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1.
Chemosphere ; 287(Pt 4): 132395, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34597628

RESUMO

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its neural and reproductive toxicity. To uncover potential effects of GLA on male reproductive health in mammals, adult male C57BL/6J mice were administered 0.2 mg/kg·d GLA for 5 weeks. After examination on fertility, testis histology and semen quality in the GLA group, we performed deep sequencing to identify repressive epigenetic marks including DNA methylation and histone modifications (H3K27me3 and H3K9me3), together with mRNA transcript levels in sperm. Then, we integrated multi-omics sequencing data to comprehensively explore GLA-induced epigenetic and transcriptomic alterations. We found no significant difference either on fertility, testis histology or semen quality-related indicators. As for epigenome, the protein level of H3K27me3 was significantly increased in GLA sperm. Next generation sequencing showed alterations of these epigenetic marks and extensive transcription inhibition in sperm. These differential repressive marks were mainly distributed at intergenic regions and introns. According to results by Gene Ontology enrichment analysis, both differentially methylated and expressed genes were mainly enriched in pathways related to synapse organization. Subtle differences in genomic imprinting were also observed between the two groups. These results suggested that GLA predominantly impaired sperm epigenome and transcriptome in mice, with little effect on fertility, testis histology or semen quality. Further studies on human sperm using similar strategies need to be conducted for a better understanding of the male reproductive toxicity of GLA.


Assuntos
Epigenoma , Transcriptoma , Aminobutiratos , Animais , Metilação de DNA , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Saúde Reprodutiva , Análise do Sêmen , Espermatozoides/metabolismo
2.
Nat Immunol ; 22(11): 1382-1390, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34663978

RESUMO

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Hereditariedade , Imunidade Inata/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Células Mieloides/imunologia , Animais , Candida albicans/patogenicidade , Candidíase/genética , Candidíase/metabolismo , Candidíase/microbiologia , Células Cultivadas , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/metabolismo , Listeriose/microbiologia , Masculino , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/microbiologia , Espermatozoides/imunologia , Espermatozoides/metabolismo , Transcrição Genética
3.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639209

RESUMO

The process of freezing cells or tissues and depositing them in liquid nitrogen at -196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.


Assuntos
Cálcio/metabolismo , Criopreservação/normas , Embrião de Mamíferos/citologia , Epigênese Genética , Células Germinativas/citologia , Infertilidade/terapia , Proteoma/metabolismo , Sobrevivência Celular , Crioprotetores/química , Feminino , Preservação da Fertilidade/normas , Fertilização In Vitro , Células Germinativas/metabolismo , Humanos , Infertilidade/metabolismo , Infertilidade/patologia , Masculino , Oócitos/citologia , Oócitos/metabolismo , Gravidez , Espermatozoides/citologia , Espermatozoides/metabolismo
4.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638585

RESUMO

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.


Assuntos
AMP Cíclico/metabolismo , Fosforilação/fisiologia , Maturação do Esperma/fisiologia , Motilidade Espermática/fisiologia , Espermatozoides/fisiologia , Animais , Epididimo/metabolismo , Epididimo/fisiologia , Feminino , Fertilização/fisiologia , Ligadura/métodos , Masculino , Camundongos , Transdução de Sinais/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo
5.
PLoS One ; 16(9): e0256701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473747

RESUMO

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10-11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode's Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10-11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.


Assuntos
Criopreservação/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Fertilização In Vitro/métodos , Expressão Gênica , Masculino , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo
7.
Int J Mol Sci ; 22(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34502434

RESUMO

Integrins are transmembrane receptors that facilitate cell adhesion and cell-extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.


Assuntos
Integrina alfaV/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica , Animais , Humanos , Masculino , Camundongos , Suínos
8.
Int J Mol Sci ; 22(18)2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-34576283

RESUMO

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.


Assuntos
Epididimo/metabolismo , Receptores de Kisspeptina-1/metabolismo , Espermatozoides/metabolismo , Animais , Líquidos Corporais/metabolismo , Contagem de Células , Cães , Epididimo/anatomia & histologia , Cinética , Kisspeptinas/metabolismo , Masculino , Testículo/anatomia & histologia
9.
Hum Reprod ; 36(10): 2638-2648, 2021 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-34486673

