Quantitative phosphoproteomic profiling of mouse sperm maturation in epididymis revealed kinases important for sperm motility.

Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6,447 phosphorylation sites in 1,407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.

Complete genome sequence of Pseudomonas aeruginosa L3, a bacterium with Mn(II) oxidative property isolated from heavy metal-contaminated soils.

Pseudomonas aeruginosa L3, isolated from heavy metal-contaminated soils, possesses the ability of Mn(II) oxidation. To further enhance the understanding of genes involved in Mn(II) oxidation, the complete genome of this strain was sequenced and annotated, which has a total size of 6.39 Mb with a G + C content of 66.39%.

Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes.

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 µg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos.

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

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.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis.

Peroxisomes are organelles enclosed by a single membrane and are present in various species. The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs. However, little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs. Through transcriptomic and proteomic analyses, we observed heterogenous peroxisomal components among different organs, as well as between testicular somatic cells and different developmental stages of germ cells. As Pex3 is expressed in both germ cells and Sertoli cells, we generated Pex3 germ cell- and Sertoli cell-specific knockout mice. While Pex3 deletion in Sertoli cells did not affect spermatogenesis, the deletion in germ cells resulted in male sterility, manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells. Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins. These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis, but not for Sertoli cells.

Oocytes orchestrate protein prenylation for mitochondrial function through selective inactivation of cholesterol biosynthesis in murine species.

Emerging research and clinical evidence suggest that the metabolic activity of oocytes may play a pivotal role in reproductive anomalies. However, the intrinsic mechanisms governing oocyte development regulated by metabolic enzymes remain largely unknown. Our investigation demonstrates that geranylgeranyl diphosphate synthase1 (Ggps1), the crucial enzyme in the mevalonate pathway responsible for synthesizing isoprenoid metabolite geranylgeranyl pyrophosphate from farnesyl pyrophosphate, is essential for oocyte maturation in mice. Our findings reveal that the deletion of Ggps1 that prevents protein prenylation in fully grown oocytes leads to subfertility and offspring metabolic defects without affecting follicle development. Oocytes that lack Ggps1 exhibit disrupted mitochondrial homeostasis and the mitochondrial defects arising from oocytes are inherited by the fetal offspring. Mechanistically, the excessive farnesylation of mitochondrial ribosome protein, Dap3, and decreased levels of small G proteins mediate the mitochondrial dysfunction induced by Ggps1 deficiency. Additionally, a significant reduction in Ggps1 levels in oocytes is accompanied by offspring defects when females are exposed to a high-cholesterol diet. Collectively, this study establishes that mevalonate pathway-protein prenylation is vital for mitochondrial function in oocyte maturation and provides evidence that the disrupted protein prenylation resulting from an imbalance between farnesyl pyrophosphate and geranylgeranyl pyrophosphate is the major mechanism underlying impairment of oocyte quality induced by high cholesterol.

Multi-omics reveal the metabolic patterns in mouse cumulus cells during oocyte maturation.

Bi-directional communication between cumulus cells and the surrounded oocytes is important for the development and functions of both compartments. However, the metabolic framework in cumulus cells has not been systematically described. In the present study, cumulus cells from cumulus-oocyte complexes (COCs) at three key time points were isolated (arrested GV stage, post-hCG 0h; meiotic resumption GVBD stage, post-hCG 3h; and metaphase II stage, post-hCG 12h), and the temporal metabolomic and proteomic profiling were performed. Integrated multi-omics analysis reveals the global metabolic patterns in cumulus cells during mouse oocyte maturation. In particular, we found the active hyaluronic acid metabolism, steroid hormone synthesis, and prostaglandin E2 (PGE2) production in cumulus cells. Meanwhile, accompanying the oocyte maturation, a progressive increase in nucleotide and amino acid metabolism was detected in the surrounding cumulus cells. In sum, the data serve as a valuable resource for probing metabolism during terminal differentiation of ovarian granulosa cells, and provide the potential biomarkers for improving and predicting oocyte quality.

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.

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.

Absence of CEP78 causes photoreceptor and sperm flagella impairments in mice and a human individual.

Cone-rod dystrophy (CRD) is a genetically inherited retinal disease that can be associated with male infertility, while the specific genetic mechanisms are not well known. Here, we report CEP78 as a causative gene of a particular syndrome including CRD and male infertility with multiple morphological abnormalities of sperm flagella (MMAF) both in human and mouse. Cep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced ERG amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted connecting cilia elongation and abnormal structures. Cep78 deletion also caused male infertility and MMAF, with disordered '9+2' structure and triplet microtubules in sperm flagella. Intraflagellar transport (IFT) proteins IFT20 and TTC21A are identified as interacting proteins of CEP78. Furthermore, CEP78 regulated the interaction, stability, and centriolar localization of its interacting protein. Insufficiency of CEP78 or its interacting protein causes abnormal centriole elongation and cilia shortening. Absence of CEP78 protein in human caused similar phenotypes in vision and MMAF as Cep78-/- mice. Collectively, our study supports the important roles of CEP78 defects in centriole and ciliary dysfunctions and molecular pathogenesis of such multi-system syndrome.

Proteomic Profiling Reveals the Molecular Control of Oocyte Maturation.

