RESUMO
Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.
Assuntos
Azoospermia/genética , Mutação , Insuficiência Ovariana Primária/genética , Adulto , Azoospermia/fisiopatologia , Pareamento Cromossômico , Códon sem Sentido , Proteínas de Ligação a DNA/metabolismo , Feminino , Homozigoto , Humanos , Masculino , Meiose , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Linhagem , Insuficiência Ovariana Primária/fisiopatologia , Espermatócitos/metabolismo , Espermatócitos/fisiologia , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Sequenciamento Completo do GenomaRESUMO
TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RTâPCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.
Assuntos
Homozigoto , Infertilidade Masculina , Mutação de Sentido Incorreto , Cauda do Espermatozoide , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Paquistão , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Adulto , Linhagem , Astenozoospermia/genética , Astenozoospermia/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Sequenciamento do Exoma , Oligospermia/genética , Oligospermia/patologia , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologiaRESUMO
Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.
Assuntos
Astenozoospermia , Consanguinidade , Homozigoto , Linhagem , Splicing de RNA , Cauda do Espermatozoide , Adulto , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patologia , Sequenciamento do Exoma , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Mutação , Splicing de RNA/genética , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Espermatozoides/ultraestrutura , Espermatozoides/patologiaRESUMO
Male infertility affects more than 20 million men worldwide and is a major public health concern. Male infertility has a strong genetic basis, particularly for those unexplained cases. Here, through genetic analysis of three Pakistani families having eight infertile men with normal parameters in routine semen analysis, we identified a novel ACTL7A variant (c.149_150del, p.E50Afs*6), recessively co-segregating with infertility in these three families. This variant leads to the loss of ACTL7A proteins in spermatozoa from patients. Transmission EM analyses revealed acrosome detachment from nuclei in 98.9% spermatozoa of patients. Interestingly, this ACTL7A variant was frequently detected in our sequenced Pakistani Pashtuns with a minor allele frequency of ~0.021 and all the carriers shared a common haplotype of about 240 kb flanking ACTL7A, indicating that it is likely originated from a single founder. Our findings reveal that a founder ACTL7A pathogenic variant confers a high genetic susceptibility for male infertility with normal routine semen parameters but acrosomal ultrastructural defects in Pakistani Pashtun descendants, and highlight that variants not rare should also be considered when trying to identify disease-causing variants in ethnic groups with the tradition of intra-ethnic marriages.
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Acrossomo , Infertilidade Masculina , Humanos , Masculino , Infertilidade Masculina/genética , Paquistão , Sêmen , Espermatozoides/metabolismoRESUMO
Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward. Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.
Assuntos
Fertilidade/genética , Proteínas do Tecido Nervoso/genética , Espermatogênese/genética , Testículo/metabolismo , Animais , Masculino , CamundongosRESUMO
Family with sequence similarity 181 (Fam181) is a gene family with two paralogues (Fam181a and Fam181b) found among vertebrates. Fam181a exhibits dynamic and stage-specific expression during murine embryo development. Furthermore, searching in the National Center for Biotechnology Information database revealed predominant expression of Fam181a in mouse and human testes, implying that it may have essential roles in spermatogenesis. In this study we investigated the invivo function of Fam181a in mouse spermatogenesis and fertility by generating Fam181a-/- mice using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 genome editing technology. The resulting Fam181a-/- mice exhibited normal growth and development. In addition, the mice were completely fertile, with no obvious differences in the testis-to-bodyweight ratio, epididymal sperm count or sperm motility compared with wild-type mice. Further examination of testicular and epididymal histology of Fam181a-/- mice found an intact seminiferous tubule structure and the presence of all types of germ cells, from spermatogonia to mature spermatozoa, similar to wild-type littermates. Similarly, analysis of meiotic prophase I progression revealed normal populations of each substage of prophase I in Fam181a+/+ and Fam181a-/- testes, suggesting that this gene is dispensable for male fertility. These negative findings will help avoid research overlap, save time and resources and allow researchers to concentrate on genes that are critical for male fertility and spermatogenesis.
RESUMO
Thousands of genes are involved in spermatogenesis, however, the functional roles of most these genes for male fertility remain to be discovered. This research focused to explore the function of evolutionarily conserved and testis-specific expressed gene 4930524B15Rik, which is known as C5orf47 in human. We generated 4930524B15Rik knockout mice by CRISPR/Cas9 technology and found 4930524B15Rik-/- mice were fertile. Furthermore, no averted abnormalities were observed in testis morphology, epididymal sperm contents and sperm morphology in 4930524B15Rik knockout mice. Subsequently, histological analysis of testicular tissue revealed intact structure of seminiferous tubules along with the presence of all types of germ cells in 4930524B15Rik-/- mice similar to wild type. Additionally, cytological analysis of spermatocytes displayed no significant differences in the prophase I progression of meiosis, further indicating that 4930524B15Rik have no essential function in mammalian spermatogenesis. Altogether, these results indicated that 4930524B15Rik is dispensable for fertility of male mice and these findings will help researchers to avoid future research overlap and to focus on genes that are crucial for spermatogenesis and reproduction.
