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.

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 prime editor efficiently repaired human induced pluripotent stem cells with AR gene mutation (c.2710G > A; p. V904M).

Prime Editor (PE) is a precise genome manipulation technology based on the CRISPR-Cas9 system, while its application in human induced pluripotent stem cells (iPSCs) remains limited. Here, we established a repaired hiPS cell line (SKLRMi001-A-1) from hiPSCs with androgen receptor (AR) mutation (c.2710G > A; p.V904M). The repaired iPSC line expressed pluripotency markers, retained normal karyotype, showed the capability of differentiating into three germ layers and was absence of mycoplasma infection. The repaired iPSC line will help to elucidate the mechanism of androgen insensitivity syndrome (AIS) and benefit treatment for AIS in the future.

Derivation of new pluripotent stem cells from human extended pluripotent stem cells with formative features and trophectoderm potential.

Previous studies have demonstrated the existence of intermediate stem cells, which have been successfully obtained from human naive pluripotent stem cells (PSCs) and peri-implantation embryos. However, it is not known whether human extended pluripotent stem cells (hEPSCs) can be directly induced into intermediate stem cells. Moreover, the ability of extra-embryonic lineage differentiation in intermediate stem cells has not been verified. In this issue, we transformed hEPSCs into a kind of novel intermediate pluripotent stem cell resembling embryonic days 8-9 (E8-E9) epiblasts and proved its feature of formative epiblasts. We engineered hEPSCs from primed hPSCs under N2B27-LCDM (N2B27 plus Lif, CHIR, DiH and MiH) conditions. Then, we added Activin A, FGF and XAV939 to modulate signalling pathways related to early humans' embryogenesis. We performed RNA-seq and CUT&Tag analysis to compare with AF9-hPSCs from different pluripotency stages of hPSCs. Trophectoderm (TE), primordial germ cells-like cells (PGCLC) and endoderm, mesoderm, and neural ectoderm induction were conducted by specific small molecules and proteins. AF9-hPSCs transcription resembled that of E8-E9 peri-implantation epiblasts. Signalling pathway responsiveness and histone methylation further revealed their formative pluripotency. Additionally, AF9-hPSCs responded directly to primordial germ cells (PGCs) specification and three germ layer differentiation signals in vitro. Moreover, AF9-hPSCs could differentiate into the TE lineage. Therefore, AF9-hPSCs represented an E8-E9 formative pluripotency state between naïve and primed pluripotency, opening new avenues for studying human pluripotency development during embryogenesis.

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.

Single-cell RNA-seq uncovers dynamic processes orchestrated by RNA-binding protein DDX43 in chromatin remodeling during spermiogenesis.

Mammalian spermatogenesis shows prominent chromatin and transcriptomic switches in germ cells, but it is unclear how such dynamics are controlled. Here we identify RNA helicase DDX43 as an essential regulator of the chromatin remodeling process during spermiogenesis. Testis-specific Ddx43 knockout mice show male infertility with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. The loss of its ATP hydrolysis activity by a missense mutation replicates the infertility phenotype in global Ddx43 knockout mice. Single-cell RNA sequencing analyses of germ cells depleted of Ddx43 or expressing the Ddx43 ATPase-dead mutant reveals that DDX43 regulates dynamic RNA regulatory processes that underlie spermatid chromatin remodeling and differentiation. Transcriptomic profiling focusing on early-stage spermatids combined with enhanced crosslinking immunoprecipitation and sequencing further identifies Elfn2 as DDX43-targeted hub gene. These findings illustrate an essential role for DDX43 in spermiogenesis and highlight the single-cell-based strategy to dissect cell-state-specific regulation of male germline development.

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.

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.

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.

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.

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.

Single-cell RNA-Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation.

The assembly of primordial follicles in mammals represents one of the most critical processes in ovarian biology. It directly affects the number of oocytes available to a female throughout her reproductive life. Premature depletion of primordial follicles contributes to the ovarian pathology primary ovarian insufficiency (POI). To delineate the developmental trajectory and regulatory mechanisms of oocytes during the process, we performed RNA-seq on single germ cells from newborn (P0.5) ovaries. Three cell clusters were classified which corresponded to three cell states (germ cell cyst, cyst breakdown, and follicle) in the newborn ovary. By Monocle analysis, a uniform trajectory of oocyte development was built with a series of genes showed dynamic changes along the pseudo-timeline. Gene Ontology term enrichment revealed a significant decrease in meiosis-related genes and a dramatic increase in oocyte-specific genes which marked the transition from a germ cell to a functional oocyte. We then established a network of regulons by using single-cell regulatory network inference and clustering (SCENIC) algorithm and identified possible candidate transcription factors that may maintain transcription programs during follicle formation. Following functional studies further revealed the differential regulation of the identified regulon Id2 and its family member Id1, on the establishment of primordial follicle pool by using siRNA knockdown and genetic modified mouse models. In summary, our study systematically reconstructed molecular cascades in oocytes and identified a series of genes and molecular pathways in follicle formation and development.

T-complex protein 1 subunit zeta-2 (CCT6B) deficiency induces murine teratospermia.

