Evaluation of the effects of different ovulation induction protocols on folliculogenesis of rat ovary tissue by using immunohistochemical method and ultrastructural level

The investigation of the effects of normal ovarian cycle and ovulation induction protocols in ovarian tissue on molecular level is important for to increase the chance of healthy pregnancies in Assisted Reproductive Techniques applications. It was aimed to evaluate the changes of ovarian tissue in rats with normal ovarian cycle which administered recombinant follicle stimulating hormone (rFSH) and clomiphene citrate (CC) and controls in the present study by determining the distribution of bone morphogenic proteins (BMP), cyclin dependent kinase (cdc), connexin (Cx), mitogen activated protein kinase (MAPK) using immunohistochemistry and ultrastructure of oocytes and granulosa cells during follicular development in ovary tissue using transmission electron microscope.Wistar Albino female rats, 230-280 gr., (n: 7, each) were divided into five groups as: proestrous, estrous, diestrous, rFSH (10 IU, sc), CC (4 mg, orally). Groups were determined according to Giemsa stained vaginal smear results and confirmed with blood FSH levels. Drugs were administered to the rats during estrous cycle. Under general anesthesia, for light microscopic evaluation, ovarian tissues were obtained and were fixed in 10% formalin solution and routine paraffin tissue procedure were executed. For electron microscopic evaluation, tissue samples were fixed in 2,5 % gluteraldehyde and then 1% osmium tetroxide solutions and routine electron microscopy procedure were executed and were embedded in araldite mixture.Five µm sections taken from paraffin blocks were stained with Hematoxylin-Eosin (H.E.) and follicle evaluation was done and the numbers of follicles were counted in the groups. Sections were stained immunohistochemically using anti-BMP-4, anti-BMP-6, anti-P34cdc2, anti-Cx43 and anti-P38MAPK primary antibodies using avidin-biotin-peroxidase technique. Immunoreactivities were scored as mild, moderate or severe and results were evaluated comparatively using ANOVA statistical test.One µm semithin sections taken from araldite blocks were prepared for staining using toluidine blue and was evaluated follicle structure evaluation was done in groups. One hundred forty nm thin sections were prepared and contrasted using uranyl acetate-lead citrate and were evaluated using ZEISS transmission electron microscopy.FSH results were consistent with vaginal smears. In evaluation of H.E. stained samples, dominantly growing follicles were seen in proestrus group, Graffian follicles in estrous group and corpus luteum in diestrous group. There was an increase in the number of growing and Graffian follicles in rFSH, dominantly, and CC groups. Immunoreactivities of MAPK, Cx43, p34cdc2 were observed in oocyte, granulose and theca cells. Immunoreactivity of BMP-6 was observed in oocyte, granulosa cells, while BMP-4 was detected in theca, stromal cells and germinal epithelium of ovary. These molecules were observed as mild/moderate in proestrous group, moderate/severe in estrous group, mild/moderate in diestrous group, severe in rFSH and moderate/severe in CC groups.In evaluation of semithin sections, growing follicles were seen in proestrus group, Graffian follicles in estrous group and corpus luteum and secondary follicles in diestrous group. Increased Graffian follicles were seen in rFSH and CC groups. In evaluation of uranyl acetat-lead citrate contrasted thin sections, lipid droplets and mitochondri were seen in the cytoplasm of oocyte and microvilli and trans zonal processes were observed within zona pellusida in all groups. While the lipid droplets and mitochondri were increased in the cytoplasms of oocytes in the estrous, rFSH and CC groups; decreased in the diestrous group.During the normal ovarian cycle, the increase of the level of FSH increases the cAMP and BMP-6 in oocytes by means of P38MAPK and ovulation process is regulated by their interactions with p34cdc2 and Cx43. BMP-4 and BMP-6 play a role in the luteinisation process of granulosa cells with inhibiting FSH-dependent progesterone syntesis. P38MAPK increases estradiol synthesis and effects the intercellular changes and survival of granulosa cells.It was observed that application of rFSH, CC caused increase in number of growing follicles in rat ovary and it was though that it is important to reach full maturation of all oocyte for interaction with molecules.