Scrutinizing the human TEX genes in the context of human male infertility.

BACKGROUND: Infertility affects around 15% of all couples worldwide and is increasingly linked to variants in genes specifically expressed in the testis. Well-established causes of male infertility include pathogenic variants in the genes TEX11, TEX14, and TEX15, while few studies have recently reported variants in TEX13B, TEX13C, FAM9A (TEX39A), and FAM9B (TEX39B). OBJECTIVES: We aimed at screening for novel potential candidate genes among the human TEX ("testis expressed") genes as well as verifying previously described disease associations in this set of genes. MATERIALS AND METHODS: To this end, we screened the exome sequencing data of 1305 men, including 1056 crypto- and azoospermic individuals, and determined cell-specific expression by analyzing testis-specific single-cell RNA sequencing data for genes with identified variants. To investigate the overarching role in male fertility, we generated testis-specific knockdown (KD) models of all 10 orthologous TEX genes in Drosophila melanogaster. RESULTS: We detected rare potential disease-causing variants in TEX10, TEX13A, TEX13B, TEX13C, TEX13D, ZFAND3 (TEX27), TEX33, FAM9A (TEX39A), and FAM9B (TEX39B), in 28 infertile men, of which 15 men carried variants in TEX10, TEX27, and TEX33. The KD of TEX2, TEX9, TEX10, TEX13, ZFAND3 (TEX27), TEX28, TEX30, NFX1 (TEX42), TEX261, and UTP4 (TEX292) in Drosophila resulted in normal fertility. DISCUSSION: Based on our findings, the autosomal dominant predicted genes TEX10 and ZFAND3 (TEX27) and the autosomal recessive predicted gene TEX33, which all three are conceivably required for germ cell maturation, were identified as novel potential candidate genes for human non-obstructive azoospermia. We additionally identified hemizygous loss-of-function (LoF) variants in TEX13B, TEX13C, and FAM9A (TEX39A) as unlikely monogenic culprits of male infertility as LoF variants were also found in control men. CONCLUSION: Our findings concerning the X-linked genes TEX13B, TEX13C, and FAM9A (TEX39A) contradict previous reports and will decrease false-positive reports in genetic diagnostics of azoospermic men.

Viscous Loading Regulates the Flagellar Energetics of Human and Bull Sperm.

The viscoelastic properties of the female reproductive tract influence sperm swimming behavior, but the exact role of these rheological changes in regulating sperm energetics remains unknown. Using high-speed dark-field microscopy, the flagellar dynamics of free-swimming sperm across a physiologically relevant range of viscosities is resolved. A transition from 3D to 2D slither swimming under an increased viscous loading is revealed, in the absence of any geometrical or chemical stimuli. This transition is species-specific, aligning with viscosity variations within each species' reproductive tract. Despite substantial drag increase, 2D slithering sperm maintain a steady swimming speed across a wide viscosity range (20-250 and 75-1000 mPa s for bull and human sperm) by dissipating over sixfold more energy into the fluid without elevating metabolic activity, potentially by altering the mechanisms of dynein motor activity. This energy-efficient motility mode is ideally suited for the viscous environment of the female reproductive tract.

Functional assessment of DMRT1 variants and their pathogenicity for isolated male infertility.

OBJECTIVE: To study the impact of Doublesex and mab-3-related transcription factor 1 (DMRT1) gene variants on the encoded protein's function and the variants' pathogenic relevance for isolated male infertility caused by azoospermia. DESIGN: This study established a novel luciferase assay for DMRT1 missense variants using 2 different target promotors and validated the assay by analyzing previously published variants associated with differences in sex development. SETTING: University genetics research institute and tertiary referral center for couples' infertility. PATIENT(S): Eleven infertile men with severely impaired spermatogenesis resulting in crypto- or azoospermia and carrying rare heterozygous missense variants in DMRT1 were identified within the Male Reproductive Genomics study. MAIN OUTCOME MEASURE(S): Luciferase assays with human DMRT1 variants to test functional effects on the CYP19A1 and Stra8 target promoters. RESULT(S): We first developed and refined luciferase assays to reliably test the functional impact of DMRT1 missense variants. Next, the assay was validated by analyzing 2 DMRT1 variants associated with differences in sex development, of which c.240G>C p.(Arg80Ser) displayed highly significant effects on both target promoters compared with the wild-type protein (-40% and +100%, respectively) and c.331A>G p.(Arg111Gly) had a significant effect on the Stra8 promoter (-76%). We then systematically characterized 11 DMRT1 variants identified in infertile men. The de novo variant c.344T>A p.(Met115Lys) showed a pronounced loss of function in both DMRT1 target promoters (-100% and -86%, respectively). Variants c.308A>G p.(Lys103Arg) and c.991G>C p.(Asp331His) showed a significant gain of function exclusively for the CYP19A1 promoter (+15% and +19%, respectively). Based on these results, 3 variants were reclassified according to clinical guidelines. CONCLUSION(S): The present study highlights the importance of functionally characterizing DMRT1 variants of uncertain clinical significance. Using luciferase assays for diagnostic purposes enables an improved causal diagnosis for isolated male infertility.

