RESUMO
The epididymis is a male reproductive organ involved in posttesticular sperm maturation and storage, but the mechanism underlying sperm maturation remains unclear. ß-Defensins (Defbs) belong to a family of small, cysteine-rich, cationic peptides that are antimicrobial and modulate the immune response. A large number of Defb genes are expressed abundantly in the male reproductive tract, especially in the epididymis. We and other groups have shown the involvement of several Defb genes in regulation of sperm function. In this study, we found that Defb23, Defb26, and Defb42 were highly expressed in specific regions of the epididymis. Rats with CRISPR/Cas9-mediated single-gene disruption of Defb23, Defb26, or Defb42 had no obvious fertility phenotypes. Those with the deletion of Defb23/ 26 or Defb23/ 26/ 42 became subfertile, and sperm isolated from the epididymal cauda of multiple-mutant rats were demonstrated decreased motility. Meanwhile, the sperm showed precocious capacitation and increased spontaneous acrosome reaction. Consistent with premature capacitation and acrosome reaction, sperm from multiple-gene-knockout rats had significantly increased intracellular calcium. These results suggest that Defb family members affect sperm maturation by a synergistic pattern in the epididymis.-Zhang, C., Zhou, Y., Xie, S., Yin, Q., Tang, C., Ni, Z., Fei, J., Zhang, Y. CRISPR/Cas9-mediated genome editing reveals the synergistic effects of ß-defensin family members on sperm maturation in rat epididymis.
Assuntos
Sistemas CRISPR-Cas , Epididimo , Fertilidade , Edição de Genes , Maturação do Esperma , Motilidade dos Espermatozoides/fisiologia , beta-Defensinas/fisiologia , Animais , Técnicas de Inativação de Genes , Genoma , Masculino , Fenótipo , Ratos , beta-Defensinas/antagonistas & inibidoresRESUMO
Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility.
Assuntos
Compostos Benzidrílicos/toxicidade , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fenóis/toxicidade , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Feminino , Fertilidade/efeitos dos fármacos , Fertilização in vitro/efeitos dos fármacos , Isoquinolinas/farmacologia , Masculino , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Sulfonamidas/farmacologia , Tirosina/metabolismoRESUMO
Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.
Assuntos
Acrossomo/metabolismo , Epididimo/metabolismo , Fertilidade , Proteínas Secretadas Inibidoras de Proteinases/fisiologia , Sequência de Aminoácidos , Androgênios/metabolismo , Animais , Anticorpos Monoclonais/química , Feminino , Fertilização in vitro , Lentivirus/genética , Masculino , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Secretadas Inibidoras de Proteinases/química , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Inibidores de Serinopeptidase do Tipo Kazal , Espermatozoides/metabolismo , Espermatozoides/fisiologiaRESUMO
Spermatozoa acquire forward motility and fertilizing capacity during their transit through the epididymis. The maturation process involves modifications of the sperm surface by different proteins that are secreted by a series of specialized regions in the epididymal epithelium. Here we show that the rat epididymis-specific ß-defensin 15 (Defb15) exhibits an androgen-dependent expression pattern, and it can bind to the acrosomal region of caput sperm. Coculture of caput spermatozoa with Defb15 antibody in vitro resulted in a significant decline in sperm motility. Moreover, the total and progressive motility of the spermatozoa dramatically decreased in rats when Defb15 was downregulated by lentivirus-mediated RNAi in vivo. Remarkably, knock down of Defb15 led to a reduction in fertility and embryonic development failure. In addition, the recombinant Defb15 showed antimicrobial activity in a dose-dependent fashion. These results suggest that Defb15 plays a dual role in both sperm maturation and pathogen defense in rat epididymis.
Assuntos
Epididimo/metabolismo , Fertilidade , Motilidade dos Espermatozoides , beta-Defensinas/análise , beta-Defensinas/metabolismo , Animais , Anti-Infecciosos/farmacologia , Epididimo/ultraestrutura , Fertilização , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , beta-Defensinas/genética , beta-Defensinas/farmacologiaRESUMO
The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.
