RESUMO
Non-obstructive azoospermia (NOA) is the most severe form of human male infertility, and the genetic causes of NOA with meiotic arrest remain largely unclear. In this study, we identified novel compound heterozygous MEIOB variants (c.814C > T: p.R272X and c.976G > A: p.A326T) and a previously undescribed homozygous non-canonical splicing variant of MEIOB (c.528 + 3A > C) in two NOA-affected individuals from two irrelevant Chinese families. MEIOB missense variant (p.A326T) significantly reduced protein abundance and nonsense variant (p.R272X) produced a truncated protein. Both of two variants impaired the MEIOB-SPATA22 interaction. The MEIOB non-canonical splicing variant resulted in whole Exon 6 skipping by minigene assay, which was predicted to produce a frameshift truncated protein (p.S111Rfs*32). Histological and immunostaining analysis indicated that both patients exhibited a similar phenotype as we previously reported in Meiob mutant mice, that is, absence of spermatids in seminiferous tubules and meiotic arrest. Our study identified three novel pathogenic variants of MEIOB in NOA patients, extending the mutation spectrum of the MEIOB and highlighting the contribution of meiotic recombination related genes in human fertility.
Assuntos
Azoospermia , Infertilidade Masculina , Humanos , Masculino , Camundongos , Animais , Azoospermia/genética , Azoospermia/patologia , Infertilidade Masculina/genética , Mutação/genética , Proteínas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Meiose/genética , Proteínas de Ligação a DNA/genéticaRESUMO
Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.
Assuntos
Mitofagia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Placenta , Pré-Eclâmpsia , Receptores dos Hormônios Tireóideos , Humanos , Pré-Eclâmpsia/induzido quimicamente , Feminino , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Gravidez , Mitofagia/efeitos dos fármacos , Placenta/efeitos dos fármacos , Placenta/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Cádmio/toxicidade , Regulação para Baixo/efeitos dos fármacos , Adulto , Transdução de Sinais/efeitos dos fármacosRESUMO
Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.
Assuntos
Predisposição Genética para Doença , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/genética , Proteínas Associadas aos Microtúbulos/deficiência , Fenótipo , Cauda do Espermatozoide/metabolismo , Animais , Biomarcadores , Consanguinidade , Modelos Animais de Doenças , Feminino , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Linhagem , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Espermatogênese/genética , Sequenciamento do ExomaRESUMO
Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.
Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: It has been long known that thyroid hormone regulates placental villi development, which is associated with the occurrence of miscarriage. However, whether abnormal thyroid hormone metabolism and transport in placental villi are involved in miscarriage is still to be verified. METHODS: Placental villi of elective terminations of pregnancies (ETPs) and miscarriage were collected. Proliferative activity and apoptosis of villi trophoblasts and angiogenesis were detected by TUNEL and immunochemistry. The expressions of thyroid hormone receptors (THRs), transthyretin (TTR), monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptides 1A1 (OATP1A1), deiodinase 2 (Dio2) and Dio3 were examined by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. JEG3 cell was treated with iopanoic acid (IOP), an inhibitor of Dio2 activity, the expressions of Dio2, placenta growth factor (PLGF) and sFlt1 were detected by RT-PCR and Western blot. RESULTS: Cell proliferation was suppressed and apoptosis was increased in placental villi cytotrophoblasts of miscarriage. CD34+ vessel number and vascular endothelial growth factor (VEGF) protein abundance were decreased in miscarriage. In miscarriage group, the gene expression of Dio2, Dio3, TTR and THRα, but not THRß, MCT8 and OATP1A1, were downregulated. The protein abundances of TTR and THRα were downregulated in miscarriage group, but not THRß. The protein abundance of Dio2 in miscarriage villi was decreased compared with that in ETP. In JEG3 cells, the gene expression of PLGF was decreased and the expression of sFlt1 was increased in IOP treatment; The protein abundance of Dio2 was downregulated but the gene expression of Dio2 was unaffected in IOP treatment. CONCLUSION: Thyroid hormone transport and metabolism in miscarriage were disturbed and may impaired angiogenesis of placental villi, which was associated with the occurrence of miscarriage.