Assuntos
Loci Gênicos , Infertilidade Masculina/genética , Espermatogênese , Animais , Sequência Conservada , Evolução Molecular , Deleção de Genes , Masculino , Camundongos , Testículo/citologia , Testículo/metabolismo , Testículo/fisiologiaRESUMO
ABSTRACT: Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.
RESUMO
BACKGROUND: Acephalic spermatozoa syndrome is a rare type of teratozoospermia causing male infertility due to detachment of the sperm head and flagellum, which precludes fertilization potential. Although loss-of-function variations in several genes, including TSGA10, have been associated with acephalic spermatozoa syndrome, the genetic cause of many cases remains unclear. RESULTS: We recruited a Pakistani family with two infertile brothers who suffered from acephalic spermatozoa syndrome. Through whole-exome sequencing (WES) followed by Sanger sequencing, we identified a novel missense variant in TSGA10 (c.1112T > C, p. Leu371Pro), which recessively co-segregated with the acephalic spermatozoa syndrome within this family. Ultrastructural analyses of spermatozoa from the patient revealed that 98% of flagellar cross-sections displayed abnormal axonemal ultrastructure, in addition to the head-flagellum detachment. Real-time quantitative PCR analysis revealed almost no detectable TSAG10 mRNA and western blot analysis also failed to detect TSAG10 protein in patient's sperm samples while TSGA10 expression was clearly detected in control samples. Consistently, immunofluorescence analysis demonstrated the presence of TSGA10 signal in the midpiece of sperm from the control but a complete absence of TSGA10 signal in sperm from the patient. CONCLUSION: Altogether, our study identifies a novel TSGA10 pathogenic variant as a cause of acephalic spermatozoa syndrome in this family and provides information regarding the clinical manifestations associated with TSGA10 variants in human.
RéSUMé: CONTEXTE: Le syndrome des spermatozoïdes acéphaliques est un type rare de tératozoospermie provoquant une infertilité masculine en raison du détachement de la tête et du flagelle des spermatozoïdes, ce qui exclut une potentielle fécondation. Bien que des variations de perte de fonction dans plusieurs gènes, y compris TSGA10, aient été associées au syndrome des spermatozoïdes acéphaliques, la cause génétique de nombreux cas reste incertaine. RéSULTATS: Nous avons recruté une famille pakistanaise avec deux frères infertiles qui souffraient du syndrome des spermatozoïdes acéphaliques. Grâce au séquençage de l'exome entier (WES) suivi du séquençage Sanger, nous avons identifié un nouveau variant faux-sens dans TSGA10 (c.1112T > C, p. Leu371Pro), qui co-ségréguait de manière récessive avec le syndrome des spermatozoïdes acéphaliques au sein de cette famille. Les analyses ultrastructurales des spermatozoïdes des patients ont révélé que 98% des coupes transversales flagellaires présentaient une ultrastructure axonémiques anormales, en plus du décollement tête-flagelle. L'analyse quantitative par PCR en temps réel n'a révélé presque aucun ARNm TSAG10 détectable; l'analyse par transfert Western n'a pas non plus réussi à détecter la protéine TSAG10 dans les échantillons de sperme des patients, tandis que l'expression de TSGA10 a été clairement détectée dans les échantillons du témoin. De manière cohérente, l'analyse par immunofluorescence a démontré la présence du signal TSGA10 dans la partie médiane des spermatozoïdes du témoin, mais une absence totale de signal TSGA10 chez ceux des patients. CONCLUSION: Dans l'ensemble, notre étude identifie un nouveau variant pathogène de TSGA10 comme cause du syndrome des spermatozoïdes acéphaliques dans cette famille et fournit des informations concernant les manifestations cliniques associées aux variants de TSGA10 chez l'homme. MOTS-CLéS: Infertilité, TSGA10, Spermatozoïdes acéphaliques, Variations faux-sens.
