Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

To identify new genes involved in male fertility, we have used a chemical treatment to induce random mutations within the DNA of mice and then screened for mice exhibiting male infertility. We then used genetic mapping techniques to identify which chromosome the responsible gene was situated within and DNA sequencing to identify the mutation, which was found in Katnal1. We tested the mutated copy of the gene in cells and determined it to be non-functional. We then used antibody detection to determine that, within the testis, KATNAL1 protein is expressed in the sperm-cell supporting Sertoli cells in both mice and humans. Examination of testicular tissue revealed that KATNAL1 functions to control Sertoli cell microtubule dynamics and retention of sperm during their maturation within the tubules of the testis; absence of KATNAL1 results in premature release of immature sperm and male infertility. The identification and characterisation of this gene, the first microtubule severing protein shown to function in Sertoli cells, will further our understanding of how male fertility is promoted, while also providing important information relevant to the development of male contraceptives.

Funding: This work was funded by independent UK Medical Research Council Programme Grants to LBS and JP ( www.mrc.ac.uk ). MKO and LO were funded by a project grant from the National Health and Medical Research Council ( www.nhmrc.gov.au ) of Australia. MKO is the recipient of an NHMRC Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Smith et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A novel ATPase protein has been named Katanin p60 subunit A-like1 (KATNAL1) when annotated in Ensembl ( www.ensembl.org : ENSMUSG00000041298), based upon its sequence homology to KATANIN p60 (66% identity, 78% conserved). Furthermore, recent in silico profile-profile matching and ab initio structure modelling using ROSETTA ( www.rosettadesigngroup.com ) [22] , along with recent over-expression studies in cell-lines [23] , [24] strongly suggests that KATNAL1 has a similar microtubule-severing role to KATANIN p60. In this paper we describe the identification of a mouse line homozygous for an ENU-induced null allele of Katnal1, and detail the first in vivo function ascribed to this novel microtubule severing protein.

Some inferences can be made through comparison to analogous systems elsewhere in the body. The SC cytoskeleton shares morphological, structural and functional properties with the neuronal cytoskeleton [4] . Within neurones, microtubule remodelling is facilitated in vivo by the ATPase microtubule severing proteins, SPASTIN [14] , [15] and KATANIN p60 [16] – [20] . These function to permit neuronal plasticity by controlling axonal growth through disruption of the microtubule lattice in an ATP-dependent reaction [16] , [17] , [20] , [21] . Given the similarities in cytoskeleton structure and plasticity between the cell-types it is logical to hypothesise the existence of a similar mechanism controlling microtubule remodelling within SCs.

Systemic treatment with chemical inhibitors of microtubule assembly such as colchicine, [8] , [9] , or promoters of microtubule assembly such as 2–5-Hexanedione [10] both induce germ cell loss from the seminiferous epithelium. Furthermore, studies specifically targeting microtubules within SCs, such as SC-specific over-expression of the microtubule nucleating protein, gamma-tubulin [11] , [12] , or SC-specific expression of a dominant-negative form of the microtubule plus end binding protein EB1 [13] induce similar germ cell loss from the seminiferous epithelium. Together these studies demonstrate that SC microtubule-dependent functions are extremely sensitive to aberrant microtubule remodelling. However, despite our detailed understanding of the significant roles microtubules play within the SCs to support male fertility, the molecular mechanisms that facilitate microtubule remodelling within SCs remain largely unknown.

The testis is a complex multi-cellular organ and it has long been known that cell-cell interactions between somatic and germ cells are essential for normal testis function [1] . During spermatogenesis, the somatic Sertoli cells (SCs), specialised polarised epithelial cells, form junctional complexes with germ cells and act to protect and support germ cells physically, metabolically, and immunologically during their maturation from diploid spermatogonia, through meiosis and during remodelling into mature spermatozoa [2] , [3] . The supporting roles undertaken by SCs are facilitated through an active cytoskeleton, with parallel arrays of microtubules arranged radially along the polarised luminal-basement membrane axis [4] , with their minus ends directed towards the apical surfaces [5] , [6] . These microtubules are involved in maintaining the shape of the highly branched and dynamic Sertoli cells, transporting and positioning organelles in the cytoplasm, and secreting seminiferous tubule fluid. Furthermore, microtubule-based transport machinery is coupled to intercellular junctions to ensure retention of spermatids to SCs, whilst also permitting translocation of spermatids in the seminiferous epithelium during remodelling (for review see [7] ).

