Study On The Biological Function Of PGAM1 And Its Correlation With Testicular Spermatogenic Function

IntroductionInfertility has been regarded as a serious medical problems in the world.50% of infertility couples are caused by male factor, while only 20% of the is correlated with both husband and wife. Male infertility is defined by the World Health Organization as that couples didn’t have a pregnant after more than one year without any contraceptive, which was caused by male factor.Recent studies showed that male infertility has become one of the major disease that impacted on people’s life quality, while its incidence was increasing in recent years. Studies showed that the occurrence of male infertility was connected with many factors, including environmental toxins, endocrine dysfunction, reproductive tract inflammation, ejaculation disorders, nutrition infertility, varicocele, cryptorchidism, obstruction and genetic defects seminiferous duct (gene mutation and chromosomal abnormalities), etc. Among them, azoospermia caused by various causes is an important factor affecting male infertility. According to the pathological changes in azoospermic patients, it is divided into two categories. One subtype is the spermatogenic dysfunction caused by testicle itself, for example:normal spermatogenesis is blocked by chromosomal abnormalities and gene mutations, which is known as non-obstructive azoospermia. Another subtype is charactered with normal function of spermatogenesis, but normal sperm can not be eliminated from the body due to the vas deferens infarction, known as obstructive azoospermia. However, current methods for the diagnosis and treatment of azoospermia is not satisfactory. In order to clarify the etiology and pathology in infertility patients, they need to accept multiple semen analysis, prostate fluid routine examination, endocrine, Doppler ultrasound, even testicular biopsy and karyotype analysis. However, these inspection methods is time-consuming, the clear diagnosis is still hard to obtain. The majority of infertility patients can only choose artificial reproduction. Obviously, the breakthrough of clinical diagnostics and treatment in infertility patients still depends on the progress in basic research to elucidate the mechanism of azoospermia.Recent studies have found that genetic factors is an important cause of male infertility, which caused about 15% of male infertility, including chromosomal abnormalities and single gene mutations. Chromosomal abnormalities accounted for 2% to 8%, with an average of 5%. Y chromosome microdeletions is an important cause of male infertility by disturbing normal spermatogenesis, which was first proposed the concept of azoospermia factor by Luciiano Tiepolo and Orsetta Zuffardi in 1976. In 1998, Vogt et al. further confirmed that the azoospermia factor was located in Yq11 and named as Y chromosome microdeletions. Azoospermia factor from near to far is divided into three different sub-regions:AZFa located Yq 11.21, AZFb and AZFc located in Yq11.23. Meanwhile, recent studies indicate that X autosomal translocations, gene mutations and deletions are also important factors associated with male infertility, including AR, SOX3, USP26, NXF2, TAF7L, FATE and AKAP4, etc., have been reported to be linked to spermatic disfunction.PGAM1 is one member of PGAM family, and is highly homologous with PGAM2. However, PGAM1 has more gene copy number, and therefore regarded as the ancestor of PGAM2. PGAM1 is an important enzyme in glycolytic pathway, which main catalytic 3-phosphoglycerate into 2-phosphoglycerate and eventually become into phosphoenolpyruvate. PGAM1 is expressed in normal brain, liver and kidney tissues, and associated with a variety of malignancies. However, the research about PGAM1 expression and male infertility has never been reported. Our primary study indicated that PGAM1 protein might be new candidate proteins associated with male infertility. In this study, to further investigate the relationship between PGAM1 expression and male infertility,02 carried out the study.OBJECT1. To investigate the expression an clinical significance of PGAM1 in different types of human testis tissues.2. To investigate the expression and significance of PGAM1 in animal models with spermatogenic dysfunction.3. To investigate the biological function and related mechanisms of PGAM1.METHODS1.40 cases of testis tissues with definite histopathological types (10 cases of normal spermatogenesis,10 cases of mild spermatogenic dysfunction,10 cases of severe