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1.
PLoS Genet ; 20(7): e1011357, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39074078

RESUMO

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.


Assuntos
Fertilidade , Hexoquinase , Infertilidade Masculina , Camundongos Knockout , Capacitação Espermática , Espermatozoides , Tirosina , Animais , Hexoquinase/genética , Hexoquinase/metabolismo , Masculino , Camundongos , Fosforilação , Espermatozoides/metabolismo , Capacitação Espermática/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Fertilidade/genética , Tirosina/metabolismo , Feminino , Testículo/metabolismo , Motilidade dos Espermatozoides/genética , Glicólise , Espermatogênese/genética
2.
Exp Cell Res ; 424(2): 113506, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36764590

RESUMO

After peripheral nerve injury, motor and sensory axons can regenerate, but the inaccurate reinnervation of the target leads to poor functional recovery. Schwann cells (SCs) express sensory and motor phenotypes associated with selective regeneration. Semaphorin 3A (Sema3A) is an axonal chemorepellent that plays an essential role in axon growth. SCs can secret Sema3A, and Sema3A presents a different expression pattern at the proximal and distal ends of injured sensory and motor nerves. Hence, in our study, the protein expression and secretion of Sema3A in sensory and motor SCs and the expression of its receptor Neuropilin-1 (Nrp1) in dorsal root ganglia (DRG) sensory neurons (SNs) and spinal cord motor neurons (MNs) were detected by Western blot and ELISA. The effect of Sema3A at different concentrations on neurite growth of sensory and motor neurons was observed by immunostaining. Also, by blocking the Nrp1 receptor on neurons, the effect of Sema3A on neurite growth was observed. Finally, we observed the neurite growth of sensory and motor neurons cocultured with Sema3A siRNA transfected SCs by immunostaining. The results suggested that the expression and secretion of Sema3A in sensory SCs are more significant than that in motor SCs, and the expression of its receptor Nrp1 in SNs is higher than in MNs. Sema3A could inhibit the neurite growth of sensory and motor neurons via Nrp1, and Sema3A has a more substantial effect on the neurite growth of SNs. These data provide evidence that SC-secreted Sema3A might play a role in selective regeneration by a preferential effect on SNs.


Assuntos
Axônios , Semaforina-3A , Semaforina-3A/metabolismo , Axônios/metabolismo , Neurônios Motores/metabolismo , Medula Espinal/metabolismo , Gânglios Espinais/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo
3.
Exp Cell Res ; 412(2): 113019, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35085549

RESUMO

Schwann cells (SCs) play a critical role in peripheral nerve (PN) regeneration because of their ability to proliferate, migrate, and provide trophic support for axon regeneration after PN injury. However, the underlying mechanism is still partially understood. Semaphorin3E (Sema3E), a member of the Sema3s family, is a secreted molecular known as a repelling cue in axon guidance and inhibitor of developmental and postischemic angiogenesis. In this study, we examined the expression of Sema3E in sciatic nerves and SCs and explored the effects of Sema3E on SCs proliferation and migration. Immunofluorescence and ELISA analyses illustrated the expression of Sema3E in SCs of Sciatic nerves and the secretion of Sema3E by cultured SCs, respectively. Exogenous Sema3E promoted SC proliferation and migration while knockdown of the endogenous Sema3E by siRNA transfection attenuated proliferation and migration of SCs. Furthermore, blocking the receptor Neuropilin 1 (Nrp1), PlexinD1 and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) by neutralizing antibody or inhibitor suppressed the promoting effects of Sema3E on SCs. This study indicated that Sema3E promoted SC proliferation and migration and the involvement of receptor PlexinD1, Nrp1, and VEGFR2 in these processes. This study extended our understanding of the mechanism that modulated SC phenotype during nerve injury and provided a potential target for promoting PN regeneration.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células de Schwann/metabolismo , Semaforinas/metabolismo , Animais , Axônios/metabolismo , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neuropilina-1/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
J Comp Neurol ; 529(6): 1240-1254, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32857427

RESUMO

Schwann cells (SCs) are myelin-forming glial cells of the peripheral nervous system. Recent studies suggested that SCs comprise two phenotypes: sensory SCs and motor SCs, which are associated with the modality-specific promotion of sensory and motor axon growth during peripheral neuronal regeneration. However, the molecular basis of the two phenotypic SCs is unclear. We established a workflow to obtain highly purified SCs derived from sensory nerve (SNdSCs) and motor nerve (MNdSCs) from B6; D2-Tg(s100B-EGFP)1Wjt/J mice. Subsequently, a quantitative proteomic analysis based on iTRAQ labeling was performed to compare the proteome of SNdSCs and MNdSCs. A total of 6,567 proteins were identified, of which 63 and 11 proteins were overexpressed in SNdSCs and MNdSCs, respectively. Three of the overexpressed proteins were further validated by western blot and immunocytochemistry: GMFB and CNPase, which were overexpressed in sensory SNdSCs, and histone H4, which was overexpressed in MNdSCs. The expression pattern of the three proteins was also validated in the dorsal roots and ventral roots. Bioinformatics analysis indicated that proteins highly expressed in SNdSCs are mainly involved in RNA processing and protein synthesis, while those overexpressed in MNdSCs are related to cell proliferation. Real-time cell analysis confirmed that the proliferation activity of MNdSCs is higher than that of SNdSCs. This study is the first to provide a proteomic view of the differential phenotype of mouse SNdSCs and MNdSCs. The data may serve as a valuable source for the study of the biological characteristics of these two SC phenotypes and their roles in nerve-specific regeneration.


Assuntos
Neurônios Motores/fisiologia , Fenótipo , Proteômica/métodos , Células de Schwann/fisiologia , Células Receptoras Sensoriais/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Neurônios Motores/química , Células de Schwann/química , Células Receptoras Sensoriais/química
5.
Anticancer Agents Med Chem ; 20(4): 417-428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31830896

RESUMO

BACKGROUND: In our previous study, we have isolated a new compound, named Fumosorinone (FU) from insect pathogenic fungi, and was found to inhibit proliferation, migration, and invasion of breast cancer MDA-MB-231 cells. OBJECTIVE: The aim of this study was to identify the underlying molecular mechanisms for FU effects on MDAMB- 231 cells. METHODS: After MDA-MB-231 cells were treated with FU for 48h, RNA sequencing was used to identify the effect of FU on the transcriptome of MDA-MB-231 cells. The validation of the relative expression of the selective genes was done using quantitative real-time PCR (qRT-PCR). RESULTS: The transcriptome results showed that 2733 genes were differentially expressed between the untreated and the FU-treated cells, including 1614 up-regulated and 1119 down-regulated genes. The multiple genes are associated with cancer cell growth, migration, and invasion. Functional analysis identified multitude of pathways related to cancer, such as cell cycle, ECM-receptor interaction, p53 signaling pathway. We selected 4 upregulated and 9 downregulated genes, which are associated with breast cancer to verify their expression using qRT-PCR. The validation showed that HSD3B1, ALOX5, AQP5, COL1A2, CCNB1, CCND1, VCAM-1, PTPN1 and PTPN11 were significantly downregulated while DUSP1, DUSP5, GADD45A, EGR1 were upregulated in FU-treated MDA-MB-231cells. CONCLUSION: These aberrantly expressed genes and pathways may play pivotal roles in the anti-cancer activity of FU, and maybe potential targets of FU treatments for TNBC. Further investigations are required to evaluate the FU mechanisms of anti-cancer action in vivo.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Piridonas/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Antineoplásicos/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fungos/química , Perfilação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/química , Insetos/microbiologia , Piridonas/química
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