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\begin{document}

\title{The heterozygous mutations of SLC26A8 are not the main actors but might
be the guest players for male infertility}



\author[1]{Mohan Liu}%
\author[1]{Jinhui Li}%
\author[1]{Yaqian Li}%
\author[1]{Chuan Jiang}%
\author[2]{Wenming Xu}%
\author[1]{Yihong Yang}%
\author[1]{Ying Shen}%
\affil[1]{Sichuan University}%
\affil[2]{Sichuan University West China Second University Hospital}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Male infertility has become a serious health and social problem
troubling approximately 15\% of couples worldwide; however, the genetic
and phenotypic heterogeneity of human infertility poses a substantial
obstacle to effective diagnosis and therapy. A previous study reported
that heterozygous mutations in solute carrier family 26 member 8
(SLC26A8, NG\_033897.1) were causatively linked to asthenozoospermia.
Interestingly, in our research, three deleterious heterozygous mutations
of SLC26A8 were separately detected in three unrelated patients who were
suffered from teratozoospermia. These three heterozygous mutations
resulted in the reduce of SLC26A8 expression in transfected cells, while
no disrupt expression of SLC26A8 was observed in sperm from the affected
individuals. Noticeably, two of the three SLC26A8 heterozygous mutations
detected in the patients were inherited from their fertile fathers.
Thus, we suggested that male infertility associated with SLC26A8
mutations should be involved in a recessive-inherited pattern,
considering the infertile homozygous Slc26a8 KO male mice. Given that
SLC26A8 heterozygous mutations were detected in the infertile patients,
and SLC26A8 is predominantly expressed in the various germ cells during
spermatogenesis, the heterozygous mutations in SLC26A8 may not be the
direct genetic cause but contribute to male infertility to a certain
degree.%
\end{abstract}%



\sloppy


\textbf{The heterozygous mutations of \emph{SLC26A8} are not the main
actors but might be the guest players for male infertility}

Mohan Liu\textsuperscript{1,2}, Jinhui Li\textsuperscript{3}, Yaqian
Li\textsuperscript{1,} Chuan Jiang\textsuperscript{1}, Wenming
Xu\textsuperscript{1 *}, Yihong Yang\textsuperscript{4*}, Ying
Shen\textsuperscript{1 *}

\textsuperscript{1} Department of Obstetrics/Gynecology, Key
Laboratory~of Obstetric, Gynecologic and Pediatric Diseases and~Birth
Defects~of~Ministry of Education, West China Second University Hospital,
Sichuan University, Chengdu 610041, China

\textsuperscript{2} State Key Laboratory of Biotherapy and Cancer
Center, West China Hospital, Sichuan University and Collaborative
Innovation Center, Chengdu 610041, China.

\textsuperscript{3}Department of Neonatology, Key Laboratory~of
Obstetric, Gynecologic and Pediatric Diseases and~Birth
Defects~of~Ministry of Education, West China Second University Hospital,
Sichuan University, Chengdu 610041, China

\textsuperscript{4} Reproduction Medical Center of West China Second
University Hospital, Key Laboratory of Obstetric, Gynecologic and
Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan
University, Chengdu 610041, China

Mohan Liu, Jinhui Li and Yaqian Li contributed equally to this work.

*Correspondence should be addressed to: Wenming Xu, Department of
Obstetrics/Gynecology, Key Laboratory~of Obstetric, Gynecologic and
Pediatric Diseases and~Birth Defects~of~Ministry of Education, West
China Second University Hospital, Sichuan University, Chengdu 610041,
China, E-mail: xuwenming@scu.edu.cn; Yihong Yang, Reproduction Medical
Center of West China Second University Hospital, Key Laboratory of
Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of
Ministry of Education, Sichuan University, Chengdu 610041, China E-mail:
yyhpumc@foxmail.com; Ying Shen, Department of Obstetrics/Gynecology, Key
Laboratory~of Obstetric, Gynecologic and Pediatric Diseases and~Birth
Defects~of~Ministry of Education, West China Second University Hospital,
Sichuan University, Chengdu 610041, China, E-mail:
yingcaishen01@163.com.

