鼠精子纤维鞘蛋白FSCB的磷酸化在获能和超活化中的作用研究

发布时间：2018-04-04 20:10

本文选题：精子　切入点：获能　出处：《第三军医大学》2012年博士论文

【摘要】：新鲜射出的精子在进入雌性生殖道内，发生一系列的生物化学改变从而获得受精能力的过程，称为精子获能[1]。包括精子质膜流动性增加，胆固醇外流，离子流动，酪氨酸磷酸化蛋白增多，精子超激活运动以及顶体反应的诱导等。PKA作用下的蛋白酪氨酸磷酸化是一重要的翻译后修饰事件，是精子获能的一个重要标志。目前的研究仍未解释清楚PKA信号机制，以及PKA作用的底物蛋白。已发现的与纤维鞘有关的蛋白有20余种，包括AKAP3，AKAP4, TAKAP-80；GAPDS，HK1-S，GSK3β，ALDOA，LDHA；SFEC，磷酸甘油醛异构酶，GAPDH，丙酮酸激酶，LDH-C，山梨醇脱氢酶；GSTM5；FS39；Ropporin；Rhophilin；SP17; PDE4A;FSIP1和FSIP2; ASP，CABYR等。但是目前的研究仍未完全阐明精子获能过程中PKA的底物蛋白及其作用机制。我们课题组最近发现了一种特异性表达于鼠精子尾部纤维鞘表面的全新蛋白FSCB[2]，前期的研究表明该蛋白可与另一种精子鞭毛上具有钙结合功能和在精子获能过程中可被酪氨酸磷酸化的精子特异性CABYR蛋白[3]结合，其本身也具有钙结合能力，在体外可被蛋白激酶A磷酸化。FSCB具有保守的PXXP基序，脯氨酸富积区和伸展蛋白样区域。FSCB在精子上的超微结构定位研究显示其位于精子鞭毛纤维鞘纵形柱状体和环状肋板表面的中等电子密度皮质层。鉴于该蛋白与某些精子纤维鞘蛋白如AKAP3，AKAP4，CABYR等具有相似的定位特征，而且具有可被磷酸化及与钙结合的功能特性，推测该蛋白可能是一种参与精子鞭毛的运动，与精子获能和超活化作用有关的重要蛋白。本课题为进一步探讨FSCB基本生物学特性，研究了该蛋白在精子的获能过程中是否被PKA磷酸化以及DB-CAMP和H-89对该蛋白磷酸化、PKA活性和精子运动特征的影响。整个课题研究分为三个部分进行。一、精子获能过程中FSCB可被PKA磷酸化并发生酪氨酸磷酸化我们的前期研究发现FSCB可在体外被PKA催化亚单位磷酸化，并存在多个磷酸化位点。为进一步研究FSCB在精子获能过程中是否发生磷酸化，，我们取成年雄性昆明小鼠（12-16周）精子于HTF（获能培养液）与M2（非获能培养液）分别培养2h，取等量样品进行裂解，蛋白定量，一维电泳后，用抗磷酸化PKA底物抗体进行Western blot分析，一抗同源免疫前血清做阴性对照。结果发现获能培养液培养2h精子裂解产物在270Kda处出现清晰阳性条带，而未获能培养液以及阴性对照中不出现免疫印迹条带。为验证该条带是否是FSCB以及该蛋白是否在精子获能过程中发生了磷酸化，我们进一步分别用抗磷酸化PKA底物抗体、抗磷酸化酪氨酸抗体、抗FSCB抗体进行小鼠获能与未获能精子免疫沉淀，沉淀产物用抗FSCB抗体进行Western blot验证，一抗同源免疫前血清做阴性对照。结果在获能后精子中，应用抗磷酸化PKA底物抗体、抗磷酸化酪氨酸抗体以及FSCB抗体免疫沉淀产物用FSCB抗体进行Western blot分析均在270Kda处出现阳性条带，而未获能精子中，应用抗磷酸化PKA底物抗体、抗磷酸化酪氨酸抗体的免疫沉淀产物未出现上述阳性条带，该条带仅出现于FSCB抗体免疫沉淀产物中。表明该270Kda蛋白即为FSCB，而且提示FSCB被PKA磷酸化，同时，其酪氨酸亦发生磷酸化。为研究FSCB磷酸化程度与精子获能时间之间的关系，我们进一步于不同时相点（1,2,5,10,30,60, min和120min）取等量HTF培养液中精子样品进行裂解，继续用抗磷酸化PKA底物抗体进行Western blot分析。发现该条带最早出现于接触获能培养液的1min，且随时间延长不断加深，60min后保持不变。结果表明FSCB在精子获能过程中可被PKA磷酸化，也可发生其酪氨酸的磷酸化，其磷酸化程度与接触获能培养液时间密切相关。二、PKA活性影响FSCB磷酸化 FSCB作为PKA的作用底物之一，在精子获能过程中可以被PKA磷酸化。研究PKA活性改变对FSCB磷酸化状态的影响，对进一步阐明精子获能过程蛋白磷酸化的分子基础和调节机制显得十分重要。我们分别用H-89和DB-cAMP处理小鼠精子，观察FSCB磷酸化状态的改变。取等量精子于HTF培养液、含0.5μM H-89的HTF培养液、M2培养液和含2.5mM DB-cAMP的M2培养液进行培养，分别于不同时相点（1,2,5,10,30,60, min和120min）取等量小鼠精子进行PKA活性分析，并于30分钟取不同培养液中精子样品裂解，蛋白定量后用抗磷酸化PKA底物抗体进行Western blot分析，一抗同源免疫前血清做阴性对照。实验中我们发现向HTF培养液中添加0.5μM H-89后，PKA活性受到明显抑制，向M2培养液中添加2.5mM DB-cAMP后，PKA活性显著增强。用抗磷酸PKA底物抗体Western blot分析HTF培养液、含2.5mM DB-cAMP的M2培养液30min精子裂解液在270KD处出现阳性条带，而在含0.5μM H-89的HTF培养液、M2培养液精子裂解液中无阳性条带。结果表明H-89可抑制PKA活性，DB-CAMP则增强PKA活性，二者可通过改变PKA活性从而影响FSCB的磷酸化。三、FSCB磷酸化程度与精子运动功能密切相关特定蛋白发生酪氨酸磷酸化是精子获能的重要标志，与精子运动速度、摆动频率等运动特征改变相关。为研究FSCB磷酸化与精子运动特征之间的关系，进行了不同时相点精子运动特征分析。我们分别取小鼠精子于HTF培养液、含0.5μM H-89的HTF培养液, M2培养液和含2.5mM DB-cAMP的M2培养液进行培养，分别于不同时相点（1,2,5,10,20,30,60和120min）取5μl小鼠精子精子活力分析仪（西班牙）分析。我们发现向HTF培养液中添加0.5μM H-89后，获能后1、5、10、20、30、60和120min精子VCL降低明显，获能后1、5、10、20、30和60min（A+B）%也显著下降；向M2培养液中添加2.5mM DB-cAMP后，获能后1、5、10、20、30min精子VCL明显增强，获能后1、5、10、20min（A+B）%也升高明显。综合分析上述实验结果，发现精子运动特征的变化与FSCB磷酸化水平改变有明显的线性关系，我们初步得出H-89通过抑制FSCB磷酸化而抑制精子运动，DB-CAMP可促进FSCB磷酸化而促进精子运动。
