RESUMEN
Microtus genus is the herbivorous animal with multiple stomachs, and some of them possess a mating system similar to human and thereby has been expected as a model animal for the large herbivory and human mating system model, respectively. Thus, it is significant to maintain Microtus as an animal genetic resource. We have studied the establishment of assisted reproductive technologies in Alexandromys. montebelli (formerly as Microtus motebelli: A. motebelli), and here, we investigated the effects of hypotaurine treatment to frozen-thawed (FT) spermatozoa and modified timing of nonsurgical artificial insemination (AI) on the number of offspring. As the results, regardless of without or with hypotaurine treatment, when the timing of nonsurgical AI was made closer to the estimated ovulation time (at 7-9 h post coitus), the total number of offspring derived from FT spermatozoa (27 and 28 pups, respectively) increased compared with AI at 4-6 h (five and six pups, respectively) and was equivalent to those of fresh spermatozoa (43 pups) or natural mating (33 pups). These results will lead to further dissemination of nonsurgical AI and could support the "3R principle," which is the standard philosophy of animal experiment because the procedure declines the stress and the recipient can be used repeatedly.
Asunto(s)
Arvicolinae , Criopreservación , Inseminación Artificial , Preservación de Semen , Espermatozoides , Animales , Masculino , Inseminación Artificial/veterinaria , Inseminación Artificial/métodos , Criopreservación/veterinaria , Criopreservación/métodos , Femenino , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Espermatozoides/fisiología , Arvicolinae/fisiología , Ovulación , Factores de Tiempo , Modelos Animales , Pueblos del Este de AsiaRESUMEN
The structural integrity of the sperm flagellum is essential for proper sperm function. Flagellar defects can result in male infertility, yet the precise mechanisms underlying this relationship are not fully understood. CCDC181, a coiled-coil domain-containing protein, is known to localize on sperm flagella and at the basal regions of motile cilia. Despite this knowledge, the specific functions of CCDC181 in flagellum biogenesis remain unclear. In this study, Ccdc181 knockout mice were generated. The absence of CCDC181 led to defective sperm head shaping and flagellum formation. Furthermore, the Ccdc181 knockout mice exhibited extremely low sperm counts, grossly aberrant sperm morphologies, markedly diminished sperm motility, and typical multiple morphological abnormalities of the flagella (MMAF). Additionally, an interaction between CCDC181 and the MMAF-related protein LRRC46 was identified, with CCDC181 regulating the localization of LRRC46 within sperm flagella. These findings suggest that CCDC181 plays a crucial role in both manchette formation and sperm flagellum biogenesis.
Asunto(s)
Ratones Noqueados , Proteínas de Microtúbulos , Cola del Espermatozoide , Animales , Masculino , Ratones , Fertilidad/fisiología , Flagelos/metabolismo , Flagelos/fisiología , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/fisiología , Espermatozoides/fisiología , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismoRESUMEN
Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.
Asunto(s)
Astenozoospermia , Proteínas Asociadas a Microtúbulos , Animales , Humanos , Masculino , Ratones , Astenozoospermia/genética , Codón sin Sentido , Homocigoto , Infertilidad Masculina/genética , Mutación , Oligospermia/genética , Motilidad Espermática/genética , Espermatozoides , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Rptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging - sperm DNA methylome rejuvenation and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.
Asunto(s)
Metilación de ADN , Células de Sertoli , Espermatozoides , Masculino , Animales , Células de Sertoli/metabolismo , Ratones , Espermatozoides/metabolismo , Espermatozoides/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Serina-Treonina Quinasas TOR/metabolismo , Ratones Noqueados , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Ratones Transgénicos , Envejecimiento/fisiología , Envejecimiento/genética , Transducción de Señal , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Epigénesis GenéticaRESUMEN
Semen quality and fertility has declined over the last 50 years, corresponding to ever-increasing environmental stressors. However, the cellular mechanisms involved and their impact on sperm functions remain unknown. In a repeated sampling human cohort study, we identify a significant effect of prior perceived stress to increase sperm motility 2-3 months following stress, timing that expands upon our previous studies revealing significant stress-associated changes in sperm RNA important for fertility. We mechanistically examine this post-stress timing in mice using an in vitro stress model in the epididymal epithelial cells responsible for sperm maturation and find 7282 differentially H3K27me3 bound DNA regions involving genes critical for mitochondrial and metabolic pathways. Further, prior stress exposure significantly changes the composition and size of epithelial cell-secreted extracellular vesicles that when incubated with mouse sperm, increase mitochondrial respiration and sperm motility, adding to our prior work showing impacts on embryo development. Together, these studies identify a time-dependent, translational signaling pathway that communicates stress experience to sperm, ultimately affecting reproductive functions.