\textbf{Abstract}

Male infertility has become a serious health and social problem
troubling approximately 15\% of couples worldwide; however, the genetic
and phenotypic heterogeneity of human infertility poses a substantial
obstacle to effective diagnosis and therapy. A previous study reported
that heterozygous mutations in \emph{solute carrier family 26 member
8}(\emph{SLC26A8} , NG\_033897.1) were causatively linked to
asthenozoospermia. Interestingly, in our research, three deleterious
heterozygous mutations of\emph{SLC26A8} were separately detected in
three unrelated patients who were suffered from teratozoospermia. These
three heterozygous mutations resulted in the reduce of SLC26A8
expression in transfected cells, while no disrupt expression of SLC26A8
was observed in sperm from the affected individuals. Noticeably, two of
the three \emph{SLC26A8} heterozygous mutations detected in the patients
were inherited from their fertile fathers. Thus, we suggested that male
infertility associated with\emph{SLC26A8} mutations should be involved
in a recessive-inherited pattern, considering the infertile homozygous
\emph{Slc26a8} KO male mice. Given that \emph{SLC26A8} heterozygous
mutations were detected in the infertile patients, and SLC26A8 is
predominantly expressed in the various germ cells during
spermatogenesis, the heterozygous mutations in\emph{SLC26A8} may not be
the direct genetic cause but contribute to male infertility to a certain
degree.

\textbf{KER WORDS:} \emph{SLC26A8} , heterozygous mutation, recessive
inheritance, male infertility, WES

\textbf{INTRODUCTION}

World Health Organization (WHO) has deemed that infertility is a global
health problem that affects about 15\% of couples in the world, and male
infertility accounts for about 30\%\textasciitilde{}50\% (Jiao et al.,
2021). Spermatogenesis is regulated by multiple gene expression
strictly, and gene variations disrupting protein function often lead to
defective sperm development (Noveski et al., 2016). Therefore, male
infertility has a strong genetic basis. In spite of some disease-related
genes that have been investigated, each gene is possibly responsible for
only a small fraction of pathogenic factors. Due to the high genetic
heterogeneity in male infertility (Kasak et al., 2021), the pathogenesis
and mechanisms of male infertility have not been comprehensively
studied.

We have noticed that Dirami T \emph{et al} . reported that three
heterozygous missense mutations(c.260G\textgreater{}A {[}p.Arg87Gln{]},
c.2434G\textgreater{}A {[}p.Glu812Lys{]} and c.2860C\textgreater{}T
{[}p.Arg954Cys{]}) in \emph{solute carrier family 26 member
8}(\emph{SLC26A8} , NG\_033897.1) could result in male infertility
associated with asthenozoospermia (Dirami et al., 2013). The authors
further demonstrated heterozygous mutations in \emph{SLC26A8} might
impair the formation of SLC26A8-CFTR complex, thus disrupting the
capacity to activate CFTR-dependent anion transport (Dirami et al.,
2013). These events finally damaged the CFTR-dependent sperm-activation
in sterile patients (Dirami et al., 2013). However,
\emph{Slc26a8}\textsuperscript{-/-} mice presented infertile phenotype
but\emph{Slc26a8} \textsuperscript{+/-} mice showed normal fertility
(Tour\selectlanguage{ngerman}éet et al., 2007; Rode et al., 2012), indicated \emph{Slc26a8} is
involved in reproduction through recessive-inheritance manner. The
disparate inherited pattern between humans and mice makes us confused.
Does \emph{SLC26A8} play diverse roles in the reproductive process of
humans and mice? More importantly, what kind of genetic model
is\emph{SLC26A8} participating in spermatogenesis? Therefore, a more
comprehensive interrogation of the function of \emph{SLC26A8} in male
infertility is needed to further boost clinical diagnosis.

Interestingly, it was a remarkable fact that we found three heterozygous
mutations of \emph{SLC26A8} respectively in three unrelated infertile
males with teratozoospermia in this study, and the aberrant sperm
morphology and ultrastructure were confirmed by electron microscope.
Surprisingly, two of these heterozygous mutations of
\emph{SLC26A8}detected in the patients were inherited from their fathers
who have no reproductive barriers. The deleterious effect of the three
heterozygous mutations on SLC26A8 expression was confirmed by western
blotting in vitro. However, no significant expression difference of
SLC26A8 was exhibited in sperm between patients and normal control.
Therefore, we suggested that the heterozygous mutations in
\emph{SLC26A8} might not be the immediate cause of asthenozoospermia but
participate in spermiogenesis to a certain extent.

