Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity.

The development and function of male gametes is dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a crucial role in this process. Here, we sought to elucidate the roles of spastin, an as-yet-unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a crucial role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis and, commonly, a catastrophic loss of nuclear integrity. This work defines an essential role for spastin in regulating microtubule dynamics during spermatogenesis, and is of potential relevance to individuals carrying spastin variants and to the medically assisted reproductive technology industry.

The katanin A-subunits KATNA1 and KATNAL1 act co-operatively in mammalian meiosis and spermiogenesis to achieve male fertility.

Katanins, a class of microtubule-severing enzymes, are potent M-phase regulators in oocytes and somatic cells. How the complex and evolutionarily crucial, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit KATNA1 and its close paralogue KATNAL1 together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagellum development, manchette regulation and sperm-epithelial disengagement. Finally, using proteomic approaches, we define the KATNA1, KATNAL1 and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal that the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for 'customised cutting' via neofunctionalisation and protective buffering via gene redundancy.

Epsilon tubulin is an essential determinant of microtubule-based structures in male germ cells.

Alpha, beta, and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon, and zeta, however, remain poorly understood and their requirement in mammalian development untested. Herein we have used a spermatogenesis model to define epsilon tubulin (TUBE1) function in mice. We show that TUBE1 is essential for the function of multiple complex microtubule arrays, including the meiotic spindle, axoneme and manchette and in its absence, there is a dramatic loss of germ cells and male sterility. Moreover, we provide evidence for the interplay between TUBE1 and katanin-mediated microtubule severing, and for the sub-specialization of individual katanin paralogs in the regulation of specific microtubule arrays.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Zinc finger RNA binding protein 2 (ZFR2) is not required for male fertility in the mouse.

BACKGROUND: Thousands of genes are expressed during spermatogenesis and male infertility has a strong genetic component. Within this study, we focus on the role of Zfr2 in male fertility, a gene previously implicated in human male fertility. To date, very little is known about the role of ZFR2 in either humans or mice. To this end, the requirement for ZFR2 in male fertility was assessed using a knockout mouse model. RESULTS: Zfr2 was found to be expressed in the testes of both humans and mice. Deletion of Zfr2 was achieved via removal of exon 2 using CRISPR-Cas9 methods. The absence of Zfr2 did not result in a reduction in any fertility parameters assessed. Knockout males were capable of fostering litter sizes equal to wild type males, and there were no effects of Zfr2 knockout on sperm number or motility. We note Zfr2 knockout females were also fertile. CONCLUSIONS: The absence of Zfr2 alone is not sufficient to cause a reduction in male fertility in mice.

DDB1- and CUL4-associated factor 12-like protein 1 (Dcaf12l1) is not essential for male fertility in mice.

Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men.

The Sertoli cell expressed gene secernin-1 (Scrn1) is dispensable for male fertility in the mouse.

BACKGROUND: Male infertility is a prevalent clinical presentation for which there is likely a strong genetic component due to the thousands of genes required for spermatogenesis. Within this study we investigated the role of the gene Scrn1 in male fertility. Scrn1 is preferentially expressed in XY gonads during the period of sex determination and in adult Sertoli cells based on single cell RNA sequencing. We investigated the expression of Scrn1 in juvenile and adult tissues and generated a knockout mouse model to test its role in male fertility. RESULTS: Scrn1 was expressed at all ages examined in the post-natal testis; however, its expression peaked at postnatal days 7-14 and SCRN1 protein was clearly localized to Sertoli cells. Scrn1 deletion was achieved via removal of exon 3, and its loss had no effect on male fertility or sex determination. Knockout mice were capable of siring litters of equal size to wild type counterparts and generated equal numbers of sperm with comparable motility and morphology characteristics. CONCLUSIONS: Scrn1 was found to be dispensable for male fertility, but this study identifies SCRN1 as a novel marker of the Sertoli cell cytoplasm.

HENMT1 is involved in the maintenance of normal female fertility in the mouse.

