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.

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.

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.

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.

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.

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.

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.

Fluoride exposure alters the ultra-structure of sperm flagellum via reducing key protein expressions in testis.

Excessive fluoride (F) ingestion via drinking water interfered with spermatogenesis and lowered sperm quality of human and animals. However, it is still unclear why the effects of fluoride on sperm quality focus on mostly sperm motility rather than sperm count. The objective of this study is to investigate the potential relationship between alteration in the structure and function of sperm flagellum and fluoride exposure in the environment. 40 male mice were allocated to four groups which were treated with 0, 25, 50, 100 mg/L NaF deionized water, respectively, for 8 weeks continuously. The testicular morphology, ultra-structure of fibrous sheath and axoneme of sperm flagellum, and eleven key function genes Akap3, Akap4, Dnah1, Eno4, Cfap43, Cfap44, Hydin, Spef2, Spag6, Spag16, and Cfap69 were examined by histology, transmission electron microscopy, and real-time PCR methods respectively. The results displayed that fluoride damaged the typical "9 + 2″ microtubule structure including fibrous sheathes and axoneme of sperm flagellum in testes of mice. Furthermore, the mRNA and protein expression levels of AKAP3 and AKAP4 related to fibrous sheathes formation, and CFAP43, CFAP44 and HYDIN in axoneme were down-regulated by fluoride exposure. Taken together, we revealed that fluoride altered the structures of the fibrous sheathes and axonemal in sperm flagellum via down-regulating the mRNA and protein expression levels of AKAP3, AKAP4, CFAP43, CFAP44, and HYDIN, which may be one of the reasons that fluoride lowered sperm quality and male reproductive function.

Identification of DNAH6 mutations in infertile men with multiple morphological abnormalities of the sperm flagella.

Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. Here we describe three men with primary infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) from two unrelated Han Chinese families. We performed whole-exome sequencing (WES) and Sanger sequencing on the proband of family 1, and found that he carried novel compound heterozygous missense mutations in dynein axonemal heavy chain 6 (DNAH6) that resulted in the substitution of a conserved amino acid residue and co-segregated with the MMAF phenotype in this family. Papanicolaou staining and transmission electron microscopy analysis revealed morphological and ultrastructural abnormalities in the sperm flagella in carriers of these genetic variants. Immunostaining experiments showed that DNAH6 was localized in the sperm tail. This is the first report identifying novel recessive mutations in DNAH6 as a cause of MMAF. These findings expand the spectrum of known MMAF mutations and phenotypes and provide information that can be useful for genetic and reproductive counseling of MMAF patients.

DNAH2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella.

Multiple morphological abnormalities of flagella (MMAF) is one kind of severe teratozoospermia. Gene mutations reported in previous works only revealed the pathogenesis of approximately half of the MMAF cases, and more genetic defects in MMAF need to be explored. In the present study, we performed a genetic analysis on Han Chinese men with MMAF using whole-exome sequencing. After filtering out the cases with known gene mutations, we identified five novel mutation sites in the DNAH2 gene in three cases from three families. These mutations were validated through Sanger sequencing and absent in all control individuals. In silico analysis revealed that these DNAH2 variations are deleterious. The spermatozoa with DNAH2 mutations showed severely disarranged axonemal structures with mitochondrial sheath defection. The DNAH2 protein level was significantly decreased and inner dynein arms were absent in the spermatozoa of patients. ICSI treatment was performed for two MMAF patients with DNAH2 mutations and the associated couples successfully achieved pregnancy, indicating good nuclear quality of the sperm from the DNAH2 mutant patients. Together, these data suggest that the DNAH2 mutation can cause severe sperm flagella defects that damage sperm motility. These results provide a novel genetic pathogeny for the human MMAF phenotype.

Characterisation of three systematic sperm tail defects and their influence on ICSI outcome.