(a) Western blot analysis and (b) quantification of stable (glu) and dynamic (tyr) α-tubulin on d22 testes from wild-type and homozygous mutant animals reveals a significant reduction in numbers of stable microtubules in testes from Katnal1 1H/1H mutants (n = 5–7). (c) Immunohistochemical localisation of glu-α-tubulin on corresponding d22 testis sections localises stable microtubules to SC cytoplasm (arrows) and developing spermatids and in Wild-type animals. Conversely stable microtubules are below detection limits in much of the SC cytoplasm in homozygous mutant testes. (WT1 = Wilms Tumour 1, SC-specific loading control).

The tubulin tyrosination cycle involves the cyclical removal and re-addition of the carboxy-terminal tyrosine residue of the α-tubulin [27] . Removal of the Tyrosine residue exposes the carboxy-terminal Glutamic acid. As a consequence of the kinetics of this system, tyrosinated (tyr)-tubulin is the main component of dynamic microtubules, whereas glutamic acid (glu)-tubulin is a marker of long-lived stable microtubules [28] . Because antibodies against glu-tubulin bind only the exposed carboxy-terminal, glu-tubulin provides an excellent surrogate marker for changes in numbers of stable microtubules. To examine the impact of loss of function of KATNAL1 on SC microtubule dynamics quantitative (Western blot) and spatial (immunohistochemical) analysis of dynamic and stable microtubules was undertaken at d22. Whilst tyr-tubulin microtubules were unaffected in testes from Katnal1 1H/1H animals, there was a statistically significant reduction in glu-tubulin in Katnal1 1H/1H testes ( Figure 9a, 9b ). Subsequent immunohistochemical localisation of glu-tubulin on d22 testis sections identified staining of similar intensity associated with developing spermatids in both WT and Katnal1 1H/1H animals ( Figure 9c ), suggesting that, at least in part, the observed reduction in glu-tubulin was likely associated with the reduction in total spermatid numbers in d22 Katnal1 1H/1H testes ( Table 1 ). However, in addition to spermatids, glu-tubulin was also widely detected throughout SCs in wild-type testes, yet fell below detection limits throughout the majority of cytoplasm of the SCs from Katnal1 1H/1H animals ( Figure 9c ).

At d35, immunohistochemical localisation of the Sertoli cell-specific isoform of beta-tubulin TUBB3 reveals an apparent disruption to the microtubule network in Katnal1 1H/1H testes, (a, b). In Wild-Type animals, KATNAL1 localisation (arrows) tracks the SC microtubule network from basal to apical regions (c, e). Conversely, the mutant KATNAL1 protein is restricted to the basal region of Sertoli cells in Katnal1 1H/1H animals (d, f). Images representative of stage VI of the spermatogenic cycle. Bars = 20 µm.

To establish the mechanism underlying the premature exfoliation of spermatids in the Katnal1 mutants, we examined colocalisation of KATNAL1 with a Sertoli cell-specific isoform of beta-tubulin TUBB3 [26] . Localisation of TUBB3 revealed an apparent disruption to the microtubule network in mutant testes compared to WT ( Figure 8a, 8b ). In testes from WT animals, KATNAL1 co-localises with SC microtubules throughout the basal and adluminal compartments of the SCs ( Figure 8a, 8c, 8e ), however, in Katnal1 1H/1H animals, the mutated KATNAL1 protein was restricted to the basal compartment of SCs ( figure 8d, 8f ).

