摘要
尿激酶型纤溶酶原激活因子(urokinase-plasmingen activator，uPA)与雄性生殖系统关系密切，能够多方面影响精子功能，涉及精子排放、运动功能的获得、活力改变、获能、顶体反应以及受精等多个方面。此外，作为内源性趋化物质诱导精子趋化性可能是uPA促雄性生育的一种新作用。精子趋化性是指精子沿着趋化物质浓度递增或递减方向改变运动方向的能力，发生于精-卵接触前的通讯中。精子趋化性对精-卵在正确时间和地点相遇有着重要意义。然而，有关趋化物质的化学特性至今还未清楚，可能是一种耐热的小分子多肽。我们根据uPA的特点推测uPA可能是一种内源性化学趋化物质，能够诱导精子趋化性。首先，uPA具有介导体细胞趋化运动的特性。uPA通过暴露uPAR的88～92位点残基以释放有趋向性的抗原表位，协助跨膜调节细胞迁移，从而介导体细胞的趋化性运动，尤其是肿瘤细胞的转移。而uPA的抑制剂PAI-1则通过阻止整合素结合玻连蛋白，抑制uPA酶活性，从而抑制细胞迁移，提示uPA系统与体细胞的迁移和趋向运动有关。其次，uPA及其受体存在于受精环境中：排卵后的卵丘细胞团能够分泌uPA，精子表面存在uPAR。推测uPA在输卵管壶腹部形成以卵细胞为中心的浓度梯度；与uPAR结合后，uPA诱导精子产生趋化性运动并沿着uPA浓度递增的方向寻找卵细胞进而使卵细胞受精。因此，我们推测uPA能够诱导精子趋化性，在精-卵接触前通讯中发挥作用；结合uPA促获能、顶体反应、增加精子活力等多方面作用，从而产生促雄性生育作用。
因此，我们研究了uPA体外对小鼠精子趋化性的诱导、以及对精-卵接触前通讯和小鼠精子体外受精能力的促进作用；然后进一步从不同环节研究uPA促雄性生育作用的分子机制，包括诱导精子趋化性的分子机制、促精子获能的能量机制、以及初步在体研究uPA在精子发生中的作用机制。从而探讨uPA多环节促雄性生育的作用，旨在为uPA在临床应用提供科学的理论基础。
一、uPA促雄性生育作用的体外观察
本部分的主要实验目的是研究uPA能否在体外诱导小鼠精子趋化性；uPAR是否参与精子趋化性反应；以及uPA在小鼠精-卵接触前通讯和体外受精中的作用。从而探讨uPA在促雄性生育中的新作用。
目前用于检测精子趋化性的方法有4类：第一类趋化性测试是在一个递增的梯度化学引诱剂上的精子聚集。在此类测试中，精子感受到一浓度梯度递增的化学趋化物质，因而在化学趋化物质或其附近聚集。化学趋化物质梯度是通过自由扩散方式建立。第二类测试与第一类测试方法相反，即观察精子在一个浓度梯度递减的化学趋化物质上离开的趋势，主要测试精子离开而不是聚集在化学引诱剂上的趋势。第三类测试包括“选择”测试。这是最常见的测试方法之一，精子在分别含有化学趋化物质和其它对照缓冲液的两个孔中选择。第四类测试是检测精子运动轨迹。其中，第二类和第四类方法可以将精子趋化性与其他能够引起精子聚集的原因区分开。在此，我们选用第一和第二种方法来检测体外小鼠精子对uPA的趋化性。首先，通过检测精子在装有浓度递增或递减uPA的毛细管内聚集量的变化来测试小鼠精子体外对uPA和卵细胞的趋化性以及uPAR在其中的作用；然后，将卵细胞分别经uPA、PAI-1以及抗uPAR抗体预处理后，检测其趋化诱导精子能力的变化来反映uPA在精-卵接触前通讯中的作用；最后，观察精子经uPA预处理后其受精能力的变化。实验结果显示：精子在递减／递增uPA浓度梯度中的聚集密度明显低于／高于在等uPA浓度梯度内的密度(P＜0.05)；而抗uPAR抗体处理精子可使精子在递增uPA浓度梯度中的聚集密度明显降低。经uPA预处理的卵细胞其聚集精子的能力显著强于未经处理卯细胞(P＜0.05)，而用PAI-1预处理卵细胞后，卵细胞聚集精子的能力则显著下降(P＜0.05)；用抗uPAR抗体预处理卵细胞并不影响卵细胞对精子的聚集作用；uPA处理后的精子与卵细胞共孵育后所得受精卵数目多于未经处理组的受精卵数目(P＜0.05)。
综上，我们得出以下结论：精子在体外对uPA和卵细胞均有趋化性；uPA能增加卵细胞对精子的趋化作用，增进精-卵接触前通讯，促进受精。推测uPA作为一种内源性的精-卵接触前的通讯物质，诱导精子改变运动方向，增加精卵接触机会，进而增加受精成功机率。本实验第一次提出了uPA作为内源性趋化物质诱导精子趋化性可能是其一种新的促雄性生育作用，并为其在临床上的应用提供一定的理论依据。
二、uPA促雄性生育作用的机制研究
从上面实验我们观察到uPA在体外可以通过诱导精子趋化性，增进精-卵接触前通讯，提高小鼠精子体外受精率，从而多途径地发挥促雄性生育作用。但uPA促雄性生育作用的机制却不清楚。由于uPA与雄性生殖系统关系密切，所涉及的细胞功能事件及相应机制极其复杂，在这里，我们仅从uPA诱导精子趋化性、促获能、促精子发生三个不同方面来研究uPA促雄性生育作用的机制。