PIWI-interacting small RNAs (piRNAs) maintain genome stability in animal germ cells, with a predominant role in silencing transposable elements. Mutations in the piRNA pathway in the mouse uniformly lead to failed spermatogenesis and male sterility. By contrast, mutant females are fertile. In keeping with this paradigm, we previously reported male sterility and female fertility associated with loss of the enzyme HENMT1, which is responsible for stabilising piRNAs through the catalysation of 3'-terminal 2'-O-methylation. However, the Henmt1 mutant females were poor breeders, suggesting they could be subfertile. Therefore, we investigated oogenesis and female fertility in these mice in greater detail. Here, we show that mutant females indeed have a 3- to 4-fold reduction in follicle number and reduced litter sizes. In addition, meiosis-II mutant oocytes display various spindle abnormalities and have a dramatically altered transcriptome which includes a down-regulation of transcripts required for microtubule function. This down-regulation could explain the spindle defects observed with consequent reductions in litter size. We suggest these various effects on oogenesis could be exacerbated by asynapsis, an apparently universal feature of piRNA mutants of both sexes. Our findings reveal that loss of the piRNA pathway in females has significant functional consequences.

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.

Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse.

The katanin microtubule-severing proteins are essential regulators of microtubule dynamics in a diverse range of species. Here we have defined critical roles for the poorly characterised katanin protein KATNAL2 in multiple aspects of spermatogenesis: the initiation of sperm tail growth from the basal body, sperm head shaping via the manchette, acrosome attachment, and ultimately sperm release. We present data suggesting that depending on context, KATNAL2 can partner with the regulatory protein KATNB1 or act autonomously. Moreover, our data indicate KATNAL2 may regulate δ- and ε-tubulin rather than classical α-ß-tubulin microtubule polymers, suggesting the katanin family has a greater diversity of function than previously realised. Together with our previous research, showing the essential requirement of katanin proteins KATNAL1 and KATNB1 during spermatogenesis, our data supports the concept that in higher order species the presence of multiple katanins has allowed for subspecialisation of function within complex cellular settings such as the seminiferous epithelium.

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.

An optimised STAPUT method for the purification of mouse spermatocyte and spermatid populations.

The purification of individual male germ cell populations is integral for the molecular and biochemical characterisation of specific spermatogenic phases. Although a number of more contemporary techniques have been developed, velocity sedimentation using the STAPUT method remains as a gold standard for this purpose. The gentle nature of the technique, wherein germ cell subpopulations are separated by sedimentation at unit gravity, results in the isolation of viable and high-purity cells. We provide an updated and simplified step-by-step version of the STAPUT protocol for the purification of mouse male germ cells. As per the original method, the protocol described herein allows for the purification of mouse spermatocyte and round spermatids, however it also allows for successful purification of elongating, and elongated spermatid populations, and is optimised for the preservation of cellular ultrastructure. This method yields sufficient numbers of high-purity cells from one adult mouse for RNA or protein extraction or for immunolocalisation studies.

Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function.

STUDY QUESTION: What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility? SUMMARY ANSWER: While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function. WHAT IS KNOWN ALREADY: CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4. STUDY DESIGN, SIZE, DURATION: In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype. MAIN RESULTS AND THE ROLE OF CHANCE: While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis. LIMITATIONS REASONS FOR CAUTION: Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated. WIDER IMPLICATIONS OF THE FINDINGS: The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

LRGUK1 is part of a multiprotein complex required for manchette function and male fertility.

Infertility occurs in 1 in 20 young men and is idiopathic in origin in most. We have reported that the leucine-rich repeat (LRR) and guanylate kinase-like domain containing, isoform (LRGUK)-1 is essential for sperm head shaping, via the manchette, and the initiation of sperm tail growth from the centriole/basal body, and thus, male fertility. Within this study we have used a yeast 2-hybrid screen of an adult testis library to identify LRGUK1-binding partners, which were then validated with a range of techniques. The data indicate that LRGUK1 likely achieves its function in partnership with members of the HOOK family of proteins (HOOK-1-3), Rab3-interacting molecule binding protein (RIMBP)-3 and kinesin light chain (KLC)-3, all of which are associated with intracellular protein transport as cargo adaptor proteins and are localized to the manchette. LRGUK1 consists of 3 domains; an LRR, a guanylate kinase (GUK)-like and an unnamed domain. In the present study, we showed that the GUK-like domain is essential for binding to HOOK2 and RIMBP3, and the LRR domain is essential for binding to KLC3. These findings establish LRGUK1 as a key component of a multiprotein complex with an essential role in microtubule dynamics within haploid male germ cells.-Okuda, H., DeBoer, K., O'Connor, A. E., Merriner, D. J., Jamsai, D., O'Bryan, M. K. LRGUK1 is part of a multiprotein complex required for manchette function and male fertility.

LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility.

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.

RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

An essential role for katanin p80 and microtubule severing in male gamete production.

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.

Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition.

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans.

Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1-/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility.