The present study demonstrates increases of Kitl expression following the preovulatory LH/hCG surge in mouse ovaries. Using isolated ovarian cells, we further showed major increase of Kitl transcripts in granulosa cells, and predominant expression of c-kit in preovulatory oocytes. In in vitro cultures, KITL directly acts on preovulatory oocytes to stimulate first polar body extrusion, but not GVBD and cytoplasmic maturation.
Previous studies on the control of oocyte maturation have focused mainly on endocrine regulation of the hypothalamic-pituitary-ovarian axis. However, it has been recognized that intraovarian factors are also important in regulating oocyte maturation . The preovulatory LH surge triggers a cascade of events in ovarian follicles, including the resumption of meiotic maturation, luteinization, expansion or maturation of the cumulus cells, and follicle rupture. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation . Thus, the LH-responsive granulosa or theca cells could secret paracrine factors to regulate oocyte functions. To identify such intraovarian factors important for oocyte maturation, we performed DNA microarray analyses of the ovarian transcriptome during the peri-ovulatory period to provide a genome-wide screen of candidate genes. Here, we identified increases in the expression of Kitl mRNA after hCG stimulation in whole ovaries and isolated ovarian granulosa cells. Although Kitl mRNA was also increased in cumulus cells following hCG treatment, its level was lower than that of granulosa cells. Furthermore, KITL protein could be detected in granulosa, but not cumulus cells of preovulatory follicles obtained from ovaries of PMSG-primed mice at 4 h after hCG treatment. Granulosa cells in preantral and early antral follicles also expressed KITL, suggesting diverse role of KITL and c-kit throughout folliculogenesis and oogenesis, including the establishment of primordial germ cells within the ovary, primordial follicle activation, oocyte survival and growth, granulosa cell proliferation, theca cell recruitment, et al. The observed increases of Kitl mRNA in whole ovaries and granulosa cells are consistent with an earlier report using PMSG-primed rats at 6 h after hCG treatment . Likewise, low expression of Kitl was shown in cumulus cells of mouse antral follicles . Our findings of the predominant expression of c-kit mRNA in preovulatory oocytes are also consistent with earlier reports [8, 27, 28]. Because c-kit is also expressed in theca cells of antral follicles, corpora lutea, and some interstitial cells of the ovary , paracrine actions of KITL on the these cells cannot be ruled out.
Completion of the nuclear maturation of oocytes involves GVBD and extrusion of the first polar body. Although treatment of cultured COCs with KITL did not affect GVBD of preovulatory oocytes, it facilitated first polar body extrusion by 55% vs. control groups without treatment. This effect is comparable to that of another ovarian factor, IGF-I, showing 57% increases in first polar body extrusion vs. controls . The dose range of KITL used in the present studies was similar to that used in the rat study[26, 31]. Furthermore, the concentrations of KITL in human preovulatory follicular fluid were comparable to those levels. Similar to the BDNF stimulation of first polar body extrusion, but not GVBD, it is apparent that sequential steps of nuclear maturation of the oocyte are controlled by different paracrine factors. Although we detected transcripts of c-kit in both cumulus cells and oocytes in COCs, KITL induced first polar body extrusion in denuded oocytes, suggesting its direct effects on oocytes. Cyclin B is the regulatory subunit of maturation promoting factor, which induces meiotic resumption of immature mammalian oocytes. Mouse oocytes do not require de novo synthesis of proteins to undergo GVBD in vitro, whereas the synthesis of cyclin B1 is indispensable for the progression of meiotic maturation after GVBD. Rate of cyclin B1 synthesis controls the length of the first meiotic M phase. Our data on the increase in cyclin B1 protein in MI oocytes following KITL treatment suggest that cyclin B1 is involved in KITL promotion of completion of meiosis I.
Although a previous study reported that treatment of isolated preovulatory oocytes with KITL transiently blocked GVBD [26, 31], we did not observe an effect of KITL on GVBD of preovulatory oocytes using a follicle culture model. Because isolated oocytes undergo spontaneous GVBD after removal from surrounding somatic cells without LH treatment, our studies utilizing the preovulatory follicle model without spontaneous GVBD of the oocyte are more likely to reflect the in vivo conditions. Recent study demonstrated oocyte growth was limited in the absence of KITL, and yet spontaneous GVBD which could be inhibited by KITL2 was observed in these oocytes. The spontaneous GVBD is regarded as premature resumption of meiosis that reflects oocyte degeneration, rather than the physiologic meiosis . Their findings further supported our results. Our unpublished data further indicated that KITL treatment did not induce cumulus cell expansion in preovulatory follicles cultured with the serum-free medium. Mutant mice with defects in KITL or c-kit showed similar phenotypes including impairment of primordial germ cell survival, migration and proliferation, as well as defective follicle development . Because these mutant mice exhibit developmental arrest of follicles at primary stage, no female mice were available for investigating the roles of KITL/c-kit signaling in the preovulatory follicles. Thus, the roles of endogenous KITL in the oocyte maturation remain to be determined.
Some oocytes competent to complete nuclear maturation are unable to develop into blastocyst stage, which is indicative of deficient or defective cytoplasmic maturation of the oocyte . During the maturation process, oocytes undergo profound cytoplasmic changes which are essential for normal fertilization and embryonic development. However, treatment of preovulatory oocytes with KITL did not improve the developmental capacity of mouse oocytes. Our data is consistent with previous findings demonstrating that the concentrations of KITL in the human follicular fluid did not associate with the fertilization rate and pregnant outcome in patients undergoing intracytoplasmic sperm injection . Thus, the processes of nuclear and cytoplasmic maturation of the oocyte are independently regulated and could be controlled by different paracrine factors.
It has been demonstrated that there are significant interactions between oocyte secreted factors and KITL. Growth differentiation factor (GDF9) from oocyte has been shown to inhibit kitl expression in granulosa cells while present study demonstrates the enhancement of nuclear maturation by KITL produced by granulosa cells. And recent studies indicated an important role of GDF9 in promoting oocyte developmental potential [35–37]. These studies suggest that there are complex regulatory loops between oocytes and follicular somatic cells. Further study is needed to clarify the role of KITL and GDF9 interaction in oocyte nuclear and cytoplasmic maturation.