Ned J. Place, Ph.D., M.D. - US grants

Effects of androgens on female sexual differentiation are often considered within a pathological context (e.g. ambiguous genitalia resulting from congenital adrenal hyperplasia), but in the spotted hyena a high androgen milieu during fetal development and sexual differentiation represents normal physiology, not pathology. Thus, the spotted hyena represents a unique system in which to study the effects of antenatal androgens on sexual differentiation. High androgen exposure in utero masculinizes female external genitalia to the extreme in spotted hyenas, but the effects of androgens on ovarian differentiation are not well studied. Specifically, androgens appear to increase the rate of follicular atresia in the developing ovary, which may explain the abundance of ovarian interstitial tissue and relative paucity of follicles in spotted hyenas. In the proposed research I will study a group of female hyenas that were previously exposed to anti-androgens while in utero. These females are now adults and the structure and function of their ovaries will be compared to that of untreated controls. The steroidogenic responsiveness of the ovaries to a gonadotropin challenge test will be assessed in vivo, as well as in vitro (after unilateral oophorectomy). Following oophorectomy, follicle number and steroidogenic enzyme activity will be measured, and steroid receptor levels will be quantified by immunocytochemistry. This research will improve our understanding of how prenatal exposure to androgens affects ovarian structure and function.

DESCRIPTION (provided by applicant): This proposal outlines a career development plan for the applicant, a clinically trained obstetrician/gynecologist, who is making the transition from clinician to basic biomedical researcher. The candidate's goals for this award are to learn modern molecular and cellular techniques as applied to his research on female reproductive aging, and to facilitate the final transition to becoming an independent investigator. Under the mentorship of established investigators and a multidisciplinary advisory committee, the candidate will pursue a program of instruction and research that maximizes the likelihood of his developing into an independent scientist. The research proposed seeks to determine how photoperiod and the pineal gland hormone melatonin affect female reproductive aging. The model species is the Siberian hamster (Phodopus sungorus), an easily maintained laboratory animal whose reproductive biology is exquisitely sensitive to day length and melatonin. Rearing female hamsters in a short day photoperiod decelerates reproductive aging. Short day females had significantly greater litter success than age-matched long day females at 9 months of age. Pregnancy failures occurred only in long day females, and maternal and fetal deaths during parturition in this group are consistent with uterine dysfunction. Ovaries from adult short day females had twice as many primordial follicles as ovaries from age-matched long day females. Three aims are proposed: 1) to determine if the photoperiodic effect on ovarian primordial follicle number is due to a decrease in follicular atresia and/or an increase in follicular renewal; 2) to identify how the hormones and factors that modulate primordial follicle activation are affected by photoperiod and melatonin; and 3) to determine if photoperiod and melatonin modulate the age-associated decline in uterine function by inhibiting cellular senescence, telomere shortening, and attrition of estrogen receptors. The proposed work is relevant to understanding the cellular and molecular mechanisms underlying reproductive aging, and how that process may be decelerated. Once the objectives of this award have been met, the candidate will work as an independent investigator, and carry out research on reproductive aging that is truly integrative.

What factors drive a female to choose a particular mate? This is an important question to scientists interested in animal behavior, sexual selection, and life history theory. Individual variation in female mate preferences and choice is common. However, our understanding of the mechanisms behind this phenomenon is still quite limited. There are a few proposed sources of individual variation in mate choice, and the purpose of this research is to investigate the role of reproductive age in female mate preference and choosiness. The research team will experimentally accelerate reproductive aging in young female hamsters to determine if reproductive age modulates mate choice behavior. This will be the first study to assess the effects of reproductive aging on behavior in mammals. The results of this project will lead to a better understanding of animal behavior, the factors that might affect individual differences in behavior, and animal reproduction.

Reproductive quality declines with age in female mammals at a more rapid rate than other bodily systems. This rate varies among individuals, making it a potential source of variation in mate choice behavior. To decouple the effects of reproductive and chronological age on choosiness, young adult female Syrian hamsters will be treated with 4-vinylcyclohexene diepoxide (VCD), which specifically accelerates ovarian aging through oocyte depletion without compromising other physiological systems or life span. VCD-treated females will be given an opportunity to choose between a dominant and subordinate male hamster, and their preferences will be compared to vehicle-injected females. Young females typically choose the dominant male in this situation. If the preference for dominant male hamsters by young VCD-treated females is reduced relative to controls, then this result will emphasize the importance of reproductive quality, independent of chronological age, in explaining the individual variation in female mate choice. The products of this research will be widely disseminated and used for educational purposes. The project will also enhance scientific training of both graduate and undergraduate students in the fields of reproductive biology and animal behavior. Data generated by this project will include video recordings of female mate choice behavior and the tabulation of those data in Excel files. These data will be stored indefinitely in eCommons, a digital repository system at Cornell University. All eCommons content will be openly accessible worldwide.

Naked mole-rats are the longest-living rodents, with a maximum lifespan of 35 years, and females of this species show no decline in fertility as they age. Some degree of reproductive aging is typical of other mammals, with the naked mole-rat being a notable exception to this rule. The principal objective of this project is to determine if the molecule that is thought to provide naked mole-rats with nearly complete resistance to cancer also contributes to the long reproductive lifespan of females. The molecule is known as very high molecular weight hyaluronan, and the naked mole-rat form can be produced by transgenic mice. Mice that express the gene responsible for the synthesis of very high molecular weight hyaluronan have been shown to live a longer and healthier life than mice lacking this naked mole-rat gene, and the hypothesis to be tested is that female reproductive aging will be reduced in these specialized mice. If the naked mole-rat form of hyaluronan maintains female fertility in mice, then these results could be applicable to species that are of conservational and agricultural importance, as well as preserving fertility in women as they age. This project will provide hands-on research opportunities to trainees ranging in experience from high school students to postdoctoral associates. It will also provide zoos and animal parks that maintain naked mole-rat colonies with educational materials to add to their informational displays about the remarkable reproductive biology of this species.

The main focus of this proposal is to discover the physiological mechanisms that underlie the remarkably long reproductive lifespan of female naked mole-rats, which show no aging-associated declines in fertility or fecundity into their third decade of life. Two of the physiological mechanisms that contribute to the naked mole-rat?s remarkable longevity and healthspan will be investigated for their impacts on ovarian structure, function and fertility as females age. These include the very high molecular weight form of hyaluronan and enhanced cytoprotective signaling by nuclear regulatory factor erythroid 2 (NRF2). Through interrogations of ovaries from naked mole-rats themselves, and from transgenic mice that express naked mole-rat hyaluronan synthase 2, this project will determine whether female reproductive aging is attenuated by very high molecular weight hyaluronan and/or enhanced NRF2 signaling. This would manifest as reduced ovarian inflammation, fibrosis and stiffness, more ovarian follicle numbers with better quality oocytes, and improved fertility in older females. The project will advance scientific knowledge about the roles that inflammation and fibrosis play in the process of ovarian aging and elucidate how long-lived mammals can maintain the size and quality of their ovarian reserves to support protracted fertility. The broader impacts include providing hands-on research experiences for trainees, including high school students enrolled in the Ithaca Youth Bureau?s College Discovery Program, establishing a Naked Mole-Rat Investigator Network (listserv), and helping North American zoos to expand their educational repertoires to include exciting information about naked mole-rat reproductive biology in order to increase public scientific literacy about these amazing animals.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.