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Studies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

BackgroundA standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these ‘historical’ criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying).MethodsReasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries.ResultsWe found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation.ConclusionsCombined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

The aim of this study was to investigate the possible protective effects of aqueous garlic extract (AGE) against naphthalene-induced oxidative changes in liver, kidney, lung and brain of mice. Balb/c mice (25-30 g) of either sex were divided into five groups each comprising 10 animals. Mice received for 30 days: 0.9% NaCl, i.p. (control); corn oil, i.p; AGE in a dose of 125 mg kg-1, i.p.; naphthalene in a dose of 100 mg kg-1, i.p. (dissolved in corn oil); and AGE (in a dose of 125 mg kg-1, i.p.) plus naphthalene (in a dose of 100 mg kg-1, i.p.). After decapitation, liver, kidney, lung and brain tissues were excised. Malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in the tissues, while oxidant-induced tissue fibrosis was determined by collagen content. Tissues were also examined microscopically. Serum aspartate aminotransferase, alanine aminotransferase levels and blood urea nitrogen and creatinine concentrations were measured for the evaluation of hepatic and renal function, respectively. MDA and GSH levels were also assayed in serum samples. In the naphthalene-treated group, GSH levels decreased significantly, while MDA levels, MPO activity and collagen content increased in the tissues (P<0.01-0.001), suggesting oxidative organ damage, which was also verified histologically. In the AGE-treated naphthalene group, all of these oxidant responses were reversed significantly (P<0.05-0.01). Hepatic and renal function test parameters, which increased significantly (P<0.001) following naphthalene administration, decreased (P<0.05-0.001) after AGE treatment. The results demonstrate the role of oxidative mechanisms in naphthalene-induced tissue damage. The antioxidant properties of AGE ameliorated oxidative organ injury due to naphthalene toxicity.

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In reproductive age women, the pool of primordial follicles is continuously depleted through the process of cyclic recruitment. AMH both inhibits the initial recruitment of primordial follicles into the growing pool and modulates the sensitivity of growing follicles to FSH. Thus, AMH may be an important modulator of female infertility and ovarian reserve; however, the mechanisms regulating AMH remain unclear.To evaluate AMH levels in the absence of H19 lncRNA, H19 knockout (H19KO) mice were evaluated for analysis of ovarian AMH gene expression, protein production, and reproductive function, including assessment of follicle numbers and litter size analysis. To further investigate regulation of AMH by the H19/let-7 axis, let-7 binding sites on AMH were predicted, and in vitro studies of the effect of H19 knockdown/overexpression with let-7 rescue were performed. Lastly, response to superovulation was assessed via oocyte counts and estradiol measurements.The H19KO mouse demonstrates subfertility and accelerated follicular recruitment with increased spontaneous development of secondary, preantral and antral follicles. Ovaries of H19KO mice have decreased AMH mRNA and protein, and AMH mRNA has a functional let-7 binding site, suggesting a plausible ncRNA-mediated mechanism for AMH regulation by H19/let-7. Lastly, in the absence of H19, superovulation results in higher estradiol and more oocytes, suggesting that H19 functions to limit the number of follicles that mature, produce estradiol, and ovulate. Thus, AMH's inhibitory actions are regulated at least in part by H19, likely via let-7, marking this ncRNA pair as important regulators of the establishment and maintenance of the follicular pool.

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