Bi-allelic variants in chromatoid body protein TDRD6 cause spermiogenesis defects and severe oligoasthenoteratozoospermia in humans.

BACKGROUND: The association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing and Sanger sequencing were employed to identify potential pathogenic variants of TDRD6 in infertile men. Histology, immunofluorescence, immunoblotting and ultrastructural analyses were conducted to clarify the structural and functional abnormalities of sperm in mutated patients. Tdrd6-knockout mice were generated using the CRISPR-Cas9 system. Total RNA-seq and single-cell RNA-seq (scRNA-seq) analyses were used to elucidate the underlying molecular mechanisms, followed by validation through quantitative RT-PCR and immunostaining. Intracytoplasmic sperm injection (ICSI) was also used to assess the efficacy of clinical treatment. RESULTS: Bi-allelic TDRD6 variants were identified in five unrelated Chinese individuals with OAT, including homozygous loss-of-function variants in two consanguineous families. Notably, besides reduced concentrations and impaired motility, a significant occurrence of acrosomal hypoplasia was detected in multiple spermatozoa among five patients. Using the Tdrd6-deficient mice, we further elucidate the pivotal role of TDRD6 in spermiogenesis and acrosome identified. In addition, the mislocalisation of crucial chromatoid body components DDX4 (MVH) and UPF1 was also observed in round spermatids from patients harbouring TDRD6 variants. ScRNA-seq analysis of germ cells from a patient with TDRD6 variants revealed that TDRD6 regulates mRNA metabolism processes involved in spermatid differentiation and cytoplasmic translation. CONCLUSION: Our findings strongly suggest that TDRD6 plays a conserved role in spermiogenesis and confirms the causal relationship between TDRD6 variants and human OAT. Additionally, this study highlights the unfavourable ICSI outcomes in individuals with bi-allelic TDRD6 variants, providing insights for potential clinical treatment strategies.

FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

The conserved fertility factor SPACA4/Bouncer has divergent modes of action in vertebrate fertilization.

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Telomere Length and Male Fertility.

Over the past decade, telomeres have attracted increasing attention due to the role they play in human fertility. However, conflicting results have been reported on the possible association between sperm telomere length (STL) and leukocyte telomere length (LTL) and the quality of the sperm parameters. The aim of this study was to run a comprehensive study to investigate the role of STL and LTL in male spermatogenesis and infertility. Moreover, the association between the sperm parameters and 11 candidate single nucleotide polymorphisms (SNPs), identified in the literature for their association with telomere length (TL), was investigated. We observed no associations between sperm parameters and STL nor LTL. For the individual SNPs, we observed five statistically significant associations with sperm parameters: considering a p < 0.05. Namely, ACYP2-rs11125529 and decreased sperm motility (p = 0.03); PXK-rs6772228 with a lower sperm count (p = 0.02); NAF1-rs7675998 with increased probability of having abnormal acrosomes (p = 0.03) and abnormal flagellum (p = 0.04); ZNF208-rs8105767 and reduction of sperms with normal heads (p = 0.009). This study suggests a moderate involvement of telomere length in male fertility; however, in our analyses four SNPs were weakly associated with sperm variables, suggesting the SNPs to be pleiotropic and involved in other regulatory mechanisms independent of telomere homeostasis, but involved in the spermatogenic process.

Deletion of the Spata3 Gene Induces Sperm Alterations and In Vitro Hypofertility in Mice.

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.

Dysregulation of the Acrosome Formation Network by 8-oxoguanine (8-oxoG) in Infertile Sperm: A Case Report with Advanced Techniques.

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10-7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility.

Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice.

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1-/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

Biallelic mutations in CFAP65 lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations.

BACKGROUND: The genetic causes for most male infertility due to severe asthenozoospermia remain unclear. OBJECTIVE: Our objective was to identify unknown genetic factors in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. METHODS: We performed whole exome sequencing of 47 individuals with severe asthenozoospermia from 45 unrelated families. Mutation screening was performed in a control cohort of 637 individuals, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia and 223 fertile controls. Ultrastructural and immunostaining analyses of patients' spermatozoa were performed to characterise the effect of variants. RESULTS: One homozygous non-sense mutation (NM_194302, c.G5341T:p.E1781X), two compound heterozygous mutations (c.C2284T:p.R762X and c.1751delC:p.P584fs) and two compound heterozygous mutations (c.5714_5721del:p.L1905fs and c.C3021A:p.N1007K) were identified in CFAP65 of three individuals with completely immotile spermatozoa, respectively. No biallelic deleterious variants of CFAP65 were detected in the control cohort of 637 individuals. Ultrastructural and immunostaining analyses of spermatozoa from two patients showed highly aberrant sperm morphology with severe defects such as acrosome hypoplasia, disruption of the mitochondrial sheath and absence of the central pair complex. CONCLUSION: To the best of our knowledge, we are the first to report that CFAP65 mutations may cause spermatozoa to be completely immotile.