RESUMO
The meiosis-specific LINC complex, composed of the KASH5 and SUN1 proteins, tethers the moving chromosomes to the nuclear envelope to facilitate homolog pairing and is essential for gametogenesis. Here, we applied whole-exome sequencing for a consanguineous family with five siblings suffering from reproductive failure, and identified a homozygous frameshift mutation in KASH5 (c.1270_1273del, p.Arg424Thrfs*20). This mutation leads to the absence of KASH5 protein expression in testes and non-obstructive azoospermia (NOA) due to meiotic arrest before the pachytene stage in the affected brother. The four sisters displayed diminished ovarian reserve (DOR), with one sister never being pregnant but still having dominant follicle at 35 years old and three sisters suffering from at least 3 miscarriages occurring within the third month of gestation. The truncated KASH5 mutant protein, when expressed in cultured cells, displays a similar localization encircling the nucleus and a weakened interaction with SUN1, as compared with the full-length KASH5 proteins, which provides a potential explanation for the phenotypes in the affected females. This study reported sexual dimorphism for influence of the KASH5 mutation on human germ cell development, and extends the clinical manifestations associated with KASH5 mutations, providing genetic basis for the molecular diagnosis of NOA, DOR, and recurrent miscarriage.
Assuntos
Aborto Habitual , Azoospermia , Reserva Ovariana , Masculino , Feminino , Gravidez , Humanos , Adulto , Mutação da Fase de Leitura , Azoospermia/genética , Aborto Habitual/genética , Meiose , Proteínas de Ciclo CelularRESUMO
STUDY QUESTION: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN SIZE DURATION: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND THE ROLE OF CHANCE: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.
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Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 (CFAP57) loss-of-function mutations (c.2872C>T, p. R958*; and c.2737C>T, p. R913*) recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs' assembly and male fertility.
Assuntos
Cílios , Dineínas , Proteínas Associadas aos Microtúbulos , Animais , Humanos , Masculino , Camundongos , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Flagelos , Sêmen/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação com Perda de FunçãoRESUMO
During meiosis, at least one crossover must occur per homologous chromosome pair to ensure normal progression of meiotic division and accurate chromosome segregation. However, the mechanism of crossover formation is not fully understood. Here, we report a novel recombination protein, C12ORF40/REDIC1, essential for meiotic crossover formation in mammals. A homozygous frameshift mutation in C12orf40 (c.232_233insTT, p.Met78Ilefs*2) was identified in two infertile men with meiotic arrest. Spread mouse spermatocyte fluorescence immunostaining showed that REDIC1 forms discrete foci between the paired regions of homologous chromosomes depending on strand invasion and colocalizes with MSH4 and later with MLH1 at the crossover sites. Redic1 knock-in (KI) mice homozygous for mutation c.232_233insTT are infertile in both sexes due to insufficient crossovers and consequent meiotic arrest, which is also observed in our patients. The foci of MSH4 and TEX11, markers of recombination intermediates, are significantly reduced numerically in the spermatocytes of Redic1 KI mice. More importantly, our biochemical results show that the N-terminus of REDIC1 binds branched DNAs present in recombination intermediates, while the identified mutation impairs this interaction. Thus, our findings reveal a crucial role for C12ORF40/REDIC1 in meiotic crossover formation by stabilizing the recombination intermediates, providing prospective molecular targets for the clinical diagnosis and therapy of infertility.
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Azoospermia is a significant cause of male infertility, with non-obstructive azoospermia (NOA) being the most severe type of spermatogenic failure. NOA is mostly caused by congenital factors, but our understanding of its genetic causes is very limited. Here, we identified a frameshift variant (c.201_202insAC, p.Tyr68Thrfs∗17) and two nonsense variants (c.1897C>T, p.Gln633∗; c.2005C>T, p.Gln669∗) in KCTD19 (potassium channel tetramerization domain containing 19) from two unrelated infertile Chinese men and a consanguineous Pakistani family with three infertile brothers. Testicular histological analyses revealed meiotic metaphase I (MMI) arrest in the affected individuals. Mice modeling KCTD19 variants recapitulated the same MMI arrest phenotype due to severe disrupted individualization of MMI chromosomes. Further analysis showed a complete loss of KCTD19 protein in both Kctd19 mutant mouse testes and affected individual testes. Collectively, our findings demonstrate the pathogenicity of the identified KCTD19 variants and highlight an essential role of KCTD19 in MMI chromosome individualization.
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Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.