Sections of epididymides from homozygous mutant and WT littermates were examined. In WT animals the epididymal lumen was filled with spermatozoa throughout the length of the epididymis (a, - boxed area enlarged in c). However, in homozygous Katnal1 1H/1H littermates, recognisable spermatozoa were almost absent, instead the caput and corpa epididymides contained immature, non-condensed, germ cells (b), which collected as a plug of proteinaceous cellular debris (*) in the cauda epididymis (b, boxed area enlarged in d). Bars = 20 µm.

To establish whether the significant reduction in spermatid number was the result of a reduction in production of spermatids, an increase in spermatid apoptosis, or the result of exfoliation of developing spermatids from the seminiferous epithelium, testes and epididymides from homozygous mutants and WT littermates were examined. Colorimetric immunohistochemistry using an antibody against the apoptotic marker Cleaved-Caspase-3 identified no difference in numbers of apoptotic germ cells in testis sections from mutant versus WT animals (n = 5, data not shown), thus increased spermatid apoptosis could not explain the observed phenotype. In WT animals the epididymal lumen was, as expected, filled with spermatozoa throughout its length ( Figure 7a, 7c ). However, in Katnal1 1H/1H littermates, recognisable spermatozoa were much reduced, and instead the caput and corpus epididymides contained exfoliated immature germ cells ( Figure 7b ) which collected as a plug of proteinaceous cellular debris in the cauda epididymis ( Figure 7d ). Together these data demonstrated that the primary phenotypic defect in Katnal1 1H/1H mutants is premature exfoliation of spermatids form the seminiferous epithelium.

(a) Testes from wild-type and Katnal1 1H/1H animals appear similar at day 22 when examined by light microscopy. However, at day 35 disruption to the seminiferous epithelium is apparent in mutant testes, consistent with a significant reduction in seminiferous tubule diameter. At day 70, the seminiferous epithelium in the Katnal1 1H/1H testis appears disorganised and elongate spermatids (arrowed in WT) are almost absent. (b) The apparent reduction in elongate spermatid numbers was confirmed (arrows) using an antibody against PGK1/2 which marks post-meiotic germ cells from step 8. (c) The reduction in elongate spermatids was associated with a clear reduction in apical ectoplasmic specialisations (marked by Espin) in Katnal1 1H/1H testes (arrows in +/+). (+/+ = Wild-type; neg = no-primary control; Bar = 100 µm unless stated).

At day 22 (early puberty) examination of testicular paraffin-sections revealed no gross histological differences between WT and homozygous null animals ( Figure 6 ), although a reduction in germ cells was apparent by stereological analysis (see below). However, at day 35 (mid-puberty), clear histological differences in the seminiferous epithelium were apparent ( Figure 6 ), including a significant reduction in seminiferous tubule diameter (p<0.001, n = 5, data not shown). At day 70, the seminiferous epithelium was severely disrupted with evidence of SC vacuolation and an apparent reduction in numbers of elongate spermatids ( figure 6a ). Quantification of SC and GC numbers at all three ages revealed no significant difference either in numbers of SCs or numbers of spermatogonia or spermatocytes at any age ( Table 1 ). However, at day 22, day 35 and day 70, a significant reduction in numbers of spermatids was observed ( Table 1 ). This reduction was confirmed histologically using antibodies against PGK1/2 which specifically marks spermatids from step eight onwards [25] ( Figure 6b ), and Espin, which marks the ectoplasmic specialisation junctional complex between SCs and spermatids ( Figure 6c ).

Once we had unequivocally established that KATNAL1 function had been ablated in this model, and localised the primary cellular site of disruption as the SC, comparative analyses against wild-type littermates were undertaken at three key ages spanning the first, and subsequent waves of spermatogenesis (days 22, 35 and 70), to establish the consequences of loss of KATNAL1 upon testicular function.

