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Background: Asthenoteratospermia with multiple morphological abnormalities in the sperm flagella (MMAF) is a significant cause of male infertility. WDR19 is a core component in the IFT-A complex and has a critical role in intraflagellar transport. However, the role of WDR19 mutations in male infertility has yet to be examined., Methods and Results: We performed whole exome sequencing (WES) for 65 asthenoteratospermia individuals and identified a proband who carried a homozygous WDR19 (c.A3811G, p.K1271E) mutation from a consanguineous family. Systematic examinations, including CT scanning and retinal imaging, excluded previous ciliopathic syndromes in the proband. Moreover, semen analysis of this patient showed that the progressive rate decreased to zero, and the sperm flagella showed multiple morphological abnormalities. Scanning and transmission electron microscopy assays indicated that the ultrastructure of sperm flagella in the patient was completely destroyed, while immunofluorescence revealed that WDR19 was absent from the sperm neck and flagella. Moreover, IFT140 and IFT88, predicted to interact with WDR19 directly, were mis-allocated in the WDR19-mutated sperm. Notably, the MMAF subject harboring WDR19 variant and his partner successfully achieved clinical pregnancy through intracytoplasmic sperm injection (ICSI)., Conclusions: We identified WDR19 as a novel pathogenic gene for male infertility caused by asthenoteratospermia in the absence of other ciliopathic phenotypes, and that patients carrying WDR19 variant can have favorable pregnancy outcomes following ICSI.

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Purpose: To identify the genetic cause of patients with primary ciliary dyskinesia (PCD) and male infertility from two unrelated Han Chinese families., Methods: We conducted whole-exome sequencing of three individuals with PCD and male infertility from two unrelated Chinese families, and performed a targeted look-up for DNAAF6 variants in our previously reported cohort of 442 individuals (219 with isolated oligoasthenospermia and 223 fertile controls). Ultrastructural and immunostaining analyses of patients' spermatozoa were performed. The pathogenicity of the variants was validated using patient's spermatozoa and HEK293T cells. Intracytoplasmic sperm injection (ICSI) treatment was conducted in two patients., Results: We identified one novel hemizygous frameshift variant (NM_173494, c.319_329del: p.R107fs) of DNAAF6 gene (previously named PIH1D3) in family 1 and one novel hemizygous missense variant (c.290G>T: p.G97V) in family 2. No hemizygous deleterious variants in DNAAF6 were detected in the control cohort of 442 individuals. Ultrastructural and immunostaining analyses of patients' spermatozoa showed the absence of outer and inner dynein arms in sperm flagella. Both variants were proven to lead to DNAAF6 protein degradation in HEK293T cells. Both patients carrying DNAAF6 variants underwent one ICSI cycle and delivered one healthy child each., Conclusion: We identified novel DNAAF6 variants causing male infertility and PCD in Han Chinese patients. This finding extended the spectrum of variants in DNAAF6 and revealed new light on the impact of DNAAF6 variants in sperm flagella.

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PVL has a financial interest in YourChoice Therapeutics, Inc., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases., Methods and Result: We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65 , encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65 -mutated male mice were consistent with human MMAF., Conclusions: Our experimental observations performed on both human subjects and on Cfap65 -mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis., (© 2019 Blackwell Verlag GmbH.)

Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that SPEF2 mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in SPEF2 (8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in SPEF2 were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a Spef2 knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female Spef2 -/- mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of SPEF2 mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.

SEPT12 is a testis-specific gene involved in the terminal differentiation of male germ cells. SEPT12 protein is required for sperm head-tail formation and acts as a fundamental constituent of sperm tail annulus. In this study, we screened genetic variations in exons 5, 6, 7 of the SEPT12 and assessed the annulus status in teratozoospermic, globozoospermic, and patients with immotile short tail sperm. DNA sequencing was performed for 90 teratozoospermic and 30 normozoospermic individuals. Immunocytochemistry, transmission electron microscopy and western blotting were conducted to evaluate annulus status and the expression level of SEPT12 in patients with a distinct exonic variation (c.474G>A), respectively. Five polymorphisms identified within the desired regions of the SEPT12, among them c.474G>A had the potential to induce aberrant splicing results in the expression of a truncated protein. The annulus was detected in most of the spermatozoa from teratozoospermic and normozoospermic men with c.474G>A. In contrast, in the patient with short tail sperm defect carrying c.474G>A, 99% of spermatozoa were devoid of the annulus. Based on our findings there would be no association between exons 5, 6, 7 polymorphisms of the SEPT12 gene and the occurrence of mentioned disease but c.474G>A would be a predisposing factor in male infertility., (© 2019 Wiley Periodicals, Inc.)

