In vitro culture of sheep lamb ovarian cortical tissue in a sequential culture medium.

PURPOSE: This study evaluates the effect of a sequential culture system on the follicular development of sheep lamb ovaries, aiming to establish an available in vitro culture system for ovarian culture. METHODS: Lamb ovarian cortical fragments were cultured on a steel mesh with a nitrocellulose membrane pre-coated by type 1 collagen. Several culture media were used for the determinations, specifically, a control medium (alpha-MEM), a constant medium (control medium supplemented with 75 ng/mL human recombinant EGF, 200 mIU/mL sheep FSH, 100 ng/mL human recombinant GDF-9, and 100 ng/mL human recombinant bFGF), and a sequential medium (control medium supplemented with sequential growth factors added on different days). Ovarian tissues, both fresh and cultured, were processed for histological and apoptotic assays, while spent culture media were processed for hormone assays. RESULTS: It was found that the growth of lamb primordial follicles can be initiated during culture in vitro. Compared to the control medium, sequential culture medium significantly increased the percentage of secondary follicles in cultures, while the follicle and oocyte diameters of primary and secondary follicles were also observed to increase in this medium. The constant medium was found to increase the number and diameter of secondary follicles only 18 days after culture. After this same period of time, some normal antral follicles were found in the sequential medium, while a few abnormal antral-like follicles were found in the control medium. Moreover, sequential medium appeared to significantly increase estradiol and inhibin production, especially 10-18 days after culture. The highest percentage of normal follicles and the lowest apoptotic cell rates were observed in the sequential medium, suggesting that a sequential addition style of culture can improve follicle and tissue viability. CONCLUSIONS: The sequential addition of FSH, EGF, GDF-9,and bFGF can stimulate primordial follicle transmittal into the later development stages, even as far as the antral stage, improve the survival rate of follicles, and maintain follicular viability.