Asunto(s)
Mitocondrias , Motilidad Espermática , Espermatozoides , Animales , Masculino , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Espermatozoides/fisiología , Humanos , Ratones , Mitocondrias/metabolismo , Respiración de la Célula , Vesículas Extracelulares/metabolismo , Epidídimo/metabolismo , Ratones Endogámicos C57BL , Estrés Fisiológico , Adulto , Células Epiteliales/metabolismo , Análisis de Semen , Estudios de CohortesRESUMEN
BACKGROUND: Centrifugation is a common procedure to improve the quality of chilled and frozen canine semen by removing debris and seminal plasma and adding semen extenders. The aim of this study was to evaluate the efficacy and influence of a second centrifugation after 48 h of storage at 5 °C on the sperm quality of canine semen. The ejaculates of 45 healthy male dogs, divided into three groups according to body weight, were analyzed for macro- and microparameters such as ejaculate volume, sperm concentration, kinematic parameters, morphology, and integrity of plasma membrane. Samples were analyzed at baseline conditions (T0), after 24 h (T24) and after 48 h (T48) to assess the effects of the different treatments on sperm quality. RESULTS: The results showed a significant effect of a second centrifugation on the improvement of chilled sperm quality compared to the other techniques, especially up to 48 h. CONCLUSIONS: Analysis of the data showed that the semen samples centrifuged and then cooled at 5 °C had acceptable semen parameters, especially in terms of motility, with a gradual decrease in serial evaluations after 24 and 48 h. A second centrifugation after 48 h of storage may lead to better semen quality and improve the kinetics of sperm parameters, the percentage of morphologically normal sperm and the percentage of sperm with intact membranes.
Asunto(s)
Centrifugación , Análisis de Semen , Preservación de Semen , Animales , Perros/fisiología , Masculino , Centrifugación/veterinaria , Centrifugación/métodos , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Análisis de Semen/veterinaria , Semen/fisiología , Factores de Tiempo , Espermatozoides/fisiología , Motilidad EspermáticaRESUMEN
BACKGROUND: The importance of parental diet in relation to eventual offspring health is increasing in prominence due to the increased frequency of parents of reproductive age consuming poor diets. Whilst maternal health and offspring outcome have been studied in some detail, the paternal impacts are not as well understood. A father's poor nutritional status has been shown to have negative consequences on foetal growth and development and ultimately impact the long-term adult health of the offspring. In this study, we examined sperm- and seminal vesicle fluid-mediated mechanisms of preimplantation embryo development alterations in response to sub-optimal paternal diets. RESULTS: Male mice were fed a diet to model either under (low-protein diet (LPD)) or over (high-fat/sugar 'Western' diet (WD)) nutrition, LPD or WD supplemented with methyl donors or a control diet (CD) before mating with age-matched females. Male metabolic health was influenced by WD and MD-WD, with significant changes in multiple serum lipid classes and hepatic 1-carbon metabolites. Sperm RNA sequencing revealed significant changes to mRNA profiles in all groups when compared to CD (LPD: 32, MD-LPD: 17, WD: 53, MD-WD: 35 transcripts). Separate analysis of the seminal vesicle fluid proteome revealed a significant number of differentially expressed proteins in all groups (LPD: 13, MD-LPD: 27, WD: 24, MD-WD: 19) when compared to control. Following mating, in vitro time-lapse imaging of preimplantation embryos revealed a significant increase in the timing of development in all experimental groups when compared to CD embryos. Finally, qPCR analysis of uterine tissue at the time of implantation identified perturbed expression of Cd14 and Ptgs1 following mating with WD-fed males. CONCLUSIONS: Our current study shows that paternal nutritional status has the potential to influence male metabolic and reproductive health, impacting on embryonic development and the maternal reproductive tract. This study highlights potential direct (sperm-mediated) and indirect (seminal vesicle fluid-mediated) pathways in which a father's poor diet could shape the long-term health of his offspring.
