The present study was the first report that revealed the supplementation of ovarian tissue culture media using CGA could improve in vitro growth and development of mouse ovarian follicles by reducing oxidative stress and enhancing antioxidant capacity.
In the current study, we first attempted to determine morphological changes across the groups. Our morphological findings (using H&E, AO/EB, and TB staining techniques) demonstrated that administration of CGA could significantly improve the survival rate of follicles, the proliferation of granulosa cells (GCs), and stimulate the growth of primordial and primary follicles to the preantral follicle stage in parallel with an increase in mean surface area of ovarian tissue. It seems these effects of CGA are dose-dependent because all mentioned parameters were gradually improved during in vitro culture up to 100 µmol/L concentration of CGA. However, although the concentration of 200 µmol/L of CGA could significantly change all mentioned parameters, the concentration of 100 µmol/L CGA showed a better function than the 200 µmol/L concentration of CGA.
There is only one related study by Nguyen et al. that indicated the supplementation of porcine oocyte culture media using 50 µmol/L CGA could significantly enhance the maturation, fertilization, and developmental competence of oocytes by preventing apoptosis. They concluded that the application of CGA during oocyte in vitro maturation (IVM) has the potential to improve the porcine in vitro production (IVP) system .
Moreover, in the present study, we employed several complementary techniques in addition to morphological staining. The result of hormonal analysis confirmed morphological findings and demonstrated that the concentration of E2 was significantly enhanced in the 100 µmol/L CGA supplemented group. This finding is in agreement with Rong Ping Zhou et al. who postulated that CGA exerted estrogenic activity and prevented estrogen deficiency-induced osteoporosis in ovariectomized rats . In addition, Wang et al. indicated that CGA exhibited an estrogen-stimulating effect on ovarian granulosa cells. They expressed that CGA may have the therapeutic potential for the treatment of postmenopausal syndrome . It seems that CGA might stimulate granulosa cells to express cytochrome P450 aromatase genes that convert the androgens to E2 and promote ovarian follicular development.
Moreover, our biochemical assessment results were in accordance with those of Ali et al. who confirmed that CGA could significantly increase activities of anti-oxidant armory such as SOD (scavenger of the superoxide anion to form H2O2), CAT (convertor of H2O2 to H2O), and GSH content . The antioxidant effects of CGA have been also demonstrated by other researchers [31–33], however, they have administered CGA in different tissues and cells. For example, Koriem et al. confirmed that caftaric acid (is the ester form of caffeic acid) enhanced GSH concentrations while it reduced MDA and catalase concentrations in brain, liver, and kidney tissues  - but in the present study, we evaluated these criteria in the ovarian tissue for the first time. These observations could be related to the antioxidant effect of CGA and it has important physiological and biological, namely, anti-oxidative and anti-inflammatory effects. It has been discerned that hydroxyl groups (OH) on phenolic acids because of their antioxidant properties act as positive moieties, associated with the number of hydroxyl groups as follows: tri-hydroxy phenolic acids > di-hydroxy (catechol) > mono-hydroxy [19, 20].
Finally, our molecular findings supported both the histological and biochemical results. Otherwise stated, molecular data amplified the result of developmental ability and functional potential of CGA during IVC of ovarian tissue. Our molecular findings corroborate Dkhil et al's investigation that showed the protective effect of CGA against the injuries induced by sodium arsenite (NaAsO2) in the liver of mice. They demonstrated that, in the presence of CGA, the mRNA levels of the pro-apoptotic markers (Bax and Caspase-3) significantly decreased, but the mRNA levels of the anti-apoptotic marker (Bcl-2) significantly increased . On the other hand, our results showed that CGA up-regulated the expression level of GDF9 and BMP15 (p < 0.05).
GDF-9 and BMP-15 direct the follicle symphony and play important roles in folliculogenesis and oocyte quality [35–37]. Overall GDF-9 has been implicated in follicular development (at the stimulation of the primary follicle to the secondary follicle stage) in parallel with an increase in GCs differentiation . BMP-15 is also another fundamental factor that is implicated in: the promotion of follicle maturation since the primordial gonadotropin independent phases, regulation of GCs sensitivity to Follicle-stimulating hormone (FSH) action, prevention of GC apoptosis, and regulation of the ovulation quota [39–41].
Up to now, there isn’t any information about the effect of CGA on the expression level of developmental genes; this study is the first report that revealed enhancing effects of CGA on the expression level of GDF9 and BMP15. There is little literature and a limited number of resources that analyzed the molecular mechanisms of CGA in culture conditions, but it seems that administration of CGA to the IVC medium regulates these molecular changes by phosphatidylinositol-3-kinase (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways. Dai Cheng et al. demonstrated that CGA has a cytoprotective effect against Aluminum, which may bring about damage to the macrophages, possibly through PI3K/Akt and MAPK signaling pathways in RAW264.7 cells .
In conclusion, the present study confirmed that the supplementation of ovarian tissue culture media using CGA, especially at 100 µmol/L, could optimize the culture system and improve the survival, developmental and functional potential of follicles in mouse whole ovarian tissues after 7 days of culture. Furthermore, the result of this study (for the first time) revealed that CGA could suppress oxidative damage and improve the culture system by enhancing the concentration of antioxidants and developmental genes expression. But, before the clinical application of CGA, further experiments are appreciated to elucidate the exact mechanism at the molecular level.