钙离子通道是一种极为常见的信号通路，已在哺乳动物精子膜上鉴定出四类Ca~(2+)通道。已有实验显示精子尾部胞浆内[Ca~(2+)]_i的变化与精子趋化运动有直接关系。此外，精子作为一种转录和翻译活动都已经终止了的终末分化细胞，蛋白磷酸化在精子生理活动中应该发挥着重要的调节作用。ERK1／2是一类细胞外信号调节激酶，在体细胞的趋化迁移活动中发挥重要作用。因此，我们通过检测对uPA产生趋化性的获能小鼠精子胞浆内[Ca~(2+)]_i和磷酸化ERK1／2蛋白水平的变化来探讨uPA体外诱导小鼠精子趋化性的可能分子机制。采用毛细管法收集对uPA产生趋化运动的小鼠获能精子；对用于检测胞内Ca~(2+)荧光强度的那部分获能精子先予以Fluo3-AM荧光染料负载。再根据uPA作用时间长短，将收集的精子分为0、5、30、60min 4个时间组，定为实验组；每个时间点都设一对照组(以BWW液替代毛细管内的uPA)。然后分别用激光共聚焦显微镜和Western Blot方法检测各组精子胞内的Ca~(2+)荧光强度(经Fluo3-AM负载过的趋化运动精子)和胞内磷酸化ERK1／2蛋白水甲(未经Fluo 3-AM负载的趋化运动精子)。实验结果显示：实验组的精子胞内Ca~(2+)荧光强度在5 min和30 min显著高于对照组(P＜0.05)；磷酸化ERK1蛋白水平在5、30 min和60 min，以及磷酸化ERK2蛋白水平在30、60 min显著高于对照组(P＜0.05)。因此我们认为uPA诱导的小鼠精子趋化运动伴随胞浆内[Ca~(2+)]_i和ERK1／2蛋白磷酸化水平升高。推测uPA体外诱导精子趋化运动作用的分子机制与此有关。
获能是精子发生顶体反应以及完成受精活动所必须的前提条件。精子伴随获能而发生的改变需要高能量支持。uPA能够促进精子获能。在此，我们通过检测uPA体外对获能小鼠精子线粒体膜电位的影响来研究uPA促精子获能作用的机制，进而探讨uPA促男性生育作用的可能机制。我们利用流式细胞仪、荧光显微镜和膜电位荧光染料JC-1来检测精子线粒体膜电位。根据uPA作用与否将获能精子分为实验组和对照组；再根据uPA作用时间的不同，各组又分为5个亚组。分别将实验组和对照组的获能精子与uPA或BWW液共孵育，于孵育的第0、5、15、30min和60 min时加入JC-1继续孵育20 min，然后用流式细胞仪和荧光显微镜检测精子线粒体膜电位状态。实验结果显示：JC-1荧光主要集中在精子尾部中段，颜色呈绿色、橙色和橙红色不等；在uPA作用第5、15 min时，实验组内精子平均荧光强度显著增加，膜电位高的精子数百分比亦相应增加(P＜0.05)；而在对照组，只有5 min时的荧光强度略有升高，且无统计学意义。实验组在uPA作用5、15 min时的膜电位高的精子数百分比，以及15、30、60 min时的精子平均荧光强度较对照组相同时间点显著增加(P＜0.05)。因此，uPA在体外可以增加获能小鼠精子的线粒体膜电位，并使线粒体膜电位升高状态维持一段时间，从而为获能精子提供充足的能量供应，以适应精子活力增加以及运动模式改变所需要的高能量消耗。推测uPA通过增加线粒体功能来促进精子获能、增加精子活力，从而提高精子受精的能力。此可能为uPA促男性生育的机制之一。
精子发生过程中的主要事件有生殖细胞有丝／减数分裂以及在生精上皮内的移位，涉及细胞增殖、减数分裂进程调控、支持细胞与支持细胞或者与生殖细胞之间紧密连接的蛋白水解等复杂事件。已有大量研究显示MAPKs同时参与调控上述两方面的生理过程。而uPA由支持细胞分泌，并通过蛋白水解作用在精子发生过程中参与生精细胞转位和精子排放的调节。在体细胞，uPA通过结合其受体uPAR，可以触发MAPKs信号途径进而调控细胞增殖、凋亡、迁移、趋化等多种细胞功能。然而在睾丸内，各级生精细胞表面有uPAR，uPA是否同样通过MAPKs信号途径而在精子发生过程中发挥作用尚不清。因此，本实验通过在体研究uPA对小鼠睾丸组织中Erk1／2 mRNA转录水平、ERK 1／2蛋白表达以及磷酸化水平的影响，来探讨在生精过程中uPA发挥作用的可能信号机制。实验中将ICD小鼠随机分为2组，分别给予uPA(200 IU／10g bw，实验组)和生理盐水(对照组)连续腹腔注射35 d。然后用荧光定量RT-PCR、Western Blot以及免疫组织化学方法结合图像分析技术分别检测2组小鼠睾丸组织中Erk1／2 mRNA的相对定量以及蛋白表达及磷酸化水平。结果显示实验组小鼠睾丸组织中Erk1／2 mRNA的相对定量、蛋白表达以及磷酸化水平均显著高于对照组(P＜0.05)；ERK1／2蛋白主要分布于支持细胞和各级生殖细胞，尤其以精原细胞和支持细胞为最；磷酸化ERK1／2主要分布于精原细胞胞核及核周边区，二者免疫着色均强于对照组。因此，我们认为在体腹腔注射uPA可以在转录和／或翻译水平上调Erk1／2基因，促进ERK1／2蛋白磷酸化。推测uPA是通过ERK1／2信号通路在精子发生过程中发挥重要作用的。
三、总结
uPA与雄性生殖系统关系密切，对多种精子功能都有促进作用。在这里，我们第一次提出并证明uPA促雄性生育的另一新作用：作为内源性趋化物质在体外诱导精子趋化性，进而增进精-卵接触前通讯以及增加小鼠精子体外受精率。同时，我们从精子趋化性、获能、以及精子发生三个方面探讨了uPA发挥促雄性生育作用的机制。实验结果显示：uPA诱导的小鼠精子趋化运动伴随胞浆内[Ca~(2+)]_i和ERK1／2蛋白磷酸化水平升高；uPA在体外可以增加获能小鼠精子的线粒体膜电位，并使线粒体膜电位升高状态维持一段时间，从而为获能精子提供充足的能量供应，以适应精子活力增加以及运动模式改变所需要的高能量消耗；在体腹腔注射uPA可以在转录和／或翻译水平上调Erk1／2基因，促进ERK1／2蛋白磷酸化。综合本研究结果和已有的相关研究，我们认为uPA能够从多环节发挥促雄生育的作用；此外，针对uPA有可能研制成为抗雄性生育药品从而在男性避孕中发挥重要作用，以及作为一种诊断试剂用于男性不育症病因的查找和为人工辅助生殖技术提供优质精子。
