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Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes globozoospermia and sterility in male mice


Abstract and Figures

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 knockout (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying a potential target for non-hormonal contraception.
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Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes
globozoospermia and sterility in male mice
SSMEM1 is essential for male fertility in mice
Absence of serine-rich single-pass membrane protein 1 (SSMEM1) leads to male infertility due to
globozoospermia in mice.
Keywords: Sperm, Spermatogenesis, Fertilization, Male infertility, Null mutation/knockout
Kaori Nozawa1,2, Qian Zhang3, Haruhiko Miyata3, Darius J. Devlin1,2,4, Zhifeng Yu1,2, Seiya
Oura3,5, Takayuki Koyano6, Makoto Matsuyama6, Masahito Ikawa3,5,7,8,*, and Martin M.
1Center for Drug Discovery, Baylor College of Medicine, Houston, TX; 2Department of Pathology &
Immunology, Baylor College of Medicine, Houston, TX; 3Department of Experimental Genome Research,
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan; 4Interdepartmental Program
in Translational Biology & Molecular Medicine, Baylor College of Medicine, Houston, TX; 5Graduate School
of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan; 6Division of Molecular Genetics, Shigei
Medical Research Institute, Minami-ku, Okayama, Japan; 7Graduate School of Medicine, Osaka University,
Suita, Osaka, Japan; 8The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
*Correspondence to: (M. I.) and (M. M. M.)
© The Author(s) 2020. Published by Oxford University Press behalf of Society for the Study of
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Grant Support
This work was supported by: the Ministry of Education, Culture, Sports, Science and Technology
(MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI grants (JP17H04987 to H.M.,
JP17H01394 and JP19H05750JP to M.I.); Japan Agency for Medical Research and Development (AMED)
grant JP18gm5010001 to M.I.; Takeda Science Foundation grants to H.M. and M.I.; Eunice Kennedy Shriver
National Institute of Child Health and Human Development (Grants R01HD088412 and P01HD087157 to
M.M.M. and M.I.); National Institute of General Medical Sciences (T32GM088129 and T32GM120011 to
D.J.D); Japan Society for the Promotion of Science Overseas Research Fellowship and Lalor Foundation (to
K.N.), and the Bill & Melinda Gates Foundation (INV-001902 to M.M.M. and M.I.).
Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective
sperm motility) are known causes of male infertility in human patients. Despite many studies, the
molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single
pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study,
we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that
Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during
spermatogenesis but not in mature sperm. The sterility of the Ssmem1 knockout (null) mice is
associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the
phenotype, we analyzed testes with transmission electron microscopy and discovered that
Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of
spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi
network, also showed delayed movement of the Golgi to the spermatid posterior region, which
causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in
the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to
ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These
studies and the pathway in which SSMEM1 functions have implications for human male infertility
and identifying a potential target for non-hormonal contraception.
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Spermatozoa transmit the genetic information to the next generation and unique morphological
features specialized for fertilization. It is reported that more than 1,000 genes are dominantly
expressed in human and mouse testes [1][2]. The deficiency of those genes has been proposed to
cause spermatogenesis defects and infertility in human patients [3]. In addition, many
testis-enriched genes were reported to be evolutionarily conserved in mammals including mouse
and human [4][5]. The analysis of knockout mouse models, which carry null gene mutations, is a
powerful strategy to study male infertility pathogenesis due to the lack of in vitro culture systems of
spermatozoa [6].
The success of spermatogenesis is critical for maintenance of male fertility and requires a
well-organized process in which spermatogonia undergo mitosis, meiosis and spermiogenesis.
Furthermore, spermiogenesis can be divided into the following main phases: Golgi, acrosome
cap/elongation, and maturation phases [7]. The Golgi apparatus produces proacrosomal vesicles
during the Golgi phase, which coalesce and result in the acrosomal vesicle adjacent to the nuclear
membrane on the opposite side of the tail. The acrosome cap phase is characterized by the spread of
the acrosome over the spermatid nucleus, which coincides with tail development [8]. As the
spermatid cytoplasm and nucleus elongates, the organelles, including Golgi and mitochondria,
move from the acrosome and migrate to the caudal aspect. During the maturation phase, the
spermatozoa are fully developed and reshaped. Eventually the mitochondria locate at the midpiece
of the tail and the Golgi is discarded into cytoplasmic droplets with other organelles so that
spermatozoa obtain the morphology suitable for fertilization [9][10]. To explain elongation of the
nuclei, the current hypothesis is that the manchette, consisting of microtubules and actin filaments,
drag and compel the nucleus to become more dense and elongate toward the tail aspect [11].
Globozoospermia, which is a spermiogenesis defect, is one cause of human male
infertility [12]. The characteristic feature of globozoospermia is an abnormal nuclear shape and
arrangement of the sperm mitochondria. Some genes, such as DPY19L2, PICK1, and SPATA16,
have been found as causative genes in human patients [13][14]. Human globozoospermic
spermatozoa have also shown an incapability of natural fertilization because they are unable to bind
the zona pellucida and fuse with oocytes [12]. However, these spermatozoa also show low
fertilization rates even after intracytoplasmic sperm injection (ICSI) treatment, which is a standard
rescue option for infertile men [15], due to the oocyte activation failure [16]. While studies have
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shown correlation between globozoospermia and the lack of acrosome, the mechanisms inducing
globozoospermia are still not fully appreciated.
In our in silico bioinformatic analysis [17][18], we identified Ssmem1 as an
evolutionarily-conserved and testis-specific gene. Here, we generated Ssmem1 knockout mice and
verified its significant role in vivo. We observed that lack of Ssmem1 in male mice alters
spermiogenesis, resulting in globozoospermia and sterility. Our ultrastructural data reveals that
absence of SSMEM1 alters transport of the Golgi during spermatid elongation, thereby uncovering
a role of SSMEM1 in this process.
