Spermatogenesis-associated Protein Research Articles

P-085 Reactive molecules drive sperm capacitation via protein posttranslational modifications

Abstract Study question Are S-sulfenylation and S-nitrosylation, two essential posttranslational modifications (PTMs), increased during sperm capacitation? Which proteins are subjected to S-sulfenylation? Summary answer S-sulfenylation and S-nitrosylation increase during sperm capacitation. S-sulfenylation affects certain proteins, including A-kinase anchor protein 13, Fibrilline-1, and Spermatogenesis-associated protein 7. What is known already There is a significant increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) production within sperm capacitation. In contrast to tyrosine phosphorylation, a well-established mark of sperm capacitation, ROS, and RNS lead to PTMs themselves and modify thiol groups of cysteines. Despite these facts, the dynamics of ROS/RNS-derived PTMs during sperm capacitation remain unknown. Therefore, we consider both S-nitrosylation and S-sulfenylation of sperm proteins as novel modifications associated with the capacitation. Study design, size, duration C57BL6 mouse sperm were isolated from the caudae epididymae of 12-20-week-old males (n = 5). The study compared non-capacitated and capacitated spermatozoa using proteomic profiling. Participants/materials, setting, methods Sperm proteins were extracted into RIPA buffer. The protein lysate was incubated with 5mM dimedone, a hapten that specifically labels S-sulfenylated proteins, and immunogenically detected with an anti-dimedone antibody (Merk, Germany). Alternatively, S-nitrosylated proteins were detected by the anti-nitrosocysteine antibody (Merk, Germany). S-sulfenylated and S-nitrosylated proteins were visualized by western blotting. S-sulfenylated proteins were simultaneously identified by mass spectrometry. Main results and the role of chance We demonstrated an increase in S-sulfenylation and S-nitrosylation along with sperm capacitation. In addition, both S-sulfenylation and S-nitrosylation are positively correlated with tyrosine phosphorylation. Using mass spectrometry, we identified S-sulfenylated proteins, mostly belonging to the cytoskeleton: A-kinase anchor protein 13, Fibrilline-1, Spermatogenesis-associated protein 7, and Rab-like protein 2A. Limitations, reasons for caution The limitation of this study is the instability of PTMs. ROS/RNS-derived PTMs hypothetically arise and cease during protein sample preparation. The experiment was limited to one animal model. Further experiments should be performed on other mammalian models. Wider implications of the findings Studying novel sperm PTMs and their mechanism of action may improve the diagnosis of male infertility. Further, it could bring a novel approach to modulate the fertilizing ability of sperm used for in vitro fertilization. Trial registration number 260 536

The SPATA2/CYLD pathway contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy

Ferroptosis is an iron-dependent cell death and contributes to doxorubicin-induced cardiotoxicity, but the mechanisms behind intracellular iron overload in cardiomyocyte after administration of doxorubicin remain largely unknown. Ferritinophagy is a selective type of autophagy and could be a novel source for intracellular free iron. Spermatogenesis-associated protein 2 (SPATA2), a member of the TNF signaling pathway, can recruit cylindromatosis (CYLD, a deubiquitinating enzyme) to regulate cell death. This study aims to explore whether ferritinophagy is the source for intracellular iron overload in cardiomyocyte upon doxorubicin treatment and whether the SPATA2/CYLD pathway is involved in regulation of nuclear receptor coactivator 4 (NCOA4) level, the selective cargo receptor for ferritinophagy. The C57BL/6J mice were subjected to a single injection of doxorubicin, which showed the compromised cardiac functions, accompanied by the upregulation of SPATA2 and CYLD and the enhanced interaction between them, the increases in ferritinophagy (reflecting by increases in NCOA4 and ratio of LC3Ⅱ/LC3Ⅰ while decreases in NCOA4 ubiquitination and ferritin) and ferroptosis (reflecting by intracellular iron overload and increase of acyl-CoA synthetase long chain family member 4). Consistently, similar results were achieved in the cultured cardiomyocytes after incubation with doxorubicin. Knocked down of SPATA2 notably reduced doxorubicin-induced cardiomyocyte injury concomitant with the attenuated ferritinophagy and the decreased ferroptosis. Based on these observations, we conclude that a novel pathway of SPATA2/CYLD has been identified, which contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy through a mechanism involving the deubiquitination of NCOA4.