This study characterized three cases of systematic sperm tail defects using electron microscopy and immunolocalisation of centrin 1 and tubulin and explored their impact on ICSI outcome. Structural sperm tail defects of possible genetic origin were suspected as the eosin test revealed a sperm viability of >70% despite severe asthenozoospermia or the absence of motility. In Patient 1, 80%-85% of axoneme cross sections was incomplete. The fluorescent signal of tubulin was weak along the entire tail; the signal of centrin 1 was normal. After ICSI, a female healthy baby was born. Patient 2 showed spermatozoa with tails reduced in length at different levels, axonemal and periaxonemal alterations and fragility of head-tail junction. Centrin 1 was altered in 80% of sperm. After ICSI, no embryos were obtained. Patient 3 showed tails reduced in length at light and fluorescence microscopy; ultrastructural study revealed a condition of dysplasia of fibrous sheath with heterogeneity of tails' length. The signal for centrin 1 was altered in 50% of spermatozoa; two embryos were transferred without pregnancy. The correct diagnosis of sperm pathology is important in case of systematic sperm defects as it enables the clinician to improve patient's management and to provide an adequate genetic counselling.

SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

Microscopic sperm head damage and abnormalities as heat stress indicators in Australian Merino rams (Ovis aries) in Northern Patagonia, Argentina / Danos e anormalidades microscópicas das cabeças espermáticas como indicadores de estresse por calor em carneiros Merino Australiano (Ovis aries) da Patagônia Norte, Argentina

In Northern Patagonia, the mating season starts on March 15th, when rams are submitted to summer temperatures. Exposure of rams to heat stress increases the prevalence of microscopic damage to spermatozoa, morphological abnormalities, and reductions in fertility. This study assesses the adaptive capabilities of six unshorn and six shorn Australian Merino rams, half of which were treated in a heat chamber for eight hours for five days, gradually reaching a temperature of up to 40 °C. Microscopic damage, abnormalities and ultramicroscopic alterations of the plasma membrane and the acrosome of sperm head were analysed. There were significant differences in the percentage of tailless spermatozoa and proximal cytoplasmic droplets between post-treatment periods. Temperature primarily affected the shorn rams and the sperm heads during spermiogenesis. Submicroscopic alterations were observed when the plasma membrane was present in the anterior segment. These alterations can be intact, waved, or dilated. When the plasma membrane was absent, the acrosome might be intact, dilated, and waved. In addition, the outer acrosomal membrane may completely lose its contents or have a nude nucleus. The plasma membrane assumes a waved shape as a result of the effect of temperature on the epididymis. According to this study, the tailless head, proximal cytoplasmic droplets, and the ultramicroscopic categories studied were robust indicators of semen heat stress. After ten weeks, the sperm head recovered its normal shape. Unshorn rams are better adapted to summer heat stress than shorn ones. Microscopy and transmission electron microscopy alterations have been shown to be excellent indicators of thermal stress in Australian Merino rams and may be useful tools to help sheep farmers choose when to begin the mating season, which will vary depending on the environmental conditions of the summer.(AU)