Urokinase-plasmin(?)gen activator (uPA) is closely relevant to male reproduction and can affect mammal sperm functions in many aspects, including spermiation, acquiring of motility capability, motility changing, capacitation, acrosomal reaction and fertility. Besides, inducing sperm chemotaxis may be another new function of uPA on promoting male fertility. Sperm chemotaxis is definited that the spermatozoa swim up or down the gradient of an attractant and change their direction, occurs during precontact sperm-egg communication and is important for sperm-egg encounter timely and punctually. However, the chemical characteristic of chemoattractant is obscure by now. It is supposed that chemoattractants may be some peptides or proteins of low molecular mass (1-20 kDa), which are heat stable and sensitive to proteases. Here, we consider that uPA may be an endogenous chemoattractant to guide sperm chemotaxis in vivo. Firstly, uPA can induce somatic cell chemotaxis by uncovering a chemotactic epitope present at residues 88-92 of uPAR, which then cooperates with a transmembrane adapter to stimulate migration; while PAI-1, the inhibitor of uPA, can block cell migration through inhibiting the combination between integrin and vitronectin, which suggested that uPA system may be related to the migration and chemotaxis of somatic cell. Secondly, both uPA and uPAR are involved in fertility. Alter ovulation, cumulus-oocytes-corps release uPA that may form a gradient from ampulla to isthmus by diffusion. When binding to uPAR of sperm, uPA with ascending gradient then guide sperm to produce chemotactic movement to encounter oocyte. Thus, it is suggested that by inducing sperm chemtaxis, uPA play a role in precontact sperm-egg communication. And considering the other effects of uPA on sperm functions, we presume that uPA may promote male reproductive capability through different pathway.
Therefore, we attempted to investigate the effects of uPA on mouse sperm chemotaxis, precontact sperm-egg communication and fertilization in vitro, and to further study the possible molecular mechanisms of uPA promoting male reproduction in different aspects including chemotaxis, sperm capacitation and spermatogenesis.
1. The in vitro study of new function of uPA on promoting male fertility
The main goal of this portion is to study the effects of uPA on sperm chemotaxis, precontact sperm-egg communication and fertilization in vitro, and to investigate the involvement of uPAR in sperm chemotaxis induced by uPA.