Characterization of MicroRNAs Associated with Reproduction in the Brown Planthopper, Nilaparvata lugens.

Insects have a robust capacity to produce offspring for propagation, and the reproductive events of female insects have been achieved at the molecular and physiological levels via regulatory gene pathways. However, the roles of MicroRNAs (miRNAs) in the reproductive development of the brown planthopper (BPH), Nilaparvata lugens, remain largely unexplored. To understand the roles of miRNAs in reproductive development, miRNAs were identified by Solexa sequencing in short-winged (SW) female adults of BPH. Small RNA libraries derived from three developmental phases (1 day, 3 days, and 5 days after emergence) were constructed and sequenced. We identified 905 miRNAs, including 263 known and 642 novel miRNAs. Among them, a total of 43 miRNAs were differentially expressed in the three developmental phases, and 14,568 putative targets for 43 differentially expressed miRNAs (DEMs) were predicted by TargetScan and miRanda. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the predicted miRNA targets illustrated the putative roles for these DEMs in reproduction. The progress events were annotated, including oogenesis, lipid biosynthetic process, and related pathways such as apoptosis, ABC transporters, and amino acid metabolism. Four highly abundant DEMs (miR-9a-5p, miR-34-5p, miR-275-3p, and miR-317-3p) were further screened, and miR-34-5p was confirmed to be involved in the regulation of reproduction. Overexpression of miR-34-5p via injecting its mimics reduced fecundity and decreased Vg expression. Moreover, target genes prediction for miR-34-5p showed they might be involved in 20E signaling cascades, apoptosis, and gonadal development, including hormone receptor 4 (HR4), caspase-1 (Cp-1), and spermatogenesis-associated protein 20 (SPATA20). These findings provide a valuable resource for future studies on the role of miRNAs in BPH reproductive development.

A large-scale genetic correlation scan between rheumatoid arthritis and human plasma protein.

AimsThe aim of this study was to explore the genetic correlation and causal relationship between blood plasma proteins and rheumatoid arthritis (RA).MethodsBased on the genome-wide association studies (GWAS) summary statistics of RA from European descent and the GWAS summary datasets of 3,622 plasma proteins, we explored the relationship between RA and plasma proteins from three aspects. First, linkage disequilibrium score regression (LD score regression) was applied to detect the genetic correlation between RA and plasma proteins. Mendelian randomization (MR) analysis was then used to evaluate the causal association between RA and plasma proteins. Finally, GEO2R was used to screen the differentially expressed genes (DEGs) between patients with RA and healthy controls.ResultsWe found that seven kinds of plasma proteins had genetic correlations with RA, such as Soluble Receptor for Advanced Glycation End Products (sRAGE) (correlation coefficient = 0.2582, p = 0.049), vesicle transport protein USE1 (correlation coefficient = 0.1337, p = 0.018), and spermatogenesis-associated protein 20 (correlation coefficient = 0.3706, p = 0.018). There was a significant causal relationship between sRAGE and RA. By comparing the genes encoding seven plasma proteins, we found that only USE1 was a DEG associated with RA.ConclusionOur study identified a set of candidate plasma proteins that showed signals correlated with RA. Since the results of this study need further experimental verification, they should be interpreted with caution. However, we hope that this paper will provide new insights for the discovery of pathogenic genes and RA pathogenesis in the future.Cite this article: Bone Joint Res 2022;11(2):134–142.

Spata2 Knockdown Exacerbates Brain Inflammation via NF-κB/P38MAPK Signaling and NLRP3 Inflammasome Activation in Cerebral Ischemia/Reperfusion Rats.

