Ovarian matrix metalloproteinases are differentially regulated during the estrous cycle but not during short photoperiod induced regression in Siberian hamsters (Phodopus sungorus)

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are implicated as mediators for ovarian remodeling events, and are involved with ovarian recrudescence during seasonal breeding cycles in Siberian hamsters. However, involvement of these proteases as the photoinhibited ovary undergoes atrophy and regression had not been assessed. We hypothesized that 1) MMPs and their tissue inhibitors, the TIMPs would be present and differentially regulated during the normal estrous cycle in Siberian hamsters, and that 2) MMP/TIMP mRNA and protein levels would increase as inhibitory photoperiod induced ovarian degeneration.</p> <p>Methods</p> <p>MMP-2, -9, -14 and TIMP-1 and -2 mRNA and protein were examined in the stages of estrous (proestrus [P], estrus [E], diestrus I [DI], and diestrus II [DII]) in Siberian hamsters, as well as after exposure to 3, 6, 9, and 12 weeks of inhibitory short photoperiod (SD).</p> <p>Results</p> <p>MMP-9 exhibited a 1.6-1.8 fold decrease in mRNA expression in DII (p < 0.05), while all other MMPs and TIMPs tested showed no significant difference in mRNA expression in the estrous cycle. Extent of immunostaining for MMP-2 and -9 peaked in P and E then significantly declined in DI and DII (p < 0.05). Extent of immunostaining for MMP-14, TIMP-1, and TIMP-2 was significantly more abundant in P, E, and DI than in DII (p < 0.05). Localization of the MMPs and TIMPs had subtle differences, but immunostaining was predominant in granulosa and theca cells, with significant differences noted in staining intensity between preantral follicles, antral follicles, corpora lutea, and stroma classifications. No significant changes were observed in MMP and TIMP mRNA or extent of protein immunostaining with exposure to 3, 6, 9, or 12 weeks of SD, however protein was present and was localized to follicular and luteal steroidogenic cells.</p> <p>Conclusions</p> <p>Although MMPs appear to be involved in the normal ovarian estrus cycle at the protein level in hamsters, those examined in the present study are unlikely to be key players in the slow atrophy of tissue as seen in Siberian hamster ovarian regression.</p

Estrogenic exposure alters the spermatogonial stem cells in the developing testis, permanently reducing crossover levels in the adult

Bisphenol A (BPA) and other endocrine disrupting chemicals have been reported to induce negative effects on a wide range of physiological processes, including reproduction. In the female, BPA exposure increases meiotic errors, resulting in the production of chromosomally abnormal eggs. Although numerous studies have reported that estrogenic exposures negatively impact spermatogenesis, a direct link between exposures and meiotic errors in males has not been evaluated. To test the effect of estrogenic chemicals on meiotic chromosome dynamics, we exposed male mice to either BPA or to the strong synthetic estrogen, ethinyl estradiol during neonatal development when the first cells initiate meiosis. Although chromosome pairing and synapsis were unperturbed, exposed outbred CD-1 and inbred C3H/HeJ males had significantly reduced levels of crossovers, or meiotic recombination (as defined by the number of MLH1 foci in pachytene cells) by comparison with placebo. Unexpectedly, the effect was not limited to cells exposed at the time of meiotic entry but was evident in all subsequent waves of meiosis. To determine if the meiotic effects induced by estrogen result from changes to the soma or germline of the testis, we transplanted spermatogonial stem cells from exposed males into the testes of unexposed males. Reduced recombination was evident in meiocytes derived from colonies of transplanted cells. Taken together, our results suggest that brief exogenous estrogenic exposure causes subtle changes to the stem cell pool that result in permanent alterations in spermatogenesis (i.e., reduced recombination in descendent meiocytes) in the adult male

Evidence for paternal age-related alterations in meiotic chromosome dynamics in the mouse

Increasing age in a woman is a well-documented risk factor for meiotic errors, but the effect of paternal age is less clear. Although it is generally agreed that spermatogenesis declines with age, the mechanisms that account for this remain unclear. Because meiosis involves a complex and tightly regulated series of processes that include DNA replication, DNA repair, and cell cycle regulation, we postulated that the effects of age might be evident as an increase in the frequency of meiotic errors. Accordingly, we analyzed spermatogenesis in male mice of different ages, examining meiotic chromosome dynamics in spermatocytes at prophase, at metaphase I, and at metaphase II. Our analyses demonstrate that recombination levels are reduced in the first wave of spermatogenesis in juvenile mice but increase in older males. We also observed age-dependent increases in XY chromosome pairing failure at pachytene and in the frequency of prematurely separated autosomal homologs at metaphase I. However, we found no evidence of an age-related increase in aneuploidy at metaphase II, indicating that cells harboring meiotic errors are eliminated by cycle checkpoint mechanisms, regardless of paternal age. Taken together, our data suggest that advancing paternal age affects pairing, synapsis, and recombination between homologous chromosomes--and likely results in reduced sperm counts due to germ cell loss--but is not an important contributor to aneuploidy

Germ cell transplantation experimental design.

<p>C3H male pups from timed-pregnant C3H females were orally exposed to placebo or 0.25 ng/g/day EE from 1–12 dpp. At 13 dpp, testes were pooled per exposure group, germ cells were isolated, and transplanted into W/W^v adult males. Placebo cells were injected into one testis, and EE-exposed cells into the contralateral testis. Different letters and numbers represent different transplantation days and individuals, respectively. Meiotic analyses occurred 8 weeks post-transplantation.</p

Analysis of metaphase I in placebo and exposed C3H males.

<p>*Data represent number of cells and percentage in parentheses from 20–27 MI cells per animal from 3 placebo and 5 EE-exposed C3H males; X² = 8.65, p<0.005 for comparison of normal and abnormal cells.</p><p>Analysis of metaphase I in placebo and exposed C3H males.</p

Recombination rates^* in placebo and exposed males.

<p>*Mean MLH1 ± SEM for 25–30 pachytene cells per male, with 5–12 males/group.</p><p>^a-d Exposure within a strain and age were compared by one-way ANOVA (CD-1, C57BL/6J) or one-tail t-test (C3H/HeJ, C3H/B6 F1). Superscript letters denote significant differences as determined by a Newman-Keuls post hoc test (p<0.05); like letters indicate no difference.</p><p>— Denotes experimental group was not performed.</p><p>Recombination rates^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004949#t001fn001" target="_blank">*</a>} in placebo and exposed males.</p

Recombination in transplanted males.

<p>¹Recipients with different letters were transplanted on different days.</p><p>²Germ cells from placebo-exposed donors in one testis.</p><p>³Germ cells from EE-exposed males in the contralateral testis.</p><p>Values represent mean MLH1 ± SEM.</p><p>n = number of cells analyzed.</p><p>*indicates significant difference between exposed and placebo cells in one-tailed t-test (p<0.05)</p><p>Recombination in transplanted males.</p

Analysis of metaphase I (MI) and metaphase II (MII) cells in C3H spermatocytes from placebo and exposed males.

<p>A) Normal MI spermatocyte from a placebo male. B) MI spermatocyte with small autosomal univalents (arrows) and sex chromosome univalents (X and Y) from an EE-exposed male. C) Normal MII spermatocyte from a placebo male. D) MII spermatocyte with an extra chromatid (arrow) from an EE-exposed male. Scale bars represent 10 μm.</p

Analysis of metaphase II in placebo and exposed males.

<p>*Data represent number of cells and percentage in parentheses from 20–25 MII cells per animal from 3 placebo and 5 EE-exposed C3H males.</p><p>Analysis of metaphase II in placebo and exposed males.</p