| There were a large number of quiescent primordial follicles in sheep ovarian tissue, but the utilization rate of them was low. This study aimed to exploid a synthesis in vitro culture system and cryopreservation method for sheep ovaian. For this purpose, the physical characters of various sheep ovarian tissue, the effects of various basic culture conditions on in vitro culture of sheep ovarian tissue, and the effectes of various growth factors and hormone, including ascorbic acid (VC). epidermal growth factor (EGF). follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF) and growth and differentiation factor 9 (GDF-9) on sheep follicle developmental and survival were studied. Based on these studies, interaction of various factors on follicle developmental was studied. At last, a sequential culture system was designed by which different concentrations of FSH, GDF-9, EGF, and bFGF were added during different culture days. In addition, ovarian cryopreservation was also studied in this study, and a solid-surface vitrification method which proved effectively for sheep ovarian tissue cryopreservation was exploied. Sheep ovarian cortial tissues, both fresh and cultured, were processed for histological and lmmunohistochemical assays (proliferating cell nuclear antigen (PCNA) and apopotic assays (TUNEL)), or observed by transmission electron microscopy (TEM), while the spent cultured medium were processed for hormone assays (Estradiol, progesterone, and inhibin). The major results were as follow:1. As the observation results by histological and TEM showed, primordial follicles were suited in the outer part of ovarian cortex, abutted to the albuginea, which had one lay of squamous granulose cells, and it had a higher density in the ovarian tissue. While developing follicles were occurred inner of cortex, near to the medulla, one or more lays of cubical granulosa cells were found in these stages. More developing follicles were found in adolescence sheep ovary, consistent with higher follicle and oocyte diameters; More egenerated follicles were observed in old sheep ovary; while more primordial follicles were seen in lamb and fetal ovary, meanwhile, smaller of follicle diameters and simple construction were also found in this two ages. Ovary with corepus luteum had more secondary follicles and lower primordial follicles than in no-corepus luteum ovary. In addition, primordial follicles number was higher in left ovary when compared to right. These results indicated that ovarian cortex was the optional test material for primordial follicle study. And adolescence sheep ovary was the best source on various factors screening experiment, because it had higher ovarian activity. While lamb and fetal were the best target for primordial follicle developmental study. In conclusion, these results indicated that the same ovarian should be compared in the same group.2. Various culture conditions were studied in a steal mesh culture system, including culture method, precoated density and concentration, temperature, cultured media, FBS and BSA, ITS system. As the resul1 showed, ovarian tissue survived and developed in such basic system:basic culture method:steel mesh system with nitrocellulose membrane which pre-coated by 3 mg/mL type 1 collagen. Basic culture medium:α-MEM medium supplemented with glutamine (2 mM), pyruvate (3 mM), BSA (3 mg/mL), ITS (insulin 10μg/mL, transferrin 6.25μg/mL, and selenium 6.25 ng/mL), penicillin G (75μg/mL), and streptomycin (50μg/mL), and at 38.6 oC in humidified air with 5% carbon dioxide (CO2)3. During in vitro culture of sheep ovarian tissue, Medium added 50ng/m L VC, stimulated sheep follicle survival and maintained the follicle structural interagency. FSH would involve in early follicle, and dynamic addition of FSH would benefit for primordial follicle developing and tissue activity maintenance.50ng/mL FSH improved the development quality of follicles and increased the follicle diameters. 100μg/mLEGF stimulated primordial follicles activation and growth, but higer concentration was harmful for follicle development. bFGF stimulated follicle development in a dose dependent style, and GDF-9 also benefited to follicle development and growth. All of these result indicated factors choosed in this study were all stimulator factors on sheep follicle developmental.4. The addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture. The sequential addition of FSH, EGF, GDF-9, and bFGF can stimulate larger proportion of primordial follicle transmittal into the later development stages, even as far as the antral stage, improve the survival rate of follicles, and maintain follicular viability5. Entire ovarian stored in both medium at 4℃and 20℃for 2 or 6 h, and ovarian fragments stored in MEM at 4℃or 20℃for 2 h can used to the study of ovarian in vitro culture. Sheep ovarian tissue can be cryopreserved in 20% DMSO+20% EG by Solid-surface vitrification. Follicle viability and tissue activity were well maintained after cryopreservation. These indicated that method used in present study is an effective, simple and inexpensive alternative forsheep ovarian tissue cryopreservation. |
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