Having identified Katnal1 expression in the testis, an immunohistochemical time-course analysis of testis sections from several pre- and post-natal ages was undertaken using a custom polyclonal antibody designed to specifically detect KATNAL1 ( Figure S1 ). This confirmed expression of KATNAL1 from 15.5dpc onwards ( Figure 5b ) and that KATNAL1 protein is restricted to Sertoli cells within the seminiferous epithelium throughout postnatal life ( Figure 5c, 5d and data not shown). In post-natal life, KATNAL1 protein was observed throughout the Sertoli cell cytoplasm, with apparent concentration in a number of discrete foci, ( Figure 5c, 5d ). Immunohistochemical interrogation of adult human testis sections (n = 3) using the KATNAL1 antibody confirmed a similar expression pattern for KATNAL1 in both human and mouse testis ( Figure 5e, 5f ).

Testicular Katnal1 expression is detected from 15.5 dpc and is SC-specific within the seminiferous epithelium throughout life. (a) RT-PCR analysis identified Katnal1 gene expression in a wide panel of tissues including the testis. To establish the cellular localisation of KATNAL1, a custom antibody was designed to specifically recognise KATNAL1. Using this antibody, an immunohistochemical time-course analysis confirmed testicular Sertoli cell (arrowed) expression of KATNAL1 from 15.5dpc onwards (b), and that KATNAL1 protein is restricted to Sertoli cells of the seminiferous epithelium throughout postnatal life where it is localised at a number of discrete foci (c) (dotted box enlarged in (d)). Immunohistochemical interrogation of adult human testis sections using the KATNAL1 antibody (e) (dotted box enlarged in (f)), confirmed a similar localization for KATNAL1 in both human and mouse testis. (B = Brain, H = Heart, Lu = Lung, K = Kidney, Li = Liver, Sp = Spleen, Sv = Seminal Vesicle, T = Testis, O = Ovary, W = water control. Bars in (c, d, e and f) = 20 µm; Neg = peptide blocked control).

Given that the loss of function of KATNAL1 resulted in a testicular phenotype, we next conducted RT-PCR analysis, interrogating from exon eight to the 3′-untranslated region of Katnal1 (which amplifies both published splice variants that contain the functional ATPase AAA Core domain, www.ensembl.org ) on RNA taken from a panel of tissues of adult C57BL/6J mice, to establish the body-wide expression pattern of Katnal1. This analysis demonstrated that Katnal1 is widely expressed, with gene expression detectable in all tissues examined including brain, heart, lung, kidney, liver, spleen, seminal vesicles, and ovary, in addition to the predicted testicular expression ( Figure 5a ).

(a, b) Transduction of HT1080 cells with lentiviral particles containing the WT cDNA of Katnal1 under the control of the CMV promoter results in a significant increase in numbers of cells arrested in mitosis (arrows) 48 hours post-transduction. Conversely, numbers of cells in mitotic arrest does not differ from controls following transduction of the mutant Katnal1 1H cDNA. (c) The arrest of cells during mitosis is followed by a significant increase in cell death in cells transduced with WT but not mutated Katnal1 1H . Together these data confirm that the observed mutation in Katnal1 1H generates a loss of function allele. Bar = 50 µm (*** = p<0.001).

To empirically establish that this mutation has functional significance, we generated Lentiviral vectors expressing either the WT or Katnal1 1H cDNA under the control of the strong CMV promoter and transduced cells in vitro. Having shown that induced-expression of a functional KATNAL1 protein increases microtubule severing, we hypothesised that disruption to microtubule dynamics through over-expression of functional KATNAL1 would lead to cellular arrest during mitosis, followed by cell death. Indeed, over-expression of the WT KATNAL1 protein in HT1080 cells significantly increased both the number of cells in mitosis (p<0.0001) and significantly increased overall cell death compared to vehicle or control lentivirus when tested at 48 hours post-infection (p<0.001), whereas over-expression of mutant Katnal1 1H cDNA failed to induce any increase in number of cells in mitosis or any change in overall cell death relative to controls ( Figure 4 ). We therefore deduced that the observed male infertility phenotype in these mice resulted from a single base-pair change encoding a conserved residue of KATNAL1, and that Katnal1 1H is a recessive loss-of-function allele.

