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

Abstract

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

Transition to the ovulatory season in mares: An investigation of antral follicle receptor gene expression in vivo

“…Previous studies have shown the importance of the epidermal growth factor (EGF) for oocyte maturation in mares (Goudet, Belin, Bézard, & Gérard, ; Lorenzo, Liu, Carneiro, Conley, & Enders, ), cows (Harper & Brackett, ; Lorenzo, Illera, Illera, & Illera, ), and sows (Ding & Foxcroft, ; Reed, Estrada, Illera, & Petters, ). Furthermore, the association of EGF and FSH has been reported to stimulate antral and preantral follicle growth in mares, cows, goats, and sows (Aguiar et al, ; Celestino et al, ; Gastal et al, ; Gutierrez, Ralph, Telfer, Wilmut, & Webb, ; Silva et al, ; Wu & Tian, ; Zhou & Zhang, ). Notwithstanding, EGF modulates steroidogenesis (Hsueh, Welsh, & Jones, ) and reduces the apoptosis (Luciano, Pappalardo, Ray, & Peluso, ) in granulosa cells.…”

“…All steps were performed at room temperature, and sections were washed three times with PBS between all steps. All primary antibodies were successfully used in previous studies with equine tissues (Aguiar et al, ; Gastal et al, ; Herrera‐Luna, Scarlet, Walter, & Aurich, ; Ishak et al, ), except for the specificity of the VEGF antibody that was tested through western immunoblotting of total proteins on mare ovaries as described previously (Feugang et al, ). Finally, histological sections were immediately counterstained with a DAPI‐contained mounting medium, and fluorescence was evaluated using a fluorescent microscope (EVOS FL Cell Imaging System; Thermo Fisher Scientific Inc.).…”