RESUMO

STUDY QUESTION: Do selective serotonin reuptake inhibitor (SSRI) antidepressants affect the function of human sperm? SUMMARY ANSWER: The SSRI antidepressant Sertraline (e.g. Zoloft) inhibits the sperm-specific Ca2+ channel CatSper and affects human sperm function in vitro. WHAT IS KNOWN ALREADY: In human sperm, CatSper translates changes of the chemical microenvironment into changes of the intracellular Ca2+ concentration ([Ca2+]i) and swimming behavior. CatSper is promiscuously activated by oviductal ligands, but also by synthetic chemicals that might disturb the fertilization process. It is well known that SSRIs have off-target actions on Ca2+, Na+ and K+ channels in somatic cells. Whether SSRIs affect the activity of CatSper is, however, unknown. STUDY DESIGN, SIZE, DURATION: We studied the action of the seven drugs belonging to the most commonly prescribed class of antidepressants, SSRIs, on resting [Ca2+]i and Ca2+ influx via CatSper in human sperm. The SSRI Sertraline was selected for in-depth analysis of its action on steroid-, prostaglandin-, pH- and voltage-activation of human CatSper. Moreover, the action of Sertraline on sperm acrosomal exocytosis and penetration into viscous media was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The activity of CatSper was investigated in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. Acrosomal exocytosis was investigated using Pisum sativum agglutinin and image cytometry. Sperm penetration in viscous media was evaluated using the Kremer test. MAIN RESULTS AND THE ROLE OF CHANCE: Several SSRIs affected [Ca2+]i and attenuated ligand-induced Ca2+ influx via CatSper. In particular, the SSRI Sertraline almost completely suppressed Ca2+ influx via CatSper. Remarkably, the drug was about four-fold more potent to suppress prostaglandin- versus steroid-induced Ca2+ influx. Sertraline also suppressed alkaline- and voltage-activation of CatSper, indicating that the drug directly inhibits the channel. Finally, Sertraline impaired ligand-induced acrosome reaction and sperm penetration into viscous media. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study. Future studies have to assess the physiological relevance in vivo. WIDER IMPLICATIONS OF THE FINDINGS: The off-target action of Sertraline on CatSper in human sperm might impair the fertilization process. In a research setting, Sertraline may be used to selectively inhibit prostaglandin-induced Ca2+ influx. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Swiss Centre for Applied Human Toxicology (SCAHT), the Département de l'Instruction Publique of the State of Geneva, the German Research Foundation (CRU326), the Interdisciplinary Center for Clinical Research, Münster (IZKF; Str/014/21), the Innovation Fund Denmark (grant numbers 14-2013-4) and the EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation. The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the research reported. TRIAL REGISTRATION NUMBER: NA.


Assuntos
Cálcio , Sertralina , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Masculino , Progesterona/farmacologia , Sertralina/metabolismo , Sertralina/farmacologia , Motilidade Espermática , Espermatozoides/metabolismo
10.
Int J Mol Sci ; 22(17)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34502430

RESUMO

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)-sperm interaction reflect the specific features of the spawning process in this species.


Assuntos
Quimiotaxia/fisiologia , Fertilização/fisiologia , Oncorhynchus mykiss/metabolismo , Ovário/metabolismo , Espermatozoides/metabolismo , Zigoto/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Feminino , Masculino , Ovário/citologia , Espermatozoides/citologia , Zigoto/citologia
11.
Cells ; 10(9)2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34572103

RESUMO

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.


Assuntos
Sistema Livre de Células/fisiologia , Fertilização , Mitofagia , Oócitos/fisiologia , Interações Espermatozoide-Óvulo , Espermatozoides/patologia , Animais , Bovinos , Feminino , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oócitos/citologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Espermatozoides/metabolismo , Suínos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
12.
Andrologia ; 53(11): e14226, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34478154

RESUMO

The measurement of protein expression level plays a pivotal role in both biological and medical studies. Housekeeping proteins, generally encoded by housekeeping genes are used as loading control proteins to normalize protein expression. Obviously, proper reference standards are essential for adequate analysis of protein expression. However, our study showed that the widely used normalisation proteins, whose expression levels varied greatly among sperm samples, were unsuitable for data standardisation. To uncover the proteins steadily expressed in sperm, we analysed several published transcriptome data of sperm. Seven proteins whose expression levels were relatively stable (co-efficient variation values less than 0.35) were selected and further evaluated by quantitative real-time polymerase chain reaction, Western Blot (WB) and immunocytochemistry. Our results showed that among the classical housekeeping proteins, only ß-tubulin remained constant in sperm samples from 85 individuals. Compared with other classical housekeeping proteins such as glyceraldehyde 3-phosphate dehydrogenase, actin and histone H3, Cullin-1 (CUL1) and F-box only protein 7 (FBXO7) seemed to be more suitable to be used as internal controls for WB in sperm protein studies. Combined with the locations of these proteins, CUL1 and FBXO7 were suggested to be used as a housekeeping protein for total proteins.


