RESUMO
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.
Assuntos
Alelos , Astenozoospermia , Sequenciamento do Exoma , Camundongos Knockout , Espermatogênese , Adulto , Animais , Humanos , Masculino , Camundongos , Acrossomo/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Oligospermia/genética , Oligospermia/patologia , Linhagem , Injeções de Esperma Intracitoplásmicas , Espermatogênese/genética , Espermatozoides/patologia , Espermatozoides/metabolismoRESUMO
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.
Assuntos
Acrossomo , Infertilidade Masculina , Proteínas dos Microfilamentos , Adulto , Humanos , Masculino , Acrossomo/patologia , Acrossomo/ultraestrutura , Actinas/metabolismo , Actinas/genética , Sequenciamento do Exoma , Fertilização/genética , Deleção de Genes , Infertilidade Masculina/genética , Cabeça do Espermatozoide/ultraestrutura , Cabeça do Espermatozoide/patologia , Injeções de Esperma Intracitoplásmicas , Espermatozoides/ultraestrutura , Espermatozoides/anormalidades , Proteínas dos Microfilamentos/genéticaRESUMO
Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss-/- male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss-/- mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.
Assuntos
Ferroptose , Glutationa Sintase , Infertilidade Masculina , Testículo , Glutationa Sintase/deficiência , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Espermatócitos/metabolismo , Infertilidade Masculina/genética , Testículo/enzimologia , Testículo/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Ferroptose/genética , Técnicas de Inativação de Genes , Células Germinativas/citologia , Meiose/genética , Espermatogênese/genética , Acrossomo/patologia , Autofagia/genética , Masculino , Feminino , Animais , Camundongos , Fatores EtáriosRESUMO
As a new widespread contaminant, nanoplastics (NPs) pose a potential risk to human health. Nevertheless, the adverse effects of NPs on the male reproductive system are poorly understood. In this study, we aimed to determine the effects of polystyrene nanoplastics (PS-NPs) (50 nm) on sperm quality, with a focus on the acrosome defects. After 35 days of intragastric administration, sperm quality was decreased and testicular structures were impaired in mice exposed to PS-NPs in both the medium (1.0 mg/kg) and high dose (10 mg/kg) groups. No significant changes were observed in the low dose (0.2 mg/kg) group. Meanwhile, acrosome parameters including acrosome integrity and acrosome reaction were decreased after the administration of PS-NPs. These findings were consistent with the disruption of acrosome biogenesis, as identified by the changed testicular ultrastructure. Additionally, the findings were further validated using seven marker genes (Gba2, Pick1, Gopc, Hrb, Zpbp1, Spaca1 and Dpy19l2) essential for acrosome formation, which showed that two of these genes (Gopc and Dpy19l2) were significantly down-regulated. Moreover, repressed autophagy was observed in the testes of PS-NPs-exposed mice based on autophagy-related protein expression. This phenomenon was further verified in GC-2spd cells treated with PS-NPs (50 µg/mL, 100 µg/mL, 200 µg/mL for 24 h). The potential role of autophagy in such acrosome defects was explored by using the autophagy inhibitor 3-methyladenine (3-MA), autophagy activator rapamycin or beclin-1 siRNA. The results showed that Golgi-associated vesicle disorganization was exacerbated with the 3-MA and beclin-1 siRNA pretreatments, but decreased with the rapamycin pretreatment, and the expression of GOPC and DPY19L2 was also altered. These results indicated that autophagy might be involved in the PS-NPs-induced acrosome lesions based on the regulation of two key acrosome-formation proteins, GOPC and DPY19L2. Altogether, our results will provide new insights into the PS-NPs-induced male reproductive impairment.
Assuntos
Acrossomo , Nanopartículas , Acrossomo/metabolismo , Acrossomo/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Administração Oral , Animais , Autofagia , Proteína Beclina-1/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Masculino , Camundongos , Microplásticos , Nanopartículas/toxicidade , Poliestirenos/metabolismo , Poliestirenos/toxicidade , RNA Interferente Pequeno/metabolismo , Sirolimo/metabolismoRESUMO
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.
Assuntos
Acrossomo/patologia , Astenozoospermia/genética , Infertilidade Masculina/genética , Reação Acrossômica/genética , Adulto , Astenozoospermia/patologia , China , Estudos de Coortes , Consanguinidade , Características da Família , Feminino , Homozigoto , Humanos , Infertilidade Masculina/patologia , Infertilidade Masculina/terapia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Masculino , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Membranas Mitocondriais/patologia , Mutação de Sentido Incorreto , Linhagem , Gravidez , Injeções de Esperma Intracitoplásmicas , Espermatozoides/anormalidades , Espermatozoides/patologiaRESUMO
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.
