DEVELOPMENTAL BIOLOGY 143,162-172 (1991) Meiotic Competence Acquisition Is Associated with the Appearance of M-Phase Characteristics in Growing Mouse Oocytes DINELI WICKRAMASINGHE, KARL M. EBERT, AND DAVID F. ALBERTINI’ Tufts University Health Science Schools, Department of Anatomy and Cellu,lar Biology, Schools of Medicine and Veterinary Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111 Accepted October 5, 1YW To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13. to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization, in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtu- bule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components. 0 1991 Academic Press, Inc INTRODUCTION Meiotic competence is defined as the ability of mam- malian oocytes to spontaneously resume and progress through meiosis, a process initiated during germinal ves- icle breakdown (GVBD)’ and completed after extrusion of the first polar body and arrest at metaphase of meio- sis-2. The expression of meiotic competence in viva is normally held in abeyance by a combination of intrafol- licular factors (Schultz, 1988). In response to periovula- tory hormonal signals, meiotic maturation ensues in the hours normally preceding ovulation (Thibault et al., 1987). The ability of isolated oocytes to resume meiosis spontaneously in culture has been exploited experimen- tally to define when during the course of oogenesis the oocyte acquires meiotic competence. In the mouse, oo- cytes from animals 15 days and older in age exhibit an increased frequency of meiotic resumption in culture (Bachvarova et al, 1980; Eppig, 1977; Szybek, 1972) that correlates with the extent of oocyte growth (Sorensen and Wassarman, 1976). While small oocytes liberated from juvenile mouse ovaries cannot resume meiosis, 1 To whom correspondence should be addressed. ’ Abbreviations used: GVBD, germinal vesicle breakdown; MPF, maturation promoting factor; PBS, phosphate-buffered saline; GV, germinal vesicle; BMOC-2, Brinster’s modified oocyte culture me- dium. Sorensen and Wassarman (1976) noted further that many growing oocytes that do undergo GVBD are un- able to form polar bodies and these authors made the suggestion that competence acquisition is expressed in two phases relative to the extent of oocyte growth and differentiation. This observation suggests that developing oocytes must first acquire the capacity to reinitiate meiosis and, at a later time, acquire the capacity to complete meio- sis-l and progress to metaphase of meiosis-2. While there is evidence to suggest that the biochemical or structural properties of developing oocytes are altered at either of these transitional states (Bornslaeger et al., 1988; Schultz and Wassarman, 1977; Wassarman and Josefowicz, 1978), very little is known with regard to modifications in the expression of cell cycle control ele- ments during the protracted period of meiotic prophase. The importance of maturation promoting factor (MPF) at the onset of M-phase in both meiotic and mi- totic cells has been well established in recent years par- ticularly with respect to critical protein phosphoryla- tion events that drive cells to the metaphase state (Ki- shimoto et al,, 1982; Murray and Kirschner, 1990). Among the events involving protein phosphorylation, chromatin condensation and microtubule assembly at sites of centrosome activation are known to be tempo- rally coordinated during entry into mitotic M-phase (Vandre and Borisy, 1989); however, these events have 0012-1606/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 162