Assuntos
Aborto Espontâneo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Gravidez , Feminino , Aborto Espontâneo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vilosidades Coriônicas/metabolismo , Linhagem Celular Tumoral , Placenta/metabolismo , Hormônios Tireóideos/metabolismoRESUMO
PURPOSE: Poor ovarian response (POR) affects approximately 9% to 24% of women undergoing in vitro fertilization (IVF) cycles, resulting in fewer eggs obtained and increasing clinical cycle cancellation rates. The pathogenesis of POR is related to gene variations. Our study included a Chinese family comprising two siblings with infertility born to consanguineous parents. Poor ovarian response (POR) was identified in the female patient who had multiple embryo implantation failures occurring in subsequent assisted reproductive technology cycles. Meanwhile, the male patient was diagnosed with non-obstructive azoospermia (NOA). METHODS: Whole-exome sequencing and rigorous bioinformatics analyses were conducted to identify the underlying genetic causes. Moreover, the pathogenicity of the identified splicing variant was assessed using a minigene assay in vitro. The remaining poor-quality blastocyst and abortion tissues from the female patient were detected for copy number variations. RESULTS: We identified a novel homozygous splicing variant in HFM1 (NM_001017975.6: c.1730-1G > T) in two siblings. Apart from NOA and POI, biallelic variants in HFM1 were also associated with recurrent implantation failure (RIF). Additionally, we demonstrated that splicing variants caused abnormal alternative splicing of HFM1. Using copy number variation sequencing, we found that the embryos of the female patients had either euploidy or aneuploidy; however, both harbored chromosomal microduplications of maternal origin. CONCLUSION: Our results reveal the different effects of HFM1 on reproductive injury in males and females, extend the phenotypic and mutational spectrum of HFM1, and show the potential risk of chromosomal abnormalities under the RIF phenotype. Moreover, our study provides new diagnostic markers for the genetic counseling of POR patients.
Assuntos
Azoospermia , Variações do Número de Cópias de DNA , Feminino , Humanos , Masculino , Gravidez , Azoospermia/genética , Aberrações Cromossômicas , DNA Helicases/genética , Implantação do Embrião/genética , Gametogênese , Isoformas de ProteínasRESUMO
Cadmium is a ubiquitous environmental pollutant, which can increase the risk of preeclampsia. This study was designed to determine the mechanism of cadmium exposure during pregnancy impaired placental angiogenesis that was associated with the occurrence of preeclampsia. The effects of cadmium exposure on placental thyroid hormone receptor signaling were explored. JEG3 cells were treated with CdCl2 (20 µM) and the Dio2 inhibitor, IOP (100 µM). Cadmium levels in maternal blood and placentae were increased in preeclampsia group. Placental angiogenesis of preeclampsia was decreased with decreased expression of PLGF and VEGF and increased expression of sFlt1. Meanwhile, the expression and nuclear translocation of thyroid hormone receptor α were decreased in preeclampsia placenta, as well as the expression of Dio2, but not the expression and nuclear translocation of thyroid hormone receptor ß. Furthermore, we found that cadmium exposure downregulated the expression of thyroid hormone receptor α and Dio2, but not the expression of thyroid hormone receptor ß in JEG3 cells. Also, we found that cadmium exposure decreased the expression of PLGF and VEGF and increased the expression of sFlt1 in JEG3 cells. IOP pretreatment decreased the expression of PLGF and increased the expression of sFlt1. In conclusion, our results elucidated that cadmium exposure would impair placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.
Assuntos
Poluentes Ambientais , Pré-Eclâmpsia , Cádmio/metabolismo , Linhagem Celular Tumoral , Poluentes Ambientais/metabolismo , Feminino , Humanos , Neovascularização Patológica , Placenta/metabolismo , Fator de Crescimento Placentário/metabolismo , Fator de Crescimento Placentário/farmacologia , Pré-Eclâmpsia/induzido quimicamente , Pré-Eclâmpsia/metabolismo , Gravidez , Receptores dos Hormônios Tireóideos/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Reduced generation of multiple motile cilia (RGMC) and the consequent primary ciliary dyskinesia (PCD) cause infertility due to a substantial reduction in the number of multiciliated cells (MCCs) in the efferent ducts (EDs)/oviducts. MCIDAS acts upstream of CCNO to regulate the biogenesis of basal bodies (BBs); therefore, both genes play a vital role in the multiciliogenesis of the reproductive tract epithelium. In this study, whole-exome sequencing was performed to identify the causative genes in 10 unrelated infertile patients with PCD: seven males and three females. Notably, homozygous frameshift mutations in MCIDAS (c.186dupT, p.Pro63Serfs*22) and CCNO (c.262_263insGGCCC, p.Gln88Argfs*8) were identified in one male and one female participant from two unrelated consanguineous families. Haematoxylin-eosin staining/scanning electron microscopy revealed abnormal MCCs in the mutated EDs/oviducts. Furthermore, transmission electron microscopy revealed significantly reduced BBs. Immunofluorescence staining showed the absence of MCIDAS and CCNO signals in the affected tissues and confirmed that MCIDAS acts upstream of CCNO in the context of multiciliogenesis in the reproductive tract epithelium. In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was successful, with a positive pregnancy outcome in both MCIDAS- and CCNO-mutated patients. Our results support the use of IVF/ICSI interventions to treat infertility due to RGMC in couples.