spermatogenic dysfunction and 10 cases of Sertoli cell syndrome) was collected for the fixation and dehydration. Then, all tissues were made into paraffin tissue blocks. Using immunohistochemical staining, PGAM1 expression and its relationship with clinicopathological parameters in different types of testicular tissue were analyzed, respectively.2. Different types of fresh testicular tissues were collected (2 cases of normal spermatogenesis,2 cases of mild hypospermatogenesis,2 cases of severe hypospermatogenesis and 2 cases of Sertoli cell syndrome) to extract total RNA by tissue grinder. Using qRT-PCR, PGAM mRNA expression level was analyzed with clinical pathological features.3.10 cases of C57 mice with 6 weeks old, weighing 19～21 g, were randomly divided into experimental and control groups. The mice in experimental groups were intraperitoneally injected through a single busulfan (30 mg/kg) and dimethyl sulfoxide (DMSO). The mice in control group were injected with an equal volume of DMSO. After 2 weeks injection, all mice were sacrificed and mouse testis tissues were collected and stained by hematoxylin-eosin (HE) for the observation of morphological changes. Meanwhile, qRT-PCR and Western blot were used to detect the expression PGAM1 and analyze the correlation between PGAM1 expression and spermatogenic disfunction.4. PGAM1 expression was detected in mouse spermatogonial cell lines GC1, GC2 and TM4 by qRT-PCR. Then, PGAM1 expression in GC1 and TM4 cell lines was knockdown by PGAM1-specific siRNA, and validated by qRT-PCR and Western blot.5. MTT assay was used to evaluate the effect of PGAM1 down-regulation on GC1 and TM4 cells. The apoptosis in GC1 and TM4 was detected by flow cytometry, when PGAM1 expression was down-regulated. The athletic ability of GC1 and TM4 cells was measured by scratches assay, after PGAM1 down-regulation.6. Using immunohistochemistry and Western blot, apoptosis-related proteins Caspase6 and caspase 9, and anti-apoptotic protein Bcl-2 were detected after PGAM1 down-regulation.RESULTS1. PGAMlexpression in different types of testis tissuesPGAM1 expression in testicular tissue with normal spermatogenesis, was mainly located in the nucleus and cytoplasm of spermatogonia, spermatocytes and Sertoli cell rather than in sperm cells. Among them,40 patients with different types of testicular tissue, high expression rates of PGAM1 in normal spermatogenesis, mild hypospermatogenesis, severe hypospermatogenesis, and Sertoli cell only syndrome were 90%(9/10),80%(8/10),10%(1/10),100%(10/10), respectively, and the difference was statistically significant (P=0.000). However, PGAM1 expression was not significantly associated with age, testicular size, and the levels of FSH, LH and T (P>0.05).2. PGAM1 expression was detected in different types of testicular tissue by qRT-PCRTo further explore the relationship between PGAM1 expression and spermatogenesis, we examined the expression of PGAM1 in testis tissues with normal spermatogenesis, mild hypospermatogenesis, severe hypospermatogenesisand and Sertoli cell only syndromeby by qRT-PCR. Results showed that the level of PGAM1 mRNA in testis tissues with normal spermatogenesis was significantly higher than those with severe hypospermatogenesisand, and the difference was statistically significant (P=0.000). The results also showed PGAM1mRNA level was not significantly correlated with patient’s age, testicular size, and the levels of FSH, LH and T (P>0.05).3. PGAM1 expression in animal models with spermatogenic dysfunctionTo verify whether busulfan with a single intraperitoneal injection could affect normal spermatogenesis, we observed the testicular morphological changes in experimental groups and control groups by HE staining. The results shown that the morphological structure was clear in seminiferous tubule in control group, and a large number of mature sperm were focused on middle seminiferous tubule lumen, suggesting that DMSO injection did not damaged spermatogenesis function. Compared with the control group, we found that the seminiferous tubules were obviously distorted and atrophy and spermatogenic cells (spermatogonia, spermatocytes, sperm cells and sperm) were significantly reduced, suggesting busulfan treatment significantly damaged normal spermatogenesis. Thus, these data indicated that animal model with hypospermatogenestic function was successfully constructed.After successfully constructed mouse models with spermatogenic dysfunction, we examined the expression of PGAM1 by immunohistochemistry. Results