Expression And Function Of Neurotrophin-4and Its Receptor In Bovine Mature Spermatozoa And Sertoli Cells

Recently, our studies have demonstrated that two members of neurotrophins, nerve growthfactor （NGF）and brain-derived neurotrophic factor（BDNF）, exist in mature bull spermatozoa,and play a crucial role in the normal function of spermatozoa. Neurotrophin-4（NT-4）is anotherneurotrophic factor that signals predominantly through the TrkB receptor tyrosine kinase, but noreports of detection of NT-4and TrkB in bovine mature spermatozoa and Sertoli cells have beenpublished.1. Detection of NT-4mRNA and protein in mature bovine spermatozoaIn the present study, the presence of NT-4in mature bull spermatozoa was investigated usingRT-PCR, immunofluorescence and Western blotting. The results showed that the TrkB transcriptswere present in bovine spermatozoa, but no RT-PCR evidence for NT-4transcripts in bovinespermatozoa. However, the NT-4and TrkB proteins were present in bovine spermatozoa, and theNT-4immunoreactivity was localized to the equatorial segment and midpiece of bovinespermatozoa, while the TrkB was localized to the midpiece and acrosome membrane of bovinespermatozoa.2. Study on the function of NT-4in mature bovine spermatozoaIn addition, effects of NT-4on the mitochondria activity, Ca²⁺level, viability and acrosomereaction of spermatozoa were studied. Significant increased mitochondria activity or Ca²⁺levelof mature bovine spermatozoa were observed in response to300-500ng/mL or100-500ng/mLexogenous NT-4（p <0.05）, respectively, in comparison with the control, while addition ofinhibitors （40ng mL k252a） specific for tyrosine protein kinase significantly blocked theincrease of mitochondria activity. However, NT-4had no effects on the viability or acrosomereaction of spermatozoa （p>0.05）. Consequently, this study provided evidence that NT-4andTrkB proteins present in the mature bull spermatozoa and can influence the mitochondrialactivity and Ca²⁺level of bovine spermatozoa through TrkB tyrosine kinase-dependent pathways. As there is no NT-4mRNA in bovine mature spermatozoa, where does the NT-4proteincome from and what is the function of it? Therefore, we hypothesized that NT-4may besynthesized in Sertoli cells, delivered to the target germ cells and exerts its function in theprocess of spermatogenesis. In the present study, two-step enzyme digest and three-step enzymedigest method were compared according to their isolation result. Results showed that nosignificant different cell density and purity were found and the purity of the isolated Sertoli cellswas all above90%. In addition, NT-4and its receptor TrkB mRNA and protein in bovine Sertolicells were found using RT-PCR, immunofluorescence and Western blotting. Moreover, TrkBproteins were also detected by immunofluorescence and Western blot analysis in themitochondria which was isolated from bovine Sertoli cells, which indicate that TrkB also exist inthe mitochondria.4. Study on the function of NT-4in bovine Sertoli cellsIn order to study the effect of NT-4on bovine Sertoli cells, significant increased Ca²⁺level inbovine Sertoli cells was observed in response to50-300ng mL exogenous NT-4（p <0.05）,while no different in the mitochondria activity were found （p>0.05）, in comparison with thecontrol. However, the addition of inhibitors （20ng/mL k252a） specific for tyrosine protein kinasesignificantly decreases of Ca²⁺level and mitochondria activity （p <0.05）. All these resultsindicate that NT-4may regulate the Ca²⁺level and mitochondrial activity of bovine Sertoli cellsthrough TrkB tyrosine kinase-dependent pathways in the cellular membrane and mitochondriaouter-membrane, respectively. Moreover, the effect of NT-4on proliferation of Sertoli cells wasalso studied. No significance differences of the proliferation rate were observed between thegroups treated with various concentrations of NT-4（p <0.05）.Meanwhile, effect of NT-4on theexpression of FSHR mRNA and protein, FSH on the NT-4and TrkB mRNA expression werealso studied. Results showed that50-300ng mL NT-4can significantly stimulate the expressionof FSHR mRNA （p <0.05）, and this effect was blocked by k252a, the inhibitor TrkB. Aftertreatment with5or50μg/mL FSH for24h, the expression of NT-4and TrkB mRNA and proteinwere significantly increased （p <0.05）.In conclusion, the present study firstly demonstrated that the expression pattern of NT-4inmature bovine spermatozoa and Sertoli cells, and NT-4can influence the mitochondrial activityand concentration of Ca²⁺in bovine spermatozoa and Sertoli cells through TrkB tyrosinekinase-dependent pathways. Meanwhile, NT-4can modulate the expression of FSHR in Sertoli cells and FSH can modulate the NT-4and TrkB expression. The findings define the maturebovine spermatozoa as one of the non-neuronal, endocrine targets of NT-4actions. These resultswill provide new insights and theory in the role of NTs in male reproduction system.