Unraveling the Kinematics of Sperm Motion by Reconstructing the Flagellar Wave Motion in 3D.

Sperm swim through the female reproductive tract by propagating a 3D flagellar wave that is self-regulatory in nature and driven by dynein motors. Traditional microscopy methods fail to capture the full dynamics of sperm flagellar activity as they only image and analyze sperm motility in 2D. Here, an automated platform to analyze sperm swimming behavior in 3D by using thin-lens approximation and high-speed dark field microscopy to reconstruct the flagellar waveform in 3D is presented. It is found that head-tethered mouse sperm exhibit a rolling beating behavior in 3D with the beating frequency of 6.2 Hz using spectral analysis. The flagellar waveform bends in 3D, particularly in the distal regions, but is only weakly nonplanar and ambidextrous in nature, with the local helicity along the flagellum fluctuating between clockwise and counterclockwise handedness. These findings suggest a nonpersistent flagellar helicity. This method provides new opportunities for the accurate measurement of the full motion of eukaryotic flagella and cilia which is essential for a biophysical understanding of their activation by dynein motors.

CRISPs Function to Boost Sperm Power Output and Motility.

Fertilization requires sperm to travel long distances through the complex environment of the female reproductive tract. Despite the strong association between poor motility and infertility, the kinetics of sperm tail movement and the role individual proteins play in this process is poorly understood. Here, we use a high spatiotemporal sperm imaging system and an analysis protocol to define the role of CRISPs in the mechanobiology of sperm function. Each of CRISP1, CRISP2, and CRISP4 is required to optimize sperm flagellum waveform. Each plays an autonomous role in defining beat frequency, flexibility, and power dissipation. We thus posit that the expansion of the CRISP family from one member in basal vertebrates, to three in most mammals, and four in numerous rodents, represents an example of neofunctionalization wherein proteins with a common core function, boosting power output, have evolved to optimize different aspects of sperm tail performance.

Evaluation of telomere length and genotoxicity among asphalt associated workers.

There are contradictory reports about bitumen exposure and malignancy risk worldwide. Also, the evidence for genotoxicity risk among workers occupationally exposed to asphalt is insufficient. The study intended to evaluate particulate matter 10 (PM10) at the workplace and biomarkers of genotoxicity effects among a group of asphalt workers in and around Bangalore, India. This study involved a total of 107 participants (54 exposed group and 53 unexposed control group). To evaluate the genotoxicity, the urinary 8-OHdG and relative telomere length as oxidative damage while micronucleus (MN) assay for cytogenetic damage was carried out during the study. The majority of workers have reported health complaints and 57.4% of them were not using any personal protective equipments (PPE's). The level of PM10 detected was 104 ± 9.5 µg/m3 and 619 ± 22.7 µg/m3 in the road paving and asphalt mixing sites respectively. The biomonitoring study observed a highly significant (p = <0.001) increase in the level of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the exposed group (23.17 ± 8.65 ng/mg creatinine) compared to the control (13.6 ± 7.12 ng/mg creatinine), revealed age significant associated and non-smoking borderline significant associated for oxidative stress. The relative telomere length (TL) analysis revealed its highly significant (p = 0.004) reduction in the exposed group, adjusted mean 0.95 (95% CI 0.83-1.07) compared to the control 1.06 (95% CI 0.91-1.26). The job category (p = 0.028), non-smoking (p = 0.026), and tobacco chewing (p = 0.013) were associated with reduced relative TL in the asphalt exposed group. In cytogenotoxicity analysis, the mean micronucleus (MN) frequency per 100 cells in the exposed group (26.46 ± 19.8) was significantly (p = <0.001) increased over the control group (8.56 ± 7.18). Neither smoking habit nor age appeared to influence the MN frequencies in either group. In the present study, we have demonstrated genetic damage in workers occupationally exposed to asphalt and particulate matter, raising concern for an increased risk of malignancy in these workers.

Mitochondrial DNA copy number and cytogenetic damage among fuel filling station attendants.