Assuntos
Acrossomo/metabolismo , Fertilidade/fisiologia , Proteínas Nucleares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab2 de Ligação ao GTP/metabolismo , Acrossomo/patologia , Animais , Genética , Complexo de Golgi/metabolismo , Infertilidade Masculina , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Ligação Proteica , Cabeça do Espermatozoide/metabolismo , Espermatogênese , Teratozoospermia/metabolismo , Testículo/metabolismoRESUMO
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.
Assuntos
Antígenos Ly/metabolismo , Fertilização , Infertilidade Masculina/patologia , Glicoproteínas de Membrana/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Interações Espermatozoide-Óvulo , Acrossomo/metabolismo , Acrossomo/patologia , Animais , Antígenos Ly/genética , Feminino , Infertilidade Masculina/etiologia , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Zona Pelúcida/metabolismo , Zona Pelúcida/patologiaRESUMO
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.
Assuntos
Acrossomo/metabolismo , Proteínas de Transporte/genética , Redes Reguladoras de Genes , Guanina/análogos & derivados , Infertilidade Masculina/genética , Tubulina (Proteína)/genética , Acrossomo/patologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Guanina/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Metaboloma , Mapeamento de Interação de Proteínas , Proteômica/métodos , Análise do Sêmen , Espermatogênese/genética , Tubulina (Proteína)/metabolismoRESUMO
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.
Assuntos
Hidrolases Anidrido Ácido/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Proteínas Serina-Treonina Quinases/genética , Ribonucleoproteínas/genética , Telômero/genética , Acrossomo/metabolismo , Acrossomo/patologia , Adulto , Feminino , Estudos de Associação Genética , Humanos , Infertilidade Masculina/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Polimorfismo de Nucleotídeo Único/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Espermatozoides/patologia , Homeostase do Telômero/genéticaRESUMO
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.
Assuntos
Infertilidade Masculina/genética , RNA Interferente Pequeno/genética , Espermatogênese/genética , Espermatozoides/patologia , Acrossomo/patologia , Animais , Cromossomos/genética , Humanos , Infertilidade Masculina/patologia , Masculino , Meiose/genética , Camundongos , Estágio Paquíteno/genética , Espermátides/crescimento & desenvolvimento , Espermátides/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
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.
Assuntos
Acrossomo/patologia , Fertilização in vitro/métodos , Deleção de Genes , Infertilidade Masculina/genética , Proteínas/genética , Acrossomo/metabolismo , Acrossomo/ultraestrutura , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Infertilidade Masculina/metabolismo , Gotículas Lipídicas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Gravidez , Proteínas/metabolismo , Motilidade dos Espermatozoides/genética , Espermatogênese/genética , Testículo/metabolismoRESUMO
Gene expression analyses have revealed that there are >2,300 testis-enriched genes in mice, and gene knockout models have shown that a number of them are responsible for male fertility. However, the functions of numerous genes have yet to be clarified. The aim of the present study was to identify the expression pattern of testis-expressed protein 33 (TEX33) in mice and explore the role of TEX33 in male reproduction. Reverse transcription-polymerase chain reaction and western blot assays were used to investigate the mRNA and protein levels of TEX33 in mouse testes during the first wave of spermatogenesis. Immunofluorescence analysis was also performed to identify the cellular and structural localization of TEX33 protein in the testes. Tex33 knockout mice were generated by CRISPR/Cas9 gene-editing. Histological analysis with hematoxylin and eosin or periodic acid-Schiff (PAS) staining, computer-assisted sperm analysis (CASA) and fertility testing, were also carried out to evaluate the effect of TEX33 on mouse spermiogenesis and male reproduction. The results showed that Tex33 mRNA and protein were exclusively expressed in mouse testes and were first detected on postnatal days 21-28 (spermiogenesis phase); their expression then remained into adulthood. Immunofluorescence analysis revealed that TEX33 protein was located in the spermatids and sperm within the seminiferous tubules of the mouse testes, and exhibited specific localization to the acrosome, flagellum and manchette during spermiogenesis. These results suggested that TEX33 may play a role in mouse spermiogenesis. However, Tex33 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. PAS staining and CASA revealed that spermatogenesis and sperm quality were normal in mice lacking Tex33. In addition, fertility testing suggested that the Tex33 knockout mice had normal reproductive functions. In summary, the findings of the present study indicate that TEX33 is associated with spermiogenesis but is not essential for sperm development and male fertility.
Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Testículo/metabolismo , Acrossomo/metabolismo , Acrossomo/patologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/patologiaRESUMO
PURPOSE: This study is intended to investigate the candidate pathogenic gene in a patient with primary infertility but without the defect in routine semen parameters from a consanguineous family and explore the potential impacts of mutations on assisted reproductive technology outcome. METHODS: Whole-exome sequencing (WES) was carried out. A variant in his family found by WES was verified by Sanger sequencing. Intracytoplasmic sperm injection (ICSI) was applied to obtain a successful outcome. RESULTS: A Cation Channel of Sperm 3(CATSPER3) homozygous variant (NM_ 178019.3:exon5:c.707T>A, p.L236*) was identified for the first time. The anti-CD46 immunofluorescence analysis revealed the failure of sperm acrosome reaction (AR) caused by the mutation. ICSI treatment was successful. CONCLUSION: This is the first report of a homozygous pathogenic CATSPER3 mutation. This mutation may cause male infertility with the failure of AR but without the defect in routine semen parameters. ICSI was supposed to be the most appropriate therapy.
Assuntos
Infertilidade Masculina/genética , Canais Iônicos/genética , Injeções de Esperma Intracitoplásmicas/métodos , Acrossomo/patologia , Adulto , Transferência Embrionária/métodos , Feminino , Homozigoto , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/terapia , Nascido Vivo , Masculino , Mutação , GravidezRESUMO
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.
Assuntos
Acrossomo/patologia , Deleção de Genes , Infertilidade Masculina/genética , Proteínas de Ligação a RNA/genética , Sus scrofa/genética , Animais , Mapeamento Cromossômico , Genótipo , Haplótipos , Masculino , Polimorfismo de Nucleotídeo Único , Suínos , Doenças dos Suínos/genéticaRESUMO
Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. The aim of this study was to synthetize magnetic nanoparticles (MNPs) coated by peanut (Arachis hypogaea) agglutinin lectin (PNA) and investigate its beneficial effect in improving of sperm characteristics. MNPs were obtained by co-precipitation method, functionalized with chitosan and coated by PNA at a concentration of 0.04 mg/mL. Semen was frozen either with glycerol-based or sucrose-based extenders. Frozen-thawed straws from five donkeys (three ejaculates per donkey) were incubated with lectin-MNPs (2 mg/mL), and then exposed to an external magnet enabling the non-bound sperm to be collected as nanopurified sperm. Sperm were evaluated post-thawing (control) and after nanopurification for motility, plasma membrane integrity, acrosome integrity, morphology, DNA fragmentation and concentration. The statistical analyses were extended to investigate the correlation between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing. The obtained MNPs were biocompatible to the sperm and significantly improved the progressive motility (P < 0.05) for the glycerol nanopurified group (43.08 ± 3.52%) in comparison to control (33.70 ± 2.64%). Acrosome-damaged sperm were reduced (P < 0.05) in both nanopurified groups (19.92 ± 2.69 for G and 21.57 ± 2.77 for S) in comparison to control (36.07 ± 3.82 for G and 35.35 ± 3.88 for S). There were no significant changes in sperm morphology and membrane integrity after nanopurification. The average sperm recovery after nanopurification was 80.1%. Sperm quality index was significantly higher (P < 0.001) in nanopurified groups regardless of the initial quality of the frozen thawed semen samples. However, in the high sperm quality group, nanopurification significantly improved the progressive motility and membrane integrity besides the increasing of acrosome-intact sperm. Sperm nanopurification using lectin-magnetic nanoparticles can be considered as a suitable method to reduce the proportion of acrosome-damaged sperm and to increase the quality of frozen thawed donkey semen.
Assuntos
Acrossomo/patologia , Equidae , Lectinas , Nanopartículas de Magnetita , Aglutinina de Amendoim , Espermatozoides , Animais , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/farmacologia , Fragmentação do DNA , Glicerol/farmacologia , Masculino , Análise do Sêmen , Sacarose/farmacologiaRESUMO
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.
Assuntos
Peso Corporal , Proteínas Alimentares , Túbulos Seminíferos/patologia , Motilidade dos Espermatozoides , Espermatozoides/patologia , Testículo/patologia , Acrossomo/patologia , Animais , Glândulas Bulbouretrais/patologia , Tamanho Celular , Sobrevivência Celular , Células Epiteliais/patologia , Glutens , Masculino , Proteínas de Vegetais Comestíveis , Próstata/patologia , Ratos , Análise do Sêmen , Proteínas de Soja , Cauda do Espermatozoide/patologia , Triticum , Zea maysRESUMO
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.