Currently, there are four kinds of techniques for sperm chemotaxis study: The first is an accumulation assay in which spermatozoa sense an ascending gradient established by diffusion of the attractant and accumulate near or at its source. The second is also an accumulation assay, but in this assay spermatozoa sense a descending gradient of the attractant. Thus, this assay measures the tendency of the spermatozoa to leave the attractant rather than to accumulate in it. The third is a 'choice' assay in which spermatozoa choose between two wells or chambers containing the attractant and the control buffer respectively. The last is the tracing of video-recorded tracks made by spermatozoa in a gradient of an attractant. Among the four types of techniques, only the second and the last one can actually distinguish chemotaxis from other causes of sperm accumulation. Therefore, we chose both the first and second techniques to investigate sperm chemotaxis in this study.
Firstly, the effects of uPA on mouse sperm chemotaxis and the involvement of uPAR in this process were assayed by measuring the sperm densities in capillaries with a descending or ascending gradient of uPA respectively. Secondly, sperm chemotaxis induced by eggs, which had been treated with uPA, PAI-1 and anti-uPAR beforehand respectively, was assayed to study the role of uPA in precontat sperm-egg communication. Lastly, the fertilization capability of spermatozoa treated with uPA was examined by counting the number of fertilized eggs. The experimental data showed: The densities of spermatozoa that migrated up / down the gradient of uPA into the capillary were significantly more / less than that into the capillary containing no-gradient uPA (P<0.05); When uPAR of spermatozoa was blocked, the density of spermatozoa that migrated into the capillary with ascending gradient of uPA decreased correspondingly; The density of spermatozoa attracted by egg, which were treated with uPA beforehand, increased significantly than that of attracted by no-treated eggs. On the contrary, the sperm density decreased correspondingly when the egg was
treated with PAI-1 (P<0.05): the number of fertilized eggs increased significantly after the spermatozoa were treated with uPA beforehand (P<0.05).