\textbf{2. METHODS}

\textbf{2.1 Study Participants}

The infertile patients were enrolled at the West China Second University
Hospital of Sichuan University. These patients all had normal somatic
karyotypes (46, XY). This study was conducted following the tenets of
the Declaration of Helsinki, and ethical approval was obtained from the
Ethical Review Board of West China Second University Hospital, Sichuan
University. Informed consent was obtained from each study participant.

\textbf{2.2 Genetic Analysis}

Peripheral blood samples were obtained from all subjects, and the
genomic DNA was isolated by DNeasy Blood and Tissue Kit (QIAGEN),
according to the manufacturer's protocol. Next-generation sequencing was
carried out using Agilent SureSelectXT Human All Exon Kit and Illumina
HiSeq X-TEN. Functional annotation was done using ANNOVAR through a
series of databases including 1000Genomes Project, dbSNP, HGMD and ExAC.
Next, PolyPhen-2, SIFT, MutationTaster and CADD were used for functional
prediction. Targeted testing of the potentially pathogenic variants in
the patients' parents was performed by Sanger sequencing. The primers
used in the PCR analysis were as follows: F1,
5'-GACATGAGCTGGCAGAGACAAG-3'; R1, 5'-CAAGGCTCTGGTGGCCTAG-3'; F2, 5'-
TGTCATGGCACCAGTCCATG-3'; R2, 5'-GGAGAATTGCTTGAACCTGGCAG-3'; F3, 5'-
AATGTGGGAATGTGTAGAGACG-3'; R3, 5'- GCAGATAAGGGAACAAATGGT-3';

\textbf{2.3 Western blotting}

The proteins of the cultured cells and sperm samples were extracted
using a universal protein extraction lysis buffer (Bioteke) containing a
protease inhibitor cocktail (Roche). Then, denatured proteins were
separated on 10\% SDS-polyacrylamide gels and transferred onto a
polyvinylidene difluoride (PVDF) membrane (Millipore) for immunoblot
analysis. The primary antibodies used were anti-SLC26A8 (1:1000, Atlas
antibody) and GAPDH (1:5000, Abcam).

\textbf{2.4 Immunofluorescence staining}

The spermatozoa samples were washed with Sperm Washing Medium
(CooperSurgical Inc.) and then fixed onto slides with 4\%
paraformaldehyde, permeabilized with 0.3\% Triton X-100, and blocked
with 5\% BSA. Next, the slides were sequentially incubated with primary
antibodies (SLC26A8, 1:50) overnight at 4 °C. The slides were washed in
1× PBS, incubated with DyLight 488- or DyLight 594-labeled secondary
antibodies (1:1000, Thermo Fisher) for 1 h at room temperature, and then
counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma- Aldrich).

\textbf{2.5} \textbf{Electron Microscopy}

For scanning electron microscopy (SEM), the sperm cells were fixed onto
slides using 4\% glutaraldehyde refrigerated overnight at 4°C. After
washing the slides with PBS three times, the slides were gradually
dehydrated with an ethanol gradient (30\%, 50\%, 75\%, 95\%, and 100\%
ethanol) and dried by a CO\textsubscript{2} critical-point dryer. After
metal spraying by an ionic sprayer meter, the samples were observed by
SEM (S-3400, Hitachi). For transmission electron microscopy (TEM), the
sperm cells were washed three times and fixed routinely. After embedded
in Epon 812, ultrathin sections were stained with uranyl acetate and
lead citrate, and observed under a TEM (TECNAI G2 F20, Philips) with an
accelerating voltage of 80 kV.