Material and methods
Ethics Statement
Mice were maintained in accordance with NIH guidelines, and all animal procedures were approved
by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine and
Osaka University.
B6D2F1 purchased from Japan SLC (Hamamatsu, Shizuoka, Japan) or CLEA Tokyo, B6D2F1 mice
were used for generating Ssmem1 mutant founder mice. In-house hybrid mice (C57BL/6J ×
129S5/SvEvBrd) were mated with Ssmem1 heterozygous mice to expand the line. For phenotypic
analysis, sexually mature male mice (6 weeks to 6 months old) were used. All mice were housed
with a 12 hours light cycle. All mouse experiments were performed according to the guidelines
from the Institutional Animal Care and Use Committee at Baylor College of Medicine (protocol
Mouse cDNA was cultivated from tissues of C57BL6J/129SvEv hybrid mice. Human multiple
tissue cDNAs were purchased from BD bioscience. The following primers were used as performed
[5]: Human SSMEM1, 5′–ATGAAGGCAGTGGGACAAG–3′ and 5′–
and 5′–TCGGTTACTGTGGAAACTTGG–3′ ; mouse Hprt, 5′–
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Generation of Ssmem1 knockout mice
The pX330 plasmids expressing hCas9 and single guide (sg)RNAs
(ACAGATGTCTGAGAGCAAAC) targeting exon 2, which shares all splicing variants of Ssmem1
were injected into pronuclei of zygotes [19]. Eggs were cultured in KSOM overnight and
subsequently transferred into the oviducts of pseudopregnant ICR females. Screening of mutant
pups was performed by direct sequencing following polymerase chain reaction (PCR) using primers
mouse with a 6 bp deletion and 1 bp insertion was used to expand the colony. The genotyping was
carried out by PCR with specific primers for the wild-type (WT) allele (primer a: 5′–
or KO allele (primer c: 5′–GACCACATCTTTCATGTCC–3′ and primer d: 5′–
Production of a monoclonal antibody against mouse SSMEM1
These procedures were performed as previously described [20]. In brief, the DNA sequence
encoding mouse SSMEM1 (aa residues 58-224, ENSMUSG00000029784) with C-terminal 8xHis
and 1D4 epitope tags were cloned into the pET15b (Novagen) plasmid vector. The plasmids were
transformed into the Rosetta Escherichia coli (E. coli) strain (Millipore). The expression of
SSMEM1-8xHis-1D4 protein was induced by adding isopropyl β-D-1-thiogalactopyranoside
(IPTG) to final concentration of 1.0 mM in LB medium. The cells were cultured at 30℃ for
overnight post-induction. After collection by centrifuge, the pellet was resuspended in Lysis buffer I
[150 mM NaCl, 20 mM Tris-HCl pH 8.0, 10 mM Imidazole, 2% (v/v) Triton X-100, 1 mM DTT,
100 µg/mL Lysozyme, protease inhibitor cocktail tablets (Merck)] and lysed by ultrasonic disruptor
(UD-201, TOMY). Triton-soluble fraction was removed by centrifugation (37,500 g, 30 min). The
purified pellet (inclusion body) was resuspended in Lysis buffer II [150 mM NaCl, 20 mM Tris-HCl
pH 8.0, 10 mM Imidazole, 8M Urea] and incubated overnight with gentle agitation. After
centrifugation (37,500 g, 30 min), the supernatant was incubated with Ni-NTA Agarose (product no.
30210, QIAGEN) for 1 hr with gentle agitation. The lysate was loaded on a column and washed
with 40 mL of column wash buffer [150 mM NaCl, 20 mM Tris-HCl pH 8.0, 40 mM Imidazole, 8M
Urea]. SSMEM1-8xHis-1D4 was eluted from the column with column elution buffer [150 mM
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NaCl, 20 mM Tris-HCl pH 8.0, 250 mM Imidazole, 8M Urea]. Recombinant SSMEM1 was used to
produce the monoclonal antibody as previously described [21]. Specifically, purified SSMEM1
protein with Freund’s complete adjuvant was injected into female rats. After 17 days post-injection,
lymphocytes were collected from iliac lymph nodes and hybridomas generated as described in
Kishiro [22]. Supernatants from hybridoma cell cultures were collected and used as antibodies. The
candidates were screened by ELISA against recombinant mouse SSMEM1.
Western blot analysis
Testis and sperm were collected from adult mice and lysed in NP40 buffer (50 mM Tris-HCl pH 7.5,
150mM NaCl, 0.5% NP40, 10% Glycerol) followed by incubation for 1 hr at 4°C with gentle
agitation. The lysate was centrifuged at 15,000 g for 5 minutes at 4°C in a tabletop centrifuge. The
supernatant was collected and subjected SDS-PAGE under reducing condition (with 5%
2-mercaptoethanol). The proteins from the gel were transferred to a PVDF membrane. Membranes
were blocked with 10% skim milk in Tris-buffered saline-Tween 20 (TBST; 20 mM Tris-HCl pH
7.6, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature (RT). Primary antibodies were
diluted in blocking solution and incubated with the membranes overnight at 4°C. Membranes were
washed three times with TBST before being incubated with anti-mouse IgG-HRP-conjugated
secondary antibody for 1 hour at RT. Membranes were then developed after washing. The blotted
membrane was stained by Coomassie Brilliant Blue (CBB) for a loading control.