[Abstract]:Fresh ejaculated sperm into the female reproductive tract, a series of biochemical changes to achieve the fertilization ability of sperm capacitation, known as [1]. including sperm plasma membrane fluidity increased cholesterol efflux, ion mobility, protein tyrosine phosphorylation increased sperm hyperactivated protein tyrosine phosphorylation induced by.PKA effect and top sports body reaction under is an important post-translational modification events, is an important sign of sperm capacitation. Research has not yet been explained PKA signal mechanism, and the role of PKA protein substrate.
Have been found associated with the fibrous sheath protein more than 20 species, including AKAP3, AKAP4, TAKAP-80; GAPDS, HK1-S, ALDOA, GSK3 beta, LDHA; SFEC, triosephosphate isomerase, GAPDH, pyruvate kinase, LDH-C, sorbitol dehydrogenase; GSTM5; FS39; Ropporin; Rhophilin; SP17; PDE4A; FSIP1 and FSIP2 ASP; CABYR, etc. but the study has not yet been fully elucidated sperm capacitation substrates of PKA protein and its mechanism.
We recently discovered a specific expression in mouse sperm tail fibrous sheath on the surface of a new protein FSCB[2], previous studies have shown that the protein and calcium binding function in sperm capacitation by sperm specific CABYR protein tyrosine phosphorylation of [3] in combination with another sperm flagellum has its own. Also has the ability of binding calcium in vitro by protein kinase A phosphorylation of.FSCB has a conserved PXXP motif, the ultrastructural localization of proline rich region and protein like region.FSCB in sperm showed its extension in sperm flagella fibrous sheath longitudinal cylindrical body and an annular rib on the surface of the secondary electron density. In view of the cortex the protein and some sperm fibrous sheath protein such as AKAP3, AKAP4, CABYR etc. with positioning similar features, but also has the characteristics of function can be phosphorylated and calcium binding, speculated that the protein may be An important protein that participates in the movement of sperm flagellum, which is related to the effect of sperm capacitation and super activation.
In order to further explore the basic biological characteristics of FSCB, we studied whether the protein was phosphorylated by PKA and the effects of DB-CAMP and H-89 on the protein phosphorylation, PKA activity and sperm motility in the process of sperm capacitation. The whole research is divided into three parts.
