207 OOCYTE SURVIVAL AND FOLLICULAR DEVELOPMENT IN Fas-KNOCKOUT AND KIT-DEFICIENT DOUBLE MUTANT MICE

Abstract

Growth factors and cytokines regulate survival and growth of mammalian oocytes via their cognate receptors. Among those receptors, KIT, a receptor tyrosine kinase, has been thought of as an essential molecule for growth and survival of oocytes and for follicular development. The defect of KIT-mediated signals leads to the loss of oocytes and impairment of follicular development. Fas is a member of the death receptor family inducing apoptosis; it expresses in the ovary. In a previous study (Sakata et al. 2003 Cell Death Differ. 10, 676–86), we generated KIT-deficient and Fas-knockout double mutant (W^v/W^v:Fas^−/−) mice to study the relation between Fas and KIT signaling in germ cell apoptosis. To further understand the role of KIT in oocyte survival and follicular development, we examined the ovaries of W^v/W^v and W^v/W^v:Fas^−/− in comparison to those of C57BL/6 (wild type) mice. We also examined the possibility of overcoming the deleterious effects of KIT deficiency by ovarian allotransplantation. One ovary of each mouse was fixed for immediate histological examination and the other was transplanted under the kidney capsule of a female SCID (severe combined immune deficiency) mouse. Ovaries and recovered grafts were fixed, embedded, serially sectioned at 5 μm, stained with hematoxylin and eosin, and examined under a microscope. Oocytes were counted in every section where the nucleus was seen, avoiding double counting in adjacent sections. Mean (with standard deviation) numbers of oocytes per graft or ovary were compared using Student's t-test. At 13 days post-coitum (dpc), ovaries of W^v/W^v fetuses contained 1104.3 ± 118.8 (n = 4) germ cells which was significantly (P < 0.05) lower than those of wild-type mice. However, at 16 dpc (n = 6) and 2-days old (n = 6), ovaries did not contain any germ cells/oocytes. After allotransplantation of the ovaries (n = 6) from W^v/W^v fetuses (13 dpc) for 2 weeks, all of the germ cells disappeared. When the ovaries from 2-day-old W^v/W^v mice (n = 6) were allotransplanted for 12 days, no oocytes appeared. On the other hand, transplanted ovaries from C57BL/6 fetuses (13 dpc) contained 2162.0 ± 97.3 (n = 6) oocytes after 2 weeks. In those ovaries, 4.7 ± 1.6% follicles developed to secondary follicles which contained growing oocytes. Importantly, ovaries of 2-day-old W^v/W^v:Fas^−/− mice (n = 4) contained 1936.0 ± 245.0 oocytes (64.0 ± 10.0% of wild-type mice), and 14-day-old mice (n = 4) still contained 911.3 ± 106.3 follicles in which 28.6 ± 6.0% and 11.4 ± 3.2% follicles developed to primary and secondary follicles, respectively. These results indicate that oocyte death due to KIT-deficiency can not be rescued by ovarian transplantation in SCID mice, and that the Fas-knockout condition partially prevents the death of oocytes induced by KIT-deficiency, and primordial follicles develop in this condition.