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.

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.

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.

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.

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.

Na,K-ATPase Atp1a4 isoform is important for maintaining sperm flagellar shape.

PURPOSE: The aim of this study is to investigate the mechanisms by which the testis specific Na,K-ATPase ion transport system (Atp1a4) controls sperm morphology and shape. METHODS: Sperm from wild-type (WT) and Atp1a4 knockout (Atp1a4 KO) mice were analyzed morphologically, using light, transmission, and scanning electron microscopy; and functionally, applying sperm osmotic challenge and viability tests. In addition, a sperm proteomic study was performed. RESULTS: Light microscopy confirmed that sperm lacking Atp1a4 present a bend at the junction of the mid- and principal piece of the flagellum. This bend had different degrees of angulation, reaching occasionally a complete flagellar retroflexion. The defect appeared in sperm collected from the cauda epididymis, but not the epididymal caput or the testis. Transmission and scanning electron microscopy revealed a dilation of the cytoplasm at the site of the bend, with fusion of the plasma membrane in overlapping segments of the flagellum. This was accompanied by defects in the axoneme and peri-axonemal structures. Sperm from Atp1a4 KO mice showed an abnormal response to hypoosmotic challenge with decreased viability, suggesting reduced capacity for volume regulation. Exposure to Triton X-100 only partially recovered the flagellar bend of Atp1a4 KO sperm, showing that factors other than osmotic regulation contribute to the flagellar defect. Interestingly, several key sperm structural proteins were expressed in lower amounts in Atp1a4 KO sperm, with no changes in their localization. CONCLUSIONS: Altogether, our results show that Atp1a4 plays an important role in maintaining the proper shape of the sperm flagellum through both osmotic control and structurally related mechanisms.

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Infertility due to defective sperm flagella caused by an intronic deletion in DNAH17 that perturbs splicing.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Novel bi-allelic variants in DNAH2 cause severe asthenoteratozoospermia with multiple morphological abnormalities of the flagella.

RESEARCH QUESTION: Multiple morphological abnormalities of the flagella (MMAF) is characterized by excessive immotile spermatozoa with severe flagellar abnormalities in the ejaculate. Previous studies have reported a heterogeneous genetic profile associated with MMAF. What other genetic variants might explain the cause of MMAF? DESIGN: Whole-exome sequencing was conducted in a cohort of 90 Chinese patients with MMAF. The pathogenicity of identified mutations was assessed through electron microscopy and immunofluorescent examinations. RESULTS: Three unrelated men with bi-allelic DNAH2 variants were identified. Sanger sequencing verified that the six novel variants originated from every parent. All these variants were located at the conserved domains of DNAH2 and predicted to be deleterious by bioinformatic tools. Haematoxylin and eosin staining and scanning electron microscopy revealed that spermatozoa harbouring DNAH2 variants displayed severely aberrant morphology mainly with absent and short flagella (≥78%). Moreover, transmission electron microscopy revealed the obvious absence of a central pair of microtubules and inner dynein arms in the spermatozoa with mutated DNAH2. Immunofluorescence data further validated these findings, showing reduced DNAH2 protein expression in the spermatozoa with DNAH2 variants, compared with normal spermatozoa. Intracytoplasmic sperm injection using spermatozoa from the three men with mutated DNAH2 resulted in blastocyst formation in all cases. Embryo transfer was carried out in two couples, both resulting in clinical pregnancy. CONCLUSIONS: These experimental and clinical data suggest that bi-allelic DNAH2 variants might induce MMAF-associated asthenoteratozoospermia, which can be overcome through intracytoplasmic sperm injection. These findings contribute to the knowledge of the genetic landscape of asthenoteratozoospermia and clinical counselling of male infertility.

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility.

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

The anatomy, movement, and functions of human sperm tail: an evolving mystery.

Sperms have attracted attention of many researchers since it was discovered by Antonie van Leeuwenhoek in 1677. Though a small cell, its every part has complex structure and different function to play in carrying life. Sperm tail is most complicated structure with more than 1000 proteins involved in its functioning. With the advent of three-dimensional microscopes, many studies are undergoing to understand exact mechanism of sperm tail movement. Most recent studies have shown that sperms move by spinning rather than swimming. Each subunit of tail, including axonemal, peri-axonemal structures, plays essential roles in sperm motility, capacitation, hyperactivation, fertilization. Furthermore, over 2300 genes are involved in spermatogenesis. A number of genetic mutations have been linked with abnormal sperm flagellar development leading to motility defects and male infertility. It was found that 6% of male infertility cases are related to genetic causes, and 4% of couples undergoing intracytoplasmic sperm injection for male subfertility have chromosomal abnormalities. Hence, an understanding of sperm tail development and genes associated with its normal functioning can help in better diagnosis of male infertility and its management. There is still a lot that needs to be discovered about genes, proteins contributing to normal human sperm tail development, movement, and role in male fertility. Sperm tail has complex anatomy, with surrounding axoneme having 9 + 2 microtubules arrangement along its entire length and peri-axonemal structures that contribute in sperm motility and fertilization. In future sperm tail-associated genes, proteins and subunits can be used as markers of male fertility.

Ultrastructure of sperm and complete mitochondrial genome in Meretrix sp. (Bivalvia: Veneridae) from Taiwan.

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified.

Ca2+ ionophore A23187 inhibits ATP generation reducing mouse sperm motility and PKA-dependent phosphorylation.

Male infertility is a global problem in modern society of which capacitating defects are a major cause. Previous studies have demonstrated that Ca2+ ionophore A23187 can make mouse sperm capable of fertilizing in vitro, which may aid in clinical treatment of capacitating defects. However, the detailed role and mechanism of Ca2+ in the capacitating process are still unclear especially how A23187 quickly renders sperm immotile and inhibits cAMP/PKA-mediated phosphorylation. We report that A23187 induces a Ca2+ flux in the mitochondria enriched sperm tail and excess Ca2+ inhibits key metabolic enzymes involved in acetyl-CoA biosynthesis, TCA cycle and electron transport chain pathways resulting in reduced ATP and overall energy production, however this flux does not destroy the structure of the sperm tail. Due to the decrease in ATP production, which is the main phosphate group donator and the power of sperm, the sperm is rendered immobile and PKA-mediated phosphorylation is inhibited. Our study proposed a possible mechanism through which A23187 reduces sperm motility and PKA-mediated phosphorylation from ATP generation, thus providing basic data for exploring the functional roles of Ca2+ in sperm in the future.

Analysis of the sperm flagellar axoneme using gene-modified mice.

Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.