3.3 The special expression pattern of SLC26A8 during spermatogenesis
The previous animal model presented that male Slc26a8-/- mice sperm showed lack of motility (Touréet et al., 2007; Rode et al., 2012), while the information of SLC26A8 in spermatogenesis still needs to be explored (Toure et al., 2001). Mouse testicular sections were used for immunofluorescence and the results revealed that SLC26A8 is detectable in the nucleus and cytoplasm of various germ cells and in the flagella of spermatozoa (Figure 3a). Additionally, we evaluated the expression of SLC26A8 in human testis, and the results demonstrated that SLC26A8 was distributed in the cytoplasm of spermatocytes, and in early spermatids, the expression began to drop off (Figure 3b). Moreover, germ cell-typing staining results revealed that SLC26A8 was detectable in the head and cytoplasm of various germ cell types, supporting that SLC26A8 is a major morphogenetic participant in the early spermatogenic process (Figure 3c and Figure S1b). In summary, our results suggest that SLC26A8 may be involved in spermatogenesis and unravels its potential role in regulating sperm morphology.
DISCUSSION
In the present study, we described the monoallelic mutations inSLC26A8 are not be the causative mutations of male infertility. Especially, although these heterozygous mutations in SLC26A8 were identified to be all deleterious in transfected cells, immunofluorescence staining and western blotting results depicted the heterozygous mutations didn’t impair SLC26A8 expression in the patients’ sperm. And two heterozygous mutations were inherited from the fertile fathers. Collectively, we provided evidence that heterozygous mutations in SLC26A8 were not directly responsible for male infertility.
SLC26A8 has a regulatory effect on cystic fiber transmembrane transduction regulatory factors (CFTR) (Dirami et al., 2013). CFTR is located in the head and mid-flagella of mature sperm and controlled sperm capacitation and motility (Rode et al., 2012). SLC26A8 protein combined with CFTR to form the SLC26A8-CFTR complex regulated the ion flux of sperm and further the movement of sperm (Dirami et al., 2013; El Khouri et al., 2014). As indicated by Dirami T et al , the heterozygous mutations of SLC26A8 were involved in asthenozoospermia (Dirami et al., 2013), which was classified into spermatogenic failure 3 (OMIM 608480) later. Regretfully, the authors did not provide the data about the inheritance of the mutations. However, in our study, it is worth stating the fact that normal fertile males also carried the dangerous heterozygous variants of SLC26A8 , strongly supporting the heterozygous variants of SLC26A8 might not be direct etiology for asthenozoospermia. Besides that, no reproductive barriers were performed on the heterozygous Slc26a8 KO male mice, while the homozygous Slc26a8 KO male mice occurred in sterility, with completely immotile and malformed spermatozoa. These findings suggest infertility associated with Slc26a8 mutations was relevant to the autosomal recessive mode of inheritance (Touréet et al., 2007; Rode et al., 2012). What’s more, the autosomal dominant inheritance of the heterozygous alterations in SLC26A8 might be in contrast to the variants previously identified in other members of the SLC26 family (SLC26A2, SLC26A3, SLC26A4, SLC26A5 ), which follow an autosomal recessive pattern of inheritance (Anwar et al., 2009; Barreda-Bonis et al., 2018; Dawson et al., 2005; Forlino et al., 2005; Höglund et al., 2001; Mutai et al., 2013; Napiontek et al., 2009;). Collectively, we suggested that the biallelic mutations of SLC26A8 might be the confidential genetic cause for male infertility. Of note, both of our patients and the patients in previous research were harboring the detrimental SLC26A8 heterozygote, which suggested that the heterozygous mutations of SLC26A8 might increase the risk of male infertility. Exactly, the heterozygous mutation of the SLC26A8 is not the main actor but might be a guest player for male infertility. Our findings would provide valuable insights into the molecular mechanism responsible for the male infertility related to SLC26A8 mutations, which is important for the diagnosis and treatment of male infertility.
In summary, our work unveiled that the SLC26A8 heterozygous mutations were not the direct causes for asthenozoospermia but may act as a risk factor to male infertility. This report thus corrected the previous work stating that the heterozygous SLC26A8 mutations led to asthenozoospermia in a dominant inherited manner. Due to the complexity of the spermatogenetic process, the etiological factors of male infertility are mysterious. We thus should be cautious about the gene mutations discovered in patients, and performed more functional experiments to constitute the relationship between the genotype and phenotype, so that we can provide more strong and accurate evidence for clinical diagnosis of male infertility.