@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2017;23(3):235-242

Background
Spermatogenesis is a process that occurs with the propagating and differentiating spermatogonial stem cells. These cells are located on the membrane of the seminal tubes and sertoli cells surround them. This set provides an environment that causes the function and survival of spermatogenesis [1].
Previous Studies
… [2-10]. Some reports confirm the positive effects of supernatant derived from adipose tissue-derived mesenchymal stem cells on the process of repairing the tissues of the body. Therefore, many problems of cellular presence in the body, such as immune response and the formation of possible tumors can be prevented using this culture medium and removing cell [11, 12].
Aim(s)
The aim of this study was to investigate the effect of secretion of these cells on histomorphometric changes of testis in adult busulfan treated rats.
Research type
This is an experimental study.
Research society, place and time
This study was conducted in the Transgenic Technology and Stem Cell Research Center of Shiraz University of Medical Science on Balb/C adult male rats.
Sampling method and number
18 adult male Balb/C mice with the weight of approximately 30±2 gram were used.
Used devices&materials
Animals were randomly divided into control, azoospermia sham, and experimental groups. The animals in the control group did not receive any treatment and the animals in the sham and experimental groups, became azoospermia after receiving intravenous busulfan injection at a dose of 10 mg/kg after 21 days. … [13, 14]. To prepare the supernatant, the inguinal fat tissue and around the testes of a mouse, which was anesthetized with ether, was isolated. The adipose tissue was separated and sliced and for digestion, was admixed with collagenase 2% at 37 ° C for 40 minutes. Collagenase was neutralized with PBS and the resulting mixture was centrifuged for 5 minutes at 1500 rpm. After dispensing the supernatant, 10 ml of DMEM (Biovet, Bulgaria) containing 10% of the embryonic serum of the cattle (Biovet, Bulgaria), and 1% penicillin and streptomycin were added to it and the suspension was transferred to a 25 ml flask and incubated in the incubator in 37 ° C, saturated moisture and carbon dioxide 5% to reach the first passage. The medium was replaced every three days, and when the cells reached 80% density, they were removed with trypsin 0.25% (Gibco, United States) and they were cultured until the fourth passage. At the end of the fourth passage, the supernatant was collected and centrifuged and concentrated for five minutes at 1500 rpm before injection to vas deferens tube of mice under treatment with culture medium. To determine the ability of differentiation, the cells in the osteogenic environment included DMEM containing 15% FBS, 50 μg / ml ascorbat-2-phosphate (Wako Chemicals, United States), 10 nm dexamethasone (Sigma, Aldrich, United States) and 10 ml Molar beta-glycerophosphate (Sigma-Alderich, USA). The separate culture medium was changed every three days and after 21 days, the differentiation cells with osteoblasts were stained by Alizarin Red (Sigma-Aldrich, The United States) and were examined with optical microscope (Nikon; Japan). 35 days after the last injection of busulfan, the experimental group was anesthetized by ketamine (5mg/kg of body weight) and xylazine (20 mg/kg of body weight), and all their abdominal areas were shaved and sterilized. Then, in the abdominal region of the mice, a shear was made, and the right and left testicles were removed to inoculate the medium. Underneath the SZN model Strereo microscope (Optika, Italy), using an insulin syringe attached to a thin pasteurized pipette by plastic tubes, a supernatant derived from adipose tissue derived mesenchymal stem cells in combination with trypan blue (1:1 ratio and 100 μL of supernatant) was injected into the vas deferens tubes. In the presence of color, the culture medium was traced to not enter into the testicular interstitial. After injection, the testicles were returned to the abdominal area, and then the abdominal region was sutured [10]. 