Hek293 cells were stably transfected with constructs encoding either KATANIN p60 or KATNAL1 protein. Separate wells were treated +/− tetracycline or vehicle for a period of 12 hours, fixed then stained for α-tubulin as a marker of microtubules. This demonstrated that, like KATANIN p60, KATNAL1 functions as a microtubule severing protein. Scale bars = 20 µm.

To establish whether KATNAL1 had similar function to KATANIN p60, HEK293T cells were stably transfected with cDNA for either Katanin p60 or Katnal1, and protein expression induced using a tetracycline-inducible promoter. Addition of tetracycline resulted in disruption of the cellular microtubule lattice in both Katanin p60 and Katnal1 transfected cells within 12 hours confirming KATNAL1 functions as a microtubule severing protein ( Figure 3 ).

The point mutation within Katnal1 1H generates a Leucine to Valine substitution at residue 286, within the conserved ATPase AAA-Core domain of the KATNAL1 protein ( Figure 2a ). Because this substitution appeared relatively conservative (both Leucine and Valine are branch-chained, hydrophobic amino acids), we undertook comparative genome analysis of KATNAL1 peptide sequence across diverse species ( www.ensembl.org ) with a view to establishing the significance of a Leucine residue at this position. The Leucine residue at this position in KATNAL1 was conserved in all species examined, suggesting that this residue has been under strong selection pressure for >400 million years ( Figure 2c ).

Genome-wide SNP linkage analysis identified a single region of distal chromosome 5 as the site of the causal mutation. Quantitative RT-PCR analysis and DNA sequencing analysis of genes within the critical region identified a single point mutational change (Thymine to Guanine) within exon seven of the gene encoding the putative microtubule severing protein Katnal1 (a, b) (The mutated allele was designated Katnal1 1H ). The point mutation within Katnal1 1H generates a Leucine to Valine substitution at residue 286, within the conserved ATPase AAA-Core domain of the KATNAL1 protein (a, b). (c) Comparative genome analysis of KATNAL1 peptide sequence across diverse species demonstrated complete conservation of the Leucine residue for at least 400 million years.

To identify the genetic lesion responsible for the observed reduction in testis weight and infertility, a genome-wide SNP linkage analysis was employed to identify homozygous regions of DNA derived from the initial ENU-treated C57BL/6J founder, in ten infertile G3 males. This highlighted a single genomic locus, covering 2 Mbp of distal chromosome 5 (between SNP rs6349247 and SNP rs13478592) common to all mutant males, which contained 17 candidate genes. To identify the causal gene, both gene expression analysis and DNA sequencing approaches were employed. Quantitative RT-PCR analysis of every splice-variant transcript from all 17 genes from WT, heterozygous and homozygous day 22 testes (onset of puberty) revealed no significant gene expression changes coincident with the infertility phenotype (n = 4–5 per genotype, data not shown). However, DNA sequencing of every exon from all 17 candidate genes within the critical region in both fertile and infertile males did identify a single homozygous point mutational change (Thymine to Guanine) within exon seven of the gene encoding the novel microtubule severing protein KATNAL1 ( Figure 2a, 2b ). This novel allele was designated Katnal1 1H . The mutation in Katnal1 1H serendipitously generated an HpyAV restriction-enzyme recognition site which was exploited for genotyping purposes (data not shown). This assay was predictive of phenotype with 100% accuracy in all animals tested (n = 186).

Infertile males were culled and examined at various ages to establish the phenotypic changes underlying the infertility trait. Infertile males displayed no significant difference in bodyweight, or ano-genital distance (AGD) when compared to WT littermates at post-natal days (day) 22, 35, and 70 (n = 5–18 per group, data not shown). However, a significant reduction in testis weight in mutant animals was noted ( Figure 1b, 1c ), which was first observed at day 35 and remained consistently 60% of wild-type weight when compared either in early adulthood or later (approximately one year) (p<0.001) ( Figure 1c ). Gross inspection revealed no other obvious phenotypic difference.

