A homozygous ARMC3 splicing variant causes asthenozoospermia and flagellar disorganization in a consanguineous family.

Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.

Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility.

BACKGROUND: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

MYCBPAP is a central apparatus protein required for centrosome-nuclear envelope docking and sperm tail biogenesis in mice.

The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.

The proximal centriole-like structure maintains nucleus-centriole architecture in sperm.

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.

Structure and Composition of Spermatozoa Fibrous Sheath in Diverse Groups of Metazoa.

The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.

A novel homozygous missense TTC12 variant identified in an infertile Pakistani man with severe oligoasthenoteratozoospermia and primary ciliary dyskinesia.

TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.

Novel mutations in LRRC23 cause asthenozoospermia in a nonconsanguineous family.

ABSTRACT: The cause of asthenozoospermia (AZS) is not well understood because of its complexity and heterogeneity. Although some gene mutations have been identified as contributing factors, they are only responsible for a small number of cases. Radial spokes (RSs) are critical for adenosine triphosphate-driven flagellar beating and axoneme stability, which is essential for flagellum motility. In this study, we found novel compound heterozygous mutations in leucine-rich repeat-containing protein 23 ( LRRC23 ; c.1018C>T: p.Q340X and c.881_897 Del: p.R295Gfs*32) in a proband from a nonconsanguineous family with AZS and male infertility. Diff-Quik staining and scanning electron microscopy revealed no abnormal sperm morphology. Western blotting and immunofluorescence staining showed that these mutations suppressed LRRC23 expression in sperm flagella. Additionally, transmission electron microscopy showed the absence of RS3 in sperm flagella, which disrupts stability of the radial spoke complex and impairs motility. Following in vitro fertilization and embryo transfer, the proband's spouse achieved successful pregnancy and delivered a healthy baby. In conclusion, our study indicates that two novel mutations in LRRC23 are associated with AZS, but successful fertility outcomes can be achieved by in vitro fertilization-embryo transfer techniques.

Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.

PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.

Identification of a novel CFAP61 homozygous splicing variant associated with multiple morphological abnormalities of the flagella.

In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple morphologically abnormal flagella (MMAF) in an infertile patient. Using exome sequencing, we identified this variant, which led to exon 12 skipping and the production of a truncated CFAP61 protein. Transmission electron microscopy analysis of the patient's spermatozoa revealed various flagellar abnormalities, including defective nuclear chromatin condensation, axoneme disorganization, and mitochondria embedded in residual cytoplasmic droplets. Despite a fertilization rate of 83.3% through ICSI, there was no successful pregnancy due to poor embryo quality.Our findings suggest a link between the identified CFAP61 variant and MMAF, indicating potential disruption in radial spokes' assembly or function crucial for normal ciliary motility. Furthermore, nearly half of the observed sperm heads displayed chromatin condensation defects, possibly contributing to the low blastulation rate. This case underscores the significance of genetic counseling and testing, particularly for couples dealing with infertility and MMAF. Early identification of such genetic variants can guide appropriate interventions and improve reproductive outcomes.

A novel homozygous nonsense variant of AK7 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.

Identification of novel homozygous asthenoteratospermia-causing ARMC2 mutations associated with multiple morphological abnormalities of the sperm flagella.

PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.

Biallelic loss-of-function mutations in SEPTIN4 (C17ORF47), encoding a conserved annulus protein, cause thin midpiece spermatozoa and male infertility in humans.

Asthenoteratozoospermia is the primary cause of infertility in humans. However, the genetic etiology remains largely unknown for those suffering from severe asthenoteratozoospermia caused by thin midpiece defects. In this study, we identified two biallelic loss-of-function variants of SEPTIN4 (previously SEPT4) (Patient 1: c.A721T, p.R241* and Patient 2: c.C205T, p.R69*) in two unrelated individuals from two consanguineous Chinese families. SEPT4 is a conserved annulus protein that is critical for male fertility and the structural integrity of the sperm midpiece in mice. SEPT4 mutations disrupted the formation of SEPT-based annulus and localization of SEPTIN subunits in sperms from patients. The ultrastructural analysis demonstrated striking thin midpiece spermatozoa defects owing to annulus loss and disorganized mitochondrial sheath. Immunofluorescence and immunoblotting analyses of the mitochondrial sheath proteins TOMM20 and HSP60 further indicated that the distribution and abundance of mitochondria were impaired in men harboring biallelic SEPT4 variants. Additionally, we found that the precise localization of SLC26A8, a testis-specific anion transporter that colocalizes with SEPT4 at the sperm annulus, was missing without SEPT4. Moreover, the patient achieved a good pregnancy outcome following intracytoplasmic sperm injection. Overall, our study demonstrated for the first time that SEPT4 variants that induced thin midpiece spermatozoa defects were directly associated with human asthenoteratozoospermia.

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described. METHODS: We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied. RESULTS: Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal "9 + 0" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI. CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

The sperm ultrastructure of members of basal Tenebrionoidea (Coleoptera).

The sperm ultrastructure of some beetles of Tenebrionoidea was studied with particular attention to those of the Ripiphoridae, Mordellidae, and Meloidae. These three groups are often thought to form a clade, which is the sister group of the remaining Tenebrionoidea. The testes of the two former families have thinner but longer spermatic cysts containing fewer and longer sperm. Within each cyst all sperm cells have the same orientation, but cross sections showed that the orientation of the axonemes alternate between adjacent cysts, possibly due to the cysts bending on themselves. In both families the sperm has a bilayered acrosome and the flagellum, which shows mitochondrial derivatives starting laterally to the nuclear base, has a typical 9 + 9+2 axoneme with accessory tubules provided with 16 protofilaments in their wall, and well-structured triangular shaped accessory bodies. In Mordellistena sp (Mordellidae) sperm, both mitochondrial derivatives and accessory bodies are somewhat asymmetrical. Moreover, the flagellum shows a very thin and long tail end provided with only accessory tubules. Meloidae species have testes with thicker sperm cysts containing numerous shorter sperm. Within the individual cysts the sperm flagella exhibit an alternating orientation of their axonemes as consequence of a peculiar spermatogenetic process. The flagellar structure is similar to that of the above-mentioned species, but the accessory bodies are not well defined and constituted by fuzzy material. In Mylabris hieracii (Meloidae) sperm, the acrosome is flat with a conspicuous perforatorium and its nucleus has a peculiar quadrangular section. Berberomeloe majalis sperm has a large acrosome with an unusual pentagonal perforatorium. The centriolar structure of Mylabris variabilis shows a complex of dense radial links connecting the microtubular structures to the plasma membrane. These results suggest that Ripiphoridae have a closer relationship with Mordellidae than with Meloidae. These findings are in agreement with results obtained with molecular data.

CFAP65 is required in the acrosome biogenesis and mitochondrial sheath assembly during spermiogenesis.

Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65-/-) mice. The results showed that Cfap65-/- male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65-/- mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65-/- male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly.

Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse.

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.

A dynamic basal complex modulates mammalian sperm movement.

Reproductive success depends on efficient sperm movement driven by axonemal dynein-mediated microtubule sliding. Models predict sliding at the base of the tail - the centriole - but such sliding has never been observed. Centrioles are ancient organelles with a conserved architecture; their rigidity is thought to restrict microtubule sliding. Here, we show that, in mammalian sperm, the atypical distal centriole (DC) and its surrounding atypical pericentriolar matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. During asymmetric tail beating, the DC's right side and its surroundings slide ~300 nm rostrally relative to the left side. The deformation throughout the DBC is transmitted to the head-tail junction; thus, the head tilts to the left, generating a kinking motion. These findings suggest that the DBC evolved as a dynamic linker coupling sperm head and tail into a single self-coordinated system.

Novel frameshift mutation in STK33 is associated with asthenozoospermia and multiple morphological abnormalities of the flagella.

Serine/threonine kinases domain-containing proteins are known to play important functions in sperm flagella and male fertility. However, the roles of these proteins in human reproduction remain poorly understood and whether their variants are associated with human asthenozoospermia have not been reported. Here, we recruited a Pakistani family having four infertile patients diagnosed with idiopathic asthenozoospermia without any ciliary-related symptoms. Whole-exome sequencing identified a novel homozygous frameshift mutation (c.1235del, p.T412Kfs*14) in serine/threonine kinase 33 (STK33), which displays a highly conserved and predominant expression in testis in humans. This variant led to a dramatic reduction of STK33 messenger RNA (mRNA) in the patients. Patients homozygous for the STK33 variant presented reduced sperm motility, frequent morphological abnormalities of sperm flagella and completely disorganized flagellar ultrastructures, which are typical for multiple morphological abnormalities of the flagella (MMAF) phenotypes. Overall, these findings present evidence establishing that STK33 is an MMAF-related gene and provide new insights for understanding the role of serine/threonine kinases domain-containing proteins in human male reproduction.

Heterotrimeric Kinesin II is required for flagellar assembly and elongation of nuclear morphology during spermiogenesis in Schmidtea mediterranea.

Development of sperm requires microtubule-based movements that drive assembly of a compact head and flagellated tails. Much is known about how flagella are built given their shared molecular core with motile cilia, but less is known about the mechanisms that shape the sperm head. The Kinesin Superfamily Protein 3A (KIF3A) pairs off with a second motor protein (KIF3B) and the Kinesin Associated Protein 3 (KAP3) to form Heterotrimeric Kinesin II. This complex drives intraflagellar transport (IFT) along microtubules during ciliary assembly. We show that KIF3A and KAP3 orthologs in Schmidtea mediterranea are required for axonemal assembly and nuclear elongation during spermiogenesis. Expression of Smed-KAP3 is enriched during planarian spermatogenesis with transcript abundance peaking in spermatocyte and spermatid cells. Disruption of Smed-kif3A or Smed-KAP3 expression by RNA-interference results in loss of spermatozoa and accumulation of unelongated spermatids. Confocal microscopy of planarian testis lobes stained with alpha-tubulin antibodies revealed that spermatids with disrupted Kinesin II function fail to assemble flagella, and visualization with 4',6-diamidino-2-phenylindole (DAPI) revealed reduced nuclear elongation. Disruption of Smed-kif3A or Smed-KAP3 expression also resulted in edema, reduced locomotion, and loss of epidermal cilia, which corroborates with somatic phenotypes previously reported for Smed-kif3B. These findings demonstrate that heterotrimeric Kinesin II drives assembly of cilia and flagella, as well as rearrangements of nuclear morphology in developing sperm. Prolonged activity of heterotrimeric Kinesin II in manchette-like structures with extended presence during spermiogenesis is hypothesized to result in the exaggerated nuclear elongation observed in sperm of turbellarians and other lophotrochozoans.