\textbf{RESULTS}

\textbf{3.1 Identification of three novel mutations of \emph{SLC26A8} in
infertile patients with teratozoospermia}

In our study, three individuals (patient A, patient B, and patient C)
were consulted for primary infertility, and then carried out semen
analysis (Table 1). The sperm count of three individuals was basically
normal, but of a high percent of morphological abnormalities in sperm.
Then we performed WES on the three patients to evaluate the potential
genetic causes for their sterile phenotype. Consequently, three latently
detrimental heterozygous mutations of \emph{SLC26A8} strikingly
attracted our attention (Figure 1). The heterozygous frameshift
mutations of c.1570\_1571del {[}p.A524*{]} detected in patient A and c.
306del {[}p.G103Afs*9{]} detected in patient C were absent in the
general population databases (Table 1). A heterozygous missense mutation
of c.2191G\textgreater{}A {[}p.V731I{]} was found in patient B and is
estimated extremely low allele frequency in public databases (Table 1).
Moreover, the site of this missense variant is 100\% conserved across
several species (Figure S1a).

According to the report of Dirami T \emph{et al.} , the heterozygous
alterations of \emph{SLC26A8} contributed to human asthenozoospermia
(Dirami et al., 2013). To confirm the patients' phenotype in detail, we
collected their sperm samples to carry out the exhausting morphologic
examination. We observed serious frequencies of pyriform-head sperm in
patient A, and round-head anomalies in sperm from patient B as well as
coiled-tail sperm from patient C under Papanicolaou staining (Figure 2a)
and SEM further confirmed that sperm of the patients possessed aberrant
head or irregular flagella (Figure 2b). Furthermore, irregular
ultrastructure either on the head or flagella was also observed in the
spermatozoa of the three patients by transmission electron microscopy
(TEM) (Figure 2c). The nucleus in most sperm of patient A was irregular,
and patient B showed the larger sperm head with unconsolidated chromatin
(Figure 2c). And disorganization of mitochondria helices was detected in
the sperm flagella of patient C (Figure 2c). Together, the three
patients carrying the heterozygous \emph{SLC26A8} mutations showed
typical teratozoospermia.

\textbf{3.2 Heterozygous \emph{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
these\emph{SLC26A8} mutations via Sanger sequencing in the three
families (Figure 1). Surprisingly, two of the harmful mutations
(c.1570\_1571del {[}p.A524*{]} and c.2191G\textgreater{}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-type\emph{SLC26A8} to transiently transfect
HEK-293Tcells, respectively. As expected, the western blotting showed
that the expression of SLC26A8 protein encoded by c.2191G\textgreater{}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 \emph{SLC26A8} copy,
although another copy is mutated. Moreover, the\emph{Slc26a8}
\textsuperscript{-/-} mouse further proofed that\emph{Slc26a8}
participated in spermatogenesis is linked to a recessive-inheritance but
not a dominant-inheritance (Touréet et al., 2007; Rode et al., 2012).
All findings demonstrated\emph{SLC26A8} mutations in male infertility is
a recessive-inheritance, and heterozygous mutations of\emph{SLC26A8}
might exhibit a certain degree of determination towards male
infertility.

\textbf{3.3 The special expression pattern of SLC26A8 during
spermatogenesis}

The previous animal model presented that male
\emph{Slc26a8}\textsuperscript{-/-} 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.

\textbf{DISCUSSION}

In the present study, we described the monoallelic mutations
in\emph{SLC26A8} are not be the causative mutations of male infertility.
Especially, although these heterozygous mutations in \emph{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 \emph{SLC26A8} were not directly responsible for male infertility.

\emph{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 \emph{et al} , the
heterozygous mutations of \emph{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 \emph{SLC26A8}
, strongly supporting the heterozygous variants of \emph{SLC26A8} might
not be direct etiology for asthenozoospermia. Besides that, no
reproductive barriers were performed on the heterozygous \emph{Slc26a8}
KO male mice, while the homozygous \emph{Slc26a8} KO male mice occurred
in sterility, with completely immotile and malformed spermatozoa. These
findings suggest infertility associated with \emph{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 \emph{SLC26A8} might be
in contrast to the variants previously identified in other members of
the \emph{SLC26} family (\emph{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
\emph{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 \emph{SLC26A8} heterozygote,
which suggested that the heterozygous mutations of \emph{SLC26A8} might
increase the risk of male infertility. Exactly, the heterozygous
mutation of the \emph{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 \emph{SLC26A8} mutations, which is important for
the diagnosis and treatment of male infertility.

In summary, our work unveiled that the \emph{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 \emph{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.

