RESUMEN
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.
Asunto(s)
Astenozoospermia , Consanguinidad , Homocigoto , Linaje , Empalme del ARN , Cola del Espermatozoide , Adulto , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Secuenciación del Exoma , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación , Empalme del ARN/genética , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Espermatozoides/ultraestructura , Espermatozoides/patologíaRESUMEN
BACKGROUND: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.
Asunto(s)
Astenozoospermia , Dineínas Axonemales , Secuenciación del Exoma , Infertilidad Masculina , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Dineínas Axonemales/genética , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Adulto , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Inyecciones de Esperma Intracitoplasmáticas , Embarazo , Espermatozoides/ultraestructura , Espermatozoides/patología , Dineínas/genéticaRESUMEN
Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.
Asunto(s)
Núcleo Celular , Centriolos , Proteínas de Drosophila , Espermatogénesis , Espermatozoides , Centriolos/metabolismo , Centriolos/ultraestructura , Masculino , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Drosophila melanogaster/metabolismo , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Cabeza del Espermatozoide/ultraestructura , Cabeza del Espermatozoide/metabolismo , Axonema/metabolismo , Axonema/ultraestructuraRESUMEN
The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.
Asunto(s)
Centrosoma , Ratones Noqueados , Membrana Nuclear , Cola del Espermatozoide , Animales , Masculino , Membrana Nuclear/metabolismo , Centrosoma/metabolismo , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Ratones , Espermatogénesis/genética , Ratones Transgénicos , Fertilidad , Axonema/metabolismo , Axonema/ultraestructura , Espermatozoides/metabolismo , Espermatozoides/ultraestructuraRESUMEN
The ultrastructural features of the mature spermatozoon of Telorchis attenuatus (Digenea, Telorchiidae), an intestinal parasite of the red-eared turtle Trachemys scripta elegans (Testudines, Emydidae), are described using transmission electron microscopy (TEM). The mature spermatozoon of T. attenuatus is a filiform cell tapered at both ends and displays Bakhoum et al.'s type IV of digenean sperm cells. Spermatozoa of T. attenuatus have: (i) two axonemes of different lengths with the 9+'1' pattern of trepaxonematan Platyhelminthes, surrounded by a continuous submembranous layer of cortical microtubules at their anterior end, (ii) an external ornamentation of the plasma membrane following Quilichini et al.'s type 2 and associated with cortical microtubules, (iii) two bundles of parallel cortical microtubules with the maximum number situated in the anterior part of the sperm cell, (iv) spine-like bodies, (v) two mitochondria, and (vi) a large number of irregularly distributed glycogen granules. Furthermore, the morphology of the posterior spermatozoon extremity in T. attenuatus corresponds to the Quilichini et al.'s fasciolidean type. The results of the current study are especially compared to the existing information from other families within the superfamily Plagiorchioidea.
Asunto(s)
Espermatozoides , Trematodos , Tortugas , Animales , Masculino , Tortugas/parasitología , Espermatozoides/ultraestructura , Trematodos/ultraestructura , Microscopía Electrónica de Transmisión , Microtúbulos/ultraestructura , Axonema/ultraestructura , Mitocondrias/ultraestructura , Intestinos/parasitología , Intestinos/ultraestructuraRESUMEN
Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.
Asunto(s)
Microscopía por Crioelectrón , Vesículas Extracelulares , Semen , Animales , Vesículas Extracelulares/ultraestructura , Vesículas Extracelulares/metabolismo , Masculino , Microscopía por Crioelectrón/métodos , Porcinos , Espermatozoides/ultraestructura , Vesículas Seminales/ultraestructuraRESUMEN
Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as 'extracystic spermiogenesis'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.
Asunto(s)
Peces , Espermatogénesis , Espermatozoides , Testículo , Animales , Masculino , Espermatogénesis/fisiología , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Testículo/ultraestructura , Testículo/fisiología , Peces/fisiologíaRESUMEN
PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.
