It is well-established that folates are important for the development of the embryo. Pregnant women are advised to take a dietary supplement of 400 mg folic acid per day from 12 weeks before conception and during the first trimester of pregnancy (Hibbard, 1964; Cawley et al., 2016). Folate deficiency has been associated with increasing the risk of neural tube defects (NTD) (Detrait et al., 2005; Pitkin, 2007), early spontaneous abortion (George et al., 2002), congenital heart defects (Rosenquist et al., 2010) and orofacial clefts (Taparia et al., 2007) amongst others.

The folate receptor is highly expressed in reproductive tissues thereby providing folate to the embryo (da Costa et al., 2003). Deficiency in folate uptake can cause pregnancy-related complication such as NTD, a well-established association (Centers for Disease Control, 1991a,b; MRC Vitamin Study Research Group, 1991; Czeizel and Dudas, 1992; Cragan et al., 1995). Interestingly, the identification of folate receptor autoantibodies in in women with recurrent NTD pregnancies might mechanistically explain how the embryo is deprived of folate (Rothenberg et al., 2004; Berrocal-Zaragoza et al., 2009; Sequeira et al., 2013). Additionally, variation in folate pathway genes are associated with female infertility (Altmae et al., 2010). In line with this, postnatally acquired FR autoantibodies blocking folate transport to the brain have been associated with the infantile-onset cerebral folate deficiency syndrome (Ramaekers et al., 2005), which in a number of patients manifests as low-functioning autism with neurological deficits (Ramaekers et al., 2007). Folic acid supplementation in pregnancy may have beneficial effects on the neurodevelopment of children, such as cerebral folate deficiency syndrome and autism spectrum disorders (Desai et al., 2016), beyond its proven effect on NTDs (Blencowe et al., 2010; De-Regil et al., 2015; Gao et al., 2016).

It is proposed that folate deficiency affects gene expression by disrupting DNA methylation patterns or by inducing base substitutions, DNA breaks, gene deletions and gene amplification (Crott et al., 2008). In line with this, mutations in the enzyme methionine synthase reductase (Mtrr), necessary for utilization of methyl groups from the folate cycle, affect the folate metabolism and cause epigenetic instability (Padmanabhan et al., 2013). The phenotypes of the Mtrr-deficient mice included congenital malformations with neural tube, heart and placental defects, which persisted for five generations, through transgenerational epigenetic inheritance (Padmanabhan et al., 2013). Folates are co-enzymes responsible for the 1-carbon (1C) unit transfer important for purine, pyrimidine and methionin synthesis, and glycine and serine metabolism (Fowler, 2001). The methionine synthesized from homocysteine in the folate cycle is the only significant source (in most cells) of the universal methyl donor, S-adenosyl methionine (SAM), and is essential for methylation processes (Stover, 2009). Although folates are recycled in the folate cycle, it is essential to accumulate as folates are degraded. Folates, where 5-methyl-THF is the predominant bioavailable form in mammals, can either be taken up directly from dietary sources or produced by metabolism of dietary folic acid (Kooistra et al., 2013).

Folate is principally taken up into cells by a reduced folate carrier, RFC1, and by two different folate receptors with high affinity for folate, FOLR1 and FOLR2 (Kooistra et al., 2013). Other members of the receptor family are FOLR3 and FOLR4. The folate receptors are attached to the extracellular side of the membrane by a glycosylphosphatidylinositol anchor (GPI-anchor). The folate receptors are heavily glycosylated and bind folic acid and 5-methyl-tetrahydrofolate (5-methyl-THF) with high affinity (Brigle et al., 1994; Chen et al., 2013). RFC1 has been reported expressed in cumulus-oocyte-complexes (COC) but not in preimplantation embryos, suggesting a role in folate accumulation in the COC cells, but without transport into the enclosed oocyte (Kooistra et al., 2013). FOLR1 has been shown to be an important receptor for the uptake of folates into the cells and has been found expressed in mouse preimplantation embryos, increasing from the 2-cell stage onwards (Kooistra et al., 2013). FOLR2 has not been reported expressed in neither COC nor preimplantation embryos (Kooistra et al., 2013) and binds folic acid with lower affinity than FOLR1 (Brigle et al., 1994). FOLR3 does bind folic acid, but it is a constitutively secreted protein rather than a membrane bound one, probably due to the lack of an efficient signal for glycosylphosphatidylinisotol (GPI)-anchor attachment (Shen et al., 1995). FOLR4 (Juno (FR-d in humans)) is not a typical member of the Folate Receptor Family, but share a high structural homology with FOLR1 (Spiegelstein et al., 2000). Juno-deficient mice are infertile (Bianchi et al., 2014).