For histological analysis, testes were collected and fixed in Bouin’s fixative (Sigma Aldrich) for
three hours at RT. After several washes in 70% ethanol to remove excess stain, testes were
embedded in paraffin. Tissues were sectioned at 5 µm thickness and stained by Periodic Acid-Schiff
(PAS)-hematoxylin, followed by imaging with a 40x objective.
Scanning Electron Microscopy (SEM)
Spermatozoa was recovered from cauda epididymis and placed in TYH medium at 37°C, 5% CO2
for 15 min [23]. Sperm were centrifuged at 300 x g for 5min and washed in PBS. After 2nd
centrifuge, sperm were resuspended and fixed in 2.5% glutaraldehyde for 30 min at RT. Sperm were
dehydrated in ethanol from 20% to 100%, incubating for 10 min in each. Sperm were transferred
and resuspended in 50% t-butanol/50% ethanol for 15 min. The sperm samples were dried, and
sputter coated with iridium, followed by imaging using Nova NanoSEM 230 at Houston Methodist
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Research Institute’s Electron Microscopy Core.
Sperm Analysis
Sperm number and motility were measured as previously [24]. Sperm were extracted by cutting
caudal epididymis 30 times and incubated in human tubal fluid (HTF) media (Millipore)
supplemented with 5% BSA under 5% CO2 at 37C. After 15- or 90-minutes incubation, sperm
samples were applied onto a chamber of 100 µm-depth analyzed counting slides (CellVision), and
the sperm number and motility were measured using the Hamilton Thorne CEROS II system.
Transmission Electron Microscopy (TEM)
Samples were prepared as previously described [25]. Epididymis and testes were quickly removed
from sacrificed mice and fixed in 2% paraformaldehyde, 2.5% glutaraldehyde and 2 mM CaCl2 in
0.1 M cacodylate at 4°C overnight. After staining and dehydration, tissues were embedded in
Spurr’s resin. Thin sections (80 nm) were stained with uranyl acetate and lead citrate, followed by
observation using a Hitachi H7500 transmission electron microscope (Hitachi-HTA) at 80 kV at
Baylor College of Medicine Integrated Microscopy Core.
Testes were fixed in 4% PFA/PBS for overnight and replaced in from 10%, 20% to 30 % sucrose at
C. The tissues were then embedded in OCT compound, freezing at -80°C. After sectioning at 10
µm, the samples were blocked with 3% BSA + 5% Normal Donkey Serum/PBS containing 0.1%
Triton X-100 on the slides, for 1 hr at RT. Primary antibodies were incubated with sections
overnight at 4oC. Sections were washed with 3% BSA + 1% Normal Donkey Serum/PBS and
incubated with secondary antibodies for 1 hr at RT. DAPI was added in the washed buffer for 10
min in the final wash. Primary antibodies used were anti-Golgin 97 antibody at a concentration of 5
µg/ml (Abcam, #ab84340) and the lectin peanut agglutinin (PNA)-FITC at a dilution of 1:500
(Sigma-Aldrich, #L7381). (Sigma). Secondary antibody was donkey anti-rabbit AF594 (Life
Technologies). Slides were observed with Zeiss LSM 880 with Airyscan FAST Confocal
Microscope at Baylor College of Medicine Optical Imaging & Vital Microscopy Core.
Statistical Analysis
Statistical significance was evaluated by using the two-tailed unpaired Student t test assuming
unequal variances.
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Ssmem1 is a highly conserved testis-specific gene
We identified Ssmem1 as a conserved testis-specific gene by a bioinformatic screen as previously
described [5]. We performed multi-tissue RT-PCR from male mice to reveal the Ssmem1 expression
profile. We obtained a single band in both human and mouse testes (Fig. 1A). To determine the
temporal expression pattern of Ssmem1, we performed RT-PCR using testis from WT mice of
increasing age from post-natal day (PND): 5, 10, 15, 20, 25, 30, 35 and 42 to capture the earliest
stage of expression of Ssmem1 in mice. Expression of Ssmem1 was observed at low levels at day 5
and became pronounced at postnatal day 15 (Fig. 1B), which is coincident with the appearance of
the first pachytene spermatocytes [26]. Protein sequence alignment analysis was performed on
representative orthologs of SSMEM1 from different species. SSMEM1, which possesses a single
transmembrane domain, is highly conserved in mammals, including human and mouse (Fig. 1C).
Generation of Ssmem1 knockout mice
To examine the physiological role of Ssmem1, we generated knockout mice using the CRISPR/Cas9
system. The gRNAs were designed to target exon 2 which is shared with all Ssmem1 splicing
variants (Fig. 2A). After an indel mutation containing a 6-bp deletion with a 1-bp insertion was
confirmed by direct sequencing analysis, specific primers for wildtype (WT) or homozygous mutant
(KO) allele were designed and used for genotyping (Fig. 2B). The net 5-bp deletion of nucleotides,
which caused a frameshift mutation, resulted in amino acid changes and a premature stop codon in
KO mice in place of the L77 residue in WT mice (Fig. 2C). We intercrossed the mouse line (-6+1
bp) to obtain subsequent generations and confirmed the absence of SSMEM1 protein in KO testes
(Fig. 2D). In addition, while SSMEM1 is present in testis, we found that the protein is absent from
mature epididymal sperm from WT mice (Fig. 2D). Observation of the KO mice revealed no
obvious developmental abnormalities or differences in sexual behavior.