Herein, we draw the conclusions as follows: both uPA and egg could induce mouse sperm chemotaxis in vitro; uPAR may involve in sperm chemotaxis induced by uPA; uPA can promote the precontact sperm-egg communication and enhance the capability of sperm fertilization in vitro. Thus, uPA may act as an endogenous attractant in precontact sperm-egg communication and therefore fertility success correspondingly. This study, for the first time, raised a new action of uPA on promoting male fertility, and in some degree provided the theory foundation for the use of uPA in clinical medicine.
2. The mechanisms of uPA promoting male reproductive capability
We have observed that uPA can guide sperm chemotaxis, promote precontact sperm-egg communication and enhance the capability of sperm fertilization in vitro. However, the mechanisms of uPA on promoting male fertility are obscure. Considering extremely complicated involvement of uPA in male reproductive functions, here, we just studied three aspects of mechanisms of uPA promoting male fertility including sperm chemotaxis, sperm capacitation and spermatogenesis.
Ca~(2+) channel is a common signal pathway and there have been four types of Ca~(2+) channel identified on sperm membrane. Recent researches have demonstrated the direct relation between sperm chemotaxis and concentration of intracellular Ca~(2+) ([Ca~(2+)]_i) of spermatozoa. In addition, the regulation of sperm functions is largely dependent on protein phosphorylation because the spermatozoa are terminal differentiation cells. ERK1/2 (extracellular signal regulate kinase) play an important role in somatic cell migration. So, by detecting the changes of [Ca~(2+)]_i and the levels of phosphorylated ERK1/2 protein in mouse capacitated-sperm, we studied the possible molecular mechanisms of uPA inducing sperm chemotaxis. Firstly, the mouse capacitated-sperm with chemotactic movement were obtained by the use of capillary with an ascending gradient of uPA in it, and Mere divided into 4 test groups according to the different time incubated with uPA(0 5, 30, 60min respectively). Another 4
groups were, designed as control groups, in which the spermatozoa were incubated with BWW. The capacitated-sperm used for assaying intracellular [Ca~(2+)]_i were incubated with Fluo 3-AM before accepting the chemotactic induction. Then the fluorescence intensity of intracellular [Ca~(2+)]_i and the levels of phosp(?)orylated ERK1/2 proteins in sperm in each group were determined by Confocal microscopy and Western Blot respectively. The data indicated that: the mean fluorescence intensities of sperm [Ca~(2+)]_i in test groups were significantly increased than that of in the control groups at 5 and 30 min (P<0.05), and the levels of phosphorylated ERK1 and ERK2 proteins were also increased obviously at 5, 30, 60 min and 30, 60 min respectively (P<0.05). Therefore, the chemotactic movement of mouse capacitated-sperm induced by uPA was accompanied with the increase of intracellular [Ca~(2+)]_i and levels of phosphorylated ERK1/2 proteins. We suggested that, at least, the part mechanisms of sperm chemotaxis induced by uPA in vitro is related to the positive effect of uPA on the intracellular [Ca~(2+)]_i and the levels of phosphorylated ERK1/2 proteins.
During capacitation, spermatozoa need an increasing energy for series function changes of themselves. Regarding that uPA can promote sperm capacitation, we aim to study the mechanism of uPA urging sperm capacitation by investigating the effect of uPA on mitochondrial function of mouse capacitated-sperm in vitro, and to explore the possible therapeutic mechanism of uPA on male infertility. Mitochondrial function of mouse capacitated-spermatozoa was evaluated through the assessment of mitochondrial membrane potential by the use of JC-1 performed by Flow CytoMeter and fluorescent microscope respectively. Test group and control group were designed according to the presence of uPA or not; and each group was divided into 5 sub-groups respectively according to the different treat time of uPA at 0, 5, 15, 30 and 60 min respectively (or BWW in control groups). Then, JC-1 was added into each sub-group. After 20 min, the mean fluorescence intensity and number of spermatozoa with different color (green / orange-red) in each group at different time points were assayed by the use of Flow CytoMeter and fluorescent microscope respectively. The results of the experiment were as follows: the fluorescence of JC-1 was mainly located to the midpiece of flagellar and with the color of green or orange-red; the
mean fluorescence intensity and the percent of spermatozoa with orange-red color of test groups were increased significantly at 5, 15 min time points respectively after sperm incubating with uPA (P<0.05); While in the control group, except for at the 5 min, there was a decline tendency of the mean fluorescence intensity and the number of spermatozoa with orange-red color during experiment. In the test group, the mean fluorescence intensity at 15, 30 and 60 min and the percent of spermatozoa with orange-red color at 5, 15 min were significantly higher than that in the control group (P<0.05). Then, to sum up, uPA could increase the mitochondrial membrane potential of mouse capacitated-spermatozoa in vitro, and sustain it at a high level for a certain periods so that provide sufficient energy for capacitated-spermatozoa to increase their motility and change their motor pattern. It is presumed that, through enhancing sperm mitochondrial function, uPA may improve sperm capacitating, motility and fertility potential. This might be one of the therapeutic mechanisms of uPA on male infertility.