Fuel filling attendants are constantly exposed to the complex mixture of gasoline and all refinery environments are probably carcinogenic for humans. These workers are considered as an unorganized group in India and unaware of the risk. The present study was focused to monitor workplace pollutants (particulate matter size 10 [PM10 µm], total volatile organic compound [VOC], and carbon monoxide [CO]), benzene exposure (phenol), and to evaluate their genotoxicity effect with reference to relative mitochondrial DNA copy number (MtDNAcn), 8-OHdG (8-hydroxy-2'-deoxyguanosine), and micronuclei (MN) frequency (%) among fuel filling attendants. This study recorded 318 ± 134 and 1,050 ± 260 µg/m3 time-weighted average concentration of PM10 and CO, respectively. However, total VOC levels recorded were below the detectable level (BDL) to 290 ± 50 µg/m3 . A total of 53 subjects (26 exposed and 27 control) participated in this study with similar sociodemographic information. It was noticed that fuel filling attendants were not using proper personal protective equipment (PPE) and are younger generation. The significantly (p = <.001) higher level of phenol, a metabolite of benzene, was detected in the exposed group. The significantly elevated level of urinary 8-OHdG (p = .01), MN frequency (p = .001), and relative MtDNAcn (p = .001) was observed in exposed group as compared to the control group. The study exemplify that workers were exposed to the benzene, workplace pollutant, and observed genotoxicity suggest malignancy risk. This study highlights the importance of biomonitoring in occupational settings to avoid malignancies. The possible engineering controls, frequent health check-ups, awareness about the risks, and PPE use can reduce health hazards.

The functions of CAP superfamily proteins in mammalian fertility and disease.

BACKGROUND: Members of the cysteine-rich secretory proteins (CRISPS), antigen 5 (Ag5) and pathogenesis-related 1 (Pr-1) (CAP) superfamily of proteins are found across the bacterial, fungal, plant and animal kingdoms. Although many CAP superfamily proteins remain poorly characterized, over the past decade evidence has accumulated, which provides insights into the functional roles of these proteins in various processes, including fertilization, immune defence and subversion, pathogen virulence, venom toxicology and cancer biology. OBJECTIVE AND RATIONALE: The aim of this article is to summarize the current state of knowledge on CAP superfamily proteins in mammalian fertility, organismal homeostasis and disease pathogenesis. SEARCH METHODS: The scientific literature search was undertaken via PubMed database on all articles published prior to November 2019. Search terms were based on following keywords: 'CAP superfamily', 'CRISP', 'Cysteine-rich secretory proteins', 'Antigen 5', 'Pathogenesis-related 1', 'male fertility', 'CAP and CTL domain containing', 'CRISPLD1', 'CRISPLD2', 'bacterial SCP', 'ion channel regulator', 'CatSper', 'PI15', 'PI16', 'CLEC', 'PRY proteins', 'ASP proteins', 'spermatogenesis', 'epididymal maturation', 'capacitation' and 'snake CRISP'. In addition to that, reference lists of primary and review article were reviewed for additional relevant publications. OUTCOMES: In this review, we discuss the breadth of knowledge on CAP superfamily proteins with regards to their protein structure, biological functions and emerging significance in reproduction, health and disease. We discuss the evolution of CAP superfamily proteins from their otherwise unembellished prokaryotic predecessors into the multi-domain and neofunctionalized members found in eukaryotic organisms today. At least in part because of the rapid evolution of these proteins, many inconsistencies in nomenclature exist within the literature. As such, and in part through the use of a maximum likelihood phylogenetic analysis of the vertebrate CRISP subfamily, we have attempted to clarify this confusion, thus allowing for a comparison of orthologous protein function between species. This framework also allows the prediction of functional relevance between species based on sequence and structural conservation. WIDER IMPLICATIONS: This review generates a picture of critical roles for CAP proteins in ion channel regulation, sterol and lipid binding and protease inhibition, and as ligands involved in the induction of multiple cellular processes.

Expression and purification of recombinant mouse CRISP4 using a baculovirus system.

Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

GLIPR1L1 is an IZUMO-binding protein required for optimal fertilization in the mouse.

BACKGROUND: The sperm protein IZUMO1 (Izumo sperm-egg fusion 1) and its recently identified binding partner on the oolemma, IZUMO1R, are among the first ligand-receptor pairs shown to be essential for gamete recognition and adhesion. However, the IZUMO1-IZUMO1R interaction does not appear to be directly responsible for promoting the fusion of the gamete membranes, suggesting that this critical phase of the fertilization cascade requires the concerted action of alternative fusogenic machinery. It has therefore been proposed that IZUMO1 may play a secondary role in the organization and/or stabilization of higher-order heteromeric complexes in spermatozoa that are required for membrane fusion. RESULTS: Here, we show that fertilization-competent (acrosome reacted) mouse spermatozoa harbor several high molecular weight protein complexes, a subset of which are readily able to adhere to solubilized oolemmal proteins. At least two of these complexes contain IZUMO1 in partnership with GLI pathogenesis-related 1 like 1 (GLIPR1L1). This interaction is associated with lipid rafts and is dynamically remodeled upon the induction of acrosomal exocytosis in preparation for sperm adhesion to the oolemma. Accordingly, the selective ablation of GLIPR1L1 leads to compromised sperm function characterized by a reduced ability to undergo the acrosome reaction and a failure of IZUMO1 redistribution. CONCLUSIONS: Collectively, this study characterizes multimeric protein complexes on the sperm surface and identifies GLIPRL1L1 as a physiologically relevant regulator of IZUMO1 function and the fertilization process.