Ssmem1 is required for male fertility
Beginning at 6 weeks of age, sexually mature heterozygous or homozygous mutant males were
housed with one female each for 6 months to test their fertility. The average number of offspring per
litter and the number of litters were counted. Six control mating pairs had 7.3 ± 1.8 pups on average,
whereas four homozygous mutant males exhibited sterility (Fig. 3A). This data demonstrates that
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Ssmem1 is essential for male fertility. To determine the cause of the sterility defect, we first
analyzed the testis and sperm from controls and Ssmem1 KO mice. The sperm count from caudal
epididymis between HET and KO mice showed no significance difference (Fig. 3B). However, the
testicular size and weight from Ssmem1 KO mice were significantly reduced compared to the
control males (Fig. 3C-D). In histological analysis of testes stained with PAS-hematoxylin, most
tubules appeared normal but atrophic changes showing absence of germ cells and vacuolation in
seminiferous tubules were occasionally observed in the Ssmem1 KO (Fig. 3E), which may explain
the slight reduction in testis weight for the Ssmem1 KO males. The epididymal histology showing
the lumen filled with sperm in both HET and KO correlated with the results of the sperm count.
Ssmem1 KO results in spermiogenesis defects and motility reduction
To examine the morphology of spermatozoa, the sperm extracted from cauda epididymis were
analyzed by SEM (Fig. 4A). All KO spermatozoa showed a round or amorphous head morphology,
and some presented with the tail coiled around the head (Fig. 4A, bottom right). To further evaluate
their infertility, we collected epididymal spermatozoa from adult Ssmem1 HET and KO males and
examined the sperm motility using a Computer Assisted Sperm Analysis (CASA) system. The
percentage of motile sperm were significantly decreased in Ssmem1 KO mice (4.1% at 15 min;
3.8% at 90 min incubation in sperm capacitation medium) compared to HET mice (45.3% at 15
min; 35.3% at 90 min) (Fig. 4B). In addition, motile sperm from KO mice showed impaired
kinematic velocities after 90 minutes of incubation in capacitation medium (Fig. 4C).
Ssmem1 influences Golgi apparatus migration during spermiogenesis
The abnormalities of KO spermatogenesis were further examined by Transmission Electron
Microscopy (TEM). Epididymal observations revealed detailed abnormalities in the KO sperm:
misshapen nuclei, disorganized mitochondria and tail entrapment in cytoplasmic droplets (Fig. 5A).
In HET testis, the Golgi in step 8 spermatids localized to the caudal region of the cell. In contrast,
the Golgi failed to undergo migration to the caudal region in all spermatids at comparable stages in
Ssmem1 KO (Fig. 5B); however, the formation of the acrosome did not appear to be disturbed. In
step 9 elongating spermatids from HET controls, the Golgi and mitochondria completely migrated
to the caudal side of the cell and the acrosomal membrane made contact with the cell plasma
membrane (Fig. 5C). In the KO spermatids, however, the Golgi remained in the rostral region of the
cell and the acrosomal membrane did not attach to the plasma membrane. In step 11 spermatids
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(maturation phase), although the migration of the Golgi to the caudal side was finally observed in
KO sperm, the shape of the nuclei and elongation of the cell were incomplete (Fig. 5D). The
abnormalities above were confirmed by immunofluorescent staining of testes, using GOLGIN-97 to
label the trans-Golgi network and PNA to label the acrosome. The delayed migration of Golgi from
acrosome side to tail side of the spermatid cytoplasm was observed in step 9 spermatids in KO
testes (Fig. 5E). These data suggest the delay of the Golgi migration during elongation of
spermatids is defective during spermatogenesis in Ssmem1 KO.
By generating an Ssmem1 KO model, we discovered that SSMEM1 is essential for male fertility.
KO mice lacking Ssmem1, which is testis-specific and well-conserved in mammals, are sterile due
to abnormal sperm head morphology (globozoospermia) and diminished motility. Absence of
SSMEM1 causes a delay in intracellular repositioning of the Golgi during the transition from round
spermatids to elongating spermatids in steps 7 9 of spermiogenesis. The Golgi is found lingering
near the acrosome in step 9 spermatids of KO testes, which is followed by disrupted spermatid
elongation, disorganization of cell organelles, derangement of sperm head morphology, and
subsequent loss of sperm motility (Fig. 6). These data imply that the mechanisms for spermatid
elongation and for intracellular organelle trafficking might be independent, and that the organelle
migration should occur before the initiation of elongation for appropriate spermatogenesis.
Studies of spermatogenesis have identified many acrosome-associated genes, such as
Zpbp1, Spaca1, and Spata46 as causes of globozoospermia [25][27][28]. Knockout of these genes
in mice showed morphological defects of the acrosome during spermatogenesis, followed by
disrupted sperm morphogenesis. In addition to the role of the mature acrosome in undergoing
exocytosis (acrosome reaction) during transit through the female reproductive tract, the acrosome
has a critical role in the development of proper sperm head shape [29][30]. Unlike the previously
mentioned genes essential for acrosome biogenesis, Ssmem1 KO spermatids displayed normal
acrosome formation in the early stages of spermatogenesis. In addition, all organelles were found to
have normal morphology using ultra-structural analysis. The earliest phenotype in mouse
spermatids lacking SSMEM1 is failure of the transfer of the Golgi from the rostral region of the
polarized spermatid (near the acrosome) to the caudal region of the cell in step 7 and step 8
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spermatids, when the spermatids begin elongation [31]. This abnormality is accompanied with the
loss of the contact between the acrosomal membrane and the plasma membrane. Although the
organelles eventually migrate to the caudal region of the cell for removal in KO testis at a later
stage, the spermatids appear to have already lost the ability to orchestrate its organelles properly by
that time, resulting in abnormal sperm morphology.
