Prenatal Androgen Exposure Alters KNDy Neurons and Their Afferent Network in a Model of Polycystic Ovarian Syndrome

Abstract

Polycystic ovarian syndrome (PCOS), the most common endocrinopathy affecting women worldwide, is characterized by elevated luteinizing hormone (LH) pulse frequency due to the impaired suppression of gonadotrophin-releasing hormone (GnRH) release by steroid hormone negative feedback. Although neurons that co-express kisspeptin, neurokinin B, and dynorphin (KNDy cells) were recently defined as the GnRH/LH pulse generator, little is understood about their role in the pathogenesis of PCOS. We used a prenatal androgen-treated (PNA) mouse model of PCOS to determine whether changes in KNDy neurons or their afferent network underlie altered negative feedback. First, we identified elevated androgen receptor gene expression in KNDy cells of PNA mice, whereas progesterone receptor and dynorphin gene expression was significantly reduced, suggesting elevated androgens in PCOS disrupt progesterone negative feedback via direct actions upon KNDy cells. Second, we discovered GABAergic and glutamatergic synaptic input to KNDy neurons was reduced in PNA mice. Retrograde monosynaptic tract-tracing revealed a dramatic reduction in input originates from sexually dimorphic afferents in the preoptic area, anteroventral periventricular nucleus, anterior hypothalamic area and lateral hypothalamus. These results reveal 2 sites of neuronal alterations potentially responsible for defects in negative feedback in PCOS: changes in gene expression within KNDy neurons, and changes in synaptic inputs from steroid hormone-responsive hypothalamic regions. How each of these changes contribute to the neuroendocrine phenotype seen in in PCOS, and the role of specific sets of upstream KNDy afferents in the process, remains to be determined.

Keywords: GnRH; KNDy; PCOS; luteinizing hormone; mouse; prenatal androgen.

Figure 1.

Multiplex detection of RNA expression in the arcuate nucleus of control and prenatal androgen-treated mice. A, Confocal images of 12 RNA targets visualized simultaneously in representative sections from the arcuate nucleus (ARC) of PNV male, PNV female, and PNA female mice using RNAscope HiPlex fluorescent in situ hybridization. Scale bar = 100 µm. B, Representative confocal section of the ARC from a PNV female mouse containing labeling for Dapi and RNA transcripts for KISS1, TAC2, PDYN, FOS, ESR1, PGR, AR, AMRH2, TAC3R, OPRK1, SLC17A6 (vGluT2), and SLC32A1 (vGaT). Scale bar = 100 μm. (i) High magnification images of insets containing representative cells depicting automated detection of Dapi borders (ii) and RNA transcripts (iii) by Cellprofiler software. Signal that did not meet the criteria for RNA transcripts are represented by purple outlines. (iv) Dapi cells (randomly pseudocolored by CellProfiler software) containing at least 3 RNA puncta (Scale bar in inset = 10μm).

Figure 2.

Androgen receptor RNA is increased whereas progesterone and dynorphin RNA is reduced in ARC KISS1 (KNDy) cells in PNA mice. A, Confocal images of representative cells in the ARC (identified by DAPI, white) containing RNA transcripts for KISS1 (i), AR (ii), PDYN (iii), PGR (iv), and OPRK1 (v). Arrows depict examples of KISS1-positive cells co-expressing AR, PGR, DYN, and OPRK1 transcripts. Scale bars = 10 µm. B, Mean percentage ± SEM of KISS1 neurons expressing AR (ii), PGR (iii), PDYN (iv), and OPRK1 (v) transcripts. C, Mean number ± SEM of KISS1 (i), AR (ii), PGR (iii), PDYN (iv), and OPRK1 (v) RNA transcripts in KISS1 cells. *P < 0.05.

Figure 3.

PNA treatment reduces PGR expression in non-KNDy ARC glutamatergic and GABAergic cells following PNA treatment. A, C, Confocal images of representative cells in the ARC (identified by DAPI, white) containing RNA transcripts for VGLUT2 (Ai) or vGAT (Ci) and PGR (Aii, Cii). Arrows depict examples of VGLUT2-positive cells co-expressing PGR transcripts. B, Mean number ± SEM of VGLUT2 (i) and PGR (ii) transcripts in glutamatergic cells. D, Mean number ± SEM of VGAT (i) and PGR (ii) transcripts in GABAergic cells. *P < 0.05. Scale bars = 10 μm.

Figure 4.

Glutamatergic and GABAergic synaptic input to KNDy neurons is reduced in PNA mice. A, C, Representative confocal images of sections triple labeled for YFP in ARC kisspeptin (Kiss) cells, (i), synaptophysin (SYN, ii) and either vGluT2 (glutamatergic inputs, Aiii) or vGaT (GABAergic inputs, Ciii) in the middle ARC of PNV male, PNV female, and PNA female mice. Arrowheads indicate synaptic terminals containing glutamate (VGLUT2 + SYN, A) or GABA (vGAT + SYN, C). B, D, Graphs depicting the mean ± SEM density of synaptic (SYP-positive) contacts immunoreactive for vGluT2 (B) or vGaT (D). *P < 0.05. Scale bars = 5 µm.

Figure 5.

Reduced synaptic input to KNDy neurons in PNA mice originates from preoptic and hypothalamic nuclei. A, Illustration of the rabies virus tract-tracing workflow. B, Representative confocal images of Kiss1-Cre cells in the ARC transfected with AAV-TVA/GFP (i), RVDG-mCherry (ii), and the merged images (iii). Scale bar = 100 µm. C, High magnification insets from Bi-Biii with examples of cells that co-express AAV-TVA/GFP and RVDG-mCherry (arrowheads), which represent either starter cells or presynaptic KNDy inputs to KNDy neurons. Scale bar = 10 µm. D, Graph illustrating no significant difference (NS) in the mean ± SEM number of GFP + mCherry cells transfected in the ARC of PNV male, PNV female, and PNA female mice. Scale bars = 100 µm. E-I, (i) Representative images of mCherry-positive cells (presynaptic neurons to KNDy cells) in the preoptic area (POA, E), anteroventral periventricular nucleus (AVPV, F), anterior hypothalamic area (AHA, G) and lateral hypothalamus (LHA, H). E-H, Graphs depicting the mean ± SEM number of mCherry-positive cells in afferent areas (ii), and the distribution of mCherry cells as shown by the mean ± SEM percentage of total mCherry cells in each brain nuclei (iii). *P < 0.05.