Equine chorionic gonadotropin induces in vitro follicular growth from the multi-layered secondary developmental stage in cats

Equine chorionic gonadotropin (eCG) has been commonly used to induce estrus in several felid species. However, the mechanisms by which this gonadotropin regulates cat folliculogenesis are still unclear. We investigated the responsiveness of cat ovarian follicles at different developmental stages to various eCG concentrations supplemented in vitro. Follicles were mechanically isolated from the ovaries of 22 cats and categorized into three developmental stages based on their morphology and diameter: 1) two-layered secondary follicle (SF), 100-150 μm (n = 139); 2) multi-layered SF, 150-300 μm (n = 154); and 3) early antral follicle (AF), ≥300-500 μm (n = 123). The follicles were then encapsulated in 0.5% (w/v) sodium alginate and cultured for 12 days in Minimum Essential Medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. Follicle and oocyte diameters were assessed every 3 days. On Day 12, mRNA expression levels of FHSR, LHCGR, GDF9, BMP15, CYP17A1, CYP19A1 and STAR were analyzed using real-time PCR. After being cultured for 12 days, follicle growth and mRNA expression of two-layered SF were not influenced by eCG at all concentrations (P > 0.05). However, the concentration of eCG at 0.05 IU/mL stimulated follicular growth and gene expressions in the multi-layered SF and early AF (P < 0.05). Correspondingly, the diameter of oocytes in the multi-layered SF and early AF treated with 0.05 IU/mL eCG was unchanged. Considering the mRNA expression, the level of STAR was enhanced in the early AF (P < 0.05) and tended to increase in the multi-layered SF (P = 0.08) cultured in 0.05 IU/mL eCG, whereas the expression of other genes was not affected. In sum, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward. The eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cats.