The localization and the molecular functions of SSMEM1 are not yet clear. Our RT-PCR
analysis demonstrates that Ssmem1 gene expression appears most drastically around postnatal day
15, which corresponds to period that expression of cytoskeleton genes is active [32]. Furthermore,
spe-26, which encodes a protein similar to actin-associated proteins, was reported in C. elegans
where the spe-26 mutant spermatocytes showed mislocalization of mitochondria, the endoplasmic
reticulum (ER) and ribosomes which remained in the spermatid cytoplasm [33]. Similarly, myosin
IV has been shown to be required for proper segregation of cell components including mitochondria
and ER/Golgi-derived organelle complexes during C. elegans spermatogenesis [34]. In mice,
intraflagellar transport protein 74 (IFT74)-deficient in testes caused a similar abnormal
reorganization of the cell organelles during spermatogenesis as in Ssmem1 KO mice likely due to a
failure of assembly of the cytoskeleton [35]. These data suggest SSMEM1 might associate with the
cytoskeleton in spermatids. We hypothesize that SSMEM1, a transmembrane protein, localizes to
the Golgi membrane and plays a role in the Golgi interaction with cytoskeletal proteins or
associated proteins responsible for organelle migration during spermiogenesis. Other
transmembrane proteins in the Golgi such as TM9SF3, TMED4/p25, and TMED7/p27 have been
reported to be involved in Golgi migration after acrosome formation [36]. In addition, KO of
TMF/ARA160 (TATA Element Modulatory Factor 1), which localizes to the Golgi in mouse testis
and associates with tubulin and microtubules, demonstrate spermiogenesis failure due to
disorientation of the Golgi and abnormal trafficking of the Golgi-derived proacrosomal vesicles
during early spermiogenic steps [37]. Thus, we believe that SSMEM1 functions to anchor Golgi and
cytoskeletal proteins for proper Golgi migration analogous to TMF/ARA160. Lastly, since
SSMEM1 protein expression was only detected in the testis as shown in our western blot analysis
(Fig. 2D), it is likely that SSMEM1 is discarded into residual bodies and degraded with the Golgi
before spermatids are released into the seminiferous tubule lumen during spermiation.
We show that generating KO mice using the CRISPR/Cas9 system is a powerful approach
to explore implicit causative genes for male infertility. Future studies will investigate the subcellular
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localization of SSMEM1, and the interaction with IFT proteins and/or other cytoskeleton-associated
proteins such as TMF. In addition, polymorphisms in human SSMEM1 have been identified via the
dbSNP database (, suggesting that this gene could be contributing to
human male infertility as well. The studies will have the potential for new insights into male
infertility diagnosis and treatment as well as the development of non-hormonal contraceptives.
We thank Dr. Rashieda J. Hatcher for critical reading of this manuscript, the Baylor College of
Medicine Pathology & Histology Core for processing for embedding histology samples, Jianhua Gu
and Huie Wang at Houston Methodist Research Institute’s Electron Microscopy Core for their
expertise in acquiring scanning electron microscopy images, Drs. Debra Townley and Michael
Mancini in the Integrated Microscopy Core for their advice with transmission electron microscopy
experiments, and Dr. Keisuke Shimada for shipping mice from Japan.
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Fig. 1. Ssmem1 is a conserved testis-specific gene.
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(A) RT-PCR in multiple tissues confirming the testis-specificity of Ssmem1 in mice (Top panels)
and humans (bottom panels). Hprt and GAPDH were used as controls. He, heart; Li, liver; Sp,
Lu, lung; Ki, kidney; Br, brain; St-stomach; In-intestine; Te, testis; Ov, ovary; Ut, Uterus; Ep,
Epididymis; H, head: B, Body; T, Tail. (B) RT-PCR from mouse testes at various postnatal days was
performed. Hprt was used a control. (C) Protein sequence alignment of SSMEM1 from several
mammalian species. Transmembrane region in human protein (asterisk) is well conserved with
other species. Black indicates fully conserved residues. Gray indicates conservation with residues in
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Fig. 2. Generating Ssmem1 KO mice.
(A) Genomic structure of mouse Ssmem1 and scheme to generate the gene KO mice using the
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CRISPR/Cas9 system. Three splice variants of Ssmem1 have been reported. White and black boxes
indicate untranslated and coding regions, respectively. Red arrow and black under bar indicate
gRNA that targets the region. The delivery of CRISPR/Cas9 system into zygotes for mutagenesis
via microinjection resulted in 5 bp (6 bp deletion and 1 bp insertion) frameshift deletion (shown in
red). (B) Genotyping of Ssmem1 alleles. Primers indicated in green color in Fig, 2A amplify specific
amplicon for the WT or KO allele. (C) Mutation in amino acid sequence in KO mouse induced by
-5 frameshift mutation is shown in red color. Asterisk indicates a premature stop codon. The
numbers above sequences are amino acid number for each. (D) Western blot analysis using testis
and sperm. SSMEM1 protein is detected only in WT testis. Equal loading of total protein was
confirmed by CBB staining.
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Fig. 3. Ssmem1 KO causes male infertility.
(A) Average litter size from natural mating of Ssmem1 HET and KO mice. Litter size was measured
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by the number of pups born. Ssmem1 KO males showed complete infertile, P<0.0001. (B)
Quantification of sperm released from the cauda epididymis. There was no significant difference
between HET and KO mice. (C) Images of testes from Ssmem1 HET and KO mice. (D) Average
weight of individual testis. KO males showed a reduction in testis volume, P<0.05. (E)
PAS-Hematoxylin staining of testis and caudal epididymis sections from HET and KO mice.
Occasional KO tubules in testis did not contain germ cells (asterisks). (Scale bar, 100 μm)
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Fig. 4. Ssmem1 KO sperm show morphology and motility defects.