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Astenozoospermia , Dineínas , Astenozoospermia/genética , Astenozoospermia/patologia , Dineínas/genética , Homozigoto , Humanos , Masculino , Proteínas Associadas aos Microtúbulos , Mutação , Mutação de Sentido Incorreto , Cauda do Espermatozoide/metabolismoRESUMO
Family with sequence similarity 114 member A2 (Fam114a2) is sperm binding protein that is highly conserved in mammals with homologs both in fungi and plants. Previous studies have demonstrated that miR-762 and P63 are two crucial players of spermatogenesis, and CricFM114A2 regulates their expression. Thus, the current study was focused on describing the role of Fam114a2 in spermatogenesis by generating Fam114a2 knockout (Fam114a2-/-) mice using CRISPR/Cas9 genome editing techniques. We identified that Fam114a2-/- mouse has normal fertility and normal morphology of sperm. Furthermore, histological investigation of testicular and epididymis tissues showed no subtle difference, and seminiferous tubules comprised of all stages of germ cells, including mature spermatozoa in Fam114a2-/- mice. Moreover, cytological investigation of spermatocytes in the progression of prophase I also did not display any notable difference in Fam114a2-/- mice. Additionally, normal expression of p63 and miR-762 was observed in Fam114a2+/+ and Fam114a2-/- testis indicating that Fam114a2 is not involved in the direct regulation of in mice spermatogenesis. Moreover, the removal of Fam114a2 in mouse did not affect the expression of its paralogue Fam114a1 in multiple tissues. Taken together our data determined that Fam114a2 is not essential for male fertility and spermatogenesis in mice.
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Fertilidade , MicroRNAs/metabolismo , Proteínas de Neoplasias , Espermatogênese/fisiologia , Transativadores/metabolismo , Animais , Evolução Biológica , Regulação da Expressão Gênica , Masculino , Meiose , Camundongos , Camundongos Knockout , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Transativadores/genéticaRESUMO
Sustaining and maintaining the intricate process of spermatogenesis is liable upon hormones and growth factors acting through endocrine and paracrine pathways. The Sertoli cells (SCs) are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. As each Sertoli cell supports a specific number of germ cells, thus, the final number of Sertoli cells determines the sperm production capacity. Similarly, sex hormones are also major regulators of spermatogenesis and they can determine the proliferation of Sertoli cells. In the present review, we have critically and comprehensively discussed the role of sex hormones and some other factors that are involved in Sertoli cell proliferation, differentiation and maturation. Furthermore, we have also presented a model of Sertoli cell development based upon the recent advancement in the field of reproduction. Hence, our review article provides a general overview regarding the sex hormonal pathways governing Sertoli cell proliferation and development.
Assuntos
Hormônios Esteroides Gonadais/sangue , Células de Sertoli/citologia , Espermatogênese/fisiologia , Testículo/metabolismo , Ativinas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Estrogênios/metabolismo , Fertilidade , Hormônio Foliculoestimulante/metabolismo , Humanos , Inibinas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Progesterona/metabolismo , Prolactina/metabolismo , Ratos , Receptor de Insulina/metabolismo , Transdução de Sinais , Testosterona/metabolismo , Hormônios Tireóideos/metabolismo , Tretinoína/metabolismo , Proteínas Wnt/metabolismoRESUMO
CCND1 encodes for Cyclin D1 protein and single-nucleotide polymorphisms (SNPs) can modulate its activity. In the present study, the impact of CCND1 SNPs on structure and/or function of Cyclin D1 protein using in silico tools was investigated. Our analysis revealed only one splice site SNP (c.1988+5G
Assuntos
Neoplasias da Mama/genética
, Biologia Computacional/métodos
, Ciclina D1/genética
, Polimorfismo de Nucleotídeo Único
, Sequência de Aminoácidos
, Ciclina D1/química
, Feminino
, Mutação da Fase de Leitura
, Regulação Neoplásica da Expressão Gênica
, Humanos
, Estimativa de Kaplan-Meier
, Mutação de Sentido Incorreto
, Mapas de Interação de Proteínas
, Processamento de Proteína Pós-Traducional
, Estrutura Terciária de Proteína
, Splicing de RNA
RESUMO
Exonuclease 5 (Exo5) belongs to a class of bi-directional, ssDNA-specific exonucleases that mainly involved in the DNA repair pathways. Exo5 has been reported to be crucial for DNA- DNA mismatch repair (MMR) in several human cell lines. However, its in vivo function in mammals still needs to be explored. Thus, to study the in vivo role of Exo5 in spermatogenesis, Exo5 knockout mice were generated using CRISPR/Cas9 technology. Unexpectedly, we found that the knockout mice are fertile despite a slight decrease in sperm count. Furthermore, Exo5-/- mice showed no detectable developmental anomalies, exhibited no remarkable differences in the epididymal histology and testis/body weight ratio. Moreover, cytological investigations on meiocytes revealed non-significant differences in chromosomal synapsis, recombination, and meiotic progression of prophase I, further demonstrating that Exo5 has no essential role in spermatogenesis in mice under normal breeding conditions. Collectively, these data indicate that Exo5 is dispensable for meiotic progression and fertility in mice.