Background: Male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated., Methods and Results: We conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 ( sperm flagellar 2 ) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2 -affected subjects., Conclusions: We identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P 2 Sperm from VSP -/- mice show more Ca 2+ influx upon capacitation than VSP +/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P 2 -sensitive K + channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca 2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P 2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P 2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P 2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P 2 distribution plays important roles in ion channel regulation as well as sperm motility., Competing Interests: The authors declare no competing interest.

Changes in calcium concentration along the sperm flagellum regulate sperm motility and hyperactivation, characterized by an increased flagellar bend amplitude and beat asymmetry, enabling the sperm to reach and penetrate the ovum (egg). The signalling pathways by which calcium increases within the flagellum are well established. However, the exact mechanisms of how calcium regulates flagellar bending are still under investigation. We extend our previous model of planar flagellar bending by developing a fluid-structure interaction model that couples the 3D motion of the flagellum in a viscous Newtonian fluid with the evolving calcium concentration. The flagellum is modelled as a Kirchhoff rod: an elastic rod with preferred curvature and twist. The calcium dynamics are represented as a 1D reaction-diffusion model on a moving domain, the flagellum. The two models are coupled assuming that the preferred curvature and twist of the sperm flagellum depend on the local calcium concentration. To investigate the effect of calcium on sperm motility, we compare model results of flagellar bend amplitude and swimming speed for three cases: planar, helical (spiral with equal amplitude in both directions), and quasi-planar (spiral with small amplitude in one direction). We observe that for the same parameters, the planar swimmer is faster and a turning motion is more clearly observed when calcium coupling is accounted for in the model. In the case of flagellar bending coupled to the calcium concentration, we observe emergent trajectories that can be characterized as a hypotrochoid for both quasi-planar and helical bending., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

Background: The nerve growth factor (NGF), a member of the neurotrophins family, plays an important role not only in the nervous but also in other non-nervous systems such as the reproductive system. The aim of the paper is to study the in vitro effect of NGF on rabbit sperm functions., Methods: Ten adult rabbit bucks were collected five times, and pooled semen samples have been analysed. NGF was quantified in seminal plasma, and the distribution of NGF receptors (TrKA and p75NTR) in sperm was established. Moreover, the dose-effect of NGF on motility rate and track speed was evaluated. Successively, the effect of the neutralisation of NGF receptors was assessed to verify the specific role of each receptor. Untreated sperm were used as control., Results: Our study identified several interesting results: i) We detected NGF in seminal plasma and TrKA and p75NTR in sperm surface. In particular, TrKA is localised in the head and p75NTR in the midpiece and tail of rabbit sperm. ii) Once the optimal dose of NGF (100 ng/mL) was established, its addition affected both kinetics and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm. (iii) The neutralisation of TrKA and p75NTR receptors affected sperm traits differently. In particular, sperm speed, apoptosis and capacitation seemed mainly modulated via p75NTR receptor, whereas motile, live cells, necrosis and acrosome reaction were modulated via TrKA., Conclusion: For the first time, we showed the presence of p75NTR in rabbit sperm. NGF affects kinetic and other physiological traits of rabbit sperm. Most of these changes are modulated by the receptors involved (TrKA or p75NTR). Considering that some seminal disorders in human have been correlated with a lower NGF concentration and no studies have been done on the possible involvement of NGF receptors, these findings also provide new insights on human fertility.

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Multiple morphological anomalies of the sperm flagella (MMAF syndrome) is a severe male infertility phenotype which has so far been formally linked to the presence of biallelic mutations in nine genes mainly coding for axonemal proteins overexpressed in the sperm flagellum. Homozygous mutations in QRICH2, a gene coding for a protein known to be required for stabilizing proteins involved in sperm flagellum biogenesis, have recently been identified in MMAF patients from two Chinese consanguineous families. Here, in order to better assess the contribution of QRICH2 in the etiology of the MMAF phenotype, we analyzed all QRICH2 variants from whole exome sequencing data of a cohort of 167 MMAF-affected subjects originating from North Africa, Iran, and Europe. We identified a total of 14 potentially deleterious variants in 18 unrelated individuals. Two unrelated subjects, representing 1% of the cohort, carried a homozygous loss-of-function variant: c.3501C>G [p.Tyr1167Ter] and c.4614C>G [p.Tyr1538Ter], thus confirming the implication of QRICH2 in the MMAF phenotype and human male infertility. Sixteen MMAF patients (9.6%) carried a heterozygous QRICH2 potentially deleterious variant. This rate was comparable to what was observed in a control group (15.5%) suggesting that the presence of QRICH2 heterozygous variants is not associated with MMAF syndrome., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality., Competing Interests: The authors have declared that no competing interests exist.

Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster , individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. Here, we show that Combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes, and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Propose: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level., Methods: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells., Results: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells., Conclusions: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.

Study Question: Is there a role for lysine glutarylation (Kglu), a newly identified protein post-translational modification (PTM), in human sperm?, Summary Answer: Kglu occurs in several proteins located in the tail of human sperm, and it was reduced in asthenozoospermic (A) men and positively correlated with progressive motility of human sperm, indicating its important role in maintaining sperm motility., What Is Known Already: Since mature sperm are almost transcriptionally silent, PTM is regarded as an important pathway in regulating sperm function. However, only phosphorylation has been extensively studied in mature sperm to date. Protein lysine modification (PLM), a hot spot of PTMs, was rarely studied except for a few reports on lysine methylation and acetylation. As a newly identified PLM, Kglu has not been well characterized, especially in mature sperm., Study Design, Size, Duration: Sperm samples were obtained from normozoospermic (N) men and A men who visited the reproductive medical center between February 2016 and January 2018. In total, 61 N men and 59 A men were recruited to participate in the study., Participants/materials, Setting, Methods: Kglu was examined by immunoblotting and immunofluorescence assays using a previously qualified pan-anti-glutaryllysine antibody that recognizes glutaryllysine in a wide range of sequence contexts (both in histones and non-histone substrates) but not the structurally similar malonyllysine and succinyllysine. The immunofluorescence assay was imaged using laser scanning confocal microscopy and super-resolution structured illumination microscopy. Sperm motility parameters were examined by computer-assisted sperm analysis., Main Results and the Role of Chance: Kglu occurs in several proteins (20-150 kDa) located in the tail of human sperm, especially in the middle piece and the latter part of the principal piece. Sperm Kglu was modulated by regulatory systems (enzymes and glutaryl-CoA) similar to those in HeLa cells. The mean level of sperm Kglu was significantly reduced in A men compared with N men (P < 0.001) and was positively correlated with progressive motility (P < 0.001). The sodium glutarate-induced elevation of Kglu levels in A men with lower Kglu levels in sperm significantly improved the progressive motility (P < 0.001). Furthermore, the reduced sperm Kglu levels in A men was accompanied by an increase in sperm glutaryl-CoA dehydrogenase (a regulatory enzyme of Kglu)., Large Scale Data: N/A., Limitations, Reasons for Caution: Although the present study indicated the involvement of sperm Kglu in maintaining progressive motility of human sperm, the underlying mechanism needs to be investigated further., Wider Implications of the Findings: The findings of this study provide an insight into the novel role of Kglu in human sperm and suggest that abnormality of sperm PLMs may be one of the causes of asthenozoospermia., Study Funding/competing Interest(s): National Natural Science Foundation of China (81 771 644 to T.L.; 31 671 204 to X.Z. and 81 871 207 to H.C.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); Natural Science Foundation of Jiangxi, China (20171ACB21006 and 20161BAB204167 to T.L.; 20165BCB18001 to X.Z.). The authors have no conflicts of interest to declare., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)-enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding., Methods: Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay., Results: pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding., Conclusions: The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. Here, we identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective "9 + 2" structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD&#95;R&#95;PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15 KO mutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella., (Copyright © 2019 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

It has been shown that sperm flagellar motility is generated and modulated by metachronal sliding and two types of synchronous sliding of the outer doublet microtubules. Metachronal sliding propagates around the axoneme circumferentially from one doublet to another along the flagellum, whereas the two types of synchronous sliding occur synchronously throughout an extended region along the doublet microtubules. Oscillatory synchronous sliding occurs between most pairs of the nine doublet microtubules, whereas non-oscillatory synchronous sliding occurs between a specific pair of the nine doublet microtubules. These types of sliding coexist in the flagellum and create beat cycles of flagellar movement. The circumferential propagation of active sliding around the nine doublet microtubules in the metachronal sliding suggests that it is easier for a flagellum to produce helical waves than planar waves. Most sperm flagellar movements are planar to a certain extent. Therefore, mechanisms that modulate the helical waves into planar waves may be present. Structures such as the central pair microtubules in 9 + 2 sperm flagella and the fusion of fibrous-sheath and 3-,8-doublet microtubules in mammalian sperm flagella partition the nine outer doublet microtubules into two groups. Accordingly, the sliding between these two groups generates planar flagellar waves. A similar effect is caused by the sliding between a specific pair of the nine doublet microtubules of the non-oscillatory synchronous sliding, occurring in a Ca2+ concentration-dependent manner. These hard- and soft-wired systems produce the nearly planar flagellar waves required for the efficient propulsion of spermatozoa., (© The Author 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