Brain inflammation induced by ischemic stroke is an important cause of secondary brain injury. The nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and NLRP3 inflammasome signaling are believed to drive the progression of brain inflammation. Spermatogenesis-associated protein2 (SPATA2) functions as a partner protein that recruits CYLD, a negative regulator of NF-κB signaling, to signaling complexes. However, the role of SPATA2 in the central nervous system remains unclear and whether it is involved in regulating inflammatory responses remains controversial. Rats were subjected to transient middle cerebral artery occlusion followed by reperfusion (tMCAO/R) surgery. The expression and localization of SPATA2 in the brain were investigated. The lentivirus-mediated shRNA was employed to inhibit SPATA2 expression. The inflammatory responses and outcomes of Spata2 knockdown were investigated. SPATA2 was co-localized with CYLD in neurons. SPATA2 expression was reduced in tMCAO/R rats. Spata2 knockdown resulted in increased microglia, increased expression of Tnfa, Il-1β, and Il-18, decreased Garcia score, and increased infarct volume. Spata2 knockdown resulted in the activation of P38MAPK and NLRP3 inflammasome and the increased activation of NF-κB signaling. These results suggest that SPATA2 plays a protective role against brain inflammation induced by ischemia/reperfusion injury. Therefore, SPATA2 could be a potential therapeutic target for treating ischemic stroke.

LC-MS/MS-Based Quantitative Proteomics Analysis of Different Stages of Non-Small-Cell Lung Cancer.

Lung cancer has a higher incidence rate and mortality rate than all other cancers. Early diagnosis and treatment of lung cancer remain a major challenge, and the 5-year survival rate of its patients is only 15%. Basic and clinical research, especially the discovery of biomarkers, is crucial for improving the diagnosis and treatment of lung cancer patients. To identify novel biomarkers for lung cancer, we used the iTRAQ8-plex labeling technology combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the serum and urine of patients with different stages of lung adenocarcinoma and healthy individuals. A total of 441 proteins were identified in the serum, and 1,161 proteins were identified in the urine. The levels of elongation factor 1-alpha 2, proteasome subunit alpha type, and spermatogenesis-associated protein increased significantly in the serum of patients with lung cancer compared with those in healthy controls. The levels of transmembrane protein 143, cadherin 5, fibronectin 1, and collectin-11 decreased significantly in the serum of patients with metastases compared with those of nonmetastatic lung cancer patients. In the urine of stage III and IV lung cancer patients, the prostate-specific antigen and prostatic acid phosphatase decreased significantly, whereas neutrophil defensin 1 increased significantly. The results of LC-MS/MS were confirmed by enzyme-linked immunosorbent assay (ELISA) for transmembrane protein 143, cadherin 5, fibronectin 1, and collectin-11 in the serum. These proteins may be a potential early diagnosis and metastasis biomarkers for lung adenocarcinoma. Furthermore, the relative content of these markers in the serum and urine could be used to determine the progression of lung adenocarcinoma and achieve accurate staging and diagnosis.

Circular RNA circSPATA6 Inhibits the Progression of Oral Squamous Cell Carcinoma Cells by Regulating TRAF6 via miR-182.

BackgroundOral squamous cell carcinoma (OSCC) has become a widely concerned social problem. Circular RNA spermatogenesis-associated protein 6 (circSPATA6) exhibited low expression in OSCC tissues, yet the regulatory mechanism of circSPATA6 remains vague.MethodsLevels of circSPATA6, linear SPATA6, microRNA-182 (miR-182), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Migration, invasion, cell cycle arrest, and apoptosis were assessed by Wound-healing, Matrigel invasion, and Flow cytometry assays. The binding relationship between miR-182 and circSPATA6 or TRAF6 was predicted by circRNA interactome or DIANA TOOL and then proved by the dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. TRAF6 protein level was measured by Western blot assay. The biological role of circSPATA6 on OSCC tumor growth was analyzed by xenograft tumor model in vivo. Exosomes were isolated and detected by differential centrifugation and a transmission electron microscope.ResultsCircSPATA6 and TRAF6 were declined, and miR-182 was elevated in OSCC cells. Functionally, circSPATA6 impeded migration and invasion, and facilitated cell cycle arrest and apoptosis of OSCC cells. Mechanistically, circSPATA6 could modulate TRAF6 expression through sponging miR-182. Moreover, circSPATA6 blocked tumor growth in the OSCC mice model. Exosomal circSPATA6 retarded the growth of OSCC cells.ConclusionCircSPATA6 curbed migration and invasion, and expedited cell cycle arrest and apoptosis in OSCC cells partly through regulating the miR-182/TRAF6 axis. These findings hinted at an underlying circRNA-targeted therapy for OSCC.

SPATA4 improves aging-induced metabolic dysfunction through promotion of preadipocyte differentiation and adipose tissue expansion.

Spermatogenesis‐associated protein 4 (SPATA4) is conserved across multiple species. However, the function of this gene remains largely unknown. In this study, we generated Spata4 transgenic mice to explore tissue‐specific function of SPATA4. Spata4 overexpression mice displayed increased subcutaneous fat tissue compared with wild‐type littermates at an old age, while this difference was not observed in younger mice. Aging‐induced ectopic fat distribution, inflammation, and insulin resistance were also significantly attenuated by SPATA4. In vitro, SPATA4 promoted preadipocyte differentiation through activation of the ERK1/2 and C/EBPβ pathway and increased the expression of adipokines. These data suggest SPATA4 can regulate lipid accumulation in a tissue‐specific manner and improve aging‐induced dysmetabolic syndromes. Clarifying the mechanism of SPATA4 functioning in lipid metabolism might provide novel therapeutic targets for disease interventions.

Comparative transcriptome analysis of three gonadal development stages reveals potential genes involved in gametogenesis of the fluted giant clam (Tridacna squamosa)

BackgroundGonad development and differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. However, the mechanisms underlying gonad development and gametogenesis remain unclear in Tridacna squamosa, a large-size bivalve of great ecological value. They are protandrous simultaneous hermaphrodites, with the male gonad maturing first, eventually followed by the female gonads. In this study, nine gonad libraries representing resting, male and hermaphrodite stages in T. squamosa were performed to identify the molecular mechanisms.ResultsSixteen thousand four hundred ninety-one unigenes were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 5091 and 7328 unigenes were assigned to Gene Ontology categories and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database, respectively. A total of 4763 differentially expressed genes (DEGs) were identified by comparing male to resting gonads, consisting of 3499 which were comparatively upregulated in males and 1264 which were downregulated in males. Six hundred-ninteen DEGs between male and hermaphroditic gonads were identified, with 518 DEGs more strongly expressed in hermaphrodites and 101 more strongly expressed in males. GO (Gene Ontology) and KEGG pathway analyses revealed that various biological functions and processes, including functions related to the endocrine system, oocyte meiosis, carbon metabolism, and the cell cycle, were involved in regulating gonadal development and gametogenesis in T. squamosa. Testis-specific serine/threonine kinases 1 (TSSK1), TSSK4, TSSK5, Doublesex- and mab-3-related transcription factor 1 (DMRT1), SOX, Sperm surface protein 17 (SP17) and other genes were involved in male gonadal development in Tridacna squamosal. Both spermatogenesis- (TSSK4, spermatogenesis-associated protein 17, spermatogenesis-associated protein 8, sperm motility kinase X, SP17) and oogenesis-related genes (zona pellucida protein, Forkhead Box L2, Vitellogenin, Vitellogenin receptor, 5-hydroxytryptamine, 5-hydroxytryptamine receptor) were simultaneously highly expressed in the hermaphroditic gonad to maintain the hermaphroditism of T. squamosa.ConclusionAll these results from our study will facilitate better understanding of the molecular mechanisms underlying giant clam gonad development and gametogenesis, which can provided a base on obtaining excellent gametes during the seed production process for giant clams.

Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway.

Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual's lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSCself-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.

Deep Proteome Profiling of Semen of Indian Indigenous Malnad Gidda (Bos indicus) Cattle.

Malnad Gidda is a dwarf indigenous cattle breed of India, which is known for its uniqueness of calving every year under a low input grazing system of rearing. Bulls of Malnad Gidda are known to be highly fertile even in stress conditions. However, the proteomic profiling of semen of this breed has not been investigated so far, which might provide a platform for a better understanding of its semen quality and male fertility. Therefore, we made an effort to characterize and quantify the proteome of seminal plasma and spermatozoa components of Malnad Gidda semen using a high-resolution mass spectrometry platform. We identified 2814 proteins from spermatozoa and 1974 proteins from the seminal plasma of this breed. Furthermore, >90% of proteins from each fraction were quantified using the intensity-based absolute quantification. We observed signal peptides in 33% of seminal plasma proteins, indicating their secretory nature. Gene Ontology analysis revealed their involvement in cytoskeletal assembly associated with sperm head, sperm motility, acrosome reaction, seminal plasma binding, and spermatogenesis-associated protein. An in-depth proteome profiling of semen of a unique indigenous cattle breed of India was carried out. Our findings could provide a reference for further studies on sperm functions, semen quality, and reproductive health of Bos indicus cattle. Mass spectrometry data generated in this study is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014172.