Asunto(s)
Flagelos , Homocigoto , Infertilidad Masculina , Mitocondrias , Mutación , Motilidad Espermática , Cola del Espermatozoide , Espermatozoides , Masculino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Humanos , Ratones , Espermatozoides/patología , Espermatozoides/ultraestructura , Espermatozoides/metabolismo , Animales , Mitocondrias/genética , Mitocondrias/ultraestructura , Mitocondrias/patología , Mitocondrias/metabolismo , Mutación/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Flagelos/genética , Flagelos/ultraestructura , Flagelos/metabolismo , Motilidad Espermática/genética , Secuenciación del Exoma , Linaje , Adulto , Análisis de SemenRESUMEN
Global coverage of living coral has declined by half since 1950s. Reef-building species have been severely impacted in this climate crisis scenario, compromising the future of coral reefs. Despite their importance, there is a lack of knowledge regarding the reproductive biology of scleractinian corals. In the present study, we evaluated through electron microscopy approaches, the gametes of the endemic Southwestern Atlantic coral Mussismilia harttii. We observed spherical oocytes with microvilli throughout the outer membrane. Fine granular material dispersed in cytoplasm, lipid granules, numerous yolk bodies, and mitochondria were identified in the oocytes. In addition, small Symbiodinium-like cells were observed, suggesting a vertical transmission from parental coral to oocytes. The spherical-head sperm presents a 9.3 ± 2.1⯵m flagellum. The nucleus is located centrally in the head, and the centrioles are positioned between the nuclear base and the flagellar insertion, which is connected to the axoneme. This axoneme has a microtubular arrangement (9+2). Vesicles, underlining the inner plasma membrane, presented the same electron-dense pattern as the Golgi complex, and mitochondria positioned surrounding the axoneme. The vesicles present in the sperm may have a role as an acrosome since the oocytes do not develop any cell specialization for fertilization.
Asunto(s)
Antozoos , Oocitos , Espermatozoides , Animales , Antozoos/ultraestructura , Masculino , Oocitos/ultraestructura , Espermatozoides/ultraestructura , Arrecifes de Coral , FemeninoRESUMEN
The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.
Asunto(s)
Cola del Espermatozoide , Espermatozoides , Masculino , Animales , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Espermatozoides/fisiología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Humanos , Axonema/ultraestructura , Axonema/metabolismo , Motilidad Espermática/fisiologíaRESUMEN
Sperm cryopreservation can lead to subfertility due to potential damage to sperm DNA, membranes, and overall motility caused by the freeze-thaw process. Interleukin-6 (IL-6) is a versatile cytokine with various roles in reproductive processes. However, the impacts of IL-6 supplementation on cryopreserved ram sperm have not been thoroughly investigated. Therefore, this study aims to assess the influence of IL-6 on the sperm quality of cryopreserved ram sperm. Ram semen was collected, pooled, and extended with tris-citrate soybean lecithin extender supplemented with 0, 50, 100, and 200 ng/mL of IL-6. The samples experienced a standard freezing protocol, and sperm quality, kinematic parameters, ultrastructure, and molecular docking of cryopreserved ram spermatozoa were evaluated. The results showed that sperm kinematics, viability, progressive motility, and membrane integrity were significantly enhanced by the addition of 100 or 200 ng of IL-6/mL (p < 0.05). Semen supplemented with 100 or 200 ng/mL of IL-6 also exhibited higher percentages of sperm kinematics, including DAP, DCL, DSL, VSL, VAP, VCL, and ALH, compared to other groups (p < 0.05). IL-6 supplementation enhanced acrosome integrity, and reduced caspase-3 activity in post-thawed ram spermatozoa (p < 0.05) compared to untreated group. Supplementation with IL-6 (200 ng/mL) significantly decreased oxidative biomarkers (NO, MDA, and H2O2) (p < 0.001) and improved total antioxidant capacity (p < 0.05). The percentage of sperm damage (tail, head, and midpiece) was significantly reduced by IL-6 supplementation (p < 0.05). Electron micrographs showed that supplementation with 100 or 200 ng/mL IL-6 protected acrosome stability, plasma membrane integrity, and sustained the ultrastructure integrity of cryopreserved ram spermatozoa. The docking exploration indicates a higher binding affinity with sperm function biomarkers, including caspase 3, BCL2, and PSMA6, with binding energies of - 52.30 kcal/mol, - 56.04 kcal/mol, and - 57.06 kcal/mol, respectively. In conclusion, the addition of IL-6 to the freezing extender can enhance the post-thaw quality of cryopreserved ram spermatozoa.