(A) SEM images of HET/KO spermatozoa from caudal epididymis. KO sperm exhibited
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globozoospermia. (B) Sperm motility at 15 and 90 min after sperm suspension. There was a
reduction in sperm motility in KO. (C) Sperm kinematic parameters measured using the CEROS II
sperm analysis system. Velocities of KO sperm were lower than those of HET. P<0.05 (*), P<0.01
(**), P<0.001 (***).
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Fig. 5. Ssmem1 KO caused a delay in organelle transfer.
(A) TEM images of HET/KO spermatozoa in epididymis. Nuclei showed abnormal morphology and
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mitochondria are located at the sperm head region, alongside the nuclei (Scale bar, 2.0 μm). (B, C)
TEM images of spermatids at same stages, step 8 (B); step 9 (C) in testis. In Ssmem1 null spermatid,
Golgi body (Red arrows) failed to undergo migration as control spermatid at step 8. In step 9,
elongating spermatids, the Golgi stayed in the caput aspect of the cell and the acrosomal membrane
did not contact the cell surface (Black arrowheads) in Ssmem1 null spermatids (Scale bar, 4.0 μm).
(D) TEM images of sperm at step 11. Golgi and mitochondria were located at caudal region of
sperm in both HET and KO sperm (Scale bar, 2.0 μm). (E) Immunostaining of HET/KO testes. The
Golgi body and acrosome were stained with Golgin 97 (red) and peanut agglutinin (PNA, green),
respectively. Arrows indicate the Golgi retained in acrosome in mutant spermatids. Migration of
Golgi from acrosome side to tail side was delayed in KO spermatids (Scale bar, 10.0 μm).
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Fig. 6. Schematic representation of the Ssmem1 deficient spermatogenesis.
The delay in organelle migration from step 8 to step 9 causes progressive changes leading to
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round-headed sperm in Ssmem1 KO males.
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... In mice, this gene has been related to late gestational embryonic lethality when a loss of function occurs [63]. Recently, Zhang, et al. [64] demonstrated the effects of NRF1 on steroidogenesis and cell apoptosis in goat luteinized granulosa cells. An attenuated expression of NRF1 led to mitochondrial dysfunction, disrupted the cellular redox balance, impaired steroid synthesis, and finally resulted in granulosa cell apoptosis through the mitochondria-dependent pathway. ...
... Serine-rich single-pass membrane protein 1 (SSMEM1) is a conserved testis-specific gene in mammals. Nozawa, et al. [64] demonstrated that SSMEM1 is essential for male fertility in mice and found that the SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the SSMEM1 KO mice was associated with globozoospermia and a loss of sperm motility, which is crucial for fertilization. ...
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In this study, we analyzed the variation of reproductive efficiency, estimated as the deviation between the optimal and real parity number of females at each stage of the cow’s life, in 12,554 cows belonging to the Retinta Spanish cattle breed, using classical repeatability and random regression models. The results of the analyses using repeatability model and the random regression model suggest that reproductive efficiency is not homogeneous throughout the cow’s life. The h2 estimate for this model was 0.30, while for the random regression model it increased across the parities, from 0.24 at the first calving to 0.51 at calving number 9. Additionally, we performed a preliminary genome-wide association study for this trait in a population of 252 Retinta cows genotyped using the Axiom Bovine Genotyping v3 Array. The results showed 5 SNPs significantly associated with reproductive efficiency, located in two genomic regions (BTA4 and BTA28). The functional analysis revealed the presence of 5 candidate genes located within these regions, which were previously involved in different aspects related to fertility in cattle and mice models. This new information could give us a better understanding of the genetic architecture of reproductive traits in this species, as well as allow us to accurately select more fertile cows.
... This includes mitochondrial rearrangement around the flagella at the midpiece of the tail, and proteins, organelles, and bulk cytoplasm that are no longer needed are discarded through the extrusion of cytoplasmic droplets, which are eventually removed from the sperm head and neck region [17,18]. Our previous studies have shown that spatiotemporal organelle migration is critical for successful spermatogenesis [19]. Similarly, improper retainment of cytoplasmic droplets during spermiogenesis has severe consequences on sperm maturation in the epididymis. ...
... Sperm number and motility were measured as described previously [19]. Sperm were extracted by mincing caudal epididymis and incubated in TYH medium (Millipore) supplemented with 4% BSA under 5% CO 2 at 37 °C. ...
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Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 ( Rnf133 ); RING finger protein 148 ( Rnf148 ); RING finger protein 151 ( Rnf151 ); and Zinc finger SWIM-type containing 2 ( Zswim2 )]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148 , Rnf151 , and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
... a previous study reported that >2,300 genes are expressed predominantly in mouse testes (13), and a number of them are identified as serving important roles in numerous events during spermiogenesis through gene-editing technology, such as the biogenesis of the acrosome, the morphology of the manchette and the assembly of the flagellum. For example, mice lacking autophagy related 7, sirtuin 1, serine-rich single-pass membrane protein 1, vacuolar protein sorting (Vps)13b, Vps54 and sperm acrosome associated 1 exhibit abnormal round-headed sperm as a result of aberrant acrosome formation (8,9,(14)(15)(16). leucine-rich repeats and guanylate kinase domain containing isoform 1, serine/threonine kinase 22, katanin-like 2, sperm flagellar 2, hook microtubule tethering protein 1 and calcium and integrin binding family member 4 are essential for the correct formation of the manchette; deficiency in any of these genes can lead to abnormal elongation of the manchette and thus, a malformed head or flagellum (11,12,(17)(18)(19)(20). ...
