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TJS thanks the British Society for Neuroendocrinology and the Society for Reproduction and Fertility for research project funds. PB acknowledges the Scottish Government for funding. Experiments investigated neuroanatomically localized 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 examine the role of ovarian steroids. Hypothalamic Dnmt3a expression was observed to significantly increase during the transition from proestrous to oestrous. A single bolus injection of diethylstilbestrol (DES) and progesterone was sufficient to increase Dnmt3a cell numbers and Dnmt3b immunoreactive intensity in the suprachiasmatic nucleus (SCN). In vitro analyses using an embryonic rodent cell line revealed that DES was sufficient to induce Dnmt3b expression. Upregulating DNA methylation in vitro reduced 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. Postprint

Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility in women of reproductive age, characterised by androgen excess, polycystic appearance of the ovary and irregular menstruation. PCOS is also frequently associated with metabolic abnormalities, including increased adiposity and insulin resistance. The origins of PCOS are unknown, however recent findings in animal models strongly implicate androgen signalling in the brain in the development of PCOS pathophysiology. Exposure to androgen excess, either acutely during prenatal development or chronically from a peripubertal timepoint, can drive the development of PCOS-like features in adulthood. Prenatally androgenized (PNA) mice exhibit the cardinal reproductive features of PCOS and increased luteinizing hormone (LH) pulse frequency. This phenotype is associated with increased GABAergic innervation of gonadotropin-releasing hormone (GnRH) neurons, postulated to drive elevated GnRH/LH release and downstream effects. Chronic exposure to di-hydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic PCOS-like features that are ameliorated by selective AR loss from the brain. Here, we aimed to determine whether chronic exposure to DHT drives a similar increase in LH pulsatility and elevated GABAergic innervation to GnRH neurons as seen following prenatal exposure to androgen excess. GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day (PND) 21 (N = 6-7/group). Serial tail tip blood sampling was used to measure pulsatile LH and fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. Chronic androgen excess from the peripubertal period resulted in acyclicity and increased body weight as expected. However, LH pulsatility was not different between DHT-treated females and controls. Similarly, the density of VGAT appositions to GnRH neurons was not different between groups. Therefore, the programmed changes in the GnRH neuronal network and hyperactive LH secretion that result from prenatal androgen excess are not affected by chronic DHT exposure initiated at 3 weeks of age. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

Polycystic ovary syndrome (PCOS) is a highly prevalent endocrine disorder associated with hyperandrogenism and anovulation. Although a spectrum disorder, many women with PCOS exhibit elevated luteinizing hormone (LH) pulse frequency and an elevated LH to follicle stimulating hormone ratio. This aberrant pattern of gonadotrophin signalling drives many of the downstream ovarian features of PCOS, including increased androgen synthesis, and indicates neuroendocrine impairments upstream. Decreased responsiveness to gonadal steroid hormone negative feedback in PCOS patients points toward dysfunction within the gonadotropin-releasing hormone (GnRH) neuronal network in the brain. Excessive androgen exposure during development or over pubertal onset can recapitulate the neuroendocrine pathology of PCOS in pre-clinical models, and these models have been fundamental in beginning to pick apart the specific central mechanisms involved. This mini-review will briefly describe the pathology of PCOS associated with high frequency GnRH/LH pulses and then highlight what is currently known, and yet to be discovered, about the central mechanisms involved.

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.

Epigenetic modifications in reproductive tissues have predominantly focused on pathological conditions, such as ovarian and uterine cancers. The contribution of DNA methylation and histone acetylation to the timing and control of fertility is not well described. Siberian hamsters provide an important model to investigate the relatively short-term regulation of fertility (e.g. estrous) as well as long-term timing of breeding (e.g. seasonal). Recent work has shown that DNA methyltransferase 3a (dnmt3a) expression is associated with reproductive involution. Here, the objectives were to identify the impact of photoperiod on hdac1–3 expression in hamster testicular, ovarian and uterine tissue. Then, we assessed the effect of E2P4 and estrous cycling on hdac1–3 expression in uterine tissue. Testicular expression of hdac1 was significantly reduced, whereas hdac3 increased in reproductively photoregressed male hamsters; hdac2 expression did not significantly change across photoperiod conditions. There was no significant photoperiodic effect on ovarian expression of hdac1–3. Uterine expression of hdac3 expression was greater in long day hamsters; exposure to short days significantly reduced uterine hdac2 expression. Ovariectomized hamsters administered a single bolus injection of oil were found to have elevated uterine hdac2 compared to E2P4 treated females 12 h and 24 h post injection. Uterine hdac1–3 expression was relatively constant across the estrous cycle. Altogether these data indicate tissue-dependent photoperiodic regulation of hdac1–3 expression and that E2P4 may inhibit uterine hdac2 over long-term breeding cycles.

The direct conversion of methane to liquid fuels and chemicals is highly sought, especially at low-scale to more effectively utilize remote natural gas resources. Direct conversion has proven to be a very difficult problem, because methane is highly unreactive, though tends to over-oxidize once activated. Methanol is primarily produced today by the steam reformation of methane at high temperatures and pressures to synthesis gas (carbon monoxide plus hydrogen), followed by catalytic conversion at intermediate temperatures and high pressures. Though highly selective, this technology is not well suited for low production wellheads. The direct conversion of methane to methanol and other liquids would be a game changer, potentially leading to much lower production costs. In our presentation we will be reporting the development of the metal oxide-based electrocatalysts, which provide an oxygen activity needed for the partial oxidation of methane to liquid fuels suitable for applications in intermediate temperature fuel cells. The tests were performed at atmospheric pressure at different CH4 flow rates and different temperatures in the 200-700oC range.The product analysis was performed by online quadrupole mass spectrometer as well as by GC-MS. Methanol is the primary product of methane conversion at temperatures below 400oC. At 600-700oC, ethane, ethylene, propane, propene, n-butane, iso-butane, 2-butyne, and benzene formation was observed. In all tests the formation of carbon oxides was observed as well. Separately, the catalysts were evaluated using diffuse reflectance infrared Fourier Transform Infrared Spectroscopy (DRIFTS), TGA and in operando XRD. The kinetics of oxygen gain appeared to be slower that the oxygen loss. Somewhat longer time was needed to re-oxidize the catalyst. Kinetics are faster and conversion of CH4 was greater at higher temperatures. The post-test analysis was done using SEM and XRD. Figure 1. Methane conversion (right y-axis) and the rates of methanol, CO, and CO2 formation (left y-axis) at 300oC. Figure 2. Relative hydrocarbon distribution at 700oC. Figure 1

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