Asunto(s)
Acrosoma , Apoptosis , Criopreservación , Interleucina-6 , Simulación del Acoplamiento Molecular , Análisis de Semen , Preservación de Semen , Espermatozoides , Masculino , Criopreservación/veterinaria , Animales , Interleucina-6/metabolismo , Interleucina-6/farmacología , Preservación de Semen/veterinaria , Ovinos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Acrosoma/efectos de los fármacos , Acrosoma/ultraestructura , Análisis de Semen/veterinaria , Apoptosis/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Crioprotectores/farmacología , Fenómenos BiomecánicosRESUMEN
In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple morphologically abnormal flagella (MMAF) in an infertile patient. Using exome sequencing, we identified this variant, which led to exon 12 skipping and the production of a truncated CFAP61 protein. Transmission electron microscopy analysis of the patient's spermatozoa revealed various flagellar abnormalities, including defective nuclear chromatin condensation, axoneme disorganization, and mitochondria embedded in residual cytoplasmic droplets. Despite a fertilization rate of 83.3% through ICSI, there was no successful pregnancy due to poor embryo quality.Our findings suggest a link between the identified CFAP61 variant and MMAF, indicating potential disruption in radial spokes' assembly or function crucial for normal ciliary motility. Furthermore, nearly half of the observed sperm heads displayed chromatin condensation defects, possibly contributing to the low blastulation rate. This case underscores the significance of genetic counseling and testing, particularly for couples dealing with infertility and MMAF. Early identification of such genetic variants can guide appropriate interventions and improve reproductive outcomes.
Asunto(s)
Homocigoto , Infertilidad Masculina , Adulto , Femenino , Humanos , Masculino , Embarazo , Secuenciación del Exoma , Flagelos/genética , Flagelos/ultraestructura , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Empalme del ARN/genética , Inyecciones de Esperma Intracitoplasmáticas , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Espermatozoides/patología , Espermatozoides/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
The ultrastructure of snake sperm has received substantial attention primarily because snakes exhibit considerable variability in reproductive characteristics between species, with a wide range of mating systems and reproductive behaviors. Variability of sperm morphology among snake species may be associated with the reproductive strategies of each taxon, such as competition or sperm storage. We provide a detailed description of the sperm ultrastructure of nine snake species (Anilius scytale, Tropidophis paucisquamis, Bothrops jararaca, Oxyrhopus guibei, Dipsas mikanii, Micrurus corallinus, Xenopholis scalaris, Acrochordus javanicus, and Cylindrophis ruffus) and compared this with sperm data from the literature for the following taxa: Liotyphlops beui, Amerotyphlops reticulatus, Trilepida koppesi, Anilios waitii, Anilios endoterus, Aspidites melanochephalus, Boa constrictor amarali, Corallus hortulana, Epicrates cenchria, Boa constrictor occidentalis, Eryx jayakari, Micrurus corallinus, Micrurus surinamensis, Micrurus frontalis, Micrurus altirostris, Oxyuranus microlepidotus, Bothrops alternatus, Bothrops diporus, Crotalus durissus, Agkistrodon contortrix, Vipera aspis, Boiga irregularis, Zamenis schrenckii, Zamenis scalaris, Stegonotus cuculatus, Nerodia sipedon, Liodytes pygaea, and Myrrophis chinensis. We found twelve polymorphic characters in the ultrastructure of sperm among the described snakes. Our work supports the importance of ultrastructural analysis of sperm morphology to understand snake reproduction, and provides sperm-derived morphological characters for phylogenetic analysis.
Asunto(s)
Serpientes , Espermatozoides , Animales , Masculino , Espermatozoides/ultraestructura , Serpientes/anatomía & histología , Microscopía Electrónica de Transmisión/métodosRESUMEN
The ultrastructural study on the female reproductive system of the beetle M. brevicauda (Mordellidae) confirmed the positive correlation between the length of the sperm and the size of the female seminal receptacle (Spermatheca). The spermatheca of the species is characterized by an apical bulb-like structure where the spermathecal duct forms numerous folds filled with sperm. At this level many bacterial cells are present intermingled with the duct folds. Some are organized in large structures, such as bacteriomes, while other are single bacteriocytes. The latter are often found near the basal lamina of duct epithelium. In addition, some bacteria are visible in the cytoplasm of the duct epithelial cells. Interestingly, bacterial cells have never been observed in the duct lumen. The possible function of the bacterial cells is discussed.