60 days after infusion of supernatant, all animals were anesthetized with ether and their testicles were removed and placed in 10% formalin for tissue fixation. After conventional tissue processes, samples were molded with paraffin and from each testicle, 5 microns were made for each testicle. The tissue sections were stained with hematoxylin and eosin, and were evaluated under an optical microscope. According to Panahi et al. [14] in each section, 10 rounded and almost rounded seminal tubes were randomly measured in terms of total diameter and lumen of seminal tubes, thickness and cell surface area of seminal tubes, lumens area and transverse section of seminal tubes, numerical densities and number of seminal tubes with a magnification of x100 using Dinocapture 2.0 software (version 1.4.3). The total diameter of semiconductor tubes (D), the lumen diameter of seminal tubes (Ld), the cell thickness of the seminal tubes (Cd), the cell surface of seminal tubes (Ca), luminal area of seminal tubes (La), , and numerical densities of seminal tubes (Nv) were measured. The spermatogenesis index is based on the spermatogenesis of the seminal tubes and the count of different cell types and the number of spermatid cells is round and long that each seminal tube was scored with a scale of 0 to 7. Statistical analysis: At the end, the results were analyzed by SPSS 18 software and after confirmation of the normal distribution of data by kolmogoronov-Smirnov test, the data was analyzed with one-way ANOVA and Tukey's post hoc test. Also, non-parametric and spermatogenesis indices were analyzed by Mann-Whitney test.
Findings by Text
The cells in the fourth passage had a ducklich and pseudo bacterial appearance, which is major characteristic of mesenchymal stem cells (Fig. 1). 21 days after the culture of cells in osteogenic environment, the presence of mineral deposit was observed due to the differentiation of mesenchymal cells into osteoblasts by the staining of Alizarin Red (Fig. 2). Based on the histological findings, spermatogenesis in the control group was active and a variety of cell lines were observed in reproductive epithelium and sertoli cells in seminal tubes. In the azoospermic sham group, the thickness of epithelium was significantly reduced and there was a large vacuolar space. There was only a few spermatogenial cells on the membrane of the animal seminal tubes. Sertoli cells were visible and spermatogenesis was completely destroyed. In the experimental group under treatment with supernatant, in most seminal tubes, there was different cells types including spermatogonia, spermatocyte, spermatid and sperm. Vacuolar space was lost and the reproductive epithelial layer thickness was higher than the spam azoospermia group. However, the thickness of the reproductive epithelium layer was relatively reduced compared to the control group, and the less number of spermatid was observed (Figure 3).Based on histomorphometric findings, the diameter and area of the lumen of seminal tubes did not differ significantly in all three groups, but the overall diameter, thickness and cell area, cross-sectional area and spermatogenesis index of seminal tubes in azoospermia sham group showed a significant decrease compared to the control group and experimental group treated with supernatant (p<0.001). In addition, in overall diameter, thickness and cell areas, cross-sectional area and spermatogenesis index of seminal tubes in the experimental group were significantly lower than the control group and significantly more than azoospermic sham group (p<0.05). Also, the number of seminal tubes per unit area and numerical densities of semen tubes in the azoospermic group was significantly higher than the control and experimental groups (p<0.001). However, both indices in the experimental group were not significantly different from control group (Table 1).
Main comparison to the similar studies
Other studies have shown that mesenchymal stem cell secretion is effective in treating some diseases such as renal failure, acute liver failure, acute myocardial infraction, brain hypoxia, wound healing, bone repair, and soft tissues [11, 12, and 21]. … [22-32].
Suggestions
It is suggested that in subsequent studies, several infusion of supernatant medium be done with a longer treatment period to achieve better results.
Limitations
One of the limitations of this study is the lack of the number of animals tested in each group, the shortage of treatment periods, and the lack of frequent intravenous infusion into the testicles of animals.
Conclusions
Injection of a culture medium derived from adipose tissue-derived mesenchymal stem cells into busulfan-induced azoospermia mice, probably due to having growth, differentiation and proliferation factors can restore spermatogenesis and improve histological and histomorphometric indices of testicles.
Acknowledgments
The authors of this article need to thank the officials of the Islamic Azad University of Shiraz, The Center of Transgenic Technology and Stem Cell Research in Shiraz University of Medical Sciences who collaborated in this study.
Conflict of interest
Non-declared
Ethical Permissions
At all stages of this study, the ethics of working with animals have been observed.
Funding Sources
This research was sponsored by Islamic Azad University of Shiraz Branch.

TABLES and CHARTS

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