Spermatogenesis involves a series of cellular transformations and thousands of regulated genes. Previously, we showed that the triploid fish (3nBY) cannot produce mature spermatozoa. In the present study, evaluation of the testis microstructure revealed that germ cells of 3nBY could develop into round spermatids, but then degenerated, resulting in male infertility. In this study we comparatively analysed the testis transcriptomes from 3nBY and its diploid parent YB and identified a series of differentially expressed genes (DEGs) that were enriched in the Wnt signalling pathway and the apoptotic and ubiquitin-mediated proteolysis processes in 3nBY. Gene ontology functional analyses revealed that some DEGs in 3nBY were directly associated with the process of gamete generation, development and sperm flagellum assembly. In addition, the expression of a number of genes related to meiosis (Inhibitor Of DNA Binding 2 (ID2), Ovo Like Transcriptional Repressor 1 (OVOL1)), mitochondria (ATP1b (ATPase Na+/K+ Transporting Subunit Beta 1), ATP2a (ATPase, Ca++ Transporting, Cardiac Muscle, Slow Twitch 2), ATP5a (ATP Synthase F1 Subunit Alpha), Mitochondrially Encoded Cytochrome C Oxidase I (COX1), NADH Dehydrogenase Subunit 4 (ND4)) and chromatin structure (Histone 1 (H1), Histone 2a (H2A), Histone 2b (H2B), Histone 3 (H3), Histone 4 (H4)) was lower in the testes of 3nBY, whereas the expression of genes encoding ubiquitin (Ubiquitin Conjugating Enzymes (UBEs), Ring Finger Proteins (RNFs)) and apoptosis (CASPs (Caspase 3, Caspase 7,Caspase 8), BCLs (B-Cell Lymphoma 3, B-Cell CLL/Lymphoma 2, B Cell CLL/Lymphoma 10)) proteins involved in spermatid degeneration was higher. These data suggest that the disrupted expression of genes associated with spermatogenesis and the increased expression of mitochondrial ubiquitin, which initiates cell apoptosis, may result in spermatid degeneration in male 3nBY. This study provides information regarding the potential molecular regulatory mechanisms underlying male infertility in polyploid fish.

Multiple morphological abnormalities of flagella (MMAF) are human reproduction disorders due to the dysplastic development of sperm flagella. The spermatozoa of men with MMAF manifest absent, short, coiled, bent, and/or irregular-caliber flagella. Previous studies revealed genetic contributions to human MMAF, but known MMAF-associated genes only explained approximately 50% MMAF cases. In this study, we employed human whole-exome sequencing for genetic analysis and identified biallelic mutations of CFAP251 (cilia- and flagella-associated protein 251, also known as WDR66) in three (5%) of 65 Han Chinese men with MMAF. All these CFAP251 mutations are loss-of-function. The population genome data suggested that these CFAP251 mutations are extremely rare (only heterozygous) or absent from human populations. Our functional assays of gene expression and immunofluorescence staining in a CFAP251-deficient man, together with previous experimental evidence from model organisms, suggested that CFAP251 is involved in flagellar functions. Our observations suggested that CFAP251 is associated with sperm flagellar development and human male infertility.

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 μM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca ++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility., (© 2017 Wiley Periodicals, Inc.)