Deletion of porcine BOLL is associated with defective acrosomes and subfertility in Yorkshire boars.

A recessive sperm defect of Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain spermatozoa with defective acrosomes, resulting in low fertility. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re-analyzed microarray-derived genotypes of affected boars and confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15. To detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11-fold. Read depth analysis revealed a 55 kb deletion that is associated with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, an RNA binding protein which is an evolutionarily conserved member of the DAZ (Deleted in AZoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Boars that carry the deletion in the homozygous state produce sperm but their acrosomes are defective, suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the role of BOLL during spermatogenesis and sperm maturation in pigs.

The effects of different dietary protein sources on live weight, sperm quality and the histology of the testes and accessory glands in male rats.

This study was aimed at determining the effects of corn and wheat gluten, used as dietary protein sources, on live weight gain, sperm quality and the histology of the testes and accessory glands in male rats. For this purpose, 20-day-old 24 male Wistar albino rats were randomly assigned to three groups. Group 1 (Control), Group 2 and Group 3 were fed on a basal ration supplemented with high levels of soybean meal, corn gluten and wheat gluten, respectively, as a protein source. At the end of the study, when compared to Group 1, live weight values were determined to have increased in Group 3 and to have decreased in Group 2 (p < .05). Furthermore, sperm density, sperm motility, the dead/ live sperm ratio and testes weight were determined to have significantly decreased in Group 2, in comparison to Groups 1 and 3 (p < .05). The percentages of abnormal spermatozoon, and head, acrosome, mid-piece and tail abnormalities were high in Group 2 (p < .05). Histological examination demonstrated that, in Group 2, the diameter of the Tubulus Seminiferous Contortus (TSC) and the size of the Tubular Epithelial Cells (TE) were small, and the tubular and anatomical structure of the testes were shrunken and altered. Group 2 also presented with connective tissue increase and alveolar lumen enlargement in the prostate gland, and with connective tissue thickening, muscle tissue increase and secretory capacity decrease in the seminal vesicle (p < .05). Moreover, in Group 2, the Gl. Bulbourethral (Cowper's gland) presented with a decreased size and dilatations in the mucous structures. In a result, based on the findings obtained in this study, it is suggested that high levels of dietary corn gluten adversely affect live weight, sperm quality, and the testes and accessory glands.

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.

The histone methyltransferase SETD2 is required for expression of acrosin-binding protein 1 and protamines and essential for spermiogenesis in mice.

Spermatogenesis is precisely controlled by complex gene expression programs and involves epigenetic reprogramming, including histone modification and DNA methylation. SET domain-containing 2 (SETD2) is the predominant histone methyltransferase catalyzing the trimethylation of histone H3 lysine 36 (H3K36me3) and plays key roles in embryonic stem cell differentiation and somatic cell development. However, its role in male germ cell development remains elusive. Here, we demonstrate an essential role of Setd2 for spermiogenesis, the final stage of spermatogenesis. Using RNA-seq, we found that, in postnatal mouse testes, Setd2 mRNA levels dramatically increase in 14-day-old mice. Using a germ cell-specific Setd2 knockout mouse model, we also found that targeted Setd2 knockout in germ cells causes aberrant spermiogenesis with acrosomal malformation before step 8 of the round-spermatid stage, resulting in complete infertility. Furthermore, we noted that the Setd2 deficiency results in complete loss of H3K36me3 and significantly decreases expression of thousands of genes, including those encoding acrosin-binding protein 1 (Acrbp1) and protamines, required for spermatogenesis. Our findings thus reveal a previously unappreciated role of the SETD2-dependent H3K36me3 modification in spermiogenesis and provide clues to the molecular mechanisms in epigenetic disorders underlying male infertility.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation.

Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8-/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8-/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception.

Association between total globozoospermia and sperm chromatin defects.