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Gene expression analyses have revealed that there are >2,300 testis-enriched genes in mice, and gene knockout models have shown that a number of them are responsible for male fertility. However, the functions of numerous genes have yet to be clarified. The aim of the present study was to identify the expression pattern of testis-expressed protein 33 (TEX33) in mice and explore the role of TEX33 in male reproduction. Reverse transcription-polymerase chain reaction and western blot assays were used to investigate the mRNA and protein levels of TEX33 in mouse testes during the first wave of spermatogenesis. Immunofluorescence analysis was also performed to identify the cellular and structural localization of TEX33 protein in the testes. Tex33 knockout mice were generated by CRISPR/Cas9 gene-editing. Histological analysis with hematoxylin and eosin or periodic acid-Schiff (PAS) staining, computer-assisted sperm analysis (CASA) and fertility testing, were also carried out to evaluate the effect of TEX33 on mouse spermiogenesis and male reproduction. The results showed that Tex33 mRNA and protein were exclusively expressed in mouse testes and were first detected on postnatal days 21-28 (spermiogenesis phase); their expression then remained into adulthood. Immunofluorescence analysis revealed that TEX33 protein was located in the spermatids and sperm within the seminiferous tubules of the mouse testes, and exhibited specific localization to the acrosome, flagellum and manchette during spermiogenesis. These results suggested that TEX33 may play a role in mouse spermiogenesis. However, Tex33 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. PAS staining and CASA revealed that spermatogenesis and sperm quality were normal in mice lacking Tex33. In addition, fertility testing suggested that the Tex33 knockout mice had normal reproductive functions. In summary, the findings of the present study indicate that TEX33 is associated with spermiogenesis but is not essential for sperm development and male fertility.
The formation of fertilization-competent sperm requires spermatid morphogenesis (spermiogenesis), a poorly understood program that involves complex coordinated restructuring and specialized cytoskeletal structures. A major class of cytoskeletal regulators are the actin-related proteins (ARPs), which include conventional actin variants, and related proteins that play essential roles in complexes regulating actin dynamics, intracellular transport, and chromatin remodeling. Multiple testis-specific ARPs are well-conserved among mammals, but their functional roles are unknown. One of these is actin-like 7b (Actl7b) which encodes an orphan ARP highly similar to the ubiquitously expressed beta actin (ACTB). Here we report ACTL7B is expressed in human and mouse spermatids through the elongation phase of spermatid development. In mice, ACTL7B specifically localizes to the developing acrosome, within the nucleus of early spermatids, and to the flagellum connecting region. Based on this localization pattern and high level of sequence conservation in mice, humans and other mammals, we examined the requirement for ACTL7B in spermiogenesis by generating and characterizing the reproductive phenotype of male Actl7b KO mice. KO mice were infertile, with severe and variable oligoteratozoospermia (OAT) and multiple morphological abnormalities of the flagellum (MMAF) and sperm head. These defects phenocopy human OAT and MMAF, which are leading causes of idiopathic male infertility. In conclusion, this work identifies ACTL7B as a key regulator of spermiogenesis that is required for male fertility.
Background: The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known. Objective: The goals of our studies were to understand the mechanism of action of TSSK3. Materials and methods: We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells. Results: TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels. Conclusions: We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.
Background: Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa. Objective: We generated phosducin-like 2 (PDCL2) knockout (-/-) mice to investigate the essential roles of PDCL2 in mammalian reproduction. Materials and methods: We used RT-PCR to demonstrate that PDCL2 was expressed exclusively in the male reproductive tract in mice and humans. We created Pdcl2-/- mice using the CRISPR-Cas9 system and analyzed their fertility. Pdcl2 null spermatozoa underwent further evaluation using computer-assisted sperm analysis (CASA), light microscopy, and ultrastructural microscopy. We used immunoblot analysis and immunofluorescence to elucidate relationships between PDCL2 and other acrosomal proteins. Results: The PDC family is highly conserved in eukaryotes. Mouse and human PDCL2 are testis-enriched and localized to the testicular endoplasmic reticulum. Loss of the protein causes sterility due to abnormal acrosome biogenesis during spermiogenesis and immotility. Furthermore, Pdcl2 null spermatozoa have rounded heads, similar to globozoospermia in humans. Observation of the knockout testis shows a lack of acrosomal cap formation, aberrant localization of mitochondria in the sperm head, and misshapen nuclei. Conclusion: PDCL2 is essential for sperm acrosome development and male fertility in mice and is a putative contraceptive target in men This article is protected by copyright. All rights reserved.
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As the basis of animal reproductive activity, normal spermatogenesis directly determines the efficiency of livestock production. An in-depth understanding of spermatogenesis will greatly facilitate animal breeding efforts and male infertility treatment. With the continuous development and application of gene editing technologies, they have become valuable tools to study the mechanism of spermatogenesis. Gene editing technologies have provided us with a better understanding of the functions and potential mechanisms of action of factors that regulate spermatogenesis. This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis-related genes and disease treatment. The problems of gene editing technologies in the field of spermatogenesis research are also discussed.
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Infertility afflicts up to 15% of couples globally each year with men a contributing factor in half of these cases. Globozoospermia is a rare condition found in infertile men that is characterized by defective acrosome biogenesis leading to the production of round shaped sperm. Here, we report a novel gene, Fam209 (Family with sequence similarity 209), that is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 result in fertility defects secondary to abnormalities in acrosome biogenesis during spermiogenesis reminiscent of globozoospermia. Proteomic analysis of the FAM209 proteome identified DPY19L2, a protein involved in the majority of globozoospermia cases. While mutations in human and mouse DPY19L2 have been shown to cause globozoospermia, no in vivo interacting partners of DPY19L2 have been identified until now. FAM209 colocalizes with DPY19L2 to the inner nuclear membrane to maintain the developing acrosome. This report identifies FAM209 as the first interacting partner of DPY19L2 and the second protein that is essential for acrosome biogenesis and that co-localizes with DPY19L2 to the inner nuclear membrane.