Assuntos
Alelos , Proteínas de Ciclo Celular/genética , DNA Glicosilases/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Infertilidade/diagnóstico , Infertilidade/genética , Mutação , Fatores de Transcrição/genética , Adulto , Proteínas de Ciclo Celular/metabolismo , Consanguinidade , DNA Glicosilases/metabolismo , Análise Mutacional de DNA , Epitélio/metabolismo , Epitélio/patologia , Epitélio/ultraestrutura , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Linhagem , Fatores de Transcrição/metabolismo , Sequenciamento do ExomaRESUMO
BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility; more than half of the NOA patients are idiopathic. Although many NOA risk genes have been detected, the genetic factors for NOA in majority of the patients are unknown. In addition, it is difficult to retrieve sperm from these patients despite using the microsurgical testicular sperm extraction (microTESE) method. Therefore, we conducted this genetic study to identify the potential genetic factors responsible for NOA and investigate the sperm retrieval rate of microTESE for genetically deficient NOA patients. METHODS: Semen analyses, sex hormone testing, and testicular biopsy were performed to categorize the patients with NOA. The chromosome karyotypes and Y chromosome microdeletion analyses were used to exclude general genetic factors. Whole exome sequencing and Sanger sequencing were performed to identify potential genetic variants in 51 patients with NOA. Hematoxylin and eosin staining (H&E) and anti-phosphorylated H2AX were used to assess the histopathology of spermatogenesis. Quantitative real time-polymerase chain reaction, western blotting, and immunofluorescence were performed to verify the effects of gene variation on expression. RESULTS: We performed whole exome sequencing in 51 NOA patients and identified homozygous helicase for meiosis 1(HFM1) variants (NM_001017975: c.3490C > T: p.Q1164X; c.3470G > A: p.C1157Y) in two patients (3.9%, 2/51). Histopathology of the testis showed that spermatogenesis was completely blocked at metaphase in these two patients carrying the HFM1 homozygous variants. In comparison with unaffected controls, we found a significant reduction in the levels of HFM1 mRNA and protein expression in the testicular tissues from these two patients. The patients were also subjected to microTESE treatment, but the sperms could not be retrieved. CONCLUSIONS: This study identified novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, and microTESE did not aid in retrieving sperms from these patients.
Assuntos
Azoospermia/genética , DNA Helicases/genética , Espermatogênese/genética , Testículo/patologia , Adulto , DNA Helicases/metabolismo , Homozigoto , Humanos , Masculino , Metáfase , Microcirurgia , Recuperação Espermática , Espermatozoides/patologia , Testículo/metabolismo , Testículo/cirurgiaRESUMO
BACKGROUND: Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported. METHODS: Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied. RESULTS: By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A > G, p.Asp2319Gly; F2 II-1: c.6294 T > A, p.Phe2098Leu / c.10571 T > A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy. CONCLUSIONS: We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.
