神经营养素-4及其受体在牛成熟精子和支持细胞中的表达与功能研究
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摘要
最近的研究表明，神经营养素（neurotrophins，NTs）在神经系统以外的心血管、肌肉和生殖等一些非神经系统中发挥着重要作用。本实验室曾首次发现了牛成熟精子能够表达并分泌神经生长因子（nerve growth factor，NGF）和脑源性神经营养因子（brain-derivedneurotrophic factor, BDNF）两种神经营养素。发现了BDNF参与牛成熟精子胰岛素和瘦素的分泌，NGF的异常表达与男性不育密切相关等。然而牛成熟精子及支持细胞中是否表达神经营养素-4（neurotrophine-4，NT-4）及其受体酪氨酸激酶受体B（tyrosine kinase B，TrkB），如果表达，其功能又有哪些？本文将对此进行研究。研究结果如下：
1、牛成熟精子中NT-4及其受体TrkB的表达
首先利用RT-PCR方法检测了牛成熟精子中是否有NT-4及其受体TrkBmRNA的表达。结果表明，牛成熟精子中虽然没有NT-4mRNA的存在，但却可以表达受体TrkB的mRNA。通过Western blot方法发现牛成熟精子中存在NT-4和TrkB的蛋白，利用细胞免疫荧光方法确定了NT-4蛋白主要存在于牛成熟精子尾部的中段及赤道部，而TrkB则存在于牛成熟精子的顶体膜及尾部中段。
2、NT-4对牛成熟精子功能的影响
为了进一步验证牛成熟精子中的NT-4的作用，本试验通过向牛成熟精子体外孵育系统中加入0，50，100，300和500ng mL的NT-4重组蛋白处理牛成熟精子30min后，检测了NT-4对牛精子线粒体膜电位、Ca~(2+)水平、顶体反应和活率的影响。结果表明：发现300ng或500ng mL的外源NT-4可显著的提高牛成熟精子的线粒体膜电位和Ca~(2+)水平(p <0.05)。若用NT-4处理牛成熟精子的同时添加20ng mL的TrkB的阻断剂K252a，牛成熟精子的线粒体膜电位和Ca~(2+)水平将会显著的下降(p <0.05)。此外，还检测了NT-4对牛成熟精子顶体反应及精子活率的影响，结果表明NT-4对牛成熟精子顶体反应及精子活率无影响(p>0.05)。这说明NT-4可以通过与牛成熟精子线粒体膜上的TrkB的结合影响牛成熟精子的Ca~(2+)水平及线粒体膜电位。
3、NT-4及其受体TrkB在牛支持细胞中的表达
牛成熟精子不能表达NT-4，NT-4蛋白是如何到达牛成熟精子上的呢？由于支持细胞是与精子直接接触的唯一的体细胞，因此，牛支持细胞可能是NT-4的分泌来源。首先，本试验比较了二步酶消化法和三步酶消化法对牛支持细胞的分离效果，结果显示差异不显著，都可以获得纯度大于90%以上的牛原代支持细胞。在此基础上，利用RT-PCR、Westernblot和细胞免疫荧光的方法发现牛支持细胞中可以表达NT-4及其受体TrkB。此外，通过免疫荧光和Western blot方法在牛支持细胞中分离得到的线粒体中发现了TrkB的蛋白，这充分说明TrkB不仅存在于支持细胞的膜上，而且还在于线粒体上。
4、NT-4对牛睾丸支持功能的影响
为了进一步验证支持细胞中的NT-4的作用，通过向牛原代支持细胞体外培养体系中加入不同浓度（0,50,100和300ng mL）的NT-4重组蛋白，检测了NT-4对牛支持细胞线粒体膜电位、Ca~(2+)水平、增殖和mRNA水平的影响。结果表明：发现50~300ng/mL NT-4可以显著的提高支持细胞内的Ca~(2+)水平(p <0.05)，但对线粒体膜电位没有影响(p>0.05)。若在应用NT-4处理牛支持细胞过程中添加20ng mL的TrkB的阻断剂K252a，支持细胞的Ca~(2+)水平和线粒体膜电位均显著的下降(p <0.05)。以上结果说明NT-4可以与细胞膜上的TrkB结合影响细胞内的Ca~(2+)水平，NT-4还可以与线粒体膜上的TrkB结合影响线粒体的活性。此外，还研究了NT-4是否影响牛支持细胞的增殖，结果发现，不同浓度的NT-4对牛支持细胞的增殖无影响(p <0.05)。同时，还研究了NT-4对促卵泡素受体（folliclestimulating hormone receptor, FSHR）的表达，以及FSH对NT-4及其受体TrkB表达的影响。结果表明，50~300ng/mL的NT-4均可显著的提高支持细胞内FSHR的mRNA及蛋白的表达(p <0.05)，且这种促进作用依赖于NT-4与TrkB结合。此外，5和50μg/mL的FSH处理支持细胞12h和24h均可以显著的提高NT-4及TrkB的mRNA的表达及NT-4的分泌(p<0.05)。
综上所述，本文首次发现了牛成熟精子和支持细胞中NT-4及其受体TrkB的表达及功能。发现了（1）NT-4在促进牛成熟精子线粒体膜电位及Ca~(2+)水平等等方面有着重要的作用。（2）NT-4可以调节牛支持细胞中FSHR表达、线粒体膜电位及细胞内的Ca~(2+)水平。（3）牛支持细胞中NT-4及其受体TrkB的表达受FSH的调控。本研究的结果丰富了有关NT-4对牛雄性生殖影响的认识。
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 spermatozoa
In 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 spermatozoa
In addition, effects of NT-4on the mitochondria activity, Ca~(2+)level, viability and acrosomereaction of spermatozoa were studied. Significant increased mitochondria activity or Ca~(2+)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 Ca~(2+)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 cells
In order to study the effect of NT-4on bovine Sertoli cells, significant increased Ca~(2+)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 Ca~(2+)level and mitochondria activity (p <0.05). All these resultsindicate that NT-4may regulate the Ca~(2+)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 Ca~(2+)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.