Identification and quantification of coding and long non-coding RNAs in stallion spermatozoa separated by density.

It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. Spermatozoa from five stallions were separated into more dense and less dense populations by density gradient centrifugation. Complementary DNA libraries were made from each of the ten total RNA samples after ribosomal RNA removal. Next-generation sequencing characterized the RNA populations in more and less dense spermatozoa. Quantitative reverse transcription-PCR was used to confirm differential expression of selected RNAs. Stallion spermatozoa contain 11215 RNAs, with the most prevalent RNA being a 1492 base long non-coding RNA. The levels of 159 RNAs were greater in more dense spermatozoa, while levels of seven other RNAs were greater in less dense spermatozoa. Quantitative reverse transcription-PCR confirmed the threefold greater levels of solute carrier family 26 member 8 (SLC26A8)mRNA in less dense spermatozoa, and sixfold and threefold greater expression levels of the SCP2 sterol binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1)mRNAs in more dense spermatozoa, respectively. We identified 11215 RNAs in stallion spermatozoa and 166 with differential expression between more dense and less dense fractions. Many prevalent RNAs were also found in bull, boar, and human spermatozoa. Many differentially expressed RNAs are known to be testis- or spermatozoa-specific. Our results may lead to identification of an RNA population in spermatozoa that is optimal for establishing successful pregnancies.

TNFα signalling predicts poor prognosis of patients with endometrial cancer.

Endometrial cancer (EC) is the most common gynaecologic tumour in the Western world. Previous studies have implicated an imbalance of oestrogens and progestogens in the development of most ECs, while the role of low-grade tissue inflammation remains largely unexplored. We investigated the impact of tumour necrosis factor alpha (TNFα), a central mediator of inflammation and spermatogenesis-associated protein 2 (SPATA2), a regulator of TNF receptor signalling, on clinical outcomes in EC. We evaluated TNFA and SPATA2 transcript levels in 239 EC patients and 25 non-malignant control tissues. Findings were validated in a cohort of 332 EC patients from The Cancer Genome Atlas (TCGA). Expression of TNFA and SPATA2 was increased in EC when compared with control tissues (P < 0.001). TNFA expression correlated with SPATA2 expression in non-malignant (P = 0.003, rS = 0.568) and EC tissue (P = 0.005, rS = 0.179). High TNFA and SPATA2 expression were associated with poor recurrence-free survival (RFS; P = 0.049 and P = 0.018) and disease-specific (P = 0.034 and P = 0.002) survival. Increased SPATA2 expression was also associated with decreased overall survival (OS; P = 0.013). In multivariate analysis, both TNFA and SPATA2 were predictors of clinical outcome. The impact of SPATA2 on RFS and OS could be validated in the TCGA cohort. Our study demonstrates that ECs exhibit a TNF signature which predicts clinical outcome. These findings indicate that TNF signalling modulates the course of EC, which could be therapeutically utilized in the future.

The guanine nucleotide exchange factor, Spata13, influences social behaviour and nocturnal activity

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light–dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.

Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction

BackgroundIncilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species.ResultsThe visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi.ConclusionsThis is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.

Tumor necrosis factor receptor modulator spermatogenesis-associated protein 2 is a novel predictor of outcome in ovarian cancer.

