The Effect of Swimming Training, Cell and Laser on the Expression of Genes Involved in Autophagy (LC3 and Beclin-1) in Azoospermia Mice

Infertility in men is 10-20% of cases due to azoospermia and usually due to disorders of the reproductive system. Normally, the population of men with azoospermia is estimated to be around 2% (1) and it has been shown that in 20% of infertile men, azoospermia is the main cause of infertility (2). Non-obstructive azoospermia is a state in which no sperm is observed in ejaculation and is related to intra-testicular disorders and as a result spermatogenesis disorders, while in obstructive azoospermia, spermatogenesis is normal and the defect is related to obstruction in the ejaculatory ducts (3). In a study, decreased expression of autophagy pathway genes (Lc3B, Beclin1) was observed in patients with azoospermia (4). The aim of this study was to investigate the effect of swimming, cell and laser training on the expression of genes involved in autophagy in azoospermia model mice.

The samples of the present study were male Wistar laboratory rats, which were under control in the laboratory in terms of many variables, therefore, the present study was of an experimental type. 30 6 to 8 week old rats from the research center and Reproduction of laboratory animals in Tehran were randomly selected. In order to create azoospermia model, the drug busulfan was injected intraperitoneally for each rat at a dose of 40 mg/kg of body weight (15). After a month of inducing the model, the rats in each group were divided as follows: Patient control group (one month after creating the model until the end of the study, they remained for 8 weeks), healthy control group (kept for 8 weeks), patient control group + low power laser (one month after creating the model, low laser Power with a wavelength of 632.8 nm and a power of 10 mW and an energy of 3 joules was applied three times in the entire study period with an interval of one week in the testicular region of azoospermic rats and rats until the end of the study for 8 weeks. were maintained), the patient control group + exercise (one month after creating azoospermic mice for 8 weeks, they swam daily for 30 minutes a day, 5 days a week), the patient control group + cell (one month later From the creation of azoospermic mice, stem cells were transplanted in the vas deferens region at the rate of one million cells per mouse in the right testis and the rats were kept for 8 weeks until the end of the study), patient control group + cells + low laser power + exercise (one month after the creation of azoospermic mice, stem cells were transplanted once in the vas deferens in the amount of one million cells for each mouse, then after a week of low power laser cell transplantation with wavelength 632.8 nm and power of 10 mW and energy of 3 joules were applied three times during the entire study period with an interval of one week and after the healing of the wound of the cell graft area on the abdomen, it was applied topically for 30 minutes a day. They swam 5 days a week, which lasted for 8 weeks). Tissue sampling was done from the testicular tissue of mice under completely similar conditions and in basic conditions (two days after the end of the training period). In order to eliminate the acute effect of training, sampling of the animals was done 48 hours after the last swimming training program. For this purpose, the animals were anesthetized by peritoneal injection of ketamine (30-50 mg/kg) and xylazine (5-3 mg/kg) and then killed, and after killing the transplanted tissues, they were evaluated for genetic studies. To check the expression of the studied genes in each group, real time PCR technique was used to examine the tissues. First, primer design was done and then total RNA was extracted from the tissues and converted into cDNA. Then, the cDNA was amplified by PCR and analyzed for the expression of the mentioned genes. To analyze the findings of this research, Smirnov's Kalmograph tests, one-way analysis of variance and Tukey's were used to compare between different groups. All calculations were done using SPSS/22 statistical software and at a significant level of P≤0.05.

The results showed that there is a significant difference between the average expression of the Beclin gene in testicular tissue in different research groups; The control group has a significant difference with the model group at the confidence level of 0.01, and the model group has a significant difference with the cell, laser and training group at the confidence level of 0.05. Also, the results show that cells, laser and exercise simultaneously had an effect on the expression of Beclin gene in testicular tissue in azoospermia model mice. Also, the next finding showed that there is a significant difference between the average LC3 gene expression of testicular tissue in different research groups; The control group has a significant difference with the model group at the confidence level of 0.01, and the model group has a significant difference with the cell, laser and training group at the confidence level of 0.05. Also, the results show that cells, laser and training simultaneously had an effect on the expression of LC3 gene in testicular tissue in azoospermia model mice.

The results of the research showed that by inducing the azoospermia model, the expression of LC3 and Beclin-1 genes in the testicular tissue of mice increased significantly compared to the control group. On the other hand, with the implementation of exercise, cell and laser treatment methods, the expression of the mentioned genes in mice decreased compared to the azoospermic group, showing that this decrease was significant only in the combined exercise, cell and laser group. Research has shown that exercise can be a key factor in the regulation of proteins involved in the autophagy pathway, and the regulation of autophagy by exercise can be a key process in cellular and molecular mechanisms (11,10). The main path of the proteins involved in the autophagy process is regulated by complex regulatory mechanisms, which can be different input signals such as nutrients, growth factors, hormones, intracellular calcium concentration, ATP level, hypoxia, etc. The main regulatory pathway of these factors is the mTORC1 pathway, which is activated in cellular responses such as growth, proliferation, protein synthesis, and autophagy (11). The mTORC1 pathway can activate or inhibit the autophagy mechanism by regulating ULK1, ATG13 and FIP200 proteins (25). Various studies have shown that LC3 can increase cell death or its survival (26), it is also known as one of the effective factors in autophagy, which according to the available evidence can have an apoptotic function (27). It has been reported that decreased autophagic activity due to decreased expression of LC3 and Beclin-1 can be associated with tumorigenesis and increased tumor growth (28). No research was found that investigated the effect of stem cells and laser therapy on the expression of genes involved in autophagy. The results of the present study showed that cell therapy and laser therapy alone and in combination do not have a significant effect on the expression of LC3 and Beclin-1 genes in the testicular tissue of azoospermia model mice, but cell therapy and laser therapy in combination with exercise caused a significant decrease in these genes. And somehow it inhibited autophagy in testicular tissue of azoospermia model mice. Therefore, it is possible that exercise combined with cell therapy and laser therapy by inhibiting autophagy in the testicular tissue of azoospermia model mice induced with busulfan exerts its protective effect and in this way causes the fertility of azoospermia model mice, but a definite opinion needs more research in this regard. It is the context. In general, it has been shown in the research that autophagy is activated in pathological conditions and may have destructive effects on the tissue. In the present study, the induction of the azoospermia model by busulfan leads to an increase in the genes involved in autophagy and in some way activates the pathway that exercise Exercise in combination with cells and laser therapy by reducing genes and somehow inhibiting autophagy may exert its protective effect and thus prevent infertility in mice.