No abstract

Oocyte-granulosa cell communication, mediated by paracrine factors, is essential for oocyte development. Kit ligand (KITL) is expressed in granulosa cells as soluble (KITL1) or membrane-associated (KITL2) proteins. However, the relative biopotency of each isoform during oocyte development is unknown. Our initial results showed that Kitl2 was down-regulated in cultured granulosa cells. To determine the effect of the two isoforms of KITL on oocyte growth, Kitl-deficient fibroblasts were transfected with constructs expressing either KITL1 or KITL2, and growing oocytes were isolated from 12-day-old mice and cultured on the transfected fibroblasts for 2 days. At the end of culture, oocyte diameters were measured, the incidence of spontaneous germinal vesicle breakdown (GVBD) was noted, and oocytes were analyzed for KIT receptor expression. Oocyte growth occurred only in the presence of the KITL2-producing fibroblasts, and suppression of KITL2 expression impaired oocyte growth. Up-regulation of KIT expression occurred in the presence of KITL2 but not KITL1. The presence of KITL2 inhibited spontaneous GVBD. Meiosis inhibitors did not attenuate the GVBD that occurred in the absence of KITL2, suggesting that this process reflects oocyte degeneration rather than meiotic progression. These results indicate that KITL2 is the principal KITL isoform required for oocyte growth and survival in vitro.

Mutations in the genes encoding the Kit tyrosine kinase receptor or kit ligand (KL) cause numerous phenotypic defects, including sterility. In the postnatal ovary, Kit is expressed on the oocyte surface and KL is produced by the surrounding granulosa cells, but its function in these cells is still unknown. The purpose of this study was to determine the role KL/Kit interactions play in the regulation of oocyte meiosis. Here, we demonstrate that meiotically arrested rat oocytes that are microinjected with Kit antisense oligonucleotides have decreased Kit expression. This decreased expression is associated with an increased ability of these oocytes to resume meiosis compared with those microinjected with missense oligonucleotides or buffer alone. In addition, oocytes cultured in the presence of KL were delayed in their resumption of meiosis, but KL could not enhance the meiosis inhibitory effects of dibutyryl cAMP, suggesting that KL operates through a mechanism that is independent of cAMP. Human chorionic gonadotropin-induced meiotic resumption in oocytes was accompanied by a shift in follicular granulosa cell KL expression from membrane-bound to soluble forms and a loss of expression of both forms of KL in cumulus cells. Thus, KL-activated Kit inhibits meiotic progression, and the in vivo luteinizing hormone-stimulated resumption of meiosis may negate Kit activity by a localized decrease in KL expression and by altering the form of KL produced within the follicle.

In preparation for ovulation, paracrine communication between the preovulatory follicle and overlying theca/ stromal cells and ovarian surface epithelium (OSE) must take place to facilitate the degradative and apoptotic events associated with ovulation. Kit tyrosine kinase receptors and their ligand, kit ligand (KL) are expressed within ovarian follicles, and ligand-induced receptor activation appears to account for some of the cell ± cell interactions important for oocyte development. We investigated the expression of Kit receptors and KL in OSE cells and the possibility that modulation of their expression could a ect OSE cell activity. KL mRNA and protein were detected in the OSE cell layer of rat ovaries, and primary cultures of rat OSE as well as the immortalized rat OSE cell line, ROSE 199, expressed KL, but not Kit receptors. Both primary and immortalized OSE cells preferentially expressed KL-1, rather than KL-2, transcripts, suggesting that these cells produce predominantly the soluble form of KL. Activation of the cAMP signalling pathway using dibutyryl cAMP decreased proliferation of ROSE 199 cells and elicited a threefold increase in KL expression. TGF-b similarly inhibited ROSE 199 cell proliferation, but strongly inhibited dibutyryl cAMP-induced KL expression, indicating that changes in KL expression were not directly associated with OSE cell proliferation. The expression of mostly soluble KL in the surface epithelium suggests that this cytokine may be acting in a paracrine fashion, perhaps interacting with nearby Kit receptorbearing theca cells.