A screen for ENU-induced recessive mutations that cause male-specific infertility was undertaken. (a) In one pedigree (PED-JP5), six infertile males were identified through matings to a total of four or more CD1 females each. A significant reduction in testis weight in mutant animals was noted (b, c), which was first observed at day 35 and remained consistently 60% of wild-type weight when compared at both day 70 (adulthood) and at around one year of age (days 300–365) (c). (+/+ = Wild-type, m/m = recessive mutant, bar = 2 mm, ** p = 0.0022).

To identify novel genes important for the promotion of male fertility, a screen for recessive mutations that cause male-specific infertility was undertaken as part of a screen for developmental phenotypes (for details see Materials and Methods ). In one pedigree (PED-JP5), two G2 females out of the four tested gave rise to a total of six infertile males, confirmed through consecutive matings to a total of four or more CD1 females per male ( Figure 1a ). Subsequent pedigree analysis showed that the infertility trait had an autosomal-recessive mode of inheritance with complete penetrance and expressivity in homozygous mutant males (m/m). Conversely, heterozygous males and females of all genotypes were fertile (data not shown).

Discussion

Utilising random ENU mutagenesis, we have identified a new mouse line displaying male-specific infertility due to a point mutation in the highly conserved ATPase domain of the novel microtubule severing protein KATNAL1. We demonstrate that Katnal1 is expressed in testicular Sertoli cells from 15.5 dpc onwards, co-localises with SC microtubules in vivo, and that loss of function leads to male-specific infertility through disruption of microtubule dynamics and premature exfoliation of post-meiotic germ cells from the seminiferous epithelium. This is the first description of an in vivo function for this novel microtubule severing protein, a finding that has implications for our understanding of SC microtubule dynamics, the promotion of male fertility, and as a potential target for male contraceptive development.

The use of the chemical mutagen ENU to produce novel mouse models of male infertility is now well established [29], [30]. This approach has several advantages, not least as it requires no a priori information about the causal gene, which can be identified by a relatively straightforward combination of positional cloning and DNA sequencing. We utilised this approach to identify a mouse line displaying male-specific infertility, associated with a significant reduction in testis weight. However, the reduction in testis weight remained consistent through to approximately one year of age suggesting that the genetic lesion did not result in cumulative degeneration with aging, and thus that the infertility phenotype was likely to be due to repeated failure at a specific aspect of spermatogenesis.

Using both classical and molecular genetic analysis we identified Katnal1 as the gene responsible for the infertility phenotype. KATNAL1 derives from the same gene family as KATANIN p60 (66% identity, 78% conserved), a well described microtubule severing protein important in neuronal plasticity [18]. Functional katanin complexes are assembled from two distinct gene products, KATANIN p60 and KATANIN p80, which oligomerise to form homo-hexamers before combining to produce a functional protein [31]. The KATANIN p60 peptide has three functional domains, an N-terminal Microtubule Interacting and Trafficking (MIT) domain [22], an AAA ATPase domain [32], and a C-Terminal Vsp4_C domain, important in oligomerisation [32]. Interpro analysis (www.ebi.ac.uk/interpro) of KATNAL1 identified both the AAA ATPase domain and the Vsp4_C domain, based upon sequence homology, but the N-terminal primary sequence of KATNAL1 is less conserved when compared to KATANIN p60 suggesting KATNAL1 has a related, yet distinct function from KATANIN p60. Ab intitio structure modelling using ROSETTA confirmed the presence of a Microtubule Interacting and Transport (MIT) domain at the N-terminal of KATNAL1 [22], We (this study), and recently others [23], have now unequivocally confirmed that KATNAL1 functions as a microtubule severing protein.

Whilst the mutation in the Katnal11H allele results in a relatively conservative amino acid substitution (Leucine-Valine), comparative analysis across diverse species demonstrates the significance of the Leucine residue at this position, with complete conservation across 400 million years of evolution. The mutation is localised to the ATPase Core domain of the KATNAL1 peptide. Hydrolysis of ATP is known to be essential for microtubule severing function of the related KATANIN p60 [18], suggesting that the mutation in Katnal11H may result in a disruption to ATP hydrolysis function in KATNAL1. This requires further investigation. Nevertheless, both the recessive nature of the phenotype and our examination of protein function through in vitro over-expression studies support a ‘loss-of-function’ (LOF) model for the mutated Katnal11H allele.

