Kelly A. Young - US grants

[unreadable] DESCRIPTION (provided by applicant): Following ovulation, the corpus luteum (CL) forms from the ruptured follicle, secreting progesterone (P) to permit intrauterine pregnancy. In nonfertile cycles, P production subsides and the CL regresses, ensuring return of reproductive potential. Changes in matrix metalloproteinase (MMP) expression and activity are associated with the substantial tissue remodeling required for both formation and regression of the CL. However, the regulation of MMP dynamics, plus the specific roles of MMPs in CL development or regression are unknown. Recent studies have identified gonadotropin and local Pdependent processes in the periovulatory follicle, including regulation of specific MMPs and their tissue inhibitors (TIMPs). Therefore the current experiments were designed to test the hypothesis that (1) gonadotropins and steroids, notably P. regulate MMPs and TIMPs in the CL during the menstrual cycle, and (2) MMPs are essential for CL development and regression in primates. Molecular and biochemical techniques, such as 'real time' PCR, Western analysis, immunohistochemistry, and zymography will be employed to detect changes in MMPs and lIMPs throughout the luteal lifespan and during hormone ablation/replacement. The proposed studies will provide the first comprehensive examination of MMP regulation and function in the CL, further our knowledge of and treatment for luteal phase defects in infertility, and may recommend novel forms of contraception for women.

DESCRIPTION (provided by applicant): Ovarian function, including follicle development, ovulation, and corpus luteum formation/degradation, is dependent upon tissue remodeling events, many of which are associated with a family of Zn+dependent endopeptidases, the matrix metalloproteinases (MMPs). Extreme remodeling of ovarian tissue is observed in photoperiodic species where seasonal changes in day length can either inhibit or stimulate hypothalamic/ pituitary secretion of GnRH/gonadotropins, leading to atrophy or resumption of ovarian function. Photo- responsive individuals are therefore excellent models for basic ovarian function, because modifications of day length naturally induce and then reverse ovarian atrophy. In photoperiodic Siberian hamsters, ovarian remodeling prompted by changes in day length is associated with differential expression of MMPs. Indeed, photostimulated return to ovarian function (recrudescence) can be impeded following in vivo administration of a broad-spectrum MMP inhibitor, GM6001. While this suggests an important role for these proteases, the action of MMPs during recrudescence and the process of how the quiescent ovary can resume cycling following weeks of atrophy is unknown. This proposal hypothesizes that 1) inhibition of recrudescence in response to GM6001 occurs because cleavage of MMP substrates that normally mediate return to ovarian function is impeded, 2) that the GM6001-treated ovary fails to return to function because key ovarian processes such as angiogenesis, granulosa cell proliferation, and steroidogenesis are dependent on MMP activity, and 3) that because gelatinases (MMPs-2/-9) are key players in ovarian cyclicity, and gelatinase activity is significantly down regulated following GM6001 administration, much of the remodeling can be attributed to gelatinases. Initial experiments will use a hypothesis-driven proteomics approach to identify substrates cleaved by MMPs during recrudescence by comparing the proteome of GM6001-treated vs. control ovaries. Examination of both mRNA and protein for key markers of angiogenesis (e.g., CD34, VEGF-R1), proliferation of granulosa cells (PCNA), and steroidogenesis (e.g., Cyp19, 32HSD) will reveal a mechanism of MMP action during recrudescence, and define a role for MMPs in these processes as GM6001 treated tissue is compared to controls. Finally, a gelatinase-specific inhibitor (SB-3CT) will be administered in vivo during photostimulated recrudescence to provide direct evidence of gelatinase action and function in the recovering ovary. Together, these studies should provide novel insight into the cellular and molecular regulation of recrudescence of ovarian function. In addition, these data will provide the first evidence of the targets of MMP action as ovarian cyclicity returns. Understanding the role that MMPs play in mammalian ovarian function by using a photoperiodic model helps to elucidate critical clinical questions of how to shut down (contraception) and restart (assisted reproduction, premature menopause) ovarian activity with non-hormonal mechanisms. PUBLIC HEALTH RELEVANCE: Ovarian recrudescence is a natural process stimulated by changes in photoperiod (the number of hours of light per day) whereby the atrophied and non-functional ovary returns to a fully functional organ in 2-8 weeks. While the hormonal initiation of recrudescence has been thoroughly examined, the cellular changes that occur at the level of the ovary remain unknown. Understanding the cellular/molecular pathways of mammalian ovarian recrudescence, such as matrix metalloproteinase (MMP) action, is novel and timely, as new non-hormonal methods of contraception and assisted reproduction techniques are clinically sought. Our studies will provide potential non-hormonal targets for clinically- geared studies focused on "turning on" and "turning off" ovarian function.

? DESCRIPTION (provided by applicant): Understanding ovarian function in adults is a requirement to maximize female reproductive health, including providing women sufficient options in fertility regulation and maintenance. When ovarian function fails in young adult women, a condition known as premature ovarian failure (POF), their future fertility is at risk. However, most studies that examine how follicle development can be initiated in the ovary do so in neonatal or peri-pubertal females, not adults. Siberian hamsters (Phodopus sungorus) serve as an ideal model for investigating how a non-functional adult mammalian ovary can resume activity, because exposing these hamsters to different photoperiods, the number of hours of light per day, can turn ovarian function on or off. We have shown that exposure of adult Siberian hamsters to long day photoperiod (LD; 16 hours of light per day, 8 hours of dark per day) maintains ovarian function, whereas exposure to inhibitory short day lengths (SD, 8L:16D) for 14 weeks induces ovarian regression. Subsequent transfer of photo-regressed females to LD stimulates ovarian recrudescence, a process that restarts the non-cycling ovaries. Multiple mechanisms regulate these transitions in ovarian function, although how folliculogenesis actually resumes during recrudescence has not been explored. This proposal hypothesizes that signaling factors promoting folliculogenesis are 1) present in Siberian hamster ovaries and regulated by photoperiod, and 2) differentially regulated by non-gonadotropin factors and FSH in regressed and recrudescing ovaries. The aims of this proposal first use a systematic genomic screening of adult ovaries to identify transcripts up- and down- regulated during recrudescence. Next, an in vivo ablation and restoration of FSH during photo-stimulation examines FSH independent factors triggered by photo-stimulation that may restore follicle growth. Finally, an in vitro culture system will be used to expose recrudescing ovaries to inhibitors of key folliculogenic factors (e.g., AMH, IGF-1, KitL, FoxO3, Src), determining individual contributions of these factors to recrudescence. Culture of regressed ovaries with factors known to systemically mediate photoperiodic change (prolactin, thyroid hormone, kisspeptin) will follow, examining non-gonadotropin triggers of ovarian recrudescence to determine if these factors potentially prime the regressed ovary for rapid return to function. Importantly, while signaling factors have been broadly assessed during early stages of folliculogenesis, particularly in peri-pubertal females, no studies have done this in adult tissue with a naturally quiescent ovary as it resumes function. Taken together, the work proposed here will provide a better understanding of the how a non-cycling ovary returns to function, and how folliculogenesis is restored in the adult ovary. Our unique model may yield discovery of novel signaling factors critical for restoration of folliculogenesis, particularly at its early stages. This knowledge is fundamental to our understanding of fertility, considering that follicle activation occurs across the reproductive lifespan, not just during ovarian development.