TrkB agonist antibody ameliorates fertility deficits in aged and cyclophosphamide-induced premature ovarian failure model mice.

Premature ovarian failure (POF) is a leading cause of women's infertility without effective treatment. Here we show that intravenous injection of Ab4B19, an agonistic antibody for the BDNF receptor TrkB, penetrates into ovarian follicles, activates TrkB signaling, and promotes ovary development. In both natural aging and cyclophosphamide-induced POF models, treatment with Ab4B19 completely reverses the reduction of pre-antral and antral follicles, and normalizes gonadal hormone. Ab4B19 also attenuates gonadotoxicity and inhibits apoptosis in cyclophosphamide-induced POF ovaries. Further, treatment with Ab4B19, but not BDNF, restores the number and quality of oocytes and enhances fertility. In human, BDNF levels are high in granulosa cells and TrkB levels increase in oocytes as they mature. Moreover, BDNF expression is down-regulated in follicles of aged women, and Ab4B19 activates TrkB signaling in human ovary tissue ex vivo. These results identify TrkB as a potential target for POF with differentiated mechanisms, and confirms superiority of TrkB activating antibody over BDNF as therapeutic agents.

Melatonin rescues the reproductive toxicity of low-dose glyphosate-based herbicide during mouse oocyte maturation via the GPER signaling pathway.

Glyphosate-based herbicides (GBHs) are a group of widely used broad-spectrum agricultural pesticides. Due to the recalcitrance of GBH, it has been found in food and environment as a contaminant, posing a threat to public health. The health risks associated with GBH have been indicated by reporting acute toxicity data (an acute exposure of GBH at a 0.5% dose), which primarily discuss toxicity in relation to accidental high-rate exposure. Currently, there is little information regarding the toxicity of GBH at environmentally relevant levels. In this study, we used mature mouse oocytes to study the toxic effects of low-dose GBH exposure in vitro (0.00001%-0.00025%) and in vivo (0.0005%, orally administered through daily drinking water) during meiotic maturation. GBH exposure led to meiotic maturation failure with spindle defects and chromosome misalignment. In addition, GBH treatment severely reduced sperm-binding ability and disrupted early embryo cleavage. Moreover, GBH exposure significantly increased the reactive oxygen species (ROS) levels and apoptotic rates. Evidence indicates that such effects in GBH-exposed oocytes are likely due to overexpression of the G-protein estrogen receptor (GPER/GPR30). Remarkably, we found that melatonin administration elicited significant protection against GBH-induced oocyte deterioration via preserving the expression of GPR30, along with activation of its downstream signaling event (pERK/ERK). Taken together, these results revealed that low-dose glyphosate has a certain adverse effect on oocyte maturation and early embryo cleavage, and highlight the protective roles of melatonin.

Nicotine exposure impairs germ cell development in human fetal ovaries cultured in vitro.

In the present paper, we found that human fetal ovaries (at ~16 weeks) express the transcripts for several subunits of the nicotinic acetylcholine receptor (nAChR). Exposure to the drug in vitro resulted in the marked increase of apoptosis in the ovaries in a time and dose-dependent manner. Evidence that adverse nicotine effects are potentially due to an increased level of reactive oxygen species (ROS) and consequent DNA damage, both in the ovarian somatic cells and germ cells, are reported. After 4 days of culture, exposure to 1 mM and 10 mM nicotine caused a 50% and 75% decrease, respectively, in the number of oogonia/oocytes present in the fetal ovaries. These results represent the first indication that nicotine may directly cause apoptosis in cells of the fetal human ovary and may lead to a reduction of the ovarian reserve oocytes and consequent precocious menopause in mothers smoking during pregnancy.

In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells.