Meiotic maturation is an intricate and precisely regulated process orchestrated by various pathways and numerous proteins. However, little is known about the proteome landscape during oocytes maturation. Here, we obtained the temporal proteomic profiles of mouse oocytes during in vivo maturation. We successfully quantified 4694 proteins from 4500 oocytes in three key stages (germinal vesicle, germinal vesicle breakdown, and metaphase II). In particular, we discovered the novel proteomic features during oocyte maturation, such as the active Skp1-Cullin-Fbox pathway and an increase in mRNA decay-related proteins. Using functional approaches, we further identified the key factors controlling the histone acetylation state in oocytes and the vital proteins modulating meiotic cell cycle. Taken together, our data serve as a broad resource on the dynamics occurring in oocyte proteome and provide important knowledge to better understand the molecular mechanisms during germ cell development.

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.

Multi-omics profiling visualizes dynamics of cardiac development and functions.

Cardiogenesis is a tightly regulated dynamic process through a continuum of differentiation and proliferation events. Key factors and pathways governing this process remain incompletely understood. Here, we investigate mice hearts from embryonic day 10.5 to postnatal week 8 and dissect developmental changes in phosphoproteome-, proteome-, metabolome-, and transcriptome-encompassing cardiogenesis and cardiac maturation. We identify mitogen-activated protein kinases as core kinases involved in transcriptional regulation by mediating the phosphorylation of chromatin remodeling proteins during early cardiogenesis. We construct the reciprocal regulatory network of transcription factors (TFs) and identify a series of TFs controlling early cardiogenesis involved in cycling-dependent proliferation. After birth, we identify cardiac resident macrophages with high arachidonic acid metabolism activities likely involved in the clearance of injured apoptotic cardiomyocytes. Together, our comprehensive multi-omics data offer a panoramic view of cardiac development and maturation that provides a resource for further in-depth functional exploration.

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.

Homeodomain-interacting protein kinase HIPK4 regulates phosphorylation of manchette protein RIMBP3 during spermiogenesis.

Nonobstructive azoospermia (NOA) is the most serious form of spermatogenesis abnormalities in male infertility. Genetic factors are important to consider as elements leading to NOA. Although many pathogenic genes have been reported, the causative genes of NOA for many patients are still unknown. In this study, we found ten point mutations in the gene encoding homeodomain-interacting protein kinase 4 (HIPK4) in patients with NOA, and using in vitro studies, we determined a premature termination point mutation (p. Lys490∗, c.1468A>T) that can cause decreased expression of HIPK4. Our phosphoproteomic analysis of Hipk4-/- testes revealed phosphorylation of multiple proteins regulated by HIPK4 during spermiogenesis. We also confirmed that a substrate of HIPK4 with four downregulated phosphorylation sites matching the xSPx motif is the known manchette-related protein RIMS-binding protein 3, which is required for sperm head morphogenesis. Therefore, we conclude HIPK4 regulates the phosphorylation of manchette protein RIMS-binding protein 3 and plays essential roles in sperm head shaping and male fertility.

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro-Matured Oocytes.

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.

SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2ZYG11B activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.

DeepSCP: utilizing deep learning to boost single-cell proteome coverage.

Multiplexed single-cell proteomes (SCPs) quantification by mass spectrometry greatly improves the SCP coverage. However, it still suffers from a low number of protein identifications and there is much room to boost proteins identification by computational methods. In this study, we present a novel framework DeepSCP, utilizing deep learning to boost SCP coverage. DeepSCP constructs a series of features of peptide-spectrum matches (PSMs) by predicting the retention time based on the multiple SCP sample sets and fragment ion intensities based on deep learning, and predicts PSM labels with an optimized-ensemble learning model. Evaluation of DeepSCP on public and in-house SCP datasets showed superior performances compared with other state-of-the-art methods. DeepSCP identified more confident peptides and proteins by controlling q-value at 0.01 using target-decoy competition method. As a convenient and low-cost computing framework, DeepSCP will help boost single-cell proteome identification and facilitate the future development and application of single-cell proteomics.

BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription.

OBJECTIVES: Studies have demonstrated that B lymphoma Mo-MLV insertion region 1 (BMI1) plays an important role in male reproductive function and the regulation of spermatogonia proliferation. However, whether BMI1 exerts a similarly important function in spermatocyte development remains unclear. METHODS: In this study, we investigated the role of BMI1 in spermatocyte development using a mouse spermatocyte-derived cell line (GC-2) and a Bmi1-knockout (KO) mouse model. RESULTS: We demonstrated that BMI1 promoted the proliferation and inhibited the apoptosis of GC-2 cells. Mechanistically, we presented in vitro and in vivo evidence showing that BMI1 binds to the promoter region of the forkhead box L1 (Foxl1) gene, sequentially driving chromatin remodeling and gene silencing. BMI1, which functions as a classical polycomb protein, was found to direct the transcriptional repression of Foxl1 through increasing the H2AK119ub level and reducing that of H3K4me3 in the promoter region of Foxl1. Our results further indicated that the knockdown of Foxl1 expression significantly enhanced cell proliferation via activating ß-catenin signaling in BMI1-deficient GC-2 cells. CONCLUSIONS: Collectively, our study revealed for the first time the existence of an epigenetic mechanism involving BMI1-mediated gene silencing in GC-2 cells development and provided potential targets for the treatment of male infertility.