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 PCNApositive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNELpositive 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. ResumoO extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram ...

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

“…In this study, we observed a significant increase in mRNA levels for GDF9 , CCNB1 , PARN , cMOS , eIF4E , and H1FOO in oocytes derived from secondary follicles grown for 18 days. Several variables may affect the outcome of follicle culture in vitro (Mbemya et al, ), such as the culture base media composition (McNatty, Reader, Smith, Heath, & Juengel, ; Santos et al, ) and supplementation (Ferreira et al, ), the animal model (Magalhães et al, ), the culture system (Araújo, Gastal, Figueiredo, & Gastal, ), the follicular category (Magalhães‐Padilha et al, ), reactive oxygen species (ROS) production (Leiva‐Revilla et al, ), and activation or silencing of genes encoding antioxidant defense enzymes, growth and progression of meiosis (Dalton, Shertzer, & Puga, ). Therefore, an increase of the expression of these genes in oocytes may be a good indicator of the viability of in vitro culture secondary follicles, providing support to oocyte development.…”

Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis

“…Several variables may affect the outcome of in vitro follicle culture such as the culture base media composition [9, 10] and supplementation [11]; the animal model [8]; culture system [1, 12]; follicular category [13, 14] and reactive oxygen species (ROS) production [15], leading to oxidative stress. Many authors have shown that an increase of glutathione peroxidase (GPx) can represent a cellular transcriptional response to ROS [16], such as an activation or silencing of genes encoding antioxidant defense enzymes, growth and progression of meiosis [17].…”