Globozoospermia is a severe sperm morphological anomaly leading to primary infertility and low fertilisation following intracytoplasmic sperm injection (ICSI). This phenotype is observed in less than 0.1% of infertile men and is determined by small, round-headed spermatozoa with absence of an acrosomal cap, acrosome protease and also cytoskeletal proteins. Failure of oocyte activation is considered as the main cause of fertilisation failure in these individuals post-ICSI. Therefore, artificial oocyte activation (AOA) along with ICSI is commonly implemented. However, based on previous report, fertilisation rate remains low despite implementation of ICSI-AOA. Therefore, other mechanisms like sperm chromatin packaging and DNA fragmentation may account for low fertilisation and development post-ICSI-AOA. Therefore, this study aims to assess and compare the degree of sperm protamine deficiency and DNA fragmentation in large population of infertile men with total globozoospermia (30 globozoospermic men presenting with 100% round-headed spermatozoa) with 22 fertile individuals using chromomycin A3 and TUNEL assay respectively. Results clearly show that mean of sperm concentration and percentage of sperm motility were significantly lower, while percentage of sperm abnormal morphology, protamine-deficient and DNA-fragmented spermatozoa were significantly higher in infertile men with globozoospermia compared to fertile men. Therefore, increased sperm DNA damage in globozoospermia is likely related to defective DNA compaction and antioxidant therapy before ICSI-AOA could be recommended as an appropriate option before ICSI-AOA.

Sperm membrane integrity and stability after selection of cryopreserved ovine semen on colloidal solutions.

The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.

SNP g.1007A>G within the porcine DNAL4 gene affects sperm motility traits and percentage of midpiece abnormalities.

The flagellar beating of a spermatozoa's axoneme is caused by the varying activation and inactivation of dynein molecules. Dynein, axonemal, light chain 4 (DNAL4) is a functional candidate gene for sperm motility as it encodes a small subunit of the dyneins. We resequenced the porcine DNAL4 using three artificial insemination (AI) boars each with high (>68%) or low (<60%) motility, and detected 23 SNP. These were then genotyped for 82 AI boars. Using spermatological records, significantly negative genetic correlations between ejaculate volume (VOL) and the further spermatological parameters concentration (CONC) (r = -.43), motility of undiluted semen (MOTUD) (r = -.09), motility after 24 h (MOT1) (r = -.17) and after 48 hr (MOT2) (r = -.23) were estimated. Significantly positive correlations existed between CONC and MOT1 (r = .07) as well as MOT2 (r = .10), between MOTUD and MOT1 (r = .33), between MOTUD and MOT2 (r = .36), and finally between MOT1 and MOT2 (r = .70). Significantly negatively correlated were all motility traits with the parameters abnormal acrosome (AA) (MOTUD r = -.06; MOT1 r = -.08, and MOT2 r = -.1) and presence of cytoplasmic droplet (CD) (MOTUD r = -.07; MOT1 r = -.08; MOT2 r = -.07). Association analyses (single marker regression model; SMR) propose that SNP g.1007A>G, located in the second intron, reduces motility significantly (MOTUD -4.59%; MOT1 -10.33%; MOT2 -19.37%). According to the dominant-recessive model (DRM), genotype AA is always superior compared to genotypes AG and GG (i.e. MOTUD 67.67%, 64.16% and 53.91%; MOT1 54.17%, 43.75% and 28.44%; MOT2 44.12%, 24.91% and 4.97%). The average effect of gene substitution (g.1007A>G) on abnormal midpiece (AM) was 0.71%, the genotypic values-as expressed by LSmeans-were 0.1 (AA) and 0.81 (AG).

Effect of acute/subchronic samarium exposure on the concentration, motility, and morphology of sperm in male mice.

Male ICR mice were orally administered samarium nitrate [Sm(NO3)3] to investigate its effects on sperm concentration and sperm quality. After acute exposure to ≥2880.00 mg/kg Sm(NO3)3 via intragastric gavage, sperm motility and acrosome integrity were decreased, and the sperm malformation percentage was increased (P < 0.05). After subchronic exposure to ≥500.00 mg/L Sm(NO3)3 administered via drinking water for 90 days, relative gonad weight, sperm concentration, and sperm quality significantly decreased (P < 0.05). Sperm malformation also increased after subchronic exposure to Sm, which was found to be the most sensitive index. Sperm head malformation accounted for the largest proportion of all types of sperm malformations evaluated. Of the six different subtypes of head malformation, irregular shape accounted for the largest proportion.