Na Patagônia Norte, os ovinos têm sua estação de acasalamento iniciada em 15 de março, portanto, ficam sujeitos às temperaturas do verão. A exposição de carneiros a estresse térmico aumenta a prevalência de danos microscópicos e anomalias morfológicas nos espermatozoides, que implica uma redução na fertilidade. Este trabalho avaliou a capacidade adaptativa de carneiros Merino Australiano com lã (N = 6) e tosquiados (N = 6): metade ficou ao ar livre e outra metade foi mantida em uma câmara climática por oito horas, durante cinco dias, chegando gradualmente a uma temperatura máxima de 40 °C. Foram analisados danos microscópicos, anormalidades e alterações ultramicroscópicas da membrana plasmática e do acrossoma da cabeça dos espermatozoides. Os resultados microscópicos confirmaram a existência de diferença significativa na porcentagem de espermatozoides sem cauda e com gota citoplasmática proximal, entre os ejaculados pós-tratamento. A temperatura afetou os carneiros tosquiados, principalmente a cabeça de seus espermatozoides, durante a espermatogênese. Alterações submicroscópicas foram observados na membrana plasmática quando ela estava presente no segmento anterior: quando não intacta, ficava ondulada ou dilatada. Quando a membrana plasmática estava ausente, o acrossoma podia se apresentar ondulado ou dilatado. Além disso, sob efeito do calor, a membrana acrossomal externa pode perder completamente seu conteúdo ou apresentar núcleo desnudo. A membrana plasmática assume uma forma ondulada pelo efeito da temperatura no epidídimo. Depois de dez semanas, a cabeça dos espermatozoides recuperou sua forma normal. Como demonstrado neste estudo, a cabeça sem cauda, as gotas citoplasmáticas proximais e as categorias ultramicroscópicas estudadas são indicadores do efeito do estresse térmico no sêmen, e os carneiros com maior cobertura de lã se adaptam melhor ao estresse por calor. Alterações de microscopia e de microscopia eletrônica de transmissão têm se mostrado excelentes indicadores de estresse por calor em carneiros Merino Australiano e podem ser ferramentas úteis para ajudar criadores de ovelhas a escolher quando começar a época de acasalamento, o que irá variar de acordo com as condições ambientais do verão.(AU)

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha, Marchalinidae).

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.

Major regulatory mechanisms involved in sperm motility.

The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

Detection of a mutation in the intron of Sperm-specific glyceraldehyde-3-phosphate dehydrogenase gene in patients with fibrous sheath dysplasia of the sperm flagellum.

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibres of the sperm flagellum. Dysplasia of the fibrous sheath (DFS) is a defect of spermatozoa observed in severe asthenozoospermic patients and characterised by morphologically abnormal flagella with distorted fibrous sheaths. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is a glycolytic enzyme that is tightly associated with the fibrous sheath of the sperm flagellum. The enzymatic activity of GAPDS was investigated in sperm samples of seven patients with DFS and compared to that of normal spermatozoa (n = 10). The difference in GAPDS activity in DFS and normal spermatozoa was statistically significant (0.19 ± 0.11 and 0.75 ± 0.11 µmol NADH per min per mg protein respectively). Immunochemical staining revealed irregular distribution of GAPDS in the flagellum of DFS spermatozoa. Other five samples with typical alterations in the fibrous sheath were assayed for mutations within human GAPDS gene. In all five cases, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of GAPDS. It is assumed that the deficiency in GAPDS observed in most DFS sperm samples is ascribable to a disorder in the regulation of GAPDS expression caused by the mutation in the intron region of GAPDS gene.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

[Genetically Determined and Functional Human Sperm Motility Decrease].

Motility is the most important property of mammalian sperm required for fertilization. Axoneme and axoneme surrounding tail components are the morphological substrate of sperm motility. Quantitative research methods of human spermatozoa motility allowed the definition of the normative parameters for fertile men. Exogenous factors, and, rarely, genetic defects may cause a significant reduction in sperm motility. Axonemal anomalies (absence of external and/or internal dynein arms, central pair of microtubules absence) may be the cause of primary ciliary dyskinesia (PCD). PCD­a severe systemic disease of the reduction of sperm motility­is just one symptom. Dysplasia of the fibrous sheath (DFO) is also genetically determined sperm motility decrease. PCD and the DFO are multigene diseases that are inherited in an autosomal recessive manner. Modern molecular biological research methods are used to identify candidate genes. Assisted reproduction technologies (ART) allow men suffering from PCD and DFO to produce offspring. PCD and DFO symptoms appear in the homozygote. Children born after ART have the probability of being mutation carriers. We do not have complete information about etiological factor of genetically determined spermpathology. So we cannot assess the genetic risk degree. However, the possibility of mutations accumulation, which can be a risk factor for distant offspring, should be considered.