Asunto(s)
Desarrollo Embrionario , Hipernutrición , Semen , Animales , Masculino , Ratones , Hipernutrición/fisiopatología , Femenino , Desnutrición/fisiopatología , Blastocisto , EspermatozoidesRESUMEN
Infertility rates have risen significantly, one of which is due to monosodium glutamate (MSG) consumption. Recent studies have shown that flavonoids in black garlic (Allium sativum) act as antioxidants. The aim of this study was to assess the effect of black garlic extract (BGE) on gonadosomatic index, follicle-stimulating hormone (FSH) levels, and spermatozoa quality in rats exposed to MSG. Twenty-five healthy rats, aged ten to twelve weeks, were divided equally into five experimental groups: (1) negative control (NC), no intervention; (2) positive control (PC), fed with MSG 8 mg/kg; and (3) fed with MSG + BGE 200 mg/kg; (4) fed with MSG + BGE 400 mg/kg; and (5) fed with MSG + BGE 600 mg/kg. Oral MSG was administered once a day for two weeks before BGE administration was started for two weeks. The measured endpoints were gonadosomatic index, FSH levels, and spermatozoa concentration and quality (spermatozoa motility and abnormality). Analysis of variance (ANOVA) followed by Duncan's post hoc analysis was used to assess the measurement differences. The result suggested that the administration of BGE did not significantly affect the gonadosomatic index (p=0.513). Significant decreases in FSH levels (p=0.005) and spermatozoa concentration were observed in the PC group compared to other groups (p<0.001). Additionally, spermatozoa motility was significantly lower in the PC group compared to NC, BGE200, BGE400, and BGE600 (p<0.001), with higher motility noted in BGE200, BGE400, and BGE600 compared to PC (p<0.001). Furthermore, PC had significantly higher spermatozoa abnormalities compared to NC, BGE200, BGE400, and BGE600 (p<0.001). In conclusion, administration of BGE had a significant effect on the improvement of FSH levels and the quality of spermatozoa in rats exposed to MSG.
Asunto(s)
Hormona Folículo Estimulante , Ajo , Extractos Vegetales , Glutamato de Sodio , Espermatozoides , Animales , Masculino , Ajo/química , Glutamato de Sodio/farmacología , Hormona Folículo Estimulante/sangre , Ratas , Extractos Vegetales/farmacología , Extractos Vegetales/administración & dosificación , Espermatozoides/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Ratas Wistar , Antioxidantes/farmacologíaRESUMEN
Sexual violence is a global issue affecting individuals regardless of their relationship to the perpetrator or the setting. Microscopic examination of spermatozoa from vaginal swabs is crucial in investigating cases of sexual intercourse to determine the time of the crime. Factors such as vaginal pH and neutrophil count influence the motility and persistence of spermatozoa in the vagina, particularly in conditions like candidiasis, highlighting the need for further research in this area. This study aimed to determine the effect of pH and neutrophil count on the motility and persistence of spermatozoa in the vagina with candidiasis. An experimental study was conducted using white rats (Rattus norvegicus) of the Wistar strain, with four male rats providing spermatozoa samples and 32 female rats receiving treatment. The female rats were divided into two groups: the normal group and the candidiasis model group. In both groups, the female rats were given vaginal insemination of spermatozoa. Variables measured included pH, neutrophil count, motility, and persistence of spermatozoa in the vagina. Data were analyzed using the Mann-Whitney test, followed by the Spearman correlation test. The findings revealed that spermatozoa motility lasted up to three minutes in normal rats, whereas in the candidiasis model, it was reduced to two minutes. Additionally, spermatozoa persistence in the vagina lasted up to six days in the normal group compared to up to three days in the candidiasis model. There were significant differences in pH, neutrophil count, motility, and persistence of spermatozoa in the vagina between the normal group and the candidiasis model (all had p<0.001). There was a correlation between pH and neutrophil count with the motility and persistence of spermatozoa in the rat's vagina (p<0.001). In conclusion, vaginal pH and neutrophil count influence the motility and persistence of spermatozoa in the vagina of candidiasis rat models.
