3.2 Heterozygous SLC26A8-mutated spermatozoa show normal SLC26A8 expression
To validate the putative contribution of three heterozygous mutations to the infertility of the affected individuals, we investigated theseSLC26A8 mutations via Sanger sequencing in the three families (Figure 1). Surprisingly, two of the harmful mutations (c.1570_1571del [p.A524*] and c.2191G>A [p.V731I]) in two patients were inherited from their unaffected fathers who presented that they possess the normal reproductive capability. With the striking findings noticed, the result that the sterile phenotype associated with teratozoospermia in our patients caused by the three mutations was questionable. Considering the previous observation that decreased expression of SLC26A8 resulting from those three missense mutations detected in the transfected eukaryotic expression vectors (Dirami et al., 2013), we also used eukaryotic expression vectors for each variant and wild-typeSLC26A8 to transiently transfect HEK-293Tcells, respectively. As expected, the western blotting showed that the expression of SLC26A8 protein encoded by c.2191G>A [p.V731I] mutation was significantly decreased when compared to the wild-type SLC26A8 protein (Figure 3a). No SLC26A8 expression was detected in the cells transfected with the vector carrying c.1570_1571del [p.A524*] mutation and c. 306del [p.G103Afs*9] mutation respectively (Figure 2a). Nevertheless, using immunostaining, we confirmed that there was no difference in SLC26A8 amounts in sperm between the three infertile individuals and normal control (Figure 2c). In addition, western blotting results of spermatozoa lysates further confirmed the similar expression of SLC26A8 between patients and normal control (Figure 2b). Thus so, we deduced that no differential expression of SLC26A8 between the patients and normal control might be explained by the compensation of the maintenance of one normal SLC26A8 copy, although another copy is mutated. Moreover, theSlc26a8 -/- mouse further proofed thatSlc26a8 participated in spermatogenesis is linked to a recessive-inheritance but not a dominant-inheritance (Touréet et al., 2007; Rode et al., 2012). All findings demonstratedSLC26A8 mutations in male infertility is a recessive-inheritance, and heterozygous mutations ofSLC26A8 might exhibit a certain degree of determination towards male infertility.