Assuntos
Astenozoospermia/genética , Dineínas do Axonema/genética , Adulto , Astenozoospermia/patologia , China , Análise Mutacional de DNA , Predisposição Genética para Doença , Humanos , Infertilidade Masculina/genética , Masculino , Mutação , Linhagem , Índice de Gravidade de Doença , Sequenciamento do ExomaRESUMO
RESEARCH QUESTION: Multiple morphological abnormalities of the flagella (MMAF) is characterized by excessive immotile spermatozoa with severe flagellar abnormalities in the ejaculate. Previous studies have reported a heterogeneous genetic profile associated with MMAF. What other genetic variants might explain the cause of MMAF? DESIGN: Whole-exome sequencing was conducted in a cohort of 90 Chinese patients with MMAF. The pathogenicity of identified mutations was assessed through electron microscopy and immunofluorescent examinations. RESULTS: Three unrelated men with bi-allelic DNAH2 variants were identified. Sanger sequencing verified that the six novel variants originated from every parent. All these variants were located at the conserved domains of DNAH2 and predicted to be deleterious by bioinformatic tools. Haematoxylin and eosin staining and scanning electron microscopy revealed that spermatozoa harbouring DNAH2 variants displayed severely aberrant morphology mainly with absent and short flagella (≥78%). Moreover, transmission electron microscopy revealed the obvious absence of a central pair of microtubules and inner dynein arms in the spermatozoa with mutated DNAH2. Immunofluorescence data further validated these findings, showing reduced DNAH2 protein expression in the spermatozoa with DNAH2 variants, compared with normal spermatozoa. Intracytoplasmic sperm injection using spermatozoa from the three men with mutated DNAH2 resulted in blastocyst formation in all cases. Embryo transfer was carried out in two couples, both resulting in clinical pregnancy. CONCLUSIONS: These experimental and clinical data suggest that bi-allelic DNAH2 variants might induce MMAF-associated asthenoteratozoospermia, which can be overcome through intracytoplasmic sperm injection. These findings contribute to the knowledge of the genetic landscape of asthenoteratozoospermia and clinical counselling of male infertility.
Assuntos
Astenozoospermia/genética , Dineínas do Axonema/genética , Adulto , Astenozoospermia/patologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Gravidez , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/ultraestrutura , Sequenciamento do ExomaRESUMO
BACKGROUND: Asthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes. METHODS AND RESULTS: Two asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF. CONCLUSION: Our study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Astenozoospermia/genética , Anormalidades Múltiplas/patologia , Animais , Astenozoospermia/patologia , Exoma/genética , Células HEK293 , Homozigoto , Humanos , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/metabolismo , Espermatozoides/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases. METHODS AND RESULT: We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF. CONCLUSIONS: Our experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.
Assuntos
Anormalidades Múltiplas/genética , Infertilidade Masculina/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Cauda do Espermatozoide/metabolismo , Anormalidades Múltiplas/patologia , Adulto , Alelos , Animais , Flagelos/genética , Flagelos/patologia , Humanos , Infertilidade Masculina/patologia , Irã (Geográfico) , Masculino , Camundongos , Mutação/genética , Fenótipo , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/patologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: Male infertility is a major issue of human reproduction health. Asthenoteratospermia can impair sperm motility and cause male infertility. Asthenoteratospermia with multiple morphological abnormalities of the flagella (MMAF) presents abnormal spermatozoa with absent, bent, coiled, short and/or irregular-calibre flagella. Previous studies on MMAF reported that genetic defects in cilia-related genes (eg, AKAP4, DNAH1, CFAP43, CFAP44 and CFAP69) are the major cause of MMAF. However, the known MMAF-associated genes are only responsible for approximately 30% to 50% of human cases. We further investigated the cases with MMAF in search of additional genes mutated in this condition. METHODS AND RESULTS: We conducted whole exome sequencing in a male individual with MMAF from a consanguineous Han Chinese family. Sanger sequencing was also conducted in additional individuals with MMAF. Intriguingly, a homozygous frameshift mutation (p.Leu357Hisfs*11) was identified in the gene encoding CFAP69 (cilia and flagella-associated protein 69), which is highly expressed in testis. The subsequent Sanger sequencing of the CFAP69 coding regions among 34 additional individuals with MMAF revealed a case with homozygous nonsense mutation (p.Trp216*) of CFAP69. Both of these CFAP69 loss-of-function mutations were not present in the human population genome data archived in the 1000 Genomes Project and ExAC databases, nor in 875 individuals of two Han Chinese control populations. Furthermore, we generated the knockout model in mouse orthologue Cfap69 using the CRISPR-Cas9 technology. Remarkably, male Cfap69-knockout mice manifested with MMAF phenotypes. CONCLUSION: Our experimental findings elucidate that homozygous loss-of-function mutations in CFAP69 can lead to asthenoteratospermia with MMAF in humans and mice.