Asunto(s)
Modelos Animales de Enfermedad , Neutrófilos , Ratas Wistar , Motilidad Espermática , Vagina , Animales , Femenino , Masculino , Concentración de Iones de Hidrógeno , Ratas , Vagina/microbiología , Vagina/patología , Espermatozoides , Recuento de Leucocitos , Candidiasis/microbiologíaRESUMEN
Introduction: Epididymal lumen fluids provides a stable microenvironment for sperm maturation. Ca2+ binding protein CABS1 is known to maintain structural integrity of mouse sperm flagella during epididymal transit of sperm. Besides, CABS1 was reported to contain anti-inflammatory peptide sequences and be present in both human saliva and plasma. However, little is known about the role of CABS1 in regulation of the microenvironment of epididymal lumen fluids. Methods: To further confirm the role of CABS1 in epididymis, we identified the expression of CABS1 in epididymal lumen fluids. Moreover, high performance liquid chromatography, coupled with tandem mass spectrometry technique was used to analyze the metabolic profiles and in vivo microperfusion of the cauda epididymis and inductively coupled plasma mass spectrometry (ICP-MS) assays was used to detect the concentration of metal ion of mouse cauda epididymal lumen fluids in CABS1 deficient and normal mice. Results: The results showed that CABS1 is present in epididymal lumen fluids, and the concentration of calcium in epididymal lumen fluids is not changed in Cabs1-/- male mice. Among 34 differential metabolites identified in cauda epididymis, 21 were significantly upregulated while 13 were significantly downregulated in KO cauda epididymis. Pathway analysis identified pyrimidine metabolism, inositol phosphate metabolism, arachidonic acid metabolism, purine metabolism and histidine metabolism as relevant pathways in cauda epididymis. Discussion: The perturbations of mitochondrial dysfunction and inflammation may be the crucial reason for the poor performance of Cabs1-/- sperm.
Asunto(s)
Epidídimo , Metabolómica , Ratones Noqueados , Espermatozoides , Animales , Masculino , Epidídimo/metabolismo , Ratones , Espermatozoides/metabolismo , Metabolómica/métodos , Proteínas de Unión al Calcio/metabolismo , Ratones Endogámicos C57BL , Maduración del Esperma/fisiologíaRESUMEN
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: Inflammation-induced testicular damage is a significant contributing factor to the increasing incidence of infertility. Traditional treatments during the inflammatory phase often fail to achieve the desired fertility outcomes, necessitating innovative interventions such as cell therapy. METHODS: We explored the in vivo properties of intravenously administered Sertoli cells (SCs) in an acute lipopolysaccharide (LPS)-induced inflammatory mouse model. Infiltrating and resident myeloid cell phenotypes were assessed using flow cytometry. The impact of SC administration on testis morphology and germ cell quality was evaluated using computer-assisted sperm analysis (CASA) and immunohistochemistry. RESULTS: SCs demonstrated a distinctive migration pattern, importantly they preferentially concentrated in the testes and liver. SC application significantly reduced neutrophil infiltration as well as preserved the resident macrophage subpopulations. SCs upregulated MerTK expression in both interstitial and peritubular macrophages. Applied SC treatment exhibited protective effects on sperm including their motility and kinematic parameters, and maintained the physiological testicular morphology. CONCLUSION: Our study provides compelling evidence of the therapeutic efficacy of SC transplantation in alleviating acute inflammation-induced testicular damage. These findings contribute to the expanding knowledge on the potential applications of cell-based therapies for addressing reproductive health challenges and offer a promising approach for targeted interventions in male infertility.