Assuntos
Fumar Cigarros/efeitos adversos , Infertilidade Masculina/imunologia , Sêmen/química , Proteínas de Plasma Seminal/análise , Varicocele/complicações , Acrossomo/efeitos dos fármacos , Acrossomo/imunologia , Acrossomo/patologia , Adulto , Brasil , Estudos Transversais , Fragmentação do DNA/efeitos dos fármacos , Epididimo/irrigação sanguínea , Epididimo/efeitos dos fármacos , Epididimo/imunologia , Humanos , Infertilidade Masculina/patologia , Masculino , Pessoa de Meia-Idade , não Fumantes/estatística & dados numéricos , Proteômica/estatística & dados numéricos , Sêmen/imunologia , Sêmen/metabolismo , Análise do Sêmen/estatística & dados numéricos , Proteínas de Plasma Seminal/metabolismo , Transdução de Sinais/imunologia , Fumantes/estatística & dados numéricos , Testículo/irrigação sanguínea , Testículo/efeitos dos fármacos , Testículo/imunologia , Nicotiana/toxicidade , Varicocele/imunologia , Adulto JovemRESUMO
The exposure and health effects of fluoride are an ongoing topic that has attracted worldwide attention. Fluoride exposure disturbs the testicular development, sexual hormone levels and spermatogenesis. However, as to whether fluoride interferes with acrosome formation which is essential for production of capable spermatozoa during spermatogenesis still remains unclear. The objective was to determine the effects of fluoride on the acrosome formation and to further elucidate the potential mechanism of impaired reproductive function. For this, forty adult rats were assigned into four groups. The control group received distilled water, while the other three groups were treated with 25, 50 and 100â¯mg NaF/L via drinking water for 56â¯d, respectively. Testes were processed for total RNA extraction and western blot analysis. Three samples of each group were fixed in 2.5% glutaraldehyde solution for transmission electron microscopy analysis. From the results, we first found that fluoride decreased the expression of mRNA and protein levels of Zpbp1, Spaca1 and Dpy19l2 of seven markers during acrosome biogenesis in testes. Furthermore, fluoride damaged not only the acrosome structure, but also the structure of the nuclear lamina which was observed to be discontinuous and partially missing by transmission electron microscopy. Moreover, the results indicated that the altered structure in nuclear lamina maybe due to reduced LMNB2 expression in testis induced by fluoride. In a nutshell, fluoride exposure could restrain acrosome biogenesis during spermatogenesis and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.
Assuntos
Acrossomo/patologia , Fluoretos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/patologia , Testículo/patologia , Acrossomo/efeitos dos fármacos , Acrossomo/metabolismo , Animais , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
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.
Assuntos
Acrossomo/patologia , Astenozoospermia/genética , Proteínas do Citoesqueleto/genética , Flagelos/genética , Infertilidade Masculina/genética , Mutação/genética , Adulto , Alelos , Axonema/genética , Exoma/genética , Homozigoto , Humanos , Masculino , Cauda do Espermatozoide/patologia , Espermatozoides/patologia , Sequenciamento do Exoma/métodosRESUMO
Autophagy is a subcellular process that is extensively involved in spermiogenesis. In this study, we observed ultrastructural malformation of acrosome and mitochondrial cristae during the spermiogenesis of Chinese soft-shelled turtle due to the inhibition of autophagy. Autophagy was blocked with 3-MA, and the inhibition of autophagy was confirmed through western blot analysis. The morphological abnormalities of acrosomes and mitochondria were observed under transmission electron microscopy (TEM). In the early spermiogenesis (Golgi and cap phases), damaged macrovesicle was observed, and its proper expansion over the nucleus failed to be form a normal acrosomal cap. As spermiogenesis proceeded, the malformation of the acrosome in spermatids became more severe. In the late spermiogenesis (acrosomal and maturation phases), defective acrosome with damaged acrosomal membrane that was detached from the nucleus was observed. Along with malformed acrosome, elongation failed nucleus having oval or round shaped morphology was also observed. Moreover, morphological damage to the mitochondrial cristae was observed. Lacuna formation, half and complete loss of cristae were observed in the mitochondria of developing spermatids. We proposed that autophagy is required for normal formation of the acrosome and mitochondrial cristae during turtle spermiogenesis.