Cumulus cells act as a vital regulator of oocyte maturation [20, 21] as oocytes having compromised cumulus expansion have inadequate potential for implantation [22]. This advocates the use of cumulus cells to gain comprehensive knowledge of the reproductive potential and viability of the oocytes. GDF-9 secreted by the oocyte has been reported to act as a key factor in cumulus expansion [23], which improves oocyte developmental competence by regulating several crucial granulosa cell enzymes involved in cumulus cell expansion [24]. Prostagladin-Endoperoxide Synthase 2 (Ptgs2) and Pentraxin 3 (Ptx3) are downstream GDF-9 target genes present in cumulus cells. The transcripts of these genes and others with related functions has been suggested as biomarkers of oocyte maturation [25–30]. Our results showed that at 50µM folic acid supplementation, the degree of cumulus expansion was significant (Fig. 2), which was further supported by high blastocyst development rate (Fig. 4) at this concentration. Interestingly, supplementation of 150µM folic acid decreased the expression of transcripts for Ptgs2 as well as Ptx3, indicating towards the deleterious effects of high folic acid on oocyte maturation and developmental competence. The interaction between folic acid and GDF9 downstream pathway is not known and this study opens a new area of research.
The experiment involving the evaluation of polar body extrusion rate following oocyte maturation demonstrated no significant variation between different folic acid supplementation groups (Fig. 3). The results implies that folic acid supplementation did not improve the nuclear maturation of oocytes. On the contrary, supplementation of 50 µM folic acid during oocyte maturation increases the development rate to the blastocyst stage (Fig. 4). However, there is no apparent effect of folic acid supplementation on the number of cleaved embryos. A positive effect of folic acid supplementation in maturation medium has been reported in recent studies on different animal models [3, 4, 31]. On comparison of previous studies with present study, it is noteworthy that, there are species-specific differences in the apt concentration of folic acid used for supplementation in maturation medium. The present data also shows that the development rate to the blastocyst stage greatly decreased after 150µM folic acid supplementation (Fig. 4), indicating towards the detrimental role of high levels of folic acid in embryo development. The results are consistent with the observations of a previous study which shows that both low and high levels of folic acid before conception is detrimental for oocyte follicular growth [5]. Even moderately high levels of folic acid supplementation in maternal diet is linked with defects in neural tube closure, embryonic delays, embryonic loss, ventricular septal defects, and thinner left and right ventricular walls and neural tube closure [32, 33]. Moreover, supplementation of 50 µM folic acid during oocyte maturation decreased the intracellular ROS levels in mature oocytes (Fig. 5) signifying the potential role of folic acid as an antioxidant. In total, our work suggests that folic acid supplementation during maturation may increase the developmental competence of oocytes by influencing cytoplasmic maturation through promoting cumulus expansion and reducing ROS.
As per our knowledge, this is the first study to evaluate the dynamics of folate transporters and enzymes during pre-implantation embryo development in response to change in folate concentration during oocyte maturation. The notable observation was of Folr1, whose transcript expression significantly (P < 0.05) increased in mature oocytes, 2–4 cell stage embryos and 8–16 cell stage embryos after 50µM supplementation (Fig. 7). This observation is consistent with the significant increase in blastocyst development rate (Fig. 4) and oocyte maturation at 50µM folic acid supplementation (Fig. 2). It signifies that an increased expression of Folr1 transcripts is required for enhanced blastocyst development rate. A tendency of Rfc1 to express at high rates in oocytes during maturation in the presence of 50µM folic acid and returning to default levels at subsequent stages has been observed. RFC1 may possibly be acting as transport channel to fill the internal folic acid stores of oocytes during maturation for later cell divisions. On the contrary, the levels of Folr1 transcripts remain consistently elevated throughout the developmental stages, which indicates that Folr1 may be additionally involved in other vital pathways except performing solely as folate transporter.
DHFR (Dihydrofolate Reductase) catalyses THF (Tetrahydrofolate) regeneration by reduction of dihydrofolate, and is a target for the antifolate chemotherapeutic drug MTX (Methotrexate). In the present study, the upregulation of Dhfr transcripts observed in oocytes, cumulus and pre-implantation embryos (Fig. 7), after the folic acid supplementation, may be the result of elevated levels of internal folic acid (due to increased Folr1 and Rfc1 expression) which was needed to be further metabolized in cells. AHCY (adenosylhomocysteinase), which converts S-Adenyl-L-homocysteine to adenosine and homocysteine, was also found to be upregulated in oocytes and cumulus. The resulting adenosine is required for energy transfer via ATP and ADP as well as in signal transduction by cAMP [34]. Similarly, Methionine synthase, MTR, is necessary for normal development of the embryo by metabolizing the potentially harmful homocysteine and production of SAM [35]. In present study, the upregulation of Mtr by several folds in oocytes, cumulus and 2–4 cell stage embryos after folic acid supplementation in oocyte maturation medium probably ensures a check on the levels of homocysteine produced by AHCY. The elevated levels of Folr1 and Mtr in developmental stages, higher maturation and higher blastocyst production after 50 µM folic acid supplementation indicates a vital role of these two genes in oocyte maturation and embryo development. Moreover, homozygous Mtr knockout mice pups failed to survive after birth thereafter [36]. Folr1 and Mtr expression can thus be used for assessment of embryo quality. These results infer that folate supplementation improved the overall folate–methionine cycle metabolism inside the cells resulting in better embryonic metabolism and development. The exhaustion of cellular folic acid as the embryos approaches blastocyst stage may have resulted in unaltered expression of Folr1, Rfc1, Dhfr, Ahcy and Mtr. Therefore, a study involving supplementation of folic acid throughout pre-implantation embryo development is also required to deeply deduce the pathway.
In our study, the caprine oocytes (mature and immature) and pre-implantation embryos were found to express transcripts for Folr1, Folr2 and Rfc1 (Fig. 6). Although, the expression of Folr2 was very low in mature oocytes, mature cumulus cells, 2–4 cell stage embryos and 8–16 cell stage embryos, as indicated by very faint bands (Fig. 6). The specific reason for comparatively high Folr2 expression only in blastocysts cannot be explained because of the lack of any specific function known to be associated with Folr2 except for the folic acid transport. It is noteworthy that cumulus cells of immature as well as mature oocytes expressed Folr2, but not Folr1. The RT-PCR expression patterns of folate transporters for caprine oocytes, cumulus cells and pre-implantation embryos in present study partially disagree with the results previously reported for cow and mice [15, 16]. Kwong et al. reported that bovine Rfc1 is present in oocytes and pre-implantation embryos, comparable to the pattern we found in goat. Moreover, the expression of bovine Folr1 was found to be present in oocytes and pre-implantation embryos [16]; however, in mice its expression was absent in mature oocyte and 1-cell stage embryo [15]. In the present study, transcripts for Folr1 were detected in all the stages starting from oocyte (mature as well as immature) to blastocyst, except for cumulus cells. Contrary to the bovine and murine orthologue, caprine Folr2 was present in all the stages from oocyte to blastocyst. Human embryonic stem cells, which are analogous to the blastocyst inner cell mass, express Folr1 but not Folr2 [37], inconsistent with the expression pattern we found in goat blastocyst. Therefore, a species-specific variation can be concluded to exist in the folate transport. These differences may have been appeared evolutionarily due to the variations of folate levels in natural diet of different species. Detailed studies are required to gain the knowledge behind this observation.