First, FSCB can be phosphorylated by PKA and tyrosine phosphorylation during the process of sperm capacitation
Our previous study found that FSCB is the catalytic subunit of PKA phosphorylation in vitro, and the presence of multiple phosphorylation sites. For the further study of FSCB phosphorylation in sperm capacitation is in the process, we take the adult male Kunming mice (12-16 weeks) in HTF (sperm capacitation medium) and M2 (a non can medium) were cultured in 2H, take the same amount of sample lysis, protein, SDS-PAGE, Western blot analysis with anti phospho PKA antibody substrate, a homologous anti pre immune serum as a negative control. Results showed that capacitation of cultured 2H sperm lysates appear clear positive bands at 270Kda, and not to the culture medium and negative western blot bands do not appear in the control.
In order to verify whether the band is FSCB and whether the protein in sperm capacitation occurred in the phosphorylation process, we further respectively with anti phospho PKA substrate antibody, anti phosphotyrosine antibody, anti FSCB antibody in mice and sperm capacitation has not been able to immunoprecipitation, Western blot confirmed by anti FSCB antibody producing precipitation a homologous, anti pre immune serum as a negative control. Results after capacitation, using anti phospho PKA antibody substrate, tyrosine phosphorylation of FSCB antibody and antibody immunoprecipitation products Western blot analysis with FSCB antibody showed positive bands at 270Kda, but can not use anti sperm, P phosphorylation of the PKA substrate antibody, immune antibody against tyrosine phosphorylation of the precipitate does not appear the positive bands, the bands only appeared in FSCB antibody immunoprecipitation products. Showed that the 270Kda protein is FSCB, and the It is suggested that FSCB is phosphorylated by PKA, and its tyrosine is phosphorylated.
For the study of FSCB phosphorylation and capacitation time, we further in different time points (1,2,5,10,30,60, min and 120min) take the same amount of HTF sperm culture liquid samples pyrolysis, continue Western blot analysis with anti phospho PKA antibody. The substrate bands appeared in contact with capacitation the medium 1min, and with the extension of time continues to deepen, remained unchanged after 60min. The results showed that FSCB in sperm capacitation by PKA phosphorylation can occur the tyrosine phosphorylation, the phosphorylation level of contact was closely related to the medium.
Two, PKA activity affects the phosphorylation of FSCB
FSCB as the substrate of PKA, the sperm capacitation can be phosphorylated by PKA process. To study the change of the activity of PKA on the phosphorylation state of FSCB, to further elucidate the molecular basis of sperm capacitation and protein phosphorylation in the regulation mechanism is very important. We use H-89 and DB-cAMP treated mice sperm, observation FSCB the phosphorylation state changes. Take the same amount of HTF medium in sperm, containing 0.5 M H-89 HTF medium, M2 medium and M2 medium containing 2.5mM DB-cAMP were cultured in different time points (1,2,5,10,30,60, min and 120min) take the same amount of mouse sperm for PKA activity analysis, and in 30 minutes cultured for different sample of sperm lysates, Western blot analysis with anti phospho PKA antibody protein substrate, a homologous anti pre immune serum as a negative control.
In the experiment we found to add 0.5 M H-89 HTF culture medium, the activity of PKA was inhibited, M2 cultured with 2.5mM DB-cAMP, PKA activity was significantly enhanced. HTF medium with anti phospho PKA antibody Western substrate blot analysis, DB-cAMP M2 medium containing 2.5mM 30min sperm lysates in 270KD there was a strap and medium containing 0.5 M H-89 HTF, M2 free culture positive band liquid sperm lysates. The results showed that H-89 can inhibit the activity of PKA, DB-CAMP increased the activity of PKA two can change the activity of PKA by affecting the phosphorylation of FSCB.
Three, the degree of FSCB phosphorylation is closely related to sperm motility
Specific protein tyrosine phosphorylation is an important sign of sperm capacitation, sperm motility and velocity, the oscillation frequency of motion characteristics change. In order to study the relationship between FSCB phosphorylation and sperm motion characteristics, the sperm movement characteristics at different time points were taken. We analysis the mouse sperm in HTF culture solution, containing 0.5 M H-89 HTF medium, M2 medium and M2 medium containing 2.5mM DB-cAMP were cultured in different time points (1,2,5,10,20,30,60 and 120min) from 5 L mice sperm motility analyzer (Spain) analysis.
We found HTF to 0.5 M H-89 added to the culture medium, 1,5,10,20,30,60 and 120min after capacitation of sperm VCL decreased significantly after capacitation, 1,5,10,20,30 and 60min (A+B) also decreased to M2%; the medium adding 2.5mM DB-cAMP, 1,5,10,20,30min VCL after capacitation, sperm capacitation significantly enhanced after 1,5,10,20min (A+B%) was also higher. The comprehensive analysis of the experimental results, found that the changes of sperm movement characteristics and the level of FSCB phosphorylation changes have obvious linear relationship, we draw the inhibition of sperm motility by inhibiting the phosphorylation of FSCB and H-89, DB-CAMP can promote FSCB phosphorylation and promote sperm motility.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R339.2

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