Asunto(s)
Inflamación , Células de Sertoli , Espermatozoides , Masculino , Animales , Células de Sertoli/metabolismo , Ratones , Inflamación/patología , Inflamación/terapia , Espermatozoides/metabolismo , Lipopolisacáridos/toxicidad , Ratones Endogámicos C57BL , Testículo , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Motilidad Espermática , Macrófagos/metabolismoRESUMEN
The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender 1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate). In Experiment 2, 50 mM L-carnitine and 10 mM pyruvate were added post-thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L-carnitine and pyruvate, nor between the samples after the post-thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk-based extender with the addition of L-carnitine and pyruvate after thawing. The addition of 50 mM L-carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing.
Asunto(s)
Carnitina , Criopreservación , Crioprotectores , Fragmentación del ADN , Peroxidación de Lípido , Ácido Pirúvico , Preservación de Semen , Espermatozoides , Animales , Masculino , Caballos , Criopreservación/veterinaria , Criopreservación/métodos , Carnitina/farmacología , Carnitina/administración & dosificación , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Ácido Pirúvico/farmacología , Crioprotectores/farmacología , Peroxidación de Lípido/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Análisis de Semen/veterinaria , Acrosoma/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Antioxidantes/farmacologíaRESUMEN
Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.
Asunto(s)
Acuaporina 3 , Dorada , Motilidad Espermática , Espermatozoides , Animales , Masculino , Espermatozoides/metabolismo , Acuaporina 3/metabolismo , Acuaporina 3/genética , Dorada/metabolismo , Femenino , RatonesRESUMEN
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
Human Papillomavirus (HPV), a prevalent sexually transmitted infection, comprises high-risk (HR-HPV) and low-risk (LR-HPV) viruses, the former posing a high risk for developing malignancies whereas the latter mainly for benign warts. Despite increasing awareness of HPV's impact on men's health, the influence of HR-HPV and LR-HPV urogenital infections on male fertility potential remains uncertain. This study aimed to investigate whether male urogenital infection with HR- or LR-HPV associates with impaired sperm quality, oxidative stress, and inflammation. A total of 205 male patients attending an urology clinic were enrolled. Semen samples were analyzed for HPV using PCR and genotyped by RFLP. Semen quality was evaluated following WHO guidelines. Semen leukocytes, reactive oxygen species (ROS), and sperm viability were analyzed using flow cytometry. HPV was detected in 19% (39/205) of semen samples. HR-HPV infections were more prevalent, with HPV-16 being the most frequent genotype. Neither HR-HPV nor LR-HPV were associated with significant alterations in routine sperm quality parameters. However, HR-HPV+ individuals showed significantly higher levels of sperm necrosis and exhibited increased proportions of ROS+ spermatozoa compared to LR-HPV+ or control individuals. Furthermore, no significant semen inflammation was detected in patients infected with either HR-HPV or LR-HPV, and unexpectedly reduced semen leukocytes and inflammatory cytokines (IL-6 and IL-1ß) were observed in HR-HPV+ patients compared to controls. These observations underscore the importance of comprehensive HPV screening, including genotyping, in urology and fertility clinics to understand the progression of the infection, potential adverse effects on reproductive health, and the oncogenic risks involved.