Assuntos
Biomarcadores , Western Blotting , Espermatozoides , Actinas/genética , Actinas/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Perfilação da Expressão Gênica , Histonas , Humanos , Masculino , Padrões de Referência , Espermatozoides/metabolismo , Tubulina (Proteína)/genética
13.
Nat Commun ; 12(1): 4926, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389728

RESUMO

The sperm head-to-tail coupling apparatus (HTCA) ensures sperm head-tail integrity while defective HTCA causes acephalic spermatozoa, rendering males infertile. Here, we show that CENTLEIN is indispensable for HTCA integrity and function, and that inactivation of CENTLEIN in mice leads to sperm decapitation and male sterility. We demonstrate that CENTLEIN directly interacts with both SUN5 and PMFBP1, two proteins localized in the HTCA and related with acephalic spermatozoa syndrome. We find that the absence of Centlein sets SUN5 and PMFBP1 apart, the former close to the sperm head and the latter in the decapitated tail. We show that lack of Sun5 results in CENTLEIN and PMFBP1 left in the decapitated tail, while disruption of Pmfbp1 results in SUN5 and CENTLEIN left on the detached sperm head. These results demonstrate that CENTLEIN cooperating with SUN5 and PMFBP1 participates in the HTCA assembly and integration of sperm head to the tail, indicating that impairments of CENTLEIN might be associated with acephalic spermatozoa syndrome in humans.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Cabeça do Espermatozoide/metabolismo , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Mutação , Ligação Proteica , Espermatozoides/citologia , Teratozoospermia/genética , Teratozoospermia/metabolismo
14.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445637

RESUMO

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.


Assuntos
Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , DNA Forma Z/metabolismo , Escherichia coli/imunologia , Micrococcus luteus/imunologia , Placenta/imunologia , Espermatozoides/imunologia , Animais , Anticorpos Monoclonais/imunologia , DNA Forma Z/química , DNA Forma Z/imunologia , Escherichia coli/metabolismo , Feminino , Humanos , Masculino , Micrococcus luteus/metabolismo , Conformação de Ácido Nucleico , Placenta/metabolismo , Gravidez , Salmão , Ovinos , Especificidade da Espécie , Espermatozoides/metabolismo
15.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360620

RESUMO

BACKGROUND: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. METHODS: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. RESULTS: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. CONCLUSIONS: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.


Assuntos
Dor Crônica/metabolismo , Dor Pélvica/metabolismo , Prostatite/metabolismo , Protaminas/metabolismo , Espermatozoides/metabolismo , Adulto , Estudos de Casos e Controles , Fragmentação do DNA , Humanos , Masculino , Pessoa de Meia-Idade , Análise do Sêmen , Adulto Jovem
16.
Toxicology ; 460: 152886, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34352348

RESUMO

Arsenic intoxication affects male reproductive parameters of prepubertal rats. Besides, morphological and functional alterations in their testis and epididymis may remain after withdrawal of arsenic insult, causing potential impairment in male fertility during adulthood. In this study, we aimed at analyzing the effect of prepubertal arsenic exposure on the fecundity of epididymal sperm from sexually mature Wistar rats, assessing fertility indexes, sperm parameters, and sperm phosphoproteins content. Male pups on postnatal day (PND) 21 received filtered water (controls, n = 10) and 10 mg L-1 arsenite (n = 10) daily for 30 days. From PND52 to PND81, rats from both groups received filtered water. During this period, the males mated with non-exposed females between PND72 and PND75. Our results showed that sexually mature rats presented low sperm production, epididymal sperm count, motility, and quality after prepubertal arsenic exposure. These findings possibly contributed to the low fertility potential and high preimplantation loss. Epididymal sperm proteome detected 268 proteins, which 170 were found in animals from both control and arsenic groups, 27 proteins were detected only in control animals and 71 proteins only in arsenic-exposed rats. In these animals, SPATA 18 and other five proteins were upregulated, whereas keratin type II cytoskeletal 1 was downregulated (q < 0.1). The results of KEGG pathway analysis demonstrated an enrichment of pathways related to dopaminergic response, adrenergic signaling, protein degradation, and oocyte meiosis in arsenic-exposed animals. Moreover, 26 proteins were identified by phosphoproteomic with different phosphorylation pattern in animals from both groups, but SPATA18 was phosphorylated only in arsenic-exposed animals. We concluded that prepubertal exposure to arsenic is deleterious to sperm quality and male fertility, altering the sperm phosphoproteins profile.


