Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue.

This study investigates the impact of FSH (follicle-stimulating hormone) supplementation to a growth-enriched medium containing insulin and EGF (epidermal growth factor) on equine ovarian tissue during in vitro culture. The results show that, with or without FSH, the enriched medium maintains the ovarian tissue’s functionality over prolonged in vitro culture periods.

Research Methods

• The investigators collected ovarian tissue fragments from horses (10 mares) and divided them into three groups: noncultured control, cultured control, and cultured + FSH.
• The cultured groups were maintained for either 7 or 15 days. They used various measures to evaluate the tissues after the culture period, including follicular morphology, estradiol levels in the medium, fluorescence intensity for TUNEL, EGFR (epidermal growth factor receptor) and Ki-67 detection, and gene expression of GDF-9 (growth differentiation factor 9), BMP-15 (bone morphogenetic protein 15), and Cyclin-D2.

Findings

• After seven days in culture, medium supplemented with FSH showed a similar percentage of normal looking follicles compared to the noncultured control group – indicating that the addition of FSH does not adversely affect follicle development.
• For both cultured groups, estradiol levels increased from day 7 to day 15, showing that the cultured tissues remained active in producing hormones over time.
• There was no significant difference between the noncultured control group and the treated groups in terms of TUNEL and EGFR intensity after 15 days, suggesting maintained ejection of EGFR in cultured tissues.
• There was no difference between groups treated with or without FSH for Ki-67 intensity after 15 days, but there was a decrease when compared with the noncultured control group – pointing to possible reduction in cell proliferation in cultured tissues.
• The gene expressions for GDF-9, BMP-15, and Cyclin-D2 showed similar levels among all treatments after 15 days of culture, suggesting sustained gene activity, which is vital for normal ovarian function.

Conclusions

• This study demonstrated that an enriched medium, with or without FSH supplementation, was successful in maintaining the functionality of equine ovarian tissues during long-term in vitro culture. The findings suggest that ovarian function persists in culture conditions, paving the way to further examine the effects of other growth factors or hormones on equine ovarian cells.