Influence of TCM 199B, α-MEM, Waymouth MB 752/1 culture media, VEGF, Estradiol-17ß, GDF-9 and FGF on in vitro development of preantral follicles in sheep.

A total of 2792 preantral follicles (PFs') isolated from 750 ovaries of sheep were cultured in four different experiments. The efficacy of three commercially available culture media viz., TCM 199B, α-MEM and Waymouth MB 752/1 on the growth of sheep PFs' was tested in experiment I. Among the three media TCM 199B supported better development of PFs' in culture. The remaining experiments established the best concentrations of vascular endothelial growth factor (VEGF), Estradiol-17ß (E2), GDF-9, Fibroblast growth factor (FGF) and their best combinations for the in-vitro development of PFs'. Inclusion of VEGF at 10 ng/mL, Estradiol-17ß at 5 ng/mL, GDF-9 at 10 ng/mL or FGF at 10 ng/mL individually in a standard medium (SM) (containing FSH, IGF-I, GH and T4) supported better nuclear maturation of the oocytes to MII stage. Different combinations of VEGF, Estradiol-17ß, GDF-9 and FGF supplemented in the SM promoted similar overall follicular growth. However, (a) SM + VEGF(10 ng/mL) + E2(5 ng/mL) supported higher increase in the diameter, (b) SM without any supplements induced antrum formation in greater proportion of follicles, and (c) SM + VEGF(10 ng/mL) + GDF 9(10 ng/mL) or SM + E2 (5 ng/mL) + FGF(10 ng/mL) supported high proportion of oocytes to reach MII stage. To conclude, TCM 199B appeared to be a better medium for development of sheep PFs'. VEGF, Estradiol-17 ß, GDF-9 and FGF have beneficial influence on the development of sheep PFs' when supplemented in TCM 199B.

Effect of leptin on in vitro development of ovine preantral ovarian follicles.

The influence of human or ovine leptin on in vitro culture of preantral follicles (PFs) isolated from sheep ovaries was investigated. Among the 12 different concentrations (0-1000 ng/mL) of human leptin tested, proportion of PFs exhibiting growth, mean increase in diameter, antrum formation, and maturation of the oocytes to MII stage were the best in 10 ng/mL. Culture of sheep ovarian PFs in TCM 199 supplemented with 10 ng/mL of human or ovine leptin FSH (2.5 µg/mL), thyroxine (1 µg/mL), insulinlike growth factor I (10 ng/mL), and GH (1 mIU/mL) resulted in significantly (P ≤ 0.05) greater average increase in diameter (11 and 9 vs. 6 µm), better proportions of PFs exhibiting growth (66% and 58% vs. 48%), antrum formation (51% and 51% vs. 34%), and maturation of oocytes to MII stage (24% and 22% vs. 7%) than the control medium. It is concluded that (1) the optimum dose of leptin for the growth of sheep PFs in vitro was 10 ng/mL, (2) human or ovine leptin supported similar development in vitro of PFs in sheep, (3) inclusion of leptin along with FSH, thyroxine, insulinlike growth factor I, and GH resulted in only a marginal further improvements in in vitro development of sheep PFs'.

Quantitative expression patterns of GDF9 and BMP15 genes in sheep ovarian follicles grown in vivo or cultured in vitro.

Quantitative patterns of expression of the growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) genes in different development stages of in vivo and in vitro grown ovarian follicles in sheep were studied for the first time. Both GDF9 and BMP15 were expressed in the cumulus cells and oocytes at all the development stages of in vivo and in vitro grown ovarian follicles. Growth differentiation factor 9 and bone morphogenic protein 15 exhibited stage-specific undulations in the expression in the cumulus cells and oocytes isolated from in vivo grown ovarian follicles. These undulations could be related to discrete development events during the ovarian follicle development. The expression of GDF9 and BMP15 was highest (3.38 ± 0.02 and 2.69 ± 0.06, respectively; P ≤ 0.05) in the primordial follicles compared with preantral, early antral, antral, and large antral stages. Similarly, GDF9 and BMP15 expression in the cumulus cells (0 ± 0.16 and 0 ± 0.07) and oocytes (1.47 ± 0.07 and 1.32 ± 0.03) was lowest (P ≤ 0.05) in the in vivo grown antral follicles. In the cultured follicles, the stage-specific undulations observed in the expression of GDF9 and BMP15 in the in vivo grown follicles were either different or abolished. For example, in the oocytes from in vitro grown follicles, the expression of BMP15 did not change as the development progressed all the way from preantral to large antral follicle stage although in the oocytes from in vivo grown follicles BMP15 expression exhibited stage-specific variations. It is concluded that GDF9 and BMP15 follow a stage-specific pattern of expression during the in vivo development of ovarian follicles in sheep, and in vitro culture altered the stage-specific changes in the expression of these two genes.

Quantitative expression of antiapoptotic and proapoptotic genes in sheep ovarian follicles grown in vivo or cultured in vitro.

To test the hypothesis that the poor development of the oocytes in cultured ovarian follicles of mammals is due to aberrant expression of developmentally important genes, quantitative expression patterns of Bcl2 (B-cell leukemia/lymphoma 2; antiapoptotic) and Bax (Bcl2-associated X protein; proapoptotic) genes in preantral, early antral, antral, large antral follicles, and cumulus-oocyte complexes (COCs) grown in vivo or cultured in vitro were studied. The level and pattern of expression of Bcl2 in the cumulus cells isolated from different development stages of in vivo- and in vitro-grown ovarian follicles were similar suggesting that in vitro culture did not alter the expression of this antiapoptotic gene in the cumulus cells. However, between the in vivo- and in vitro-grown ovarian follicles (1) Bcl2 expression levels in the oocytes from antral follicles (2.21 ± 0.14 vs. 0.87 ± 0.19), large antral follicles (0 ± 0.35 vs. 1.56 ± 0.13), and COCs (0.45 ± 0.31 vs. 2.69 ± 0.15), Bax expression levels in the (2) cumulus cells from early antral (2.09 ± 0.11 vs. 0.98 ± 0.13) and large antral follicle (0.63 ± 0.44 vs. 0 ± 0.21), and (3) oocytes from antral follicles (1.65 ± 0.20 vs. 0.97 ± 0.15), large antral follicles (0.93 ± 0.18 vs. 2.08 ± 0.11), and COCs (1.03 ± 0.17 vs. 2.09 ± 0.11) were significantly different (P ≤ 0.05). Similarly, Bcl2 to Bax ratios were also significantly different between some but not all stages of in vivo and in vitro development. From the present results, it is concluded that imbalance in the expression of proapoptotic and antiapoptotic genes may be an important cause for the compromised development potential of the oocytes in cultured ovarian follicles of sheep.