Chronic androgen excess in female mice does not impact luteinizing hormone pulse frequency or putative GABAergic inputs to GnRH neurons

Abstract

Polycystic ovary syndrome (PCOS) is associated with androgen excess and, frequently, hyperactive pulsatile luteinizing hormone (LH) secretion. Although the origins of PCOS are unclear, evidence from pre-clinical models implicates androgen signalling in the brain in the development of PCOS pathophysiology. Chronic exposure of female mice to dihydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic impairments that are ameliorated by selective androgen receptor (AR) loss from the brain. This suggests centrally driven mechanisms in hyperandrogen-mediated PCOS-like pathophysiology that remain to be defined. Acute prenatal DHT exposure can also model the hyperandrogenism of PCOS, and this is accompanied by increased LH pulse frequency and increased GABAergic innervation of gonadotrophin-releasing hormone (GnRH) neurons. We aimed to determine the impact of chronic exposure of female mice to DHT, which models the hyperandrogenism of PCOS, on pulsatile LH secretion and putative GABAergic input to GnRH neurons. To do this, GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day 21 (n = 6 or 7 per group). Serial tail-tip blood sampling was used to measure LH dynamics and perfusion-fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. As expected, chronic DHT resulted in acyclicity and significantly increased body weight. However, no differences in LH pulse frequency or the density of VGAT appositions to GnRH neurons were identified between ovary-intact DHT-treated females and controls. Chronic DHT exposure significantly increased the number of AR expressing cells in the hypothalamus, whereas oestrogen receptor α-expressing neuron number was unchanged. Therefore, although chronic DHT exposure from 3 weeks of age increases AR expressing neurons in the brain, the GnRH neuronal network changes and hyperactive LH secretion associated with prenatal androgen excess are not evident. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

Keywords: GABA; GnRH; LH; androgens; membrane/nuclear; receptors.

FIGURE 1

Chronic androgen excess causes an increase in body weight and loss of oestrous cyclicity. (A) Mean ± SEM weekly body weight (g) of animals with blank (n = 6) or dihydrotestosterone (DHT) (n = 7) capsules measured over 13 weeks. Asterisks above each weekly data point indicate the significance from Sidaks post‐hoc test; asterisks between lines indicate the mean effect of the capsule over 13 weeks (repeated measures two‐way ANOVA with Sidak's post‐hoc test). (B) Example oestrous cycle data collected over a 3‐week sampling window identifying when mice are in pro‐oestrus (P), dioestrus (D) or oestrus (E) for mice with Blank (B, black line) or DHT (B, magenta line) capsules. (C) Mean ± SEM percentage of time in each oestrous cycle phase. (D) Experimental timeline from capsule placement at postnatal day (PND)21. Schematic created using BioRender.com. Asterisks denote significant differences between treatment groups from two‐way ANOVA with Bonferroni's post‐hoc test. *p < .05, **p < .01, ****p < .0001

FIGURE 2

Chronic exposure to dihydrotestosterone (DHT) does not impact pulsatile lutenizing hormone (LH) secretion. (A) Diagram of tail‐tip bleeding protocol. (B) Representative patterns of LH secretion over 2 h from mice with either a blank (black) or DHT (magenta) capsule in dioestrus. Pulses indicated by a red or black data point, respectively. (C) Average LH area under the curve (AUC) over the 2‐h bleed. (D) Average basal LH concentration. (E) Average pulse frequency. (F) Average LH pulse amplitude for pulses identified by PULSAR. Schematic created using BioRender.com. Blank, n = 5 and DHT, n = 7 for all measurements; (C, D, F) Unpaired Student's t tests; (E) Mann–Whitney U test

FIGURE 3

Chronic dihydrotestosterone (DHT) exposed females do not exhibit changes in gonadotrophin‐releasing hormone (GnRH) neuron spine density or GABAergic close appositions. (A, B) Representative confocal images of green fluorescent protein expressing GnRH neurons (cyan) and vesicular GABA transporter (VGAT) immunoreactive puncta (magenta) from animals with Blank (A) or DHT (B). Images depicting GnRH neuronal cell body and the proximal primary dendrite are collapsed maximum projection images; Scale bars =10 μm. Inset images of the soma and primary dendrite (framed in dotted line) are a single plane equating to a thickness of 0.5 μm; Scale bars =3 μm. Closed arrowheads denote some close VGAT immunoreactive appositions (magenta) onto the GnRH neuron; Open arrowheads denote some GnRH neuron spines. (C) Mean ± SEM number of VGAT immunoreactive appositions per micrometre of the total neuron, and in somatic and 15‐μm regions of the primary dendrite. Mean ± SEM number of spines per micrometre for the whole GnRH neuron and in somatic and 15 μm regions of the primary dendrite. n = 6 per group; 52–54 neurons per animal

FIGURE 4

The number of oestrogen receptor α (ERα) expressing neurons in the hypothalamus is not changed following chronic dihydrotestosterone (DHT) exposure. (A) Representative images of ERα labelling in the rostral periventricular nucleus of the third ventricle, including the anteroventral periventricular nucleus (AVPV) and periventricular nucleus (PeN) in female mice treated for 3 months with either a blank or DHT containing capsule; Scale bars =200 μm. (B) Representative images of ERα labelling in the rostral arcuate nucleus (rARN), middle arcuate nucleus (mARN), and caudal arcuate nucleus (cARN) in females treated for 3 months with either a blank or DHT containing capsule; Scale bars =200 μm. (C) Mean ± SEM number of ERα immunoreactive cells quantified in each hypothalamic area. n = 6 or 7 per group, unpaired Students t test

FIGURE 5

The number of androgen receptor (AR) expressing neurons is significantly increased in the female hypothalamus following chronic dihydrotestosterone (DHT) exposure. (A) Representative images of AR labelling in the rostral periventricular nucleus of the third ventricle, including the anteroventral periventricular nucleus (AVPV) and periventricular nucleus (PeN) in female mice treated for 3 months with either a blank or DHT containing capsule; Scale bars =200 μm. (B) Representative images of AR labelling in the rostral arcuate nucleus (rARN), middle arcuate nucleus (mARN), and caudal arcuate nucleus (cARN) in females treated for 3 months with either a blank or DHT containing capsule; Scale bars =200 μm. (C) Mean ± SEM number of AR immunoreactive cells quantified in each hypothalamic area. n = 4–7 per group, unpaired Students t test, ****p < .0001

FIGURE 6

Representative low magnification images of androgen receptor (AR) immunolabelling in anterior (A) and posterior (B) hypothalamic coronal sections. Scale bar in (A) = 250 μm; scale bar in (B) = 400 μm. Ac, anterior commissure; AVPV, anteroventricular periventricular nucleus; LS, lateral septum; BNST, bed nucleus of the stria terminalis; ARN, arcuate nucleus; VMH, venteromedial nucleus of the hypothalmus; DMH, dorsomedial nucleus of the hypothalamus; MEA, medial amygdala