Assuntos
Arsênio/toxicidade , Epididimo/metabolismo , Fertilidade/fisiologia , Fosfoproteínas/metabolismo , Maturidade Sexual/fisiologia , Espermatozoides/metabolismo , Animais , Arsênio/administração & dosagem , Bovinos , Epididimo/efeitos dos fármacos , Epididimo/patologia , Fertilidade/efeitos dos fármacos , Humanos , Masculino , Camundongos , Ratos , Ratos Wistar , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Maturidade Sexual/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia
17.
Redox Biol ; 46: 102071, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34340027

RESUMO

To date 15% of couples are suffering from infertility with 45-50% of males being responsible. With an increase in paternal age as well as various environmental and lifestyle factors worsening these figures are expected to increase. As the so-called free radical theory of infertility suggests, free radicals or reactive oxygen species (ROS) play an essential role in this process. However, ROS also fulfill important functions for instance in sperm maturation. The aim of this review article is to discuss the role reactive oxygen species play in male fertility and how these are influenced by lifestyle, age or disease. We will further discuss how these ROS are measured and how they can be avoided during in-vitro fertilization.


Assuntos
Infertilidade Masculina , Fertilização In Vitro , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo
18.
Cells ; 10(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440724

RESUMO

The etiology of human asthenozoospermia is multifactorial. The need to unveil molecular mechanisms underlying this state of infertility is, thus, impelling. Circular RNAs (circRNAs) are involved in microRNA (miRNA) inhibition by a sponge activity to protect mRNA targets. All together they form the competitive endogenous RNA network (ceRNET). Recently, we have identified differentially expressed circRNAs (DE-circRNAs) in normozoospermic and asthenozoospermic patients, associated with high-quality (A-spermatozoa) and low-quality (B-spermatozoa) sperm. Here, we carried out a differential analysis of CRISP2, CATSPER1 and PATE1 mRNA expression in good quality (A-spermatozoa) and low quality (B-spermatozoa) sperm fractions collected from both normozoospermic volunteers and asthenozoospermic patients. These sperm fractions are usually separated on the basis of morphology and motility parameters by a density gradient centrifugation. B-spermatozoa showed low levels of mRNAs. Thus, we identified the possible ceRNET responsible for regulating their expression by focusing on circTRIM2, circEPS15 and circRERE. With the idea that motility perturbations could be rooted in quantitative changes of transcripts in sperm, we evaluated circRNA and mRNA modulation in A-spermatozoa and B-spermatozoa after an oral amino acid supplementation known to improve sperm motility. The profiles of CRISP2, CATSPER1 and PATE1 proteins in the same fractions of sperm well matched with the transcript levels. Our data may strengthen the role of circRNAs in asthenozoospermia and shed light on the molecular pathways linked to sperm motility regulation.


Assuntos
Astenozoospermia/metabolismo , Canais de Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Membrana/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Adulto , Aminoácidos/administração & dosagem , Astenozoospermia/diagnóstico , Astenozoospermia/tratamento farmacológico , Astenozoospermia/genética , Canais de Cálcio/genética , Estudos de Casos e Controles , Moléculas de Adesão Celular/genética , Suplementos Nutricionais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Motilidade Espermática , Espermatozoides/efeitos dos fármacos , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
19.
Dev Cell ; 56(16): 2284-2294.e6, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34363758

RESUMO

Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent "hub" cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.


Assuntos
Proteínas de Drosophila/metabolismo , Folistatina/metabolismo , Nicho de Células-Tronco , Fatores de Transcrição/metabolismo , Ativinas/metabolismo , Animais , Proliferação de Células , Transdiferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Masculino , Espermatozoides/citologia , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Testículo/citologia , Fatores de Transcrição/genética
20.
Elife ; 102021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34409938

RESUMO

For a sperm to successfully fertilize an egg, it must first undergo capacitation in the female reproductive tract and later undergo acrosomal reaction (AR) upon encountering an egg surrounded by its vestment. How premature AR is avoided despite rapid surges in signaling cascades during capacitation remains unknown. Using a combination of conditional knockout (cKO) mice and cell-penetrating peptides, we show that GIV (CCDC88A), a guanine nucleotide-exchange modulator (GEM) for trimeric GTPases, is highly expressed in spermatocytes and is required for male fertility. GIV is rapidly phosphoregulated on key tyrosine and serine residues in human and murine spermatozoa. These phosphomodifications enable GIV-GEM to orchestrate two distinct compartmentalized signaling programs in the sperm tail and head; in the tail, GIV enhances PI3K→Akt signals, sperm motility and survival, whereas in the head it inhibits cAMP surge and premature AR. Furthermore, GIV transcripts are downregulated in the testis and semen of infertile men. These findings exemplify the spatiotemporally segregated signaling programs that support sperm capacitation and shed light on a hitherto unforeseen cause of infertility in men.


Assuntos
Fertilidade , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/genética , Transdução de Sinais/genética , Capacitação Espermática/genética , Proteínas de Transporte Vesicular/genética , Animais , Regulação para Baixo , Feminino , Fertilidade/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Espermatócitos/metabolismo , Espermatozoides/metabolismo , Testículo/citologia , Testículo/patologia
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