Investigating GABA Neuron-Specific Androgen Receptor Knockout in two Hyperandrogenic Models of PCOS.

Androgen excess is a hallmark feature of polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Clinical and preclinical evidence links developmental or chronic exposure to hyperandrogenism with programming and evoking the reproductive and metabolic traits of PCOS. While critical androgen targets remain to be determined, central GABAergic neurons are postulated to be involved. Here, we tested the hypothesis that androgen signaling in GABAergic neurons is critical in PCOS pathogenesis in 2 well-characterized hyperandrogenic mouse models of PCOS. Using cre-lox transgenics, GABA-specific androgen receptor knockout (GABARKO) mice were generated and exposed to either acute prenatal androgen excess (PNA) or chronic peripubertal androgen excess (PPA). Females were phenotyped for reproductive and metabolic features associated with each model and brains of PNA mice were assessed for elevated GABAergic input to gonadotropin-releasing hormone (GnRH) neurons. Reproductive and metabolic dysfunction induced by PPA, including acyclicity, absence of corpora lutea, obesity, adipocyte hypertrophy, and impaired glucose homeostasis, was not different between GABARKO and wild-type (WT) mice. In PNA mice, acyclicity remained in GABARKO mice while ovarian morphology and luteinizing hormone secretion was not significantly impacted by PNA or genotype. However, PNA predictably increased the density of putative GABAergic synapses to GnRH neurons in adult WT mice, and this PNA-induced plasticity was absent in GABARKO mice. Together, these findings suggest that while direct androgen signaling in GABA neurons is largely not required for the development of PCOS-like traits in androgenized models of PCOS, developmental programming of GnRH neuron innervation is dependent upon androgen signaling in GABA neurons.

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

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.

Ovarian hormones induce de novo DNA methyltransferase expression in the Siberian hamster suprachiasmatic nucleus.

The present study investigated neuroanatomically localised changes in de novo DNA methyltransferase expression in the female Siberian hamster (Phodopus sungorus). The objectives were to identify the neuroendocrine substrates that exhibit rhythmic Dnmt3a and Dnmt3b expression across the oestrous cycle and also examine the role of ovarian steroids. Hypothalamic Dnmt3a expression was observed to significantly increase during the transition from pro-oestrous to oestrous. A single bolus injection of diethylstilbestrol and progesterone was sufficient to increase Dnmt3a cell numbers and Dnmt3b immunoreactive intensity in the suprachiasmatic nucleus. In vitro analyses using an embryonic rodent cell line revealed that diethylstilbestrol was sufficient to induce Dnmt3b expression. Up-regulating DNA methylation in vitro reduced the expression of vasoactive intestinal polypeptide, Vip, and the circadian clock gene, Bmal1. Together, these data indicate that ovarian steroids drive de novo DNA methyltransferase expression in the mammalian suprachiasmatic nucleus and increased methylation may regulate genes involved in the circadian timing of oestrous: Vip and Bmal1. Overall, epigenetically mediated neuroendocrine reproductive events may reflect an evolutionarily ancient process involved in the timing of female fertility.

Activation of arcuate nucleus GABA neurons promotes luteinizing hormone secretion and reproductive dysfunction: Implications for polycystic ovary syndrome.

BACKGROUND: Enhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined. METHODS: We employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features. FINDINGS: Acute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABAA-dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels. INTERPRETATION: ARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits.

Cyclical DNA Methyltransferase 3a Expression Is a Seasonal and Estrus Timer in Reproductive Tissues.