Assuntos
Astenozoospermia/genética , Proteínas do Citoesqueleto/genética , Mutação com Perda de Função , Cauda do Espermatozoide/patologia , Animais , Astenozoospermia/patologia , Feminino , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testículo/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: Asthenoteratospermia with multiple morphological abnormalities in the sperm flagella (MMAF) is a significant cause of male infertility. WDR19 is a core component in the IFT-A complex and has a critical role in intraflagellar transport. However, the role of WDR19 mutations in male infertility has yet to be examined. METHODS AND RESULTS: We performed whole exome sequencing (WES) for 65 asthenoteratospermia individuals and identified a proband who carried a homozygous WDR19 (c.A3811G, p.K1271E) mutation from a consanguineous family. Systematic examinations, including CT scanning and retinal imaging, excluded previous ciliopathic syndromes in the proband. Moreover, semen analysis of this patient showed that the progressive rate decreased to zero, and the sperm flagella showed multiple morphological abnormalities. Scanning and transmission electron microscopy assays indicated that the ultrastructure of sperm flagella in the patient was completely destroyed, while immunofluorescence revealed that WDR19 was absent from the sperm neck and flagella. Moreover, IFT140 and IFT88, predicted to interact with WDR19 directly, were mis-allocated in the WDR19-mutated sperm. Notably, the MMAF subject harboring WDR19 variant and his partner successfully achieved clinical pregnancy through intracytoplasmic sperm injection (ICSI). CONCLUSIONS: We identified WDR19 as a novel pathogenic gene for male infertility caused by asthenoteratospermia in the absence of other ciliopathic phenotypes, and that patients carrying WDR19 variant can have favorable pregnancy outcomes following ICSI.
Assuntos
Astenozoospermia/genética , Proteínas do Citoesqueleto/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cauda do Espermatozoide/metabolismo , Adulto , Astenozoospermia/patologia , Exoma/genética , Feminino , Homozigoto , Humanos , Infertilidade Masculina/patologia , Masculino , Microtúbulos/genética , Microtúbulos/patologia , Mutação/genética , Gravidez , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/patologia , Espermatozoides/patologia , Sequenciamento do ExomaRESUMO
PURPOSE: To investigate the relation between mutations in ciliopathy-related SPAG6 and RSPH3 and male infertility with severe asthenoteratospermia characterized by multiple flagellar malformations and reveal the intracytoplasmic sperm injection (ICSI) outcomes of those primary ciliary dyskinesia (PCD) patients. METHODS: Whole-exome sequencing was applied to identify the pathogenic genes for the five PCD patients. The ICSI outcomes of those patients were compared with eight DNAH1-mutated patients and 215 oligo-asthenospermia (OAT) patients. RESULTS: We identified, for the first time, the compound heterozygous SPAG6 mutations (c.143_145del: p.48_49del, c.585delA: p.Lys196Serfs*6) in a sporadic PCD patient. Further, a novel homozygous nonsynonymous RSPH3 mutation (c.C799T: p.Arg267Cys) was identified in another PCD patient with consanguineous parents. The pathogenicity of these mutations in the assembly of sperm flagella was confirmed by flagellar ultrastructure analysis, immunofluorescence, and quantitative real-time PCR. All five patients underwent six ICSI cycles. The fertilization rate, blastocyst development rate, and clinical pregnancy rate were 69.3%, 50.0%, and 66.7%, respectively. Four of the five couples, including the subjects carrying mutations in SPAG6 or RSPH3, got healthy children born after ICSI. Additionally, the ICSI outcomes of the five PCD couples were statistically comparable with those of the eight DNAH1-mutated couples and the 215 OAT couples. CONCLUSIONS: Mutations in ciliopathy-related SPAG6 and RSPH3 cause severe asthenoteratospermia characterized by multiple flagellar malformations, resulting in sterility. ICSI is an optimal management with a positive pregnancy outcome.
Assuntos
Astenozoospermia/genética , Dineínas/genética , Infertilidade Masculina/genética , Proteínas dos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Adulto , Astenozoospermia/diagnóstico , Astenozoospermia/patologia , Feminino , Homozigoto , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/patologia , Masculino , Mutação/genética , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas , Espermatozoides/patologia , Sequenciamento do Exoma , Adulto JovemRESUMO
PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.
