Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Effects of tumour necrosis factor-alpha and interleukin-1 beta on in vitro development of bovine secondary follicles.

The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.