Inflammation plays a crucial role in the pathogenesis of cancer with tumor necrosis factor‐α (TNF‐α) as a key mediator. Recently, spermatogenesis‐associated protein 2 (SPATA2) was identified as a TNF receptor modulator which is required for TNF‐induced inflammation and apoptosis. The available data on TNF‐α in ovarian cancer (OC) are inconsistent, and SPATA2 is completely uncharacterized in tumorigenesis. We analyzed expression of SPATA2 and TNFA by quantitative real‐time polymerase chain reaction in tissues of 171 patients with low‐grade serous (LGSOC), high‐grade serous (HGSOC), endometrioid and clear cell OC compared with 28 non‐malignant control tissues. We stimulated OC cells (OVCAR3) with pro‐inflammatory (TNF‐α, interleukin [IL]‐1β) and mitogenic stimuli (IL‐6, lysophosphatidic acid) to establish a direct effect between inflammatory signaling and SPATA2. Pro‐inflammatory, but not mitogenic stimuli, potently induced SPATA2 expression in OC cells. Expression of TNFA and SPATA2 was higher in OC compared with control tissues (P = 0.010 and P = 0.001, respectively) and correlated with each other (P = 0.034, r s = 0.198). When compared with grade 1 cancers, SPATA2 was expressed higher in grade 2 and 3 tumors (P = 0.011) as well as in HGSOC compared with LGSOC (P = 0.024). Multivariate survival analyses revealed that OC with high SPATA2 expression were associated with reduced progression‐free survival (P = 0.048) and overall survival (P < 0.001). In conclusion, SPATA2 expression is regulated by TNF‐α and IL‐1β and is found to independently affect clinical outcome in OC patients. These data implicate a role of SPATA2 in tumorigenesis which warrants further investigation in gynecological malignancies.

Molecular characterization and expression profiling of 17-beta-hydroxysteroid dehydrogenase 2 and spermatogenesis associated protein 2 genes in endangered catfish, Clarias magur (Hamilton, 1822)

The 17-beta-hydroxysteroid dehydrogenase 2 (17β-HSD2) enzyme regulates steroid levels by the inactivation of estrogen and androgens. Spermatogenesis associated protein 2 (SPATA2) plays a vital role in spermatogenesis in vertebrates including fish. We report cloning and characterization of full cds of 17β-HSD2 and SPATA2 genes in Clarias magur. The full-length cDNA sequences of 17β-HSD2 and SPATA2 were 1187 bp (ORF 1125 bp) and 1806 bp (ORF 1524 bp) encoding 375 and 508 amino acids, respectively. Signal peptide analysis revealed SPATA2 is nonsecretory, while 17β-HSD2 is a secretory protein. Hydropathy profiles showed both proteins are hydrophilic in nature. Tissue distribution of both the genes revealed high mRNA level of SPATA2 in all tissues examined indicating its wide range of expression. 17β-HSD2 indicated higher expression in preparatory phase compared to spawning phase in ovary while it was opposite in case of testis. SPATA2 showed significantly higher expression in preparatory phase compared to spawning phase in both ovary and testis. Administration of OvatideTM (GnRH analog) resulted in upregulation of SPATA2 expression at 6 and 16 h post-injection while 17β-HSD2 showed upregulation only at 6 h post-injection. To the best of our knowledge, this is a first report on characterization of 17β-HSD2 and SPATA2 full-length cDNA in catfish.

Deletion of Spata2 by CRISPR/Cas9n causes increased inhibin alpha expression and attenuated fertility in male mice.

As somatic cells in the testis seminiferous tubule, Sertoli cells provide the medium for spermatogenesis. One of the important functions of Sertoli cells is synthesizing and secreting cell factors to affect the production of sperm; however, much of those molecular regulation mechanisms remain unknown. Here, we confirm the localization of protein SPATA2 (spermatogenesis-associated protein 2), which had previously been shown to be highly expressed in Sertoli cells of the adult mouse testis. To further conduct a functional study, we generated SPATA2 global knockout mice via use of the CRISPR/Cas9n gene editing technology. The 120-day-old knockout mice testes showed almost a 40% decrease in size and weight and variations in the histomorphology of the seminiferous epithelium, with a 40% decrease in sperm count. Further examination revealed that the proliferation of germ cells in the seminiferous tubules was attenuated by 28%. In addition, we found that SPATA2 deletion led to an approximately 70% increase in the inhibin alpha-subunit mRNA and protein level in the testes compared to that of wild-type mice. Our data revealed the impact of SPATA2 on male fertility and suggested that SPATA2 ensures the normal secretory function of Sertoli cells.

A critical role of solute carrier 22a14 in sperm motility and male fertility in mice.

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14−/− cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14−/− spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14−/− sperm cells: the annulus, a ring-like structure at the mid-piece–principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.

SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling

SummaryThe linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.