Microbial association with a host using model system C. elegans have been widely studied based on factors such as host survival, the mode of infection, disease pathogenesis and the role of various players regulated during infection. The influence of pathogenic microorganism on reproduction and associated issues has not been explored fully. The present study focuses on the impact of bacterial infection on male reproductive parameters such as spermatogenesis and spermiogenesis, including physiological aspects like tail morphology defect and underlying molecular mechanisms that have been perturbed. In order to compare the consequence of infection caused by Gram positive and negative bacteria, Staphylococcus aureus and Vibrio alginolyticus were chosen as candidate pathogens, respectively. Microscopic observations revealed notable changes in tail morphology during 24 h of infection, as along with change in sperm size and activation. The Real Time-PCR results suggest the plausible down regulation of DBL-1/TGF-β pathway suggesting the morphological change in the tail. Shotgun proteomics further lead to the identification of MAG-1, Magonashi Protein a candidate regulatory player that affects spermatogenesis and HIF-1 that regulate during stress in both Gram positive and Gram negative infection. The protein-protein interaction with detected proteins revealed RACK-1 protein and mTOR pathway in S. aureus and V. alginolyticus respectively interacting with MAG-1 protein, which plays an important role in spermatogenesis termination in hermaphrodites during L4 to adult switch. This study paves a way to understand the candidate players that regulate reproduction during bacterial infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non-motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down-regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α-tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α-tubulin and protect the acetylation level of α-tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α-tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo-expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: The mechanism of intramanchette transport is crucial to the transformation of sperm tail and the nuclear condensation during spermiogenesis. Although few dysfunctional proteins could result in abnormal junction between the head and tail of spermatozoon, little is known about the genetic cues in this process., Objective: Based on patients with severe decapitated and decaudated spermatozoa (DDS) syndrome, the study aimed to validate whether new mutation exists on their Hook microtubule-tethering protein 1 ( HOOK1 ) genes and follow their results of assisted reproduction treatment (ART)., Methods: 7 severe teratozoospermia patients with DDS (proportion >95%) and three relative members in one pedigree were collected to sequence the whole genomic DNA. The fertilisation rates (FRs) of these patients were followed. Morphological observation and interspecies intracytoplasmic sperm injection (ICSI) assays were applied., Results: A novel missense mutation of A to G (p.Q286R) in patients with DDS (n=3/7) was found in the HOOK1 gene, which was inherited from the mother in one patient. This variant was absent in 160 fertile population-matched control individuals. Morphological observation showed that almost all the DDS broke into decaudated heads and headless tails at the implantation fossa or the basal plate. The clinical studies indicated that the mutation might cause reduced FRs on both ART (FR=18.07%) and interspecies ICSI (FR=16.98%)., Conclusions: An unreported mutation in HOOK1 gene was identified, which might be responsible to some patients with DDS. Further studies need to uncover the molecular mechanism of spermiogenesis for genomic therapy., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1), also named Na + /H + exchange regulatory cofactor 3 (NHERF3), which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, little is known about its role in sperm flagella formation and male fertility. IFT140 is a component of IFT-A complex. In mouse, it is highly expressed in the testis. Ift140 gene was inactivated specifically in mouse spermatocytes/spermatids. The mutant mice did not show any gross abnormalities, but all were infertile and associated with significantly reduced sperm number and motility. Multiple sperm morphological abnormalities were discovered, including amorphous heads, short/bent flagella and swollen tail tips, as well as vesicles along the flagella due to spermiogenesis defects. The epididymides contained round bodies of cytoplasm derived from the sloughing of the cytoplasmic lobes and residual bodies. Knockout of Ift140 did not significantly affect testicular expression levels of selective IFT components but localization of IFT27 and IFT88, two components of IFT-B complex, was changed. Our findings demonstrate that IFT140 is a key regulator for male fertility and normal spermiogenesis in mice. It not only plays a role in sperm flagella assembling, but is also involved in critical assembly of proteins that interface between the germ cell plasma and the Sertoli cell., (© 2017 Wiley Periodicals, Inc.)

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies., (© 2017 Wiley Periodicals, Inc.)

Study Question: What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility?, Summary Answer: FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility., What Is Known Already: Some testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function., Study Design, Size, Duration: FAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients., Participants/materials, Setting, Methods: Testes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility., Main Results and the Role of Chance: Our results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001)., Large Scale Data: N/A., Limitations, Reasons for Caution: The sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility., Wider Implications of the Findings: This is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility., Study Funding/competing Interests: This study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the 'spermatozoidal activator factor' (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

