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Sperm is the ultimate executor of male reproductive function. Normal morphology, quantity, and motility of sperm ensure the normal reproductive process. Palmitoylation is a posttranslational modification mediated by palmitoyltransferases whereby palmitoyl is added to proteins. Seven palmitoyltransferases have been identified in Saccharomyces cerevisiae and 23 in humans (including ZDHHC1–9 and ZDHHC11–24), with corresponding homologs in mice. We identified two testis-specific palmitoyltransferases ZDHHC11 and ZDHHC19 in mice. The Zdhhc11 and Zdhhc19-knockout mouse models were constructed, and it was found that the Zdhhc11 knockout males were fertile, while Zdhhc19 knockout males were sterile. ZDHHC19 is located in the cell membrane of step 4–9 spermatids in the mouse testis, and phenotypic analysis showed that the testicular weight ratio in the Zdhhc19−/− mice decreased along with the number and motility of the sperm decreased, while sperm abnormalities increased, mainly due to the “folded” abnormal sperm caused by sperm membrane fusion, suggesting the involvement of ZDHHC19 in maintaining membrane stability in the male reproductive system. In addition, Zdhhc19−/− mice showed abnormal sperm morphologies and apoptosis during spermatogenesis, suggesting that spermatogenesis in the Zdhhc19−/− mice was abnormal. These results indicate that ZDHHC19 promotes membrane stability in male germ cells.

Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV‐VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2 −/− mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2‐interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility.

Orderly and stage‐specifically expressed proteins are essential for spermatogenesis, and proteases play a key role in protein activation and function. The present study aimed to investigate serine protease 55 (PRSS55), which was reported to play a role in sperm‐uterotubal junction (UTJ) migration and sperm‐zona pellucida (ZP) binding. We found that PRSS55 was specifically expressed in testicular spermatids and epididymal spermatozoa. By constructing knockout mice targeting all transcripts of Prss55, we demonstrated that deletion of Prss55 resulted in a serious decline of male fertility, with significantly increased sperm malformation and decreased sperm motility. In Prss55 −/− mice, increased structural abnormality, including deficient “9 + 2” microtubules, damaged peripheral dense fibre, and defective mitochondrial cristae, were found in sperm. In addition, sperm showed decreased expression of electron transfer chain molecules and lower ATP contents. These could be the potential causes of the astheno/teratozoospermia phenotype of the Prss55 −/− mice, and provided new evidence for the previously reported impaired sperm‐UTJ migration. Moreover, preliminary studies allowed us to speculate that PRSS55 might function by activating type II muscle myosin in the testis, which is involved in many processes requiring motivation and cytoskeleton translocation. Thus, PRSS55 is essential for the structural differentiation and energy metabolism of sperm, and might be a potential pathogenic factor in astheno/teratozoospermia. Our results provide an additional explanation for the male sterility of Prss55 −/− mice, and further reveal the role of PRSS55.

During meiosis, telomere attachment to the inner nuclear envelope is required for proper pairing of homologous chromosomes and recombination. Here, we identified F-box protein 47 (FBXO47) as a regulator of the telomeric shelterin complex that is specifically expressed during meiotic prophase I. Knockout of Fbxo47 in mice leads to infertility in males. We found that the Fbxo47 deficient spermatocytes are unable to form a complete synaptonemal complex. FBXO47 interacts with TRF1/2, and the disruption of Fbxo47 destabilizes TRF2, leading to unstable telomere attachment and slow traversing through the bouquet stage. Our findings uncover a novel mechanism of FBXO47 in telomeric shelterin subunit stabilization during meiosis.

Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood.We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system.Four low-frequency variants were identified in four genes (Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

Purpose To investigate the differences in protein expression between Dpy19l2-deficient human globozoospermia and normozoospermia. Experimental design Human sperm samples from three globozoospermic donors with Dpy19l2 deletion and three normal controls are subjected to TMT quantitative technology. SPESP1, HIST1H4A, and LYZL1 are randomly selected for western blotting analysis. GO annotations are performed using the Database for Annotation, Visualization, and Integrated Discovery. Results A total of 2567 proteins are identified, of which 2510 proteins are quantified, and 491 are differentially expressed (fold-change > 2), with 370 upregulated and 121 downregulated in globozoospermic patients. The levels of several important proteins, including SPACA 1, IZUMO1, ZPBP1, and PLCZ1, are decreased in globozoospermic sperm. Bioinformatics analysis indicates the Dpy19l2-deficient sperm presented molecular defects in acrosome, chromatin, sperm-egg interaction, and fertilization. Conclusions and clinical relevance The present study is the first to analyze total globozoospermia with Dpy19l2 deletion using high-throughput proteomics. This study may provide insights into the mechanism of globozoospermia.