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Significance The sperm−oocyte fusion step is important to transport the male genome into oocytes. So far, IZUMO1 and FIMP have been identified as fusion-related proteins in spermatozoa, but the molecular mechanisms underpinning sperm−oocyte fusion and all of the proteins required for this essential process remain unclear. In this study, using CRISPR-Cas9−mediated gene knockouts in mice, we discover that sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion and male fertility. As these genes are conserved among mammals including human, they may explain not only the sperm−oocyte fusion process but also idiopathic male infertility and be unique targets for contraception.
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There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.
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Significance Infertility is a global problem that afflicts 15% of couples, and in 50% of cases, the attributing factor is linked to men. Among these infertile men, 18% specifically exhibit decreased motility of sperm (asthenozoospermia). Sperm motility is dependent on the formation and functioning of the flagellum, a modified cilium used for locomotion. Cilia are present in almost every cell of vertebrates and are essential for proper organ functioning. Defects in cilia formation lead to severe syndromic diseases, termed ciliopathies, affecting numerous tissues (e.g., polycystic kidney disease), wherein male infertility is often comorbid. Advances in mouse genetics implicate several genes responsible for ciliopathies observed in humans. Here, we identify a nonsyndromic flagellum protein, TCTE1, that is required for sperm motility in mice.
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Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of the prominence and importance of actin structures in mammalian spermiogenesis, we investigated whether MVI was associated with actin-mediated maturation events in mammals. Both immunofluorescence and ultrastructural analyses using immunogold labeling showed that MVI was strongly linked with key structures involved in sperm development and maturation. During the early stage of spermiogenesis, MVI is associated with the Golgi and with coated and uncoated vesicles, which fuse to form the acrosome. Later, as the acrosome spreads to form a cap covering the sperm nucleus, MVI is localized to the acroplaxome, an actin-rich structure that anchors the acrosome to the nucleus. Finally, during the elongation/maturation phase, MVI is associated with the actin-rich structures involved in nuclear shaping: the acroplaxome, manchette, and Sertoli cell actin hoops. Since this is the first report of MVI expression and localization during mouse spermiogenesis and MVI partners in developing sperm have not yet been identified, we discuss some probable roles for MVI in this process. During early stages, MVI is hypothesized to play a role in Golgi morphology and function as well as in actin dynamics regulation important for attachment of developing acrosome to the nuclear envelope. Next, the protein might also play anchoring roles to help generate forces needed for spermatid head elongation. Moreover, association of MVI with actin that accumulates in the Sertoli cell ectoplasmic specialization and other actin structures in surrounding cells suggests additional MVI functions in spermatid movement across the seminiferous epithelium and in sperm release.
Male infertility is a heterogenous disease process requiring the proper functioning and interaction of thousands of genes. Given the number of genes involved, it is thought that genetic causes contribute to most cases of infertility. Identifying these causes, however, is challenging. Infertility is associated with negative health outcomes, such as cancer, highlighting the need to further understand the genetic underpinnings of this condition. This paper describes the genetic and genomic tests currently available to identify the etiology of male infertility and then will discuss emerging technologies that may facilitate diagnosis and treatment of in the future.
IFT74 is a component of the core intraflagellar transport (IFT) complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile associated with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axoneme in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88 and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 (ODF2) and sperm-associated antigen 16L (SPAG16L) proteins were also not changed. However, the processed A-Kinase anchor protein (AKAP), a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.
Sperm malformation is one of the main reasons for male infertility, but the precise mechanisms of this process remain undiscovered. The major process of spermiogenesis is sperm head shaping. Cytoskeleton is a crucial unit in this process, as the acroplaxome and manchette are two kinds of momentous structures cooperated with various functional proteins to insure the formation of acrosome and nucleus. One is primarily formed by filamentous actin (F-actin) and responsible for transverse acrosome extension and concentration, another plays as the mainstay of nuclear deformation through circular arrangement of microtubules (MTs). We suspect that the acroplaxome alone cannot maintain such a spatial framework of the acrosome. Previous studies have also revealed that a nucleus without acrosome could not induce the formation of ectoplasmic specialization. In this review, we integrated most of the key proteins that have been proven to participate in the essential developmental steps of post-meiosis. We also propose that the ambient MTs of the acrosome might be emanated from the Golgi apparatus. They form a novel cytoskeleton termed acroframosome (AFS) to transport vesicles and proteins during acrosome biogenesis. The hypothesis of the acroframosome-acroplaxome-manchette (AAM) cytoskeletal system is likely to be the axis of head-to-tail spermiogenesis.
The TYH medium was first reported as a medium for in vitro fertilization (IVF) of mouse eggs with epididymal spermatozoa, by Toyoda, Yokoyama and Hosi, in 1971. It was a modified Krebs-Ringer bicarbonate solution, supplemented with glucose, Na-pyruvate, antibiotics and bovine serum albumin. In TYH medium, almost all eggs are fertilized within 1 h, if the spermatozoa are pre-incubated for 2 h before insemination, while the sperm penetration is delayed for approximately 1 h when fresh epididymal spermatozoa are used. These findings showed that sperm capacitation can be induced in a chemically defined medium without requiring the female reproductive tract. Although the medium was not specifically named in the original paper, it was later called “TYH” after the initials of the three authors of the original paper. The IVF method using TYH medium is widely used due to its high reproducibility. In this mini-review, we describe the early efforts to develop the TYH medium and briefly discuss the related areas.