The interaction between angiogenic factors and related receptors is closely associated with follicular angiogenesis. The present study was performed to determine the relationships between the capillary network and mRNA expression of several angiogenic factors and related receptors during porcine follicular development. Ovaries in gilts were collected 72 h after eCG (1250 IU) treatment for histological observation. Granulosa cells and thecal tissues in small (diameter, <4 mm), medium (diameter, 4-5 mm), or large (diameter, >5 mm) individual follicles were collected for detection of mRNA expression of vascular endothelial growth factor (VEGF) 120, VEGF 164, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in granulosa cells and fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), bFGF receptor (bFGF-R), and EGF receptor (EGF-R) in thecal tissue by semiquantitative reverse transcription-polymerase chain reaction. The eCG treatment resulted in the emergence of healthy preovulatory follicles (diameter, >6.0 mm) that possessed more capillaries in the thecal cell layer and a significant increase in the percentage of atretic follicles of 1.0-2.9 mm in diameter. The number of capillaries in the thecal cell layer increased significantly in healthy follicles larger than 3 mm in diameter in the eCG group compared with those in controls. The expression of VEGF 120, VEGF 164, and bFGF mRNAs increased in granulosa cells of medium and large follicles from ovaries of prepubertal gilts after eCG treatment. The Flt-1, Flk-1/KDR, and bFGF-R mRNA expression increased in theca cells of medium and large follicles after eCG treatment. The expression of EGF mRNA increased in granulosa cells of small, medium, and large follicles from ovaries after eCG treatment, but the mRNA expression of EGF-R in thecal tissue did not change. These data indicate that preovulatory follicles possessed a larger capillary network and expressed more mRNAs of angiogenic factors in granulosa cells and related receptors in thecal tissue. We concluded that VEGF 120, VEGF 164, bFGF, and EGF may be greatly involved in the angiogenic process of follicular development in prepubertal gilts with eCG treatment.

The transient synthesis and accumulation of hyaluronan (HA), an extracellular matrix component of cumulus cells, brings about expansion of cumulus-oocyte complexes (COCs) in preovulatory mammalian follicles. In this study, we investigated the mRNA expressions of hyaluronan synthase 2 (has2), hyaluronan synthase 3 (has3), and CD44, as well as the responsiveness to eCG and porcine follicular fluid (pFF) of these genes, in porcine COCs, oocytectomized complexes (OXCs), and oocytes during in vitro maturation. Immunolocalization of CD44 was also analyzed in COCs. After 12 h of culture, the area of cumulus expansion in medium 199 supplemented with both 10 IU/ml eCG and 10% (v/v) pFF was significantly greater than that in the medium supplemented with eCG or pFF. Oocytectomy reduced the expansion area in the group supplemented with eCG. In reverse transcription-polymerase chain reaction analysis, all transcripts were identified in COCs, but has3 transcript was not found in OXCs. Only has3 mRNA was detectable in oocytes, indicating that cumulus cells express has2 and CD44 mRNAs, and oocytes express has3 mRNA. The expression levels of has2 and CD44 mRNAs in COCs and OXCs increased in the presence of eCG and pFF after 24 h of culture, suggesting that these genes have a positive dependency on eCG and pFF. In contrast, the high level of has3 mRNA was detected in COCs cultured in the medium alone. Oocytectomy slightly reduced the expression level of has2 mRNA. On immunostaining for CD44, CD44 was expressed apparently in COCs cultured with eCG and pFF for 24 h. The positive staining was distributed on cytoplasm along the perimembrane of cumulus cells and at the junctions between cumulus cells and oocytes. CD44 was also localized on cytoplasm of some oocytes. These results indicate that 1) porcine oocytes promote eCG-dependent cumulus expansion and the expression of has2 mRNA in cumulus cells, but these are not essential for expansion of cumulus cells and the expression of has2 mRNA; 2) HAS2 is involved in HA synthesis during cumulus expansion, and eCG and pFF up-regulate its expression; 3) the expression profile of the has3 mRNA that is transcribed in oocytes is different from those of has2 and CD44 mRNA; and 4) CD44 may participate in the interaction between cumulus cells and oocytes.

In Xenopus oocytes, Mos activates the mitogen-activated protein kinase (MAPK) signal transduction cascade and regulates meiosis. In mammalian oocytes, however, the functions of Mos are still unclear. In the present study, we used c-mos knockout mouse oocytes and examined the roles of Mos in mouse oocyte maturation and fertilization, including whether Mos controls MAPK and maturation promoting factor (MPF) activity. The kinetics of germinal vesicle breakdown (GVBD) and the first polar body emission were similar in wild-type, heterozygous mutant, and homozygous mutant mice. Activities of MPF were also not significantly different among the three genotypes until the first polar body emission. In contrast, MAPK activity in c-mos knockout oocytes did not significantly fluctuate throughout maturation, and the oocytes had abnormal diffused spindles and loosely condensed chromosomes, although a clear increase in MAPK activities was observed after GVBD in wild-type and heterozygous mutant oocytes that had normal spindles and chromosomes. After the first polar body emission, 38% of c-mos knockout oocytes formed a pronucleus instead of undergoing second meiosis, indicating the crucial role of Mos in MPF reactivation after first meiosis. When oocytes that reached second metaphase were fertilized or stimulated by ethanol, many c-mos knockout oocytes emitted a second polar body and progressed into third meiotic metaphase instead of interphase, although all fertilized or activated oocytes in the heterozygote progressed to interphase, indicating that Mos deletion leads to compensatory factors that might not be degraded after fertilization or parthenogenetic activation. These results suggest that Mos is located upstream of MAPK in mouse oocytes as in Xenopus oocytes but is independent of MPF activity, and that Mos/MAPK is not necessary go GVBD and first polar body emission. Our results also suggest that Mos plays a crucial role in normal spindle and chromosome morphology and the reactivation of MPF after first meiosis.

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