Identification of microRNAs as potential markers of ovarian toxicity.

Exposure to environmental toxicants has been associated with ovarian dysfunction yet sensitive biomarkers of adverse effect are lacking. We previously demonstrated that cigarette smoke exposure induced decreased relative ovarian weight, increased follicle loss and granulosa cell autophagy in mice. We postulate that cigarette smoke exposure will induce changes in the epigenome that can be used to reveal potential sensitive biomarkers of ovarian toxicity. Therefore, we evaluated differences in expression of 940 microRNAs (miRNAs), environmentally responsive small non-coding genes that regulate expression of genes at the post-transcriptional level, in ovarian tissue from 8-week-old female C57BL/6 mice exposed to room air or cigarette smoke 5 days per week for 8 weeks. A total of 152 miRNAs were dysregulated in expression, 17 of which were examined with quantitative polymerase chain reaction analysis. Using an online miRNA database tool, complete lists of predicted miRNA gene targets were generated, 12 of which were measured for their expression levels with quantitative polymerase chain reaction. An online bioinformatics resource database, DAVID generated functional classification lists of the target genes and their associated biological pathways. Results of the present pilot study suggest that miR-379, miR-15b, miR-691, miR-872 and miR-1897-5p are potentially useful markers of ovarian toxicity and dysfunction. Examination of the expression pattern of the target mRNA for these miRNA species demonstrated that cigarette smoke exposure induced significant changes that affect mitogen-activated protein kinase signaling pathways. We therefore suggest that miRNAs could serve as sensitive markers of ovarian toxicity and elucidate affected pathways.

Effects of in vitro exposure to butylparaben and di-(2 ethylhexyl) phthalate, alone or in combination, on ovarian function.

Parabens and phthalates are commercial chemicals widely used in the manufacture of industrial and consumer products frequently found as contaminants in biological fluids. We evaluated the effects of di-(2-ethylhexyl) phthalate (DEHP) (ranging from 10(-9) to 10(-7) m [1-100 nm; 0.39-39 ng ml(-1) ]) and butylparaben (BP) (ranging from 10(-8) to 10(-5) m [10 nm-10 µm; 1.9 ng ml(-1) to 1.9 µg ml(-1) ]), alone and in combination, on isolated mouse preantral follicle and human granulosa cell (hGC) cultures to study direct effects on follicle growth and ovarian steroidogenesis. Our results revealed that, in follicle culture, DEHP and BP attenuate estradiol output but only when present together. DEHP decreases progesterone concentrations in the spent media of hGC cultures, an effect that was attenuated when BP was added together with DEHP. Although changes in steroidogenesis were observed, no effects on follicular development or survival were noted in the culture systems. We suggest that BP and DEHP act with additive effect to decrease estradiol production whereas at later stages of follicle development BP blocks the effect of DEHP in hGCs resulting in decreased progesterone output. Taken together our results suggest that DEHP and BP adversely affect steroidogenesis from the preantral stage onward and the effects of these chemicals are both stage-dependent and modified by co-exposure. Copyright © 2016 John Wiley & Sons, Ltd.

Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.