Assuntos
Epididimo/metabolismo , Glutationa Peroxidase/fisiologia , RNA Interferente Pequeno/metabolismo , Envelhecimento/metabolismo , Animais , Regulação para Baixo , Epididimo/enzimologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
DICER1 is a key enzyme responsible for the maturation of microRNAs. Recent evidences suggested that DICER1 and microRNAs expressed in epididymis were involved in the control of male fertility. However, the exact mechanism remains to be elucidated. Here, we created a mouse line by targeted disruption of Dicer1 gene in the principal cells of distal caput epididymis. Our data indicated that a set of ß-defensin genes were downregulated by DICER1 rather than by microRNAs. Moreover, DICER1 was significantly enriched in the promoter of ß-defensin gene and controlled transcription. Besides, the antibacterial ability of the adult epididymis significantly declined upon Dicer1 deletion both in vitro and in vivo. And a higher incidence of reproductive defect was observed in middle-aged Dicer1-/- males. These results suggest that DICER1 plays an important role in transcription of ß-defensin genes, which are associated with the natural antibacterial properties in a microRNA-independent manner, and further impacts the male fertility.
Assuntos
RNA Helicases DEAD-box/metabolismo , Epididimo/metabolismo , Ribonuclease III/metabolismo , beta-Defensinas/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Epididimo/citologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Ribonuclease III/deficiência , Ribonuclease III/genética , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Transcrição Gênica , beta-Defensinas/genéticaRESUMO
Pancreatic-derived factor (PANDER) is a cytokine-like peptide highly expressed in pancreatic beta-cells. PANDER was reported to promote apoptosis of pancreatic beta-cells and secrete in response to glucose. Here we explored the effects of glucose on PANDER expression, and the underlying mechanisms in murine pancreatic beta-cell line MIN6 and primary islets. Our results showed that glucose up-regulated PANDER mRNA and protein levels in a time- and dose-dependent manner in MIN6 cells and pancreatic islets. In cells expressing cAMP response element-binding protein (CREB) dominant-negative construct, glucose failed to induce PANDER gene expression and promoter activation. Treatment of the cells with calcium chelator [EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM)], the voltage-dependent Ca(2+) channel inhibitor (nifedipine), the protein kinase A (PKA) inhibitor (H89), the protein kinase C (PKC) inhibitor (Go6976), or the MAPK kinase 1/2 inhibitor (PD98059), all significantly inhibited glucose-induced PANDER gene expression and promoter activation. Further studies showed that glucose induced CREB phosphorylation through Ca(2+)-PKA-ERK1/2 and Ca(2+)-PKC pathways. Thus, the Ca(2+)-PKA-ERK1/2-CREB and Ca(2+)-PKC-CREB signaling pathways are involved in glucose-induced PANDER gene expression. Wortmannin (phosphatidylinositol 3-kinase inhibitor), ammonium pyrrolidinedithiocarbamate (nuclear factor-kappaB inhibitor and nonspecific antioxidant), and N-acetylcysteine (antioxidant) were also found to inhibit glucose-induced PANDER promoter activation and gene expression. Because there is no nuclear factor-kappaB binding site in the promoter region of PANDER gene, these results suggest that phosphatidylinositol 3-kinase and reactive oxygen species be involved in glucose-induced PANDER gene expression. In conclusion, glucose induces PANDER gene expression in pancreatic beta-cells through multiple signaling pathways. Because PANDER is expressed by pancreatic beta-cells and in response to glucose in a similar way to those of insulin, PANDER may be involved in glucose homeostasis.
Assuntos
Citocinas/genética , Citocinas/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
Transections of the entorhinal afferent fibers to hippocampus, perforant path (PP), result in the denervation in specific hippocampal subregions, which is followed by a series of plastic events including axon sprouting and reactive synaptogenesis. Many growth-associated molecules are thought to participate in these events. In the present study, we proved the upregulation of ephrin-A2 in the denervated areas of the ipsilateral hippocampus following PP transections. Interestingly, when the elevation of ephrin-A2 reached the maximum axon sprouting in the denervated areas almost finished, implying the possible inhibitory effect of ephrin-A2 on sprouting. In addition, ephrin-A2 expression was observed in synapses during reactive synaptogenesis, suggesting that this molecule might also be implicated in the formation and maturation of synapses in the denervated areas.
