Follicular responsiveness to equine chorionic gonadotropin in cat species: study in in vitro culture

Abstract

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. In this present study, we investigated 1) the in vitro responsiveness of cat ovarian follicles at different follicle developmental stages to various eCG concentration; 2) tiger antral follicle responsiveness to eCG; 3) the influence of eCG combination with insulin-like growth factor I (IGF-I) and/or stem cell factor (SCF) on cat ovarian follicles at different developmental stages. Study 1, the isolated follicles from the ovaries of 22 cats were classified 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 culture medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. After being cultured for 12 days, follicle growth and gene 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 gene 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. Therefore, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward and the eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cat. Study 2, the optimal concentration of eCG supplementation from the experiment 1 was selected to investigate the responsiveness of the tiger follicle to eCG. Six frozen-thawed ovarian tissue from a tiger obtained post-mortem were evaluated. Twelve isolated antral follicles recovered were randomly allocated into two culture conditions (control and 0.05 IU/mL eCG supplementation) and cultured in an alginate hydrogel for 3 days. The follicle diameters in the control group significant decreased (P < 0.05) after 3 days of culture, while the size of those in eCG supplementation group were remained constant (P > 0.05). Follicle survival was 100% in both groups. However, the oocyte retrieval rate was significant different between the two treatments (control, 33%, n = 6; eCG, 67%, n = 6) (P < 0.05). The nuclear status of all recovered oocytes was remained in germinal vesicle phase. The present study showed that the tiger frozen-thawed antral follicles could not maintain their morphology and function without eCG supplementation in the culture medium. We concluded that eCG plays an important role on tiger antral follicle growth and the survival of the oocyte. Study 3, the influence of growth factor supplementations (IGF-I, SCF and the combination of these two factors) on cat ovarian follicles at different follicle developmental stages were examined. The follicles obtained from twelve cats, encapsulated in a fibrin-alginate hydrogel and cultured for 18 days in the culture medium contained 0.05 IU/mL eCG without growth factor supplementation (control group) or supplemented with 1 ng/mL IGF-I (IGF-I), 50 ng/mL SCF (SCF50), 100 ng/mL SCF (SCF100), 1 ng/mL IGF-I + 50 ng/mL SCF (IGF-SCF50) or 1 ng/mL IGF-I + 100 ng/mL SCF (IGF-SCF100). The growth factors supplementations had no effect to two-layered SF and multi-layered SF growth, whereas SCF100 supported the growth of early AF throughout the culture period (P<0.05). However, the oocyte growth was varied among developmental stages. In the two-layered SF, IGF-I failed to maintain oocyte size after culture for 12 days. Oocytes of multi-layered SF were sustained in their initial size until the end of the study in all growth factor treated follicles. However, the present or absence of growth factor showed no effect to the oocyte of early AF. SCF50 stimulated antral cavity formation and gene regulating follicle and oocyte growth and steroidogenesis in multi-layered SF. While IGF-SCF50 was the best supplementation to early AF because it increased FSHR, GDF9 and steroidogenic genes. Therefore, SCF influenced all stages of follicle development in cat. In conclusions, the responsiveness of cat follicles to eCG was apparent from the multi-layered SF stage onward and the eCG supplementation at 0.05 IU/mL appeared to be optimal for the in vitro follicle culture in the domestic cats. The addition of SCF to culture medium supported cat folliculogenesis from multi-layered SF to early AF stage. Furthermore, similar to the domestic cat, tiger follicles also respond to 0.05 IU/mL eCG supplementation.