Study Of The Roles And Mechanisms Of Urokinase-type Plasminogen Activator In Promoting Male Fertility

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 fertilityThe 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 wastreated 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 capabilityWe 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.Ca²⁺ channel is a common signal pathway and there have been four types of Ca²⁺ channel identified on sperm membrane. Recent researches have demonstrated the direct relation between sperm chemotaxis and concentration of intracellular Ca²⁺ ([Ca²⁺]_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 [Ca²⁺]_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 4groups were, designed as control groups, in which the spermatozoa were incubated with BWW. The capacitated-sperm used for assaying intracellular [Ca²⁺]_i were incubated with Fluo 3-AM before accepting the chemotactic induction. Then the fluorescence intensity of intracellular [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_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; themean 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 mousetestes 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. SummaryUPA 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 [Ca²⁺]_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 spermchemotaxis by uPA may be a way to evaluate male sperm functions in clinical medicine.