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.