PARENT SESSION
BIOLOGY OF MALE AND FEMALE GAMETES
Harborside C
7:30 AM-10:00 AM

(182) EFFECTS OF ROSCOVITINE ON G2/M TRANSITION IN MOUSE OOCYTES.

Sanfins, Alexandra^1,2, Overstrom, Eric³, Albertini, David¹, ¹ Department of Anatomy and Cellular Biology, Boston, MA² Departamento de Histologia e Embriologia, Lisboa, Portugal³ Department of Biomedical Sciences, Grafton, MA

ABSTRACT- The use of M-phase arresting compounds to enhance the developmental potential of mammalian oocytes matured in vitro prompted investigation of cell cycle markers known to be modified during the G2/M transition in mouse oocytes. Cumulus enclosed or growing mouse oocytes were collected from eCG primed 7-8 week old cycling adults or unprimed 3 week old prepubertal animals and were exposed to control (1% DMSO) or 50 M roscovitine containing media for 3, 6 or 18 hours. Following roscovitine treatment, oocytes were washed and subsequently matured in vitro for 16-19 hours, and subsequently fixed and analysed by immunofluorescence microscopy. MPM-2, pericentrin, tubulin and Hoechst staining were used to ascertain chromatin configuration (NSN, SN), microtubule (MT) arrays and centrosome/nuclear phosphorylation. NSN growing oocytes exposed to 50 M roscovitine for 3, 6, or 18 hours loose nuclear MPM-2 staining, retain interphase MTs, and uniformly exhibit a SN chromatin configuration. SN oocytes treated under the same conditions regress to a G2 phenotype as evidenced by dephosphorylation of centrosomes and restoration of an interphase MT array. In addition, an increase in the number of perinuclear centrosomes was observed under these conditions. Upon removal of roscovitine and IVM for 16-19 hours, oocytes from unprimed animals blocked at metaphase-I whereas oocytes from eCG primed cycling animals progressed to metaphase-II. Both populations of oocytes showed an increased number of cytoplasmic centrosomes that were heterogeneous with respect to their phosphorylation as evidenced by MPM-2 epitope expression. These results demonstrate that roscovitine (1) causes transcriptional repression in growing oocytes, (2) causes loss of M-phase markers in competent oocytes, and (3) that the M-phase arresting action is fully reversible in oocytes from cycling primed animals but only partially reversible in oocytes from pre-pubertal unprimed animals. The effects observed on oocyte transcription and expression of meiotic competence require further analysis if roscovitine or related drugs are to be used successfully in ARTs. (Supported by USDA #2001-35205-09966, March of Dimes Birth Defects Foundation and Fundacao para a Ciencia e Tecnologia)

KEY WORDS: roscovitine, oocytes, cell cycle, meiotic competence