BACKGROUND: The CCT complex is an important mediator of microtubule assembly and intracellular protein folding. Owing to its high expression in spermatids, CCT knockdown can disrupt spermatogenesis. In the present report, we therefore evaluated the in vivo functionality of the testis-specific CCT complex component CCT6B using a murine knockout model system. METHODS: A CRISPR/Cas9 approach was used to generate Cct6b-/- mice, after which candidate gene expression in these animals was evaluated via qPCR and Western blotting. Testicular and epididymal phenotypes were assessed through histological and immunofluorescent staining assays, while a computer-assisted sperm analyzer was employed to assess semen quality. RESULTS: Cct6b-/- mice were successfully generated, and exhibited no differences in development, fertility, appearance, testis weight, or sperm counts relative to control littermates. In addition, no differences in spermatogenesis were detected when comparingCct6b+/+ and Cct6b-/- testes. However, when progressive motility was analyzed, the ratio of normal sperm was significantly decreased in Cct6b-/- male mice, with nuclear base bending being the primary detected abnormality. In addition, slight decreases in Cct4 and Cct7 expression were detected. CONCLUSION: These data indicated that CCT6B is an important regulator of murine spermatogenesis, with the loss of this protein resulting in CCT complex dysfunction, providing a foundation for further studies.

TULP2, a New RNA-Binding Protein, Is Required for Mouse Spermatid Differentiation and Male Fertility.

Spermatogenesis requires a large number of proteins to be properly expressed at certain stages, during which post-transcriptional regulation plays an important role. RNA-binding proteins (RBPs) are key players in post-transcriptional regulation, but only a few RBPs have been recognized and preliminary explored their function in spermatogenesis at present. Here we identified a new RBP tubby-like protein 2 (TULP2) and found three potential deleterious missense mutations of Tulp2 gene in dyszoospermia patients. Therefore, we explored the function and mechanism of TULP2 in male reproduction. TULP2 was specifically expressed in the testis and localized to spermatids. Studies on Tulp2 knockout mice demonstrated that the loss of TULP2 led to male sterility; on the one hand, increases in elongated spermatid apoptosis and restricted spermatid release resulted in a decreased sperm count; on the other hand, the abnormal differentiation of spermatids induced defective sperm tail structures and reduced ATP contents, influencing sperm motility. Transcriptome sequencing of mouse testis revealed the potential target molecular network of TULP2, which played its role in spermatogenesis by regulating specific transcripts related to the cytoskeleton, apoptosis, RNA metabolism and biosynthesis, and energy metabolism. We also explored the potential regulator of TULP2 protein function by using immunoprecipitation and mass spectrometry analysis, indicating that TUPL2 might be recognized by CCT8 and correctly folded by the CCT complex to play a role in spermiogenesis. Our results demonstrated the important role of TULP2 in spermatid differentiation and male fertility, which could provide an effective target for the clinical diagnosis and treatment of patients with oligo-astheno-teratozoospermia, and enrich the biological theory of the role of RBPs in male reproduction.

SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation.

T helper type 17 (Th17) cells have important functions in the pathogenesis of inflammatory and autoimmune diseases. Retinoid-related orphan receptor-γt (RORγt) is necessary for Th17 cell differentiation and functions. However, the transcriptional regulation of RORγt expression, especially at the enhancer level, is still poorly understood. Here we identify a novel enhancer of RORγt gene in Th17 cells, RORCE2. RORCE2 deficiency suppresses RORγt expression and Th17 differentiation, leading to reduced severity of experimental autoimmune encephalomyelitis. Mechanistically, RORCE2 is looped to RORγt promoter through SRY-box transcription factor 5 (SOX-5) in Th17 cells, and the loss of SOX-5 binding site in RORCE abolishes RORCE2 function and affects the binding of signal transducer and activator of transcription 3 (STAT3) to the RORγt locus. Taken together, our data highlight a molecular mechanism for the regulation of Th17 differentiation and functions, which may represent a new intervening clue for Th17-related diseases.

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.

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.

The testis-specifically expressed gene Trim69 is not essential for fertility in mice.

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis. The tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activity, are highly conserved in mammals. They are involved in important cellular processes such as embryonic development, immunity, and fertility. Our previous studies indicated that Trim69, a testis-specific expressed TRIM family gene, potentially participates in the spermatogenesis by mediating testicular cells apoptosis. In this study, we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology. Here, we reported that the male Trim69 knockout mice had normal fertility. The adult knockout mice have shown that the appearance of testes, testis/body weight ratios, testicular histomorphology, and the number and quality of sperm were consistent with wild-type mice. These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility, and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis. This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.

The heat shock protein family gene Hspa1l in male mice is dispensable for fertility.

BACKGROUND: Heat shock protein family A member 1 like (Hspa1l) is a member of the 70kD heat shock protein (Hsp70) family. HSPA1L is an ancient, evolutionarily conserved gene with a highly conserved domain structure. The gene is highly abundant and constitutively expressed in the mice testes. However, the role of Hspa1l in the testes has still not been elucidated. METHODS: Hspa1l-mutant mice were generated using the CRISPR/Cas9 system. Histological and immunofluorescence staining were used to analyze the phenotypes of testis and epididymis. Apoptotic cells were detected through TUNEL assays. Fertility and sperm motilities were also tested. Quantitative RT-PCR was used for analyzing of candidate genes expression. Heat treatment was used to induce heat stress of the testis. RESULTS: We successfully generated Hspa1l knockout mice. Hspa1l -/- mice exhibited normal development and fertility. Further, Hspa1l -/- mice shown no significant difference in spermatogenesis, the number of apoptotic cells in testes epididymal histology, sperm count and sperm motility from Hspa1l +/+ mice. Moreover, heat stress does not exacerbate the cell apoptosis in Hspa1l -/- testes. These results revealed that HSPA1L is not essential for physiological spermatogenesis, nor is it involved in heat-induced stress responses, which provides a basis for further studies.

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.