The mitosis, meiosis and the translocation of germ cells in the seminiferous epithelium are the key events during spermatogenesis, involving in proliferation of spermatogonial cell, regulation of spermatocyte meiotic progression and proteolysis events between Serloli cell-Sertoli cell and Sertoli cell-germ cell. Increasing evidences indicated that MAPKs are closely relevant to the above cell events. UPA is secreted by Sertoli cells and plays the important roles in regulating germ cells translocation and spermiation during spermatogenesis by proteolysis. In somatic cells, binding uPA to uPAR may control cell proliferation, apoptosis, migration and chemotaxis by activating MAPKs signal pathway. However, whether can uPA activate MAPKs signal pathway to play the roles during spermatogenesis is obscure. Hence, we aimed to study the effects of uPA on the transcriptive and / or translative levels of Erk 1/2 gene in mouse testes treated with uPA, and to explore the action mechanism of uPA during spermatogenesis. The mRNA and protein levels of ERK1/2 in mouse testes from the test group and the control group which received an intraperitoneal injection of uPA or normal sodium for 35 days respectively, were evaluated by real time RT-PCR, Western Blot and immunohistochemistry followed by image analysis. Compared with that of in the control group, the levels of Erk 1/2 mRNA in mouse
testes were .significantly upregulated in the test group. Accordingly, the levels of ERK.1/2 and p-ERK 1/2 protein in mouse testes were also significantly higher in the test group in comparison with that in the control group. Moreover, the p sitions of ERK1/2 proteins were located to all stages of germ cells and Sertoli cells, and were especially dominant in the spermatogenic cells and Sertoli cells. Besides, pERK1/2 proteins were mainly located or near to the sperm nucleus, and the immunostaining of pERK1/2 proteins in the test group was stronger than that of in the control group. Taken together, the intraperitoneal injection of uPA in vivo may up-regulate Erk 1/2 gene in mouse testes at the transcription and / or translation levels and promote ERK1/2 activation. We presumed that it was through ERK1/2 signal pathway that did uPA play the important role during spermatogenesis.
3. Summary
UPA is closely relevant to the male fertility and can affect sperm functions in many aspects. Here, for the first time, we raise and confirm a new action of uPA on promoting male fertility, which is guiding sperm chemotaxis, promoting precontact sperm-egg communication and enhancing the fertilization capability of mouse sperm. Meanwhile, we further investigate the action mechanism of uPA promoting male fertility in different aspects including sperm chemotaxis, sperm capacitation and spermatogenesis. The experiment data demonstrate that: the chemotactic movement of mouse sperm induced by uPA in vitro is accompanied with the increase of intracellular [Ca~(2+)]_i and with the high levels of phosphorylated ERK1/2 proteins; uPA can enhance the membrane potential of mouse capacitated-sperm and sustain the membrane potential at a relatively high level for a certain time, which can provide sufficient energy for capacitated-sperm to enhance motility and to change motor pattern; the intraperitoneal injection of uPA in vivo may up-regulate Erk 1/2 gene in mouse testes at the transcription and / or translation levels and promote ERK 1/2 activation. Then, taken together, uPA can enhance male fertility through many pathways. We presumed that the roles of uPA in promoting male fertility might also be the targets of antifertility medicine for male contraception, and inducing sperm
chemotaxis by uPA may be a way to evaluate male sperm functions in clinical medicine.
引文
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