The basis of folate supplementation of folate has been administrated as oral or intravenous folinic acid (5-formyltetrahydrofolate) treatments that has been shown to improve clinical status. Folinic acid can be transported through the RFC and participate in folate dependent reactions compared to both folic acid and MTHF that needs further processing (Desai et al., 2016; Frye et al., 2016; Ramaekers et al., 2016).

Mice deficient of Folr1 are defective in early embryonic development (Piedrahita et al., 1999), in contrast to Folr2-deficient mice that developed normally (Piedrahita et al., 1999). Folr1^−/− embryos died at day E10 with severe morphological abnormalities, and interestingly, these phenotypes could be reverted by maternal supplement of Folr^+/− dams with folic acid (Piedrahita et al., 1999). The curly tail (ct) mouse provides a model for neuronal tube defects (spina bifida and exencephaly) (Van Straaten and Copp, 2001). However, folate supplement does not prevent NTD in ct/ct embryos (Burren et al., 2010), but formate supplementation enhanced the folate-dependent nucleotide biosynthesis and prevented spina bifida (Sudiwala et al., 2016). Interestingly, primary cultures of mouse embryonic fibroblasts established from Folr1^−/− embryos revealed altered signal transduction in pathways including transforming growth factor beta 1 (TGFβ1) and the canonical Wnt signaling pathways suggesting that pathways for proper development are significantly altered (Warner et al., 2011).

Besides a role in signal transduction, it has been shown that FOLR1 can act as a transcription factor. In cell lines, FOLR1 translocated to the nucleus and interacted with FGFR4 and HES1, and was suggested to regulate a wide range of developmental genes (Boshnjaku et al., 2012).

In this study, we noted the presence of maternally contributed FOLR1 during mouse and human ovarian follicle development. In mouse follicles, the presence of FOLR1 was observed in both oocytes and the surrounding granulosa cells. In contrast, FOLR1 was restricted to oocytes in follicles from human tissue. In 2-cell embryos, FOLR1-eGFP fusion protein was detected as cytoplasmic and membrane-associated dense structures, resembling the observed cellular localization in ovarian follicle development.

Microinjections of short interference (si)RNA probes targeting the Folr1 transcript revealed an efficient knock-down of the embryonic Folr1 transcript. Interestingly, siRNA-mediated knock-down of Folr1 in zygotes reduced the ability of embryos to develop to blastocyst. This indicates that maternally contributed FORLR1 protein and zygotically synthesized Folr1 transcripts sustain embryonic development combined.

Dysfunctional maternal folate metabolism caused by folate deficiency or polymorphisms are associated with congenital abnormalities, intrauterine growth restrictions, placental and cardiovascular abnormalities as well as neural tube defects in human. While parts of the abnormalities can be directly associated with the embryonic expression of FOLR1, it is likely that maternally contributed FOLR1 might already mark ovarian cells before fertilization, and influence pre- and post-implantation development.

Transcripts encoding the FOLR1 has not been investigated in ovarian follicles. However, a recent study enterrogated the transcriptomes in human oocytes from primordial and primary follicles, which did not detect expression of the FOLR1 gene (Ernst et al., 2017). Whether granulosa cells from primordial and primary follicles express the Folr1 transcript remains to be investigated.