1、牛成熟精子中NT-4及其受体TrkB的表达
首先利用RT-PCR方法检测了牛成熟精子中是否有NT-4及其受体TrkBmRNA的表达。结果表明，牛成熟精子中虽然没有NT-4mRNA的存在，但却可以表达受体TrkB的mRNA。通过Western blot方法发现牛成熟精子中存在NT-4和TrkB的蛋白，利用细胞免疫荧光方法确定了NT-4蛋白主要存在于牛成熟精子尾部的中段及赤道部，而TrkB则存在于牛成熟精子的顶体膜及尾部中段。
2、NT-4对牛成熟精子功能的影响
为了进一步验证牛成熟精子中的NT-4的作用，本试验通过向牛成熟精子体外孵育系统中加入0，50，100，300和500ng mL的NT-4重组蛋白处理牛成熟精子30min后，检测了NT-4对牛精子线粒体膜电位、Ca~(2+)水平、顶体反应和活率的影响。结果表明：发现300ng或500ng mL的外源NT-4可显著的提高牛成熟精子的线粒体膜电位和Ca~(2+)水平(p <0.05)。若用NT-4处理牛成熟精子的同时添加20ng mL的TrkB的阻断剂K252a，牛成熟精子的线粒体膜电位和Ca~(2+)水平将会显著的下降(p <0.05)。此外，还检测了NT-4对牛成熟精子顶体反应及精子活率的影响，结果表明NT-4对牛成熟精子顶体反应及精子活率无影响(p>0.05)。这说明NT-4可以通过与牛成熟精子线粒体膜上的TrkB的结合影响牛成熟精子的Ca~(2+)水平及线粒体膜电位。
3、NT-4及其受体TrkB在牛支持细胞中的表达
牛成熟精子不能表达NT-4，NT-4蛋白是如何到达牛成熟精子上的呢？由于支持细胞是与精子直接接触的唯一的体细胞，因此，牛支持细胞可能是NT-4的分泌来源。首先，本试验比较了二步酶消化法和三步酶消化法对牛支持细胞的分离效果，结果显示差异不显著，都可以获得纯度大于90%以上的牛原代支持细胞。在此基础上，利用RT-PCR、Westernblot和细胞免疫荧光的方法发现牛支持细胞中可以表达NT-4及其受体TrkB。此外，通过免疫荧光和Western blot方法在牛支持细胞中分离得到的线粒体中发现了TrkB的蛋白，这充分说明TrkB不仅存在于支持细胞的膜上，而且还在于线粒体上。
4、NT-4对牛睾丸支持功能的影响
为了进一步验证支持细胞中的NT-4的作用，通过向牛原代支持细胞体外培养体系中加入不同浓度（0,50,100和300ng mL）的NT-4重组蛋白，检测了NT-4对牛支持细胞线粒体膜电位、Ca~(2+)水平、增殖和mRNA水平的影响。结果表明：发现50~300ng/mL NT-4可以显著的提高支持细胞内的Ca~(2+)水平(p <0.05)，但对线粒体膜电位没有影响(p>0.05)。若在应用NT-4处理牛支持细胞过程中添加20ng mL的TrkB的阻断剂K252a，支持细胞的Ca~(2+)水平和线粒体膜电位均显著的下降(p <0.05)。以上结果说明NT-4可以与细胞膜上的TrkB结合影响细胞内的Ca~(2+)水平，NT-4还可以与线粒体膜上的TrkB结合影响线粒体的活性。此外，还研究了NT-4是否影响牛支持细胞的增殖，结果发现，不同浓度的NT-4对牛支持细胞的增殖无影响(p <0.05)。同时，还研究了NT-4对促卵泡素受体（folliclestimulating hormone receptor, FSHR）的表达，以及FSH对NT-4及其受体TrkB表达的影响。结果表明，50~300ng/mL的NT-4均可显著的提高支持细胞内FSHR的mRNA及蛋白的表达(p <0.05)，且这种促进作用依赖于NT-4与TrkB结合。此外，5和50μg/mL的FSH处理支持细胞12h和24h均可以显著的提高NT-4及TrkB的mRNA的表达及NT-4的分泌(p<0.05)。
综上所述，本文首次发现了牛成熟精子和支持细胞中NT-4及其受体TrkB的表达及功能。发现了（1）NT-4在促进牛成熟精子线粒体膜电位及Ca~(2+)水平等等方面有着重要的作用。（2）NT-4可以调节牛支持细胞中FSHR表达、线粒体膜电位及细胞内的Ca~(2+)水平。（3）牛支持细胞中NT-4及其受体TrkB的表达受FSH的调控。本研究的结果丰富了有关NT-4对牛雄性生殖影响的认识。
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 spermatozoa
In 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 spermatozoa
In addition, effects of NT-4on the mitochondria activity, Ca~(2+)level, viability and acrosomereaction of spermatozoa were studied. Significant increased mitochondria activity or Ca~(2+)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 Ca~(2+)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 cells
In order to study the effect of NT-4on bovine Sertoli cells, significant increased Ca~(2+)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 Ca~(2+)level and mitochondria activity (p <0.05). All these resultsindicate that NT-4may regulate the Ca~(2+)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 Ca~(2+)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.
引文
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