Assuntos
Efrina-A2/metabolismo , Hipocampo/metabolismo , Hipocampo/cirurgia , Via Perfurante/fisiologia , Regulação para Cima , Animais , Diferenciação Celular , Denervação , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos ICR , Sinapses/metabolismoRESUMO
SPARC is a matricellular protein that modulates cell-cell and cell-matrix interactions by virtue of its antiproliferative and counteradhesive properties. Here, we report the denervation-induced upregulation of SPARC mRNA and protein in the mouse hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that SPARC mRNA was upregulated in a transient manner in the deafferented mouse hippocampus. In situ hybridization and immunohistochemistry confirmed the temporal upregulation of both SPARC mRNA and protein specifically in the denervated areas, which initiated at 7 days postlesion, reached the maximum at 15 as well as 30 days postlesion, and subsided towards normal levels by 60 days postlesion. Double labeling by either a combination of in situ hybridization for SPARC mRNA with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in the hippocampus revealed that SPARC-expressing cells are reactive astrocytes. In respect to the spatiotemporal alterations of SPARC expression in the denervated hippocampus, we suggest that SPARC may be involved in modulation of the denervation-induced plasticity processes such as glial cell proliferation, axonal sprouting and subsequent synaptogenesis in the hippocampus following entorhinal deafferentation.
Assuntos
Denervação , Córtex Entorrinal , Hipocampo/metabolismo , Osteonectina/metabolismo , Regulação para Cima , Vias Aferentes/metabolismo , Vias Aferentes/patologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Córtex Entorrinal/citologia , Córtex Entorrinal/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/patologia , Hibridização In Situ , Camundongos , Osteonectina/genética , RNA Mensageiro/metabolismoRESUMO
Thymosin beta4 is a major actin-sequestering molecule. Here, we report a prominent upregulation of thymosin beta4 in the hippocampus following entorhinal deafferentation. Northern blotting displayed a transient increase of thymosin beta4 mRNA in the deafferented hippocampus by 1.8, 2.3, 1.3 and 1.1-fold of controls, respectively, at 1, 3, 7 and 15 days post-lesion. In-situ hybridization confirmed that the induction of thymosin beta4 mRNA specifically occurred in the entorhinally denervated zones of the hippocampus. The double labeling of in-situ hybridization for thymosin beta4 mRNA with isolectin B4 cytochemistry showed that isolectin B4-positive microglial cells are responsible for deafferentation-induced thymosin beta4 mRNA expression. The results suggest that thymosin beta4 may participate in the process of microglial activation, which is the earliest event in lesion-induced plasticity.
Assuntos
Hipocampo/metabolismo , Microglia/metabolismo , RNA Mensageiro/biossíntese , Timosina/biossíntese , Animais , Northern Blotting , Denervação , Córtex Entorrinal/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lectinas , Neurônios Aferentes/fisiologia , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo . Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus -mediated siRNA in the CES5A -expressing region of the rat epididymis, Ces5a -knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.
Assuntos
Carboxilesterase/fisiologia , Epididimo/enzimologia , Fertilidade/fisiologia , Capacitação Espermática/fisiologia , Animais , Carboxilesterase/deficiência , Carboxilesterase/genética , Epididimo/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Modelos Animais , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Motilidade dos Espermatozoides/fisiologiaRESUMO
Epididymal function depends on androgen signaling through the androgen receptor (AR), although most of the direct AR target genes in epididymis remain unknown. Here we globally mapped the AR binding regions in mouse caput epididymis in which AR is highly expressed. Chromatin immunoprecipitation sequencing indicated that AR bound selectively to 19,377 DNA regions, the majority of which were intergenic and intronic. Motif analysis showed that 94% of the AR binding regions harbored consensus androgen response elements enriched with multiple binding motifs that included nuclear factor 1 and activator protein 2 sites consistent with combinatorial regulation. Unexpectedly, AR binding regions showed limited conservation across species, regardless of whether the metric for conservation was based on local sequence similarity or the presence of consensus androgen response elements. Further analysis suggested the AR target genes are involved in diverse biological themes that include lipid metabolism and sperm maturation. Potential novel mechanisms of AR regulation were revealed at individual genes such as cysteine-rich secretory protein 1. The composite studies provide new insights into AR regulation under physiological conditions and a global resource of AR binding sites in a normal androgen-responsive tissue.