In agreements with another study, we found that Folr1 transcription appeared to initiate at the 2-cell stage, which is further emphasized in a study that interrogated amantidine-sensitive transcrips, which included Folr1 (Zeng and Schultz, 2005). Interestingly, this appears to be conserved between different mice strains, as experiments were performed independently on three different strains [CF1 (Crl:CF1) females (Charles River) and BDF1 (B6D2F1/Crl) males (Charles River) (Kooistra et al., 2013), superovulated female CF-1 mice (Harlan) mated to B6D2F1/J males (Jackson Laboratory) (Zeng and Schultz, 2005) and F1 [CBA (C57BL/6jxCBA) females and males (Janvier, this study).

Secreted FOLR1 proteins are either anchored to membranes via a glycosyl-phosphatidylinositol linkage or exist in a soluble form. The receptor-ligand complex is endocytosed into cytoplasmic vesicles and then recycled to the cell membrane. In our IHC, we accordingly observed FOLR1 at the cell membrane, but also noted it in the cytoplasm, resembling the intracellular transport of FOLR1. This might simply reflect the staus quo of the cell in the absence of active folate transport. Thus, in conclusion, it appears that oocyte folate accumulation must occur during follicle development, where folate is maternally supplied. The observation of the folate receptor in both oocytes and granulosa cells in the mouse follicles, but limited to oocytes in the follicles from human samples remains to be further investigated. Very low levels of the folate receptor and/or specific folate supplement might represent likely reasons that the folate receptor was not detected in human granulosa cells. FOLR1 appeared to be absent from the nucleus during follicle development, which suggest that during these stages in development, FOLR1 does not act as a transcription factor. This suggests that the function of FOLR1 during these stages is perhaps assigned predominantly to folate metabolism and signal transduction. Indeed, the role of TGFβ1 in ovarian physiology is well-know, and due to the fact that folate deficiency can alter this pathway, it might be that FOLR1 could influence this, as well, in ovarian follicles.

The FOLR1 distribution in 2-cell stage embryos was interogated using a eGFP fusion approach and the intracellular distribution of FOLR1-eGFP was comparable to that observed in the follicles. FOLR1-eGFP was concentrated at the cell membrane and further noted in cytoplasm, indicating that the FOLR1-eGFP follows the endogoues folate transport and makes this construct suitable for future studies on folate transport in embryonic cells.

Interestingly, while some of the embryos that were submitted to Folr1 RNAi were indeed able to complete development to the blastocyst stage, several arrest development during later preimplantation stages. This indicates that during preimplantation development, the levels of Folr1 transcript and FOLR1 protein are finely balanced to rely on maternally contributed proteins and the generation of zygotically-derived Folr1. Thus, this suggests that indeed the maternal supply of FOLR1 protein remain during preimplantation and is partly sufficient to support the development until blastocyst stage. Developmental competences might therefore be much more dependent on the maternal internal pool of folate than previously anticipated and variations might account for folate-associated deficiencies.

In this context, it is curious to note that most in vitro-based experiments today are performed without folate supplement to the media, even though the long term consequences of folate deficiency in the medium is unknown. It opens a question whether or not we should in fact supplement in vitro culture media with folate. Further studies are needed to address whether variations in folate storage might influence developmental potential accordingly.

The long-term effect of Folr1 RNAi is not known, and it might be that depletion of Folr1 could interfere with post-implantation. This is indeed in line with the fact that even low blood folate levels in pregnant women are correlated with higher risk of neural tube deficiency in their offspring (Smithells et al., 1976; Detrait et al., 2005), and periconceptional supplementation of folate reduces the occurrence of neural tube defects (Centers for Disease Control, 1991a,b; MRC Vitamin Study Research Group, 1991). Future studies are needed to elucidate the cellular and molecular mechanisms underlying folate and FOLR1 functions and correlation between FOLR1 in preimplantation and early post-implantation development. This could include the CRISP-Cas9 technology and/or a conditional knock-out strategy to functionally knock-down the folate receptor before fertilization.

KL conceived the study. TS performed IHC/Western blot experiments. SF performed qPCR analyse. EE contributed with human tissue. MN and KL performed confocal analysis. AH conducted qPCR of siRNA experiments. KL performed siRNA microinjections and wrote the manuscript. All authors approved the final manuscript.