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.