Asunto(s)
Escarabajos , Microscopía Electrónica de Transmisión , Animales , Escarabajos/ultraestructura , Femenino , Masculino , Genitales Femeninos/ultraestructura , Bacterias/ultraestructura , Espermatozoides/ultraestructura , Microscopía Electrónica de RastreoRESUMEN
PURPOSE: This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families. METHODS: We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients. RESULTS: We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants. CONCLUSION: Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.
Asunto(s)
Infertilidad Masculina , Linaje , Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides , Adulto , Humanos , Masculino , China , Cilios/genética , Cilios/patología , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Secuenciación del Exoma , Homocigoto , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación/genética , Fenotipo , Espermatozoides/patología , Espermatozoides/ultraestructura , Espermatozoides/metabolismoRESUMEN
RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.
Asunto(s)
Adenilato Quinasa , Astenozoospermia , Cola del Espermatozoide , Adulto , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Codón sin Sentido , Secuenciación del Exoma , Homocigoto , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mitocondrias/ultraestructura , Mitocondrias/genética , Mitocondrias/patología , Linaje , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Espermatozoides/ultraestructura , Espermatozoides/anomalías , Adenilato Quinasa/genéticaRESUMEN
PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.
Asunto(s)
Astenozoospermia , Secuenciación del Exoma , Homocigoto , Infertilidad Masculina , Cola del Espermatozoide , Espermatozoides , Adulto , Humanos , Masculino , Pueblo Asiatico/genética , Astenozoospermia/genética , Astenozoospermia/patología , Células HEK293 , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación/genética , Linaje , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Espermatozoides/patología , Espermatozoides/ultraestructuraRESUMEN
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.
Asunto(s)
Acrosoma , Infertilidad Masculina , Proteínas de Microfilamentos , Adulto , Humanos , Masculino , Acrosoma/patología , Acrosoma/ultraestructura , Actinas/metabolismo , Actinas/genética , Secuenciación del Exoma , Fertilización/genética , Eliminación de Gen , Infertilidad Masculina/genética , Cabeza del Espermatozoide/ultraestructura , Cabeza del Espermatozoide/patología , Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides/ultraestructura , Espermatozoides/anomalías , Proteínas de Microfilamentos/genéticaRESUMEN
PURPOSE: Teratozoospermia is the main pathogenic factor of male infertility. However, the genetic etiology of teratozoospermia is largely unknown. This study aims to clarify the relationship between novel variations in TENT5D and teratozoospermia in infertile patients. MATERIALS AND METHODS: Two infertile patients were enrolled. Routine semen analysis of patients and normal controls was conducted with the WHO guidelines. Whole-exome sequencing (WES) was conducted to identify pathogenic variants in the two patients. Morphology and ultrastructure analysis of spermatozoa in the two patients was determined by Papanicolaou staining, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The functional effect of the identified variants was analyzed by immunofluorescence staining and western blotting. The expression of TENT5D in different germ cells was detected by immunofluorescence staining. RESULTS: Two new hemizygous variations, c.101C > T (p.P34L) and c.125A > T (p.D42V), in TENT5D were detected in two patients with male infertility. Morphology analysis showed abnormalities in spermatozoa morphology in the two patients, including multiple heads, headless, multiple tails, coiled, and/or bent flagella. Ultrastructure analysis showed that most of the spermatozoa exhibited missing or irregularly arranged '9+2' structures. Further functional experiments confirmed the abrogated TENT5D protein expression in patients. In addition, both p.P34L and p.D42V substitutions resulted in a conformational change of the TENT5D protein. We precisely analyzed the subcellular localization of TENT5D in germ cells in humans and mice. And we found that TENT5D was predominantly detected in the head and flagellum of elongating spermatids and epididymal spermatozoa. CONCLUSIONS: Our results showed further evidence of a relationship between TENT5D mutation and human male infertility, providing new genetic insight for use in the diagnosis and treatment of male infertility.
Asunto(s)
Espermatozoides , Teratozoospermia , Humanos , Masculino , Teratozoospermia/genética , Espermatozoides/ultraestructura , Espermatozoides/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Adulto , Secuenciación del Exoma , Animales , Análisis de SemenRESUMEN
The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.