Our object was to explore direct effects and mechanism of BDE47 on GC2 (immortalized mouse spermatocyte). GC2 were exposed to DMSO, 0.1, 1, 10, 100μM BDE47 for 48h. Cell viability was detected by trypan-blue exclusion; ultrastructure by electron-microscopy; cell cycle, mitochondrial membrane motential (MMP), reactive oxygen species (ROS) by flow-cytometry; ATP production by luminometer; Atp5b, Uqcrc1, Bcl-2 level by WB. To explore whether the decreased mitochondrial proteins play an important role in apoptosis, MMP and apoptosis were detected after Atp5b or Uqcrc1 knockdown in GC2. Results showed BDE47 reduced cell viability, caused condensation of nuclear and vacuolated mitochondria, decreased MMP and ATP, induced ROS, cell cycle arrest at S and G2/M phase, reduced Atp5b, Uqcrc1, Bcl-2 in GC2. Knockdown of Atp5b or Uqcrc1 decreased MMP, induced apoptosis in GC2. Results suggested that BDE47 reduced cell viability, injured mitochondria in spermatocytes probably by decreasing mitochondrial protein Atp5b and Uqcrc1.

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile due to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as in late meiotic cells. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks. These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.

Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/β-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.

Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more site-specific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis.

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the “9+2” microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated–Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.

Our objective was to explore molecular markers and mechanism of BDE47 on spermatogenesis in mammals. Adult male SD rats were gavaged daily with corn oil containing 0, 0.001, 0.03, 1 or 20mg BDE47/kg bw for eight weeks. Testes morphology was analyzed using electron microscopy, TUNEL, immunohistochemistry and morphometry. Differential proteome profile and western blotting were applied to determine molecular markers and protein expression. GC1-spg cells (mouse spermatogonial cells) were used to verify mechanism of BDE47. Data showed BDE47 reduced tubular epithelial thickness, impaired mitochondrial function and induced apoptosis in early leptotene spermatocytes. Proteomic study identified 70 differential spots corresponding to 64 proteins. 20 proteins related to apoptosis, 15 located in mitochondria. Exposure of GC1-spg cells showed BDE47 induced apoptosis, impaired mitochondria and decreased Bcl-2 in cells. Data indicate that BDE47 disrupts spermatogenesis, impairs mitochondrial function and induces apoptosis of early leptotene spermatocytes in rats probably via mitochondrial pathway.

Protein lysine acetylation is a dynamic and reversible post-modification that is known to play diverse functions in eukaryotes. Nevertheless, the composition and function of non-histone lysine acetylation in gametes remain unknown. In humans, only capacitated sperm have the capacity to fertilize an egg. In the present study, we found complex composition of lysine acetylated proteins in capacitated human sperm. In vitro fertilization inhibition assay by anti-acetyllysine antibody showed essential roles of lysine acetylation in fertilization. And inhibition of lysine deacetylases, the histone deacetylases, by trichostatin A and nicotinamide, could significantly suppress sperm motility. After immunopurification enrichment of acetylpeptides with anti-acetyllysine antibody and high-throughput liquid chromatography–tandem mass spectrometry identification, we characterized 1206 lysine acetylated sites, corresponding to 576 lysine acetylated proteins in human capacitated sperm. Bioinformatics analysis showed that these proteins are associated with sperm functions, including motility, capacitation, acrosome reaction and sperm–egg interaction. Thus, lysine acetylation is expected to be an important regulatory mechanism for sperm functions. And our characterization of lysine acetylproteome could be a rich resource for the study of male fertility. Biological significance Mature sperm are almost transcriptionally and translationally silent, thus post-translational modifications play important roles in sperm functions. Till now, only two types of PTMs, phosphorylation and glycosylation, are well studied in normal human sperm based on large scale proteomics. In the present study, we established the acetylproteome of capacitated human sperm. Over 1000 lysine acetylated sites were identified. Bioinformatics analysis shows that lysine acetylated proteins participate in many biological events of sperm functions. We further provided functional data that the lysine acetylation is essential for sperm motility and fertilization using histone acetylase inhibitors and anti-acetyllysine antibody. These data can be strong evidences for the important function of lysine acetylation in human sperm.