estradiol and CORT and
(2.2) chromatin immunoprecipitation (ChIP-seq) to directly map the genome-wide binding profile of GR in
ARCKiss1 and AVPVKiss1 cells to test the hypothesis that GR binding sites are made accessible by estradiol
(hormone treatments described in Aim 1). Successful completion of this proposal will significantly increase our
knowledge of intracellular mechanisms that govern the physiologic responsiveness of Kiss1 neurons, which will
be tested in subsequent work and may identify druggable targets to influence the management of reproductive
disorders resulting from impaired central regulation. This application falls within the scope of the high risk-high
reward mechanism and will result in novel reagents that will impact biomedical research. All methods, hormone
treatments and the animal model (Kiss1-CrexSun1-GFP) are available within this investigative team.