shown that PGAM1 expression in testis tissues with normal spermatogenesis was located in the nucleus and cytoplasm of spermatogonia, spermatocytes and sperm cells, also in in the nucleus and cytoplasm of Leydig cells. However, the expression of PGAM1 in seminiferous tubules in the testis with spermatogenic dysfunction was significantly weakened. Meanwhile, quantitative detection of qRT-PCR and Western blot also showed PGAM1 expression in testicular tissues with spermatogenic dysfunction was significantly lower than those control groups with normal spermatogenesis, the difference was statistically significant (P<0.05).4. PGAM1 downregulation effects biological function in GC1 and TM4 cellsSubsequently, in order to further clarify the biological function of PGAM1, we knocked down the expression of PGAM1 in GC1 and TM4 cells by using siRNA interference transient. Then, the best of interference fragment was selected for the subsequent experiments by qRT-PCR and Western. The results shown that the rates interfered PGAM1 mRNA expression by the three interference fragments were 24.7%, 78.0%,32.0%, respectively, in GC1 cells. Compared with the control group, the interference section 2 showed the best effect, which could obviously knocked down PGAM1 expression (P=0.000). In TM4 cells, the rates interfered PGAM1 mRNA expression by the three interference fragments were 11.0%,84.0%,31.3%. Compared with control group, the interference section 2 also showed the best effect, which could obviously knocked down PGAM1 expression (P=0.000). Thus, we chosen interference fragment 2 for the follow-up study. Then, we used Western blot assay to validate the effect in GC1 and TM4 cells, which indicated that PGAM1 expression was significantly reduced.In order to investigate the effect of PGAM1 down-regulation on the proliferation in GC1 and TM4 cells, we performed MTT assay. The results showed that PGAM1 down-regulation in GC1 cells shown more gentle growth curve and lower OD value, compared with those controls, suggesting that PGAM1 downregulation significantly inhibited cell proliferation (.P=0.000). Meanwhile, compared with control groups, in TM4 cells with PGAM1 downregulation had more gentle cell growth curve and lower OD value, suggesting that PGAM1 downregulation suppressed the proliferation in TM4 cells (P=0.000).To investigate the influence of PGAM1 downregulation on apoptosis in GC1 and TM4 cells, we performed flow cytometry. The results showed that the apoptotic rate of control group in GC1 cells was 2.1%±0.5%, while PGAM1 downregulation resulted in 11.8%±1.5% of apoptostic rate, with a significant difference (P=0.000). Meanwhile, the apoptotic rate of control group in TM4 cells was 8.7%±2.4%, while while PGAM1 downregulation resulted in 18.1%±3.9%, with a significant difference (P=0.000).In order to investigate the effect of PGAM1 downregulation on the athletic ability in GC1 and TM4 cells, we conducted a scratch test. The results showed that after PGAM1 downregulated 24h, the migration distance of TM4 cells was significantly lower than those in control groups, the difference was statistically significant (P=0.000), indicating PGAM1 downregulation inhibited the migration of TM4 cells. However, no significant changes was found in cell migration after PGAM1 downregulation in TM4 cells, and the difference was not statistically significant (P=0.786), suggesting PGAM1 downregulation couldn’t affect cell migration in GC1 cells.5. The effect of PGAM1 downregulation on the expression of downstream apoptosis-related proteinsTo investigate the potential mechanisms of PGAM1, we examined the expression of apoptosis-related proteins Caspase 6 and Caspase 9, but also detected the expression of anti-apoptotic protein Bcl-2 by Western blot and immunohistochemistry. After PGAM1 downregulation, the expression of Caspase 6 and Caspase 9 are significantly up-regulated, but Bcl-2 expression was significantly down-regulated, suggesting that PGAM1 down-regulation activated Caspases and Bcl-2 signaling pathways.CONCLUSIONS1. PGAM1 is expressed in testis tissues with normal spermatogenesis, and its downregulation may results in spermatogenic disfunction.2. PGAM1 expression correlates with cell proliferation, apoptosis and migration, and its downregulation could inhibit the proliferation of GC1 and TM4 cells and promote the apoptosis of GC1 and TM4 cells, but only inhibit the migration of TM4 cells.3. The mechanism of PGAM1 in biological function is associated with the activation of Caspases and Bcl-2 signaling pathway.