Motility in sperm is driven by the flagella, the principal component of which is the axoneme. The microtubules which make up the 9 + 2 axoneme are composed of heterodimers of alpha and beta tubulins and undergo several post-translational modifications. We have earlier reported that HDAC6 functions as tubulin deacetylase in sperm and has a role in sperm movement. While exploring the specific tubulin acetyltransferase (TAT) in sperm, we observed the presence of Chromodomain Y-Like (CDYL), on the principal piece of rat spermatozoa which compelled us to explore its function in sperm. CDYL was observed to be colocalized with acetylated alpha-tubulin (Ac α Tubulin) in sperm flagella. Sperm axonemal fraction showed the presence of CDYL protein indicating its strong association with flagellar microtubules. Sequence alignment of CDYL chromo domain and Alpha tubulin acetyltransferase (αTAT1) revealed that of the 10 residues of αTAT1 known to be involved in α-tubulin binding, 5 residues were identical and 1 was conserved between the two proteins. Docking of CDYL chromo domain and α-tubulin showed that 6 of the 11 important binding residues of α-tubulin showed an interaction with CDYL chromo domain. The putative CDYL chromodomain -α-tubulin interaction was further confirmed by Microscale Thermophoresis. We further asserted the ability of recombinant CDYL and Sperm CDYL to acetylate soluble tubulin and microtubules in vitro. Acetylation of tubulin was increased over twofold in cells overexpressing CDYL. Thus, our studies convincingly demonstrate the ability of CDYL to moonlight as a tubulin acetyltransferase., (© 2017 Wiley Periodicals, Inc.)

The CatSper channel is known as one of the most important Ca²⁺ channels on the cell membrane of mammalian sperm and plays a key role in the motility, hyperactivation and fertilization function of sperm. The CatSper protein, expressed exclusively in the principal piece of the sperm tail, is composed of CatSper1-4 and 5 auxiliary unitsβ,γ,δ and ε, and has an essential part in the functional and structural domains of Ca²⁺as well as in the spatiotemporal regulation of the P-Tyr protein, sperm hyperactivation, efficient sperm migration in the oviduct, egg penetration, and normal fertility. Recent studies show that functional deficiency of CatSper seriously affects sperm function,and the loss of any one of its 9 subunits may lead to male reproductive dysfunction. This paper outlines recent advances in the studies of the CatSperprotein, focusing on its expression, location, structure, and regulation,as well as itsinfluence on sperm hyperactivation and male reproduction.

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Progressive retinal atrophy is a common cause of blindness in the dog and affects >100 breeds. It is characterized by gradual vision loss that occurs due to the degeneration of photoreceptor cells in the retina. Similar to the human counterpart retinitis pigmentosa, the canine disorder is clinically and genetically heterogeneous and the underlying cause remains unknown for many cases. We use a positional candidate gene approach to identify putative variants in the Hungarian Puli breed using genotyping data of 14 family-based samples (CanineHD BeadChip array, Illumina) and whole-genome sequencing data of two proband and two parental samples (Illumina HiSeq 2000). A single nonsense SNP in exon 2 of BBS4 (c.58A > T, p.Lys20*) was identified following filtering of high quality variants. This allele is highly associated ( P CHISQ = 3.425 e -14 , n = 103) and segregates perfectly with progressive retinal atrophy in the Hungarian Puli. In humans, BBS4 is known to cause Bardet-Biedl syndrome which includes a retinitis pigmentosa phenotype. From the observed coding change we expect that no functional BBS4 can be produced in the affected dogs. We identified canine phenotypes comparable with Bbs4 -null mice including obesity and spermatozoa flagella defects. Knockout mice fail to form spermatozoa flagella. In the affected Hungarian Puli spermatozoa flagella are present, however a large proportion of sperm are morphologically abnormal and <5% are motile. This suggests that BBS4 contributes to flagella motility but not formation in the dog. Our results suggest a promising opportunity for studying Bardet-Biedl syndrome in a large animal model., (Copyright © 2017 Chew et al.)

Spermatozoa sea urchin swimming behaviour is regulated by small peptides from the egg outer envelope. Speract, such a peptide, after binding to its receptor in Strongylocentrotus purpuratus sperm flagella, triggers a signaling pathway that culminates with a train of intracellular calcium oscillations, correlated with changes in sperm swimming pattern. This pathway has been widely studied but not fully characterized. Recent work on Arbacia punctulata sea urchin spermatozoa has documented the presence of the Ca 2+ CatSper channel in their flagella and its involvement in chemotaxis. However, if other calcium channels participate in chemotaxis remains unclear. Here, based on an experimentally-backed logical network model, we conclude that CatSper is fundamental in the S. purpuratus speract-activated sea urchin sperm signaling cascade, although other Ca 2+ channels could still be relevant. We also present for the first time experimental corroboration of its active presence in S. purpuratus sperm flagella. We argue, prompted by in silico knock-out calculations, that CatSper is the main generator of calcium oscillations in the signaling pathway and that other calcium channels, if present, have a complementary role. The approach adopted here allows us to unveil processes, which are hard to detect exclusively by experimental procedures.