Understanding the unique mechanisms of human oogenesis necessitates the development of an in vitro system of stem cell differentiation into oocytes. Specialized cell types and organoids have been derived from human pluripotent stem cells in vitro, but generating a human ovarian follicle remains a challenge. Here we report that human embryonic stem cells can be induced to differentiate into ovarian follicle-like cells (FLCs) in vitro. First, we find that two RNA-binding proteins specifically expressed in germ cells, DAZL and BOULE, regulate the exit from pluripotency and entry into meiosis. By expressing DAZL and BOULE with recombinant human GDF9 and BMP15, these meiotic germ cells are further induced to form ovarian FLCs, including oocytes and granulosa cells. This robust in vitro differentiation system will allow the study of the unique molecular mechanisms underlying human pluripotent stem cell differentiation into late primordial germ cells, meiotic germ cells and ovarian follicles.

Oxidative stress induced by zearalenone in porcine granulosa cells and its rescue by curcumin in vitro.

Oxidative stress (OS), as a signal of aberrant intracellular mechanisms, plays key roles in maintaining homeostasis for organisms. The occurrence of OS due to the disorder of normal cellular redox balance indicates the overproduction of reactive oxygen species (ROS) and/or deficiency of antioxidants. Once the balance is broken down, repression of oxidative stress is one of the most effective ways to alleviate it. Ongoing studies provide remarkable evidence that oxidative stress is involved in reproductive toxicity induced by various stimuli, such as environmental toxicants and food toxicity. Zearalenone (ZEA), as a toxic compound existing in contaminated food products, is found to induce mycotoxicosis that has a significant impact on the reproduction of domestic animals, especially pigs. However, there is no information about how ROS and oxidative stress is involved in the influence of ZEA on porcine granulosa cells, or whether the stress can be rescued by curcumin. In this study, ZEA-induced effect on porcine granulosa cells was investigated at low concentrations (15 µM, 30 µM and 60 µM). In vitro ROS levels, the mRNA level and activity of superoxide dismutase, glutathione peroxidase and catalase were obtained. The results showed that in comparison with negative control, ZEA increased oxidative stress with higher ROS levels, reduced the expression and activity of antioxidative enzymes, increased the intensity of fluorogenic probes 2', 7'-Dichlorodihydrofluorescin diacetate and dihydroethidium in flow cytometry assay and fluorescence microscopy. Meanwhile, the activity of glutathione (GSH) did not change obviously following 60 µM ZEA treatment. Furthermore, the underlying protective mechanisms of curcumin on the ZEA-treated porcine granulosa cells were investigated. The data revealed that curcumin pre-treatment significantly suppressed ZEA-induced oxidative stress. Collectively, porcine granulosa cells were sensitive to ZEA, which may induce oxidative stress. The findings from this study clearly demonstrate that curcumin is effective to reduce the dysregulation of cellular redox balance on porcine granulosa cells in vitro and should be further investigated for its protective role against ZEA in animals.

Di-(2-ethylhexyl) phthalate and bisphenol A exposure impairs mouse primordial follicle assembly in vitro.

Bisphenol-A (BPA) and diethylhexyl phthalate (DEHP) are estrogenic compounds widely used in commercial plastic products. Previous studies have shown that exposure to such compounds have adverse effects on various aspects of mammalian reproduction including folliculogenesis. The objective of this study was to examine the effects of BPA and DEHP exposure on primordial follicle formation. We found that germ cell nest breakdown and primordial follicle assembly were significantly reduced when newborn mouse ovaries were exposed to 10 or 100 µM BPA and DEHP in vitro. Moreover, BPA and DEHP exposure increased the number of TUNEL positive oocytes and the mRNA level of the pro-apoptotic gene Bax in oocytes. These effects were associated with decreased expression of oocyte specific genes such as LIM homeobox 8 (Lhx8), factor in the germline alpha (Figla), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and newborn ovary homeobox (Nobox). Interestingly, BPA and DEHP exposure also prevented DNA demethylation of CpG sites of the Lhx8 gene in oocytes, a process normally associated with folliculogenesis. Finally, folliculogenesis was severely impaired in BPA and DEHP exposed ovaries after transplantation into the kidney capsules of immunodeficient mice. In conclusion, BPA and DEHP exposures impair mouse primordial follicle assembly in vitro.

Exposure to diethylhexyl phthalate (DEHP) results in a heritable modification of imprint genes DNA methylation in mouse oocytes.

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 µg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring's oocytes.