Kit-Ligand/Stem Cell Factor Induces Primordial Follicle Development and Initiates Folliculogenesis*

Initiation of folliculogenesis through the induction of primordial follicle development in the ovary has an important role in determining the fertility and reproductive fitness of most mammalian species. The factors that control this critical process are largely unknown. The hypothesis tested in the current study was that kit-ligand/stem cell factor (KL) promotes the initiation and progression of primordial follicle development in the ovary. Ovaries from 4-day-old rats were maintained in organ culture for 5 and 14 days and treated with no factor (control), recombinant kit-ligand (KL), or gonadotropins (FSH and hCG). Follicles in ovarian sections were counted and histologically classified as primordial (stage 0), early primary (stage 1), primary (stage 2), transitional (stage 3), or preantral (stage 4). Fresh ovaries from 4-day-old rats contained 68% primordial follicles (stage 0) and 32% developing follicles (stages 1–4) per section. After 5 and 14 days in culture, section from control ovaries contained approximately 41% and 55%, respectively, developing follicles (stage 1–4) per section due to spontaneous development of primordial follicles. Spontaneous primordial follicle development was completely blocked by ACK-2, a c-kit antibody that blocks KL actions. This observation suggests that endogenous KL is necessary for primordial follicle development in vitro. After 14 days of KL treatment, sections from ovaries contained 17% primordial follicles (stage 0) and 83% developing follicles (stage 1–4) per section demonstrating a dramatic induction of primordial follicle development by KL. Gonadotropins (FSH and hCG) did not induce primordial follicle development but did increase the percentage of preantral follicles (stage 4) per section. This small increase in preantral follicles in response to gonadotropins was blocked by ACK-2 suggesting that KL may in part mediate gonadotropin actions after the initiation of primordial follicle development. Ovaries contained an average of 309 6 10 follicles per section. The total number of follicles per section did not significantly vary between treatments suggesting that the effects of KL were not due to an alteration in follicle number (i.e. survival). KL appears to be one of the first factors identified to be involved in the promotion of primordial follicle development. Results suggest that KL is necessary and sufficient to induce primordial follicle development and initiate folliculogenesis. (Endocrinology 140: 4262–4271, 1999) T FACTORS THAT control the onset of primordial follicle development in the ovary are not known. The pool of ovarian primordial follicles is established during embryonic development (e.g. cows, sheep, humans) or at birth (e.g. rodents). This pool of primordial follicles constitutes the complete supply of oocytes available to a female. Primordial follicles that initiate development are destined to ovulate or degenerate through atresia. Induction of primordial follicles to develop and grow (i.e. initiate folliculogenesis) is a fundamental process in ovarian biology and is essential for female reproduction. The current study investigates a factor potentially involved in this process. Kit ligand (KL) and its receptor c-kit are essential for oocyte migration during embryonic development (1–3) and follicular development in the adult ovary (4–6). Although previous analysis of the actions of KL on follicle development (6) are likely in part to be due to actions on primordial follicle development, no direct analysis of primordial follicles has been reported. To our knowledge, previous studies have not rigorously examined the role of KL in primordial follicle development and the initial stages of folliculogenesis. KL and c-kit are the products of the Steel (Sl) and White Spotting (W) loci in mice, respectively (7–13). A number of mutations at Sl or W have been described that cause sterility due to defects in oocyte migration or follicular development. Ovaries in mice carrying steel panda (Sl), steel t (Sl), and steel contrast (Sl) mutations contain follicles that arrest at early stages of follicular development (14–16). The pool of primordial follicles is established in these mutant mice, but initiation and progression of primordial follicle development is inhibited. These observations suggest that KL may be essential for initiation and/or progression of primordial follicle development in the ovary. When a follicle starts to develop, its oocyte begins to synthesize RNA (17, 18), and squamous pregranulosa cells enlarge to become a single layer of cuboidal granulosa cells. Theca cells are recruited from surrounding stromal stem cells and organize into distinct theca cell layers around the follicle. Organization of theca cells around the follicle provides structural integrity and helps to establish mesenchymal-epithelial cell interactions between theca cells and granulosa cells (19). Follicular development continues as granulosa cells and theca cells proliferate and differentiate. It has been suggested that theca cells differentiate from stromal stem cells in response to a putative “theca cell organizer” produced by granulosa cells (20). Such a factor may also be important for granulosa cell-oocyte interactions involved in the initiation and progression of primordial follicle development. Granulosa cells in developing follicles produce KL (4, 21), which can act on theca cells, stromal cells, and oocytes. Differentiated theca cells, undifferentiated stromal cells, and Received September 2, 1998. Address all correspondence and requests for reprints to: Michael K. Skinner, Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 991644231. E-mail: [email protected]. * This work was supported by grants from the United States Department of Agriculture and the National Institutes of Health. 0013-7227/99/$03.00/0 Vol. 140, No. 9 Endocrinology Printed in U.S.A. Copyright © 1999 by The Endocrine Society