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

Background: 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. Methods: 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). Results: 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. Conclusions: 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. Background Normal ovarian function is dependent on a series of tissue remodeling events taking place throughout the reproductive cycle. For a number of species, including Siberian hamsters (Phodopus sungorus), a seasonal pattern of reproduction is exhibited in response to changes in photoperiod. Long (> 12 h of light per day) photoperiods correlate with abundant environmental resources for many temporal rodents, and therefore can stimulate reproductive physiology and behavior [1]. Follicle development, ovulation, and corpus luteum formation and degradation all occur during the four-day estrous cycle in Siberian * Correspondence: kayoung@csulb.edu 1 Reproductive Biology Group, Department of Biological Sciences, California State University, Long Beach, Long Beach, CA 90840, USA Full list of author information is available at the end of the article © 2010 Vrooman and Young; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vrooman and Young Reproductive Biology and Endocrinology 2010, 8:79 http://www.rbej. com/content/8/1/79 Page 2 of 15 hamsters with long day stimulation. In contrast, exposure to short (< 12 h of light per day) photoperiod can terminate reproductive function [1,2], inducing an anestrous/ anovulatory state in regressed ovaries [3]. In Siberian hamsters, exposure to 12-14 weeks of short photoperiod results in reduced or absent ovulation and significant reductions in ovarian mass, the number of antral follicles and the number of corpora lutea (CL) [4-6]. In mammalian ovaries, the extracellular matrix (ECM) regulates cellular processes vital for follicle growth and maturation, including proliferation, differentiation, and survival [7], and its synthesis and degradation are vital to ovulation, CL formation, and luteal regression [8]. The remodeling of the ECM is mediated in part by a family of Zn+-dependent endopeptidases, matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs). Signaled by a variety of hormones, growth factors, and cytokines, MMPs and TIMPs contribute to the degradation of the ECM in the ovary by cleaving the various tissue components to clear space for new growth [8,9]. MMP and TIMP protein levels and mRNA expression show distinct differences in expression during follicle development and ovulation, and throughout luteal formation and degradation in rats, mice, pigs, cattle, sheep, and primates, suggesting that the concerted action of MMPs may regulate these ovarian events [10-16]. The MMPs specifically investigated in this study are MMPs -2, -9, and -14 and TIMPs -1 and -2. MMP-2 and MMP-9, of the gelatinase class, promote follicle growth in both rodents and goats [11,17,18], and have been implicated in the ovulatory process. MMP-2 protein is localized to the granulosa and theca cells and both protein and mRNA are increased in rats following PMSG [10] or hCG administration [19], while in mice, MMP-9 mRNA expression is increased with LH stimulation [19]. Both MMP-2 and MMP-9 mRNA expressions increase in primate granulosa cells after hCG administration [20], and MMP-2 increases as the ovary returns to function in photostimulated Siberian hamsters [6]. MMP-14 (mt-MMP1) is a transmembrane collagenase that cleaves collagens I, II, and III, as well as activates MMP-2 [21]. With the gonadotropin surge, MMP-14 mRNA is upregulated in bovine peri-ovulatory and luteal tissue [22], and increases in theca cells after ovulation is induced in mice [23]. Active MMP-14 protein is also increased in the bovine mid and late luteal periods [24], and MMP-14 mRNA is increased during photostimulated ovarian recrudescence in Siberian hamsters [6]. Both TIMP-1 and -2 are capable of inhibiting all MMP active forms, although show differential preference for certain MMPs. TIMP-1 preferentially inhibits MMP-9 [25], but is a poor inhibitor of MMP-14, and TIMP-2 forms a complex with pro-MMP-2 which can be stimulatory in conjunction with membrane bound MMPs, but effectively inhibits MMP-2 in higher concentrations [26]. TIMP-1 mRNA expression is increased after LH stimulus in rats [27], mice [23], and sheep [28], and TIMP-1 protein is localized to granulosa cells and luteal tissue following LH stimulus in sheep follicles [29]. In primates, TIMP-1 mRNA and protein and TIMP-2 mRNA are upregulated in human perifollicular ovarian stroma prior to and during ovulation [30] and TIMP-1 and -2 are upregulated in rhesus macaque periovulatory ovaries following hCG administration [20]. Finally, TIMP-1 declines during exposure to inhibitory photoperiods in Siberian hamsters and remains low throughout the bulk of recrudescence, returning to control levels only once the ovary returns to function [6]. While MMPs and TIMPs are linked to important events during the ovarian cycle across a number of species, and are dynamically expressed during photostimulated recrudescence in Siberian hamsters, the expression patterns of these MMPs/TIMPs during the hamster estrous cycle and during loss of function in photoperiodinduced ovarian regression have not been examined. In the current study, we hypothesized that MMP and TIMP mRNA and protein expression was 1) present and differentially regulated in the stages of estrous during tissue remodeling in the hamster estrous cycle, and 2) that MMPs and TIMPs are involved in the ovarian regression and loss of function. As a first step to address these hypotheses, mRNA and protein expression was determined in a select group of representative MMPs/TIMPs (MMP-2, -9, and -14 and TIMP-1 and -2) during the estrous cycle of Siberian hamsters subjected to a long day photoperiod (LD; 16L:8D) as well as in hamsters subjected to 3, 6, 9, and 12 weeks of short day photoperiod (SD; 8L:16D).