KATNAL1 is widely expressed throughout many body systems. It is perhaps surprising therefore that no obvious phenotypic difference outside of the testis was apparent in Katnal11H/1H animals suggesting a degree of functional redundancy exists to support LOF of KATNAL1 in other body systems. Given the shared characteristics of SCs and neurones [4], and the extensive work on the related proteins KATANIN p60 [16], [17], [20], [33]–[35] and SPASTIN [36]–[39] in neurones, a further, detailed investigation, focused upon potential behavioural or neuronal endpoints in the Katnal11H/1H mutants is an important next step.

Within the testis, KATNAL1 is exclusively expressed in SCs in human and in mouse from 15.5dpc. KATANIN p60 has previously been implicated in both mitosis and female meiosis where it functions to increase the pool of short microtubules required for chromosome separation [40]–[43]. As KATNAL1 protein is present in SCs from prenatal life, we could hypothesise a similar role for KATNAL1 during mitotic division of SCs. However, we observed no difference in SC number at any age in the Katnal11H/1H mutant, suggesting KATNAL1 is dispensable for completion of SC mitosis. The role for KATNAL1 in SCs prior to puberty therefore remains to be established.

Further to this, no reduction in numbers of pre-meiotic germ cells was observed at any age suggesting the systems to functionally and nutritionally support spermatogonia and spermatocytes were not affected by loss of KATNAL1. Furthermore, the phenotype does not appear to be degenerative or cumulative over time, with SCs continuing to support the pre-meiotic germ cells throughout life suggesting that the phenotype represents repeated failure coincident with a specific stage of germ cell development. With no evidence of increased germ cell apoptosis in the Katnal11H/1H mutant, and with epididymides filled with immature germ cells, we can conclude that KATNAL1 function is essential for retention of spermatids during spermiogenesis, and that fundamental failure at this specific stage of spermatogenesis is the primary cause of the observed infertility phenotype.

KATNAL1 is not uniformly distributed throughout the SC cytoplasm, but is localised in discrete foci localised throughout the basal and adluminal regions. In wild-type animals, KATNAL1 co-localises with SC microtubules, however, in Katnal11H/1H animals, mutant KATNAL1 is restricted to the basal region of SCs. Whether this mis-localisation is the direct result of the mutation, or a consequence of other changes in SC function such as changes in cell polarity brought about by the LOF of KATNAL1 requires further investigation.

Microtubules play several important roles within Sertoli cells during spermatogenesis, including maintaining SC architecture, facilitate the secretion of tubule fluid, intracellular vesicle transport and the translocation of spermatids during maturation, each of which are reliant upon dynamics changes in microtubules during the differing stages of the spermatogenic cycle (for review see [7]). Severing not only promotes microtubule destruction, but is also important for the creation of new microtubules, as severing of longer microtubules generates “seeds” for further microtubule polymerization [44], [45]. Katnal11H/1H animals display a reduction in numbers of stable microtubules with SCs, suggesting KATNAL1 function is required in SCs to seed development of stable microtubules. This reduction in numbers of stable microtubules is likely to be a key factor in the premature exfoliation of immature spermatids from the seminiferous epithelium, perhaps simply because the numbers of microtubules attached to each spermatid are insufficient in numbers to ensure the spermatid remains attached, the transport of nutrients to the developing spermatids is inadequate, or perturbation of microtubule dynamics interferes with correct development of the apical ectoplasmic specialisation – which were obviously reduced in Katnal11H/1H testes. Future studies will focus upon detailing the molecular interactions between KATNAL1 and other factors promoting correct spermiogenesis.

In conclusion, identification of KATNAL1 as an essential promoter of male fertility and functional analysis of its role within testicular SC represents an important first step towards understanding the molecular mechanisms underlying SC microtubule remodelling. Such information will be of utility both for increasing our understanding of male infertility and the development of treatments and non-hormonal male contraceptives.