Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation

Abstract

The extract of Auxemma oncocalyx (A. oncocalyx) and its main component, oncocalyxone A (onco A), possess anti-Tumoral activity that may affect fertility. There is limited literature on the action of these substances regarding caprine folliculogenesis. In this study, we evaluated the effect of A. oncocaly and onco A on the in vitro culture of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM) of oocytes from caprine antral follicles grown in vivo (Experiment 2). Isolated secondary follicles were distributed in six groups: The non-cultured control was immediately fixed in 4% paraformaldehyde. The remaining follicles were cultured for 7 days in α-minimal essential medium (α-MEM+) alone (control) or with dimethyl sulfoxide (DMSO), doxorubicin (DXR), A. oncocalyx, or onco A. After culture, the follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL), cellular proliferation (PCNA), as well as the expression of BCL2 and BAX. In addition, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for IVM: control, cultured only in maturation base medium (TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM, oocyte chromatin configuration and viability were assessed. After 7 days of culture, a reduction was noted in the percent of morphologically intact follicles, antrum formation, growth rate, and numbers of PCNA-positive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNEL-positive follicles and relative BAX:BCL2 mRNA ratio's (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx, and onco A treatments showed a greater percent (P < 0.05) of abnormal oocytes and a lower percent of viable oocytes as compared with the control group (P < 0.05). However, only DXR and onco A treatments increased the percent of alive oocytes with abnormal chromatin configuration (P < 0.05). There were no differences in the maturation rates among the control group and DXR, A. oncocalyx, and onco A treatments. In conclusion, under our culture conditions, A. oncocalyx and onco A did not exhibit toxic effect on isolated secondary follicles and on maturation rates of COCs recovered from antral follicles. However, these substances negatively affected the oocyte viability. Thus, culture methods including in vitro secondary follicle culture and in vitro oocyte maturation toxicity test are appropriate methods to evaluate the possible effects of drugs on folliculogenesis.