title: In vitro development of growing oocytes from fetal mouse oocytes: stage-specific regulation by stem cell factor and granulosa cells.
abstract: The development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte that is now beginning to be characterized. Little is known about the mechanisms underlying the beginning of the oocyte growth and the acquisition of the competence to resume meiosis by the growing oocyte. In the present study, we devised a multistep culture system for mouse oocytes obtained from 15.5- to 16.5-days postcoitum embryos (mean diameter +/- SEM, 9.7 +/- 1.3 microm), allowing three stages of the oocyte growth to be identified: (i) an early stage in which the oocyte growth is induced by direct stimulation of a soluble growth factor, namely stem cell factor (SCF), independent of the formation of gap junctions with granulosa cells; (ii) a second phase in which the oocyte growth depends on the combined action of SCF and contacts with granulosa cells; and (iii) a third phase of granulosa cell-dependent, SCF-independent growth. At each stage, key events of oocyte development and differentiation, such as the c-kit reexpression, the early zona pellucida assembly, and the beginning of follicologenesis, were observed to occur independently by the presence of SCF. At the end of the in vitro growing phases, lasting 18-20 days, oocytes reached a size (50 +/- 2.5 microm) and a chromatin differentiation (stage I-II) equivalent to those of 9- to 10-day-old preantral oocytes and were unable to complete the growth phase. About 50% of the in vitro-grown oocytes were induced to resume meiosis by okadaic acid (OA) treatment. However, a significant fraction of them (48%) showed inability to maintain the chromosome condensation in M-phase. When in vitro-grown oocytes were treated with UO126, a specific MEK inhibitor that prevents activation of mitogen-activated protein kinases (ERK-1 and ERK-2), for 1 h before, during, and following OA treatment, only 22% of oocytes underwent germinal vesicle breakdown after 24 h from the OA treatment. These studies demonstrate that SCF alone can induce the onset of the oocyte growth. This is, however, not sufficient to fully activate the mechanisms governing the acquisition of the meiotic competence previously described as a 15-day oocyte-autonomous clock starting at the onset of growth. The inability of oocytes to progress into the last stages of growth and the lack of synchrony between nuclear and cytoplasm maturation showed by a subset of them resemble the characteristics of oocytes from connexin-37- and -43-deficient mice and indicate the preantral/antral transition point as a critical stage of oocyte development requiring the coordinated differentiation of the oocyte with granulosa cells and the maintenance of adequate communication between these two cell types to assure the correct oocyte meiotic maturation.
authors.affiliation: Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, 00133, Italy.
authors.forename: Francesca Gioia
authors.initials: FG
authors.lastname: Klinger
authors.forename: Massimo
authors.initials: M
authors.lastname: De Felici
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002843:Chromatin / Q000648:ultrastructure; D018920:Coculture Techniques; D005260:Female; D005333:Fetus; D017629:Gap Junctions / Q000502:physiology; D006107:Granulosa Cells / Q000166:cytology*; D008540:Meiosis; D051379:Mice; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects; D006080:Ovarian Follicle / Q000196:embryology; D011247:Pregnancy; D019089:Stem Cell Factor / Q000494:pharmacology*
chemical_list: D002843:Chromatin; D019089:Stem Cell Factor