Assuntos
Mapeamento Cromossômico , Epididimo/fisiologia , Receptores Androgênicos/genética , Animais , Sequência Conservada , Epididimo/metabolismo , Evolução Molecular , Expressão Gênica , Doenças dos Genitais Masculinos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI/metabolismo , Ligação Proteica , Receptores Androgênicos/metabolismo , Transdução de Sinais , Maturação do Esperma/genética , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gelsolin is an actin-binding protein that regulates actin filament-severing and capping activity in the various processes of cell motilities. Here, we report the expression of gelsolin mRNA and protein in the hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that transcript of gelsolin was upregulated in a transient manner in the deafferented hippocampus by 1.3-, 2.1-, 1.7-, and 1.1- folds of controls, respectively, at 1, 3, 7, and 15 days postlesion (dpl). In situ hybridization and immunohistochemistry confirmed the temporal expression of gelsolin specifically in the entorhinally denervated zones: the stratum lacunosum-molecular (SLM) of the hippocampus and the outer molecular layer (OML) of the dentate gyrus (DG), which initiated as early as at 1 dpl, reached the maximum at 3 dpl, remained prominently elevated by 7 dpl, and discernibly higher at 15 dpl than that of controls. Double labeling of either gelsolin mRNA or protein with markers of glial cells (Griffonia simplicifolia IB4 and CD11b for microglial cells, GFAP for astroglial cells) revealed that gelsolin was highly expressed by both activated microglia and astrocytes. The results suggest that the spatiotemporal upregulation of gelsolin in the hippocampus is induced by entorhinal deafferentation, and that gelsolin would participate in the activation processes of both microglial and astroglial cells and thereby, indirectly play important roles in the subsequent lesion-induced neural reorganization in the hippocampus following entorhinal deafferentation.
Assuntos
Vias Aferentes/metabolismo , Córtex Entorrinal/metabolismo , Gelsolina/metabolismo , Hipocampo/metabolismo , Animais , Córtex Entorrinal/anatomia & histologia , Feminino , Gelsolina/genética , Hipocampo/anatomia & histologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Abstract It has been widely demonstrated that Eph receptors and their ephrin ligands play multiple pivotal roles in the development of the nervous system. However, less is known about their roles in the adult brain. Here we reported the expression of ephrin-B1 and its cognate EphB receptors in the adult mouse hippocampus at 3, 7, 15, 30 and 60 days after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent up-regulation of ephrin-B1 in the denervated areas of the hippocampus, which initiated at 3 days postlesion (dpl), reached maximal levels at 7-15 dpl, remained slightly elevated at 30 dpl and recovered to normal levels by 60 dpl. Double labeling of ephrin-B1 and glial fibrillary acidic protein revealed that ephrin-B1-expressing cells in the denervated areas were reactive astrocytes. Furthermore, a ligand-binding assay using ephrin-B1/Fc chimera protein also displayed the up-regulation of EphB receptors in the denervated areas of the hippocampus in a similar manner to that of ephrin-B1. Within the first week postlesion, the EphB receptors were expressed by reactive astrocytes. After 7 dpl, however, EphB receptors were expressed not only by reactive astrocytes but also first by sprouting axons and later by regrowing dendrites. These results suggest that the ephrin-B1/EphB system may participate in the lesion-induced plasticity processes in the adult mouse hippocampus.