\textbf{ACKNOWLEDGEMENTS}

We thank the patients and their families for their interest and
cooperation. The authors would like to thank Analytical and Testing
Center of Sichuan University for the morphology characterization and the
authors would be grateful to Guiping Yuan for her help of TEM images and
Yi He for his help of SEM images. This work was supported by the
National\selectlanguage{english} Key\selectlanguage{english} R\&D\selectlanguage{english} Program\selectlanguage{english} of\selectlanguage{english} China (No. 2018YFC1003600).

\textbf{CONFLICT OF INTEREST}

The authors declare no conflict of interest.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{WEB RESOURCE}

NCBI:\emph{https://www.ncbi.nlm.nih.gov/homologene}

1000 Genomes Project: http://www.internationalgenome.org

ENCODE: https://www.encodeproject.org

ExAC Browser: http://exac.broadinstitute.org

GnomAD: https://gnomad.broadinstitute.org

OMIM: http://www.omim.org

PolyPhen-2: http://genetics.bwh.harvard.edu/pph2/

SIFT: \emph{http://sift.jcvi.org}

The human protein altas: https://www.proteinatlas.org/

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\textbf{FIGURE LEGENDS}

\textbf{Figure 1. Three heterozygous mutations in \emph{SLC26A8} were
identified in three infertile patients.} Pedigrees of three families
with heterozygous variants in \emph{SLC26A8} . The c.1570\_1571del
mutation was exhibited by the dotted triangle in family A. The
c.2191G\textgreater{}A mutation was exhibited by the dotted box in
family B. The c.306del mutation was exhibited by the dotted triangle in
family C.

\textbf{Figure 2. The teratozoospermia phenotype of the three
patients.}(a) The Concentrations Papanicolaou staining of sperm in the
three patients and normal control. The sperm malformation of pyriform
heads, round-heads, and coiled-tail were observed in the three patients.
(b) Detailed defects in sperm head and flagella were observed in the
patients by SEM (scale bars, 5 \selectlanguage{greek}µ\selectlanguage{english}m). (c) The anomaly head and flagellum
as well as abnormal organized mitochondria of sperm from the three
individuals, compared with the normal control by TEM. (scale bars, 100
nm). ODF: outer dense fiber; OD: peripheral microtubule doublets; CP:
central microtubules.

\textbf{Figure 3. The expression analysis of the three
heterozygous\emph{SLC26A8} mutations in transfected cells and sperm from
the patients.} (a) The western blotting analysis of SLC26A8 expression
in cells transfected with plasmids carrying variants of p.A524*,
p.V731I, and p.G103Afs*9. (b) The SLC26A8 levels in sperm lysates of
patients and control. The SLC26A8 protein was present in the three
individuals and normal control without distinction. (c) The
immunofluorescence results showed no significant difference in the
SLC26A8 expression of the sperm between three individuals and normal
control.

\textbf{Figure 4. The expression of SLC26A8 in human and mouse
testes.}(a) Representative images of testicular tubules in a mouse
showing that SLC26A8 is principally localized to the cytoplasm of
different stages of spermatids (scale bar, 5 \selectlanguage{greek}μ\selectlanguage{english}m; red, SLC26A8; blue,
DAPI). (b) SLC26A8 was detected in the nucleus and cytoplasm of germ
cells at different stages (scale bar, 5 \selectlanguage{greek}μ\selectlanguage{english}m; red, SLC26A8; blue, DAPI).
(c) Immunofluorescence staining indicated that SLC26A8 was primarily
expressed in the head and cilia in human spermatogenic cells (scale bar,
5 \selectlanguage{greek}μ\selectlanguage{english}m; red, PNA; green, SLC26A8; blue, DAPI).

\textbf{Table 1.} Semen and variant analysis in the three patients
harboring heterozygous \emph{SLC26A8} mutations.

\textbf{Supplementary Information}

\textbf{Figure S1.} (a) Multiple sequence alignment of the SLC26A8
protein among different species. (b) Single-cell sequencing of SLC26A8
expression in human testis
(https://www.proteinatlas.org/ENSG00000112053-SLC26A8/celltype).\selectlanguage{english}
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\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/354197/articles/546231-the-heterozygous-mutations-of-slc26a8-are-not-the-main-actors-but-might-be-the-guest-players-for-male-infertility}

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