Antral formation and growth during folliculogenesis are fundamental step for accomplishment both the development and extrusion of competent oocyte. Although it has been suggested that aquaproteins (AQPs) may mediate in fluid pass into the antral cavity of the follicle, many parts are still unmasked. In this study, we examined the role of AQP9 in ovulation using in vitro follicle culture system and siRNA. Expression level of AQP 9 mRNA was dramatically increased from 24 hr and kept until 56 hr (8 hr post hCG injection) after eCG injection. AQP9 mRNA level was mostly high in the theca cells of tertiary follicles and preovulatory follicles compared with granulosa cells. The healthy follicles were isolated using sharp needles at 12 hr post eCG injection (3 wks old CD-1 mice). AQP9 specific siRNAs were transfected into the theca cells with lipofectamin 2000 for 24 hr and 36 hr. After 6 hr of hCG administration into the medium, we measure the diameter of the follicles. The number of ovulated oocytes was counted at 16 hr of hCG. The follicle size was increased more than 30% in control compared with that of AQP9 siRNA treatment group. On the other hand, the ovulation rate was dramatically decreased by suppression of AQP expression (41% vs 15% respectively in control and siRNA). At eCG 48 hr, we cannot find any ovulation marker changed in siRNA treatment group. From these results, we know that AQP9 accelerates the antral growth and ovulation.

Obesity is often associated with an impairment of the hypothalamic-pituitary-gonadal axis and also unites with reproductive defects and reduced fertility. The leptin-deficient ob/ob mouse is characterized by a morbid obesity and infertility. In the present study, we examined when ob/ob mouse lost their fertility activity according to aged dependent were investigated. The major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Here, ob/ob and ob lean various (4, 6, 9, 12 and 24 wks) aged mice were used. Histology (H & E staining) of ovary, vaginal smear and Reverse transcription polymerase chain reaction (RT-PCR) analysis were carried out. Hematoxylin/eosinstained sections show normal histology in ob lean type mice characterized by the presence of multiple primary and secondary follicles and well developed corpora lutea (CL). Ovaries from ob/ob mice, showed 4 and 6 wks mice similar to ob lean type but according to aged dependent ob/ob mice few primary and secondary follicles was seen. Many large empty follicles were present and no proper developed CL, especially 24 wks mice. Vaginal smear showed abnormal estrous cycle in ob/ob mice. Ovaries of ob/ob mice, mRNA results showed that ovulated gene TIMP1, Pgsh2 and Lhcgr expression levels are decreased according to aged dependent compared to ob lean type mice. These data suggest that aged dependent specifically when 24 wks of ob/ob mice lost their reproductive function.

A fertilized oocyte can get the competence for implantation through cleavage and stage-specific gene expression. These are under the control of autonomous and exogenous regulators including physiological culture condition. Endogenous and exogenous growth factors are considered as critical regulators of cleaving embryos during travel the oviduct and uterus. In this study, an effort was made to evaluate comprehensively the quality of embryos for implantation, grown in media enriched with EGF and PAF. The study evaluated developmental rates on given time, blastulation and hatching rates, and adhesion rates. Developmental rates of blastocyst to the hatching stage were significantly high in PAF treated group compared to the control in a dose-dependent manner but not in EGF group. Implantation rates were significantly high both PAF and EGF in a dose-dependent manner. H7, a PKC inhibitor, blocked the process of hatching of the blastocysts but combined treatment of EGF and PAF enhanced the hatching and implantation of blastocsyts. Based on these results it is suggested that EGF and PAF support acquirement of implantation competence at blastocyst stage through a PKC pathway.