PARENT SESSION
PLATFORM SESSION 2: REGULATION OF OVARIAN FOLLICULAR EXPRESSION
Chair: Adashi, Eli¹, ¹
Co-chair: Salvador, Lisa¹, ¹
Harborside C
4:30 PM-6:30 PM

(9) IS THERE A SPATIAL GRADIENT OF CELL CYCLE REGULATION IN THE DEVELOPING OVARIAN FOLLICLE?

Barrett, Susan^1,2, Albertini, David¹, ¹ Department of Anatomy and Cellular Biology, Boston, MA² Program in Cellular, Molecular and Developmental Biology, Boston, MA

ABSTRACT- The proliferative potential of the somatic granulosa cells appears to be modulated by both paracrine/oocyte derived factors (e.g. Kit ligand, GDF-9) and systemic gonadotropins such as follicle-stimulating hormone (FSH). We have analyzed cell cycle status of mouse granulosa cells (GCs) with respect to stage of follicle development and proximity to the oocyte. GCs were obtained by follicle puncture of ovaries from 14-day-old unprimed prepubertal or 21-day-old eCG primed animals. Mural or cumulus GCs were plated for 4d or 3d, respectively, in serum containing medium and then fixed and processed for immunofluorescence microscopy. Cultures were analyzed for mitotic index (Hoechst 333258), G1/S cell cycle progression (anti-PCNA) or terminal post mitotic differentiation status (TUJ1) and the percentages of cells in each category were determined in experimental triplicates. The majority of GCs from 14-day-old pre-antral follicles exhibit G1 arrest and no evidence of postmitotic cells. In contrast, mural GCs from 21-day-old eCG primed animals are actively proliferating through S-phase and exhibit a distinct fraction of terminally post-mitotic cells as evidenced by TUJ1 positive staining. Intact cumulus cultures from 21-day-old animals display a distinct gradient of expression in cell-cycle markers. Specifically, cells maintaining contact with the oocyte were PCNA negative and nonproliferative. Moreover, circumferential zones of cells in G1, S, or postmitotic states were apparent at progressively greater distances from the original position of the oocyte. These data suggest that a regulatory mechanism for a G1/S checkpoint control exists during the generation of cumulus and mural GC lineages. The parallel expression of a dominant G1 arrest phenotype observed in both Day 14 pre-antral GCs and a subset of cumulus cells is consistent with a restrictive but protractive influence of the oocyte on cell cycle progression at different stages of folliculogenesis. (Supplemented in part by USDA grant #2001-35205-0966)

KEY WORDS: cell cycle, granulosa cells, cumulus cells, checkpoint control