Assuntos
Azoospermia/genética , Infertilidade Masculina/genética , Doenças Urogenitais Masculinas/genética , Receptores Acoplados a Proteínas G/genética , Ducto Deferente/anormalidades , Adulto , Animais , Azoospermia/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Hemizigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Masculino , Doenças Urogenitais Masculinas/patologia , Camundongos , Injeções de Esperma Intracitoplásmicas/normas , Espermatogênese/genética , Ducto Deferente/patologia , Sequenciamento do ExomaRESUMO
Rough set theory is a suitable tool for dealing with the imprecision, uncertainty, incompleteness, and vagueness of knowledge. In this paper, new lower and upper approximation operators for generalized fuzzy rough sets are constructed, and their definitions are expanded to the interval-valued environment. Furthermore, the properties of this type of rough sets are analyzed. These operators are shown to be equivalent to the generalized interval fuzzy rough approximation operators introduced by Dubois, which are determined by any interval-valued fuzzy binary relation expressed in a generalized approximation space. Main properties of these operators are discussed under different interval-valued fuzzy binary relations, and the illustrative examples are given to demonstrate the main features of the proposed operators.
Assuntos
Análise Numérica Assistida por Computador , Algoritmos , Lógica FuzzyRESUMO
Triphenyltin chloride (TPTCL) is widely used in various industrial and agricultural applications. This study aimed to elucidate the mechanisms underlying the toxicological effects of TPTCL on oocytes. The obtained findings revealed that TPTCL exposure reduced polar body extrusion (PBE) and induced meiotic arrest. Mechanistically, TPTCL disrupted meiotic spindle assembly and chromosome alignment. Further analysis indicated a significant decrease in p-MAPK expression, and disturbances in the localization of Pericentrin and p-Aurora A in TPTCL exposed oocytes, which suggesting impaired microtubule organizing center (MTOC)function. Moreover, TPTCL exposure enhance microtubule acetylation and microtubule instability. Therefore, the spindle assembly checkpoint (SAC) remained activated, and the activity of the anaphase-promoting complex (APC) was inhibited, thereby preventing oocytes from progressing into the entering anaphase I (AI) stage. TPTCL exposure also augmented the actin filaments in the cytoplasm. Notably, mitochondrial function appeared unaffected by TPTCL, as evidenced indicated by stable mitochondrial membrane potential and ATP content. Furthermore, TPTCL treatment altered H3K27me2, H3K27me3 and H3K9me3 levels, suggesting changes in epigenetic modifications in oocytes. Taken together, our results suggest that TPTCL disrupts cytoskeleton assembly, continuously activates SAC, inhibits APC activity, and blocks meiotic progression, ultimately impair oocyte maturation.
Assuntos
Citoesqueleto , Meiose , Oócitos , Compostos Orgânicos de Estanho , Animais , Oócitos/efeitos dos fármacos , Meiose/efeitos dos fármacos , Feminino , Citoesqueleto/efeitos dos fármacos , Compostos Orgânicos de Estanho/toxicidade , Camundongos , Camundongos Endogâmicos ICR , Ciclo Celular/efeitos dos fármacosRESUMO
Dysplasia and invasive defects in early trophoblasts contribute to unexplained recurrent miscarriages (URMs). Mesencephalic astrocyte-derived neurotrophic factor (MANF) inhibits migration and invasion in some cancer cells, but its role in pregnancy-related diseases remains unresolved. Here, we found that MANF levels in the peripheral blood and aborted tissue of URM women were higher than in normal controls, irrespective of pregnancy or miscarriage. We confirm the interaction between MANF and nucleophosmin 1 (NPM1) in trophoblasts of URM patients, which increases the ubiquitination degradation of NPM1, leading to upregulation of the p53 signaling pathway and inhibition of cell proliferation, migration, and invasion ability. Using a URM mouse model, we found that MANF downregulation resulted in reduced fetal resorption; however, concomitant NPM1 downregulation led to increased abortion rates. These data indicate that MANF triggers miscarriage via NPM1 downregulation and p53 activation. Thus, MANF downregulation or disruption of the MANF-NPM1 interaction could be targets for URM therapeutics.