Asunto(s)
Papillomaviridae , Infecciones por Papillomavirus , Análisis de Semen , Semen , Espermatozoides , Humanos , Masculino , Infecciones por Papillomavirus/virología , Adulto , Espermatozoides/virología , Semen/virología , Papillomaviridae/genética , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Genotipo , Adulto Joven , Inflamación , Estrés Oxidativo , Genitales Masculinos/virología , Adolescente , Citocinas/metabolismoRESUMEN
Emerging studies suggest that various parental exposures affect offspring cardiovascular health, yet the specific mechanisms, particularly the influence of paternal cardiovascular disease (CVD) risk factors on offspring cardiovascular health, remain elusive. The present study explores how paternal hypercholesterolemia affects offspring atherosclerosis development using the LDL receptor-deficient (LDLR-/-) mouse model. We found that paternal high-cholesterol diet feeding led to significantly increased atherosclerosis in F1 female, but not male, LDLR-/- offspring. Transcriptomic analysis highlighted that paternal hypercholesterolemia stimulated proatherogenic genes, including Ccn1 and Ccn2, in the intima of female offspring. Sperm small noncoding RNAs (sncRNAs), particularly transfer RNA-derived (tRNA-derived) small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), contribute to the intergenerational transmission of paternally acquired metabolic phenotypes. Using a newly developed PANDORA-Seq method, we identified that high-cholesterol feeding elicited changes in sperm tsRNA/rsRNA profiles that were undetectable by traditional RNA-Seq, and these altered sperm sncRNAs were potentially key factors mediating paternal hypercholesterolemia-elicited atherogenesis in offspring. Interestingly, high-cholesterol feeding altered sncRNA biogenesis-related gene expression in the epididymis but not testis of LDLR-/- sires; this may have led to the modified sperm sncRNA landscape. Our results underscore the sex-specific intergenerational effect of paternal hypercholesterolemia on offspring cardiovascular health and contribute to the understanding of chronic disease etiology originating from parental exposures.
Asunto(s)
Aterosclerosis , Hipercolesterolemia , Receptores de LDL , Animales , Aterosclerosis/genética , Aterosclerosis/etiología , Masculino , Hipercolesterolemia/genética , Femenino , Ratones , Receptores de LDL/genética , Ratones Noqueados , Modelos Animales de Enfermedad , ARN Pequeño no Traducido/genética , Espermatozoides/metabolismo , Factores Sexuales , Exposición Paterna/efectos adversosRESUMEN
Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2⯵g/ml) in "TALP-Sperm" and "TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.
Asunto(s)
Antígenos de Protozoos , Fertilidad , Espermatozoides , Toxoplasma , Animales , Bovinos , Masculino , Espermatozoides/inmunología , Toxoplasma/inmunología , Toxoplasma/fisiología , Antígenos de Protozoos/inmunología , Motilidad Espermática , Fertilización In Vitro/veterinaria , Semen/parasitología , Semen/inmunologíaRESUMEN
Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.
Asunto(s)
Fertilización , Óvulo , Erizos de Mar , Animales , Fertilización/efectos de los fármacos , Erizos de Mar/efectos de los fármacos , Erizos de Mar/metabolismo , Óvulo/metabolismo , Óvulo/efectos de los fármacos , Masculino , Poliaminas/metabolismo , Poliaminas/farmacología , Femenino , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Calcio/metabolismoRESUMEN
This study aims to investigate the role of ferroptosis, an iron-dependent form of regulated cell death, in male infertility. The motivation behind this research stems from the increasing recognition of oxidative stress and iron metabolism dysregulation as critical factors in male reproductive health. In this study, 28 infertile patients (grouped by the presence of urogenital infections or varicocele) and 19 fertile men were selected. Spermiograms were performed by light microscopy (WHO, 2021). Testosterone, ferritin, transferrin-bound iron, transferrin, and F2-isoprostanes (F2-IsoPs) were detected in seminal plasma. Glutathione peroxidase 4 (GPX4) and acyl coenzyme A synthetase long chain family member 4 (ACSL4) were also assessed in sperm cells using enzyme-linked immunosorbent assays (ELISA). All the variables were correlated (statistically significant Spearman's rank correlations) in the whole population, and then the comparison between variables of the different groups of men were carried out. Seminal ferritin and transferrin positively correlated with seminal F2-IsoPs, which had positive correlations with ACSL4 detected in sperm cells. Ferritin and ACSL4 negatively correlated with the seminal parameters. No correlation was detected for GPX4. Comparing the variables in the three examined groups, elevated levels of ACSL4 were observed in infertile patients with urogenital infections and varicocele; GPX4 levels were similar in the three groups. These results suggested a mechanism of ferroptosis, identified by increased ACSL4 levels and the occurrence of lipid peroxidation. Such events appear to be GPX4-independent in reproductive pathologies such as varicocele and urogenital infections.