65. Development of molecular markers for stage II and stage III zebrafish ovarian follicles after in vitro culture

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but had not been successful in fish so far. Fish oocytes offers several advantages over embryos due to smaller size of the oocytes, low water content and absence of fully formed chorion. Studies in our lab have showed that early stage ovarian follicles are more suitable for cryopreservation than late stage oocytes. Hence ovarian tissue containing stage I and stage II ovarian follicles have been used in this study. Development of in vitro culture protocol for early stage ovarain follicles is important since cryopreserved early stage ovarian follicles would need to be matured in vitro . The results obtained from the in vitro culture study carried out in our lab demonstrated that the early stage ovarian fragments containing stage I and stage II follicles can be cultured in 90% L-15 medium (pH 9.0) with 20% feotal bovine serum (FBS) and 100 mIU/ml follicle stimulating hormone (FSH) for 24 h at 28 °C. The study demonstrated follicle growth in size from stage I to stage II or from stage II to stage III, respectively. Follicle viability following culture was not significantly different when compared to the cultured fresh fragments. Although follicle growth can be assessed by measuring the diameter of the follicles, molecular markers would provide more important information on follicle development. The aim of the present study was to develop a molecular marker to identify stage II and stage III ovarian follicles after in vitro culture. Studies on the levels of vtg1 and p450aromA genes in stage I, II and II ovarian follicles of zebrafish before and after culture were carried out. Ovarian follicles of zebrafish were collected from mature zebrafish females and the ovaries were placed in 90% L-15 medium. Ovarian follicles of different stages (I, II and III) were separated using syringe needles. Total RNA was extracted from ovarian follicles using trizol method. The quality of RNA of each sample was determined by the level of absorbance at 260 nm. A quantitative RT-PCR approach was used to investigate the level of gene expression. Preliminary results from the present study showed that prior to culture of the ovarian follicles the level of expression of vtg 1 was higher in stage III when compared to stage I and stage II follicles; and the level of expression in p450aromA was higher in stage II when compared to stage I and stage III ovarian follicles. Analysis on the level of expression of genes after in vitro culture of ovarian fragments are in progress. The development of molecular markers would provide a reliable method for assessing the growth of ovarian follicles in vitro .