It is becoming clear that epigenetic modifications such as DNA methylation can be dynamic and, in many cases, reversible. Here we investigated the photoperiod and hormone regulation of DNA methylation in testes, ovaries, and uterine tissue across multiple time scales. We hypothesized that DNA methyltransferase 3a (dnmt3a) is driven by photoperiodic treatment and exhibits natural variation across the female reproductive cycle and that melatonin increases whereas estrogen reduces DNA methylation. We used Siberian hamsters (Phodopus sungorus) due to their robust changes in reproductive physiology across seasonal and estrus time scales. Our findings indicate that short-day (SD) winter-like conditions significantly increased global DNA methylation and dnmt3a expression in the testes. Using immunohistochemistry, we confirm that increased dnmt3a expression was primarily localized to spermatogonium. Conversely, the ovaries did not exhibit variation in DNA methylation or dnmt3a/3b expression. However, exposure to SD significantly increased uterine dnmt3a expression. We then determined that dnmt3a was significantly decreased during the estrus stage. Next, we ovariectomized females and subsequently identified that a single estrogen+progesterone injection was sufficient to rapidly inhibit dnmt3a and dnmt3b expression. Finally, we demonstrate that treatment of human embryonic kidney-293 cells with melatonin significantly increased both dnmt3a and dnmt3b expression, suggesting that long-duration nocturnal signaling in SD may be involved in the regulation of DNA methylation in both sexes. Overall, our data indicate that dnmt3a shows marked photoperiod and estrus plasticity that likely has broad downstream effects on the timing of the genomic control of reproductive function.

Prospective stage-stratified approach to AIDS-related Kaposi's sarcoma.

PURPOSE: Combination antiretroviral therapy (cART) is standard of care for patients with HIV diagnosed with Kaposi's sarcoma (KS), but the current role of systemic chemotherapy is undefined. PATIENTS AND METHODS: Since 1998, a prospective stage-stratified approach has been adopted for 469 patients with HIV with KS. Patients with early-stage (T0) KS are treated with cART alone; patients with advanced-stage (T1) KS receive cART plus liposomal anthracycline chemotherapy. Clinical characteristics, overall survival, and KS progression-free survival were analyzed according to stage at presentation and treatment received. RESULTS: A total of 303 patients presented with T0 stage KS, including 237 who were not receiving cART, and 166 patients had T1 stage KS. Patients with T0 KS had higher CD4 cell counts (P < .001); 90% of patients with T0 KS who were not receiving cART and 84% of those with T1 KS were treated in accordance with the stage-stratified approach. Median follow-up was 4.6 years, and 5-year overall survival was 89%; 54 patients have died, 15 as a result of KS. Overall 5-year survival was 92% for T0 KS and 83% to T1 KS (P = .0024). On-treatment analysis of 213 cART-naive patients with T0 KS treated with cART alone revealed 5-year overall survival of 95% and progression-free survival of 77%. For 140 patients with T1 disease treated with cART and liposomal anthracycline chemotherapy, 5-year overall survival was 85%. CONCLUSION: This stage-stratified approach to the management of KS achieves high survival in patients with advanced KS and reduces exposure to chemotherapy in patients with early-stage KS.

The sexual scripts of Kenyan young people and HIV prevention.

The scripting of sexual encounters among young people in Kenyan is described using results of 28 focus group discussions conducted with young people attending primary school standard 7, from four different ethnic groups and living in 22 different communities. Sexual encounters were described as both mundane and inevitable and followed a predetermined scripted sequence of events and interactions in which girls and boys played complementary roles. These scripts were set within discourses of force and the exchange of gifts for sex. The gendered nature of the script and its social and cultural foundations are discussed. Potential strategies for developing HIV prevention programming are discussed from the perspective of existing sexual scripts.

Effects of trace metals and organic additives on porosity and dielectric constant of high purity mesoporous silica films.

The beneficial effects that alkali metal and alkylammonium salt additions to molecularly templated silica sols have on the resulting mesoporous silica films formed from evaporative-coating methods with respect to porosity, elastic modulus, dielectric constant, and film surface uniformity were investigated and identified.

Effect of humidity treatments on porosity and mechanical integrity of mesoporous silica films.

The beneficial effects humidity treatments have on molecularly templated mesoporous silica films with respect to elastic modulus, with minimal detrimental effects on porosity and dielectric constant, were identified.