Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis.

BACKGROUND: Circular RNAs (circRNAs) are a large class of mammalian RNAs. Several protein products translated by circRNAs have been reported to be involved in the development of various tissues and systems; however, their physiological functions in male reproduction have yet not been explored. RESULTS: Here, we report an endogenous circRNA (circRsrc1) that encodes a novel 161-amino-acid protein which we named Rsrc1-161aa through circRNA sequencing coupled with mass spectrometry analysis on mouse testicular tissues. Deletion of Rsrc1-161aa in mice impaired male fertility with a significant decrease in sperm count and motility due to dysfunctions of mitochondrial energy metabolism. A series of in vitro rescue experiments revealed that circRsrc1 regulates mitochondrial functions via its encoded protein Rsrc1-161aa. Mechanistically, Rsrc1-161aa directly interacts with mitochondrial protein C1qbp and enhances its binding activity to mitochondrial mRNAs, thereby regulating the assembly of mitochondrial ribosomes and affecting the translation of oxidative phosphorylation (OXPHOS) proteins and mitochondrial energy metabolism. CONCLUSIONS: Our studies reveal that Rsrc1-161aa protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility.

Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility.

BACKGROUND: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized. RESULTS: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa-/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs. CONCLUSIONS: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

Global phosphoproteomic analysis identified key kinases regulating male meiosis in mouse.

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.

ZDHHC19 localizes to the cell membrane of spermatids and is involved in spermatogenesis†.

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.

Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis.

BACKGROUND: 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. METHODS: 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. RESULTS: Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure. CONCLUSION: 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.

Mebendazole-Induced Blood-Testis Barrier Injury in Mice Testes by Disrupting Microtubules in Addition to Triggering Programmed Cell Death.

Mebendazole (MBZ) is a synthetic benzimidazole known for its antiparasitic properties. In recent years, growing evidence showed that MBZ was also used as an anti-tumor agent. However, whether (and to what extent) this drug treatment affected the male reproductive system was not well-understood. In this study, male C57BL/6 mice were injected with 40 mg/kg/day of MBZ. The treatment was for 3 and 7 days. Our results showed that the injected mice exhibited an abnormal spermatogenic phase with a significant decrease in sperm. We further detected microtubule disruption and transient functional destruction of the blood-testes barrier (BTB) in the MBZ-injected mice testes (BTB). Our data confirmed that MBZ suppressed the expression of the BTB junction-associated proteins and disrupted the Sertoli cells' function in vivo. Moreover, MBZ-treated mice demonstrated an aberrant caspase-3 signalling pathway, which resulted in the apoptosis of the germ cells. Here, we present our data, indicating that MBZ impairs BTB by reducing the expression of the microtubules' and BTB junction-associated proteins. The last leads to activating the caspase-3 pathway, which triggers extensive germ cell apoptosis.

PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice.

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.

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.

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 owing 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 the late meiotic cell stage. 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.

FBXO47 regulates telomere-inner nuclear envelope integration by stabilizing TRF2 during meiosis.

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.

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa.

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.

Deficiency of TPPP2, a factor linked to oligoasthenozoospermia, causes subfertility in male mice.

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.

CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

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/ß-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.

Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.

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.

Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.

STYXL1 regulates CCT complex assembly and flagellar tubulin folding in sperm formation.

Tubulin-based microtubule is a core component of flagella axoneme and essential for sperm motility and male fertility. Structural components of the axoneme have been well explored. However, how tubulin folding is regulated in sperm flagella formation is still largely unknown. Here, we report a germ cell-specific co-factor of CCT complex, STYXL1. Deletion of Styxl1 results in male infertility and microtubule defects of sperm flagella. Proteomic analysis of Styxl1-/- sperm reveals abnormal downregulation of flagella-related proteins including tubulins. The N-terminal rhodanese-like domain of STYXL1 is important for its interactions with CCT complex subunits, CCT1, CCT6 and CCT7. Styxl1 deletion leads to defects in CCT complex assembly and tubulin polymerization. Collectively, our findings reveal the vital roles of germ cell-specific STYXL1 in CCT-facilitated tubulin folding and sperm flagella development.

PEX3 promotes regenerative repair after myocardial injury in mice through facilitating plasma membrane localization of ITGB3.

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3ß signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.