The present study makes noteworthy contributions by providing valuable tools for the in vitro investigation of SSC proliferation which can be useful in the treatment of male infertility. In the present study, we developed SSC culture in SACS along with melatonin supplementation as the optimal culture protocol which prevented the release of free radicals during spermatogonial stem cell culture in vitro. In a previous study, the number and diameter of colonies increased in a group treated with melatonin in SACS compared with a two-dimensional culture supplemented with date palm pollen (Phoenix dactylifera), which confirms our study describing a successive maturation of pre-meiotic SSCs in the culture system. Neonatal mouse SSCs were isolated and enriched with Plzf antibody, which was also used to confirm SSC colonies in the SACS. Plzf antibody has been used in many studies as an indicator for SSC colony detection and purification studies [14, 34]. Several other studies have used different markers for the isolation and detection of these cells, including GFRα1, ID-4, PAX7, etc. [35,36,37]. However, there is no strong evidence for the efficient isolation of SSCs using these markers, and no efficient and specific SSC markers have yet been identified .
The SACS consisted of two phases of different agar concentrations: a softer upper layer and a more solid lower layer. The synthesis of SACS was performed according to the procedure applied by Stukenborg et al. .
In this study, we added SSCs to the upper layer of the agar system. Similarly, Elhija et al. , Stukenborg et al. , and Huleihel et al.  reported the addition of SSCs (106 cells per well per 200 μl) to the upper layer of the agar system before culturing in a 24-well plate during their investigations on SSC proliferation. Unlike these studies, we only used a flow cytometry technique for the assessment of the purity of SSCs using Plzf antibody; 96.1% of all cells expressed this antibody. Other cells of the testes, especially Sertoli cells, were transferred into the upper layer of the SACS which shows that these cells have positive effects on development and colony formation in SSCs . Sertoli cells are involved in the regulation of proliferation and differentiation of SSCs, particularly through paracrine- and endocrine-mediated signaling pathways. Sertoli cell growth factor, GDNF, fibroblast growth factor 2 (FGF2), Sertoli cell transcription factor, ETS variant 5, nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR) have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis . Recent studies have demonstrated the expression of melatonin receptors (MT1 and MT2) in Sertoli cells  and SSCs . Thus, the fact that melatonin, in addition to its antioxidant activity, can have complex biological functions through its receptor appears to be justified. Based on these findings, it seems likely that melatonin, through its receptor on Sertoli cells or SSCs, can directly play a role in the proliferation of SSCs. The isolated cells were cultured in SACS in the absence or presence of melatonin extract. Their viability was evaluated by MTT assay. In this study, we observed a dose-dependent (100 μM) activity of melatonin on SSCs in culture. As previously published, the presence of melatonin in a cell culture can increase the number of viable cells [20, 28]. We added melatonin to the basic culture medium in the SACS, which yielded an increase in cell viability up to 90%. Our results also show that the number of viable cells in the SACS supplemented with melatonin was higher compared with a two-dimensional culture system supplemented with catalase or alpha-tocopherol before culture . We also examined the effects of SACS in combination with LIF and GDNF on the proliferation of SSCs. Although several reports have described culturing SSCs in SACS, they have most commonly focused on stem cell differentiation [15, 17, 39]. Previous studies have shown that some growth factors, most notably GDNF, can have a long-term positive effect on the maintenance of SSCs and may also stimulate division of SSCs [7, 12, 42, 43]. Other studies reported that LIF is an essential factor for maintaining pluripotency and the self-renewing capacity of embryonic stem cells  and SSCs . On the other hand, the presence of LIF in the culture medium inhibited meiotic gene expression and increased the percentage of alkaline phosphatase-positive cells. Previous studies have also demonstrated that melatonin can play different roles in the cells of the body such as cell signaling, protection of fatty acids from oxidation, oncostatic action, and antiapoptotic and anti-aging properties in many cells [21, 46]. In recent years, much attention has focused on the role of melatonin as an antioxidant. Melatonin, as a free radical scavenger, plays a vital role in the reduction of ROS production, and prevents cellular death and potential DNA mutations resulting from oxidative damage in culture systems [20, 23, 26, 47,48,49]. One of the most common intracellular ROS molecules is H2O2 . We thus evaluated intracellular H2O2 content using a DCFDA-specific probe. Our findings demonstrate that supplementation with melatonin in SACS can contribute greatly to the prevention of the propagation of lipid peroxidation in SSC membranes caused by ROS production, and can protect SSCs from the adverse effects of these free radicals. Conversely, Morimoto et al.  reported that ROS formation plays a pivotal role in SSC self-renewal via the activation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. They suggested that the presence of ROS is somewhat necessary for SSC self-renewal in vivo .
In another study, Li et al.  claimed that melatonin promotes manganese superoxide dismutase (MnSOD) and sirtuin type 1 (SIRT1) expression, and therefore promotes busulfan-induced SSC apoptosis in the presence of high concentrations of ROS and p53.
A number of studies have demonstrated that the addition of melatonin to culture medium can reduce the potential effects of ROS-induced cell stress in cells such as oocyte and adipose-derived stem cells (ASCs) [48, 49]. Gholami et al.  carried out a number of investigations on the effects of melatonin on SSC transplantation in azoospermic mice. They showed that melatonin can improve the structure of testis tissue. In another study conducted by the same researchers , they demonstrated positive effects of melatonin supplementation in vitrified-thawed testicular germ cells of neonatal mice. They indicated that melatonin can induce cell proliferation in normal cells and apoptosis in damaged cells. Similarly, Niu et al.  found that adding melatonin to the culture medium of goat SSCs could increase SSC proliferation by stimulating the production of GDNF in the Sertoli cell. Measuring the number and diameter of colonies of SSCs can be used as morphological criteria in in vitro studies [41, 56]. In another study, we investigated the effect of melatonin on SSCs in a two-dimensional culture, and emphasized on the importance of adding melatonin to the culture medium as an antioxidant. We also showed that the culture of SSCs in SACS is more successful compared to the two-dimensional culture system .
In this study, we observed that the number and diameter of the colonies increased at the end of each week of culture in the SACS in both groups. The most striking aspect of our results is the vital antioxidant role of melatonin in the culture of SSCs in the SACS which provided protection against lipid peroxidation. Similar to our results, Elhija et al.  isolated colonies in the upper layer of SACS after 14 and 28 days of culture and classified them according to their size. In contrast to our findings, Eslahi et al.  indicated that the number and diameter of the colonies of SSCs in a poly-l-lactic acid (PLLA) nanofiber scaffold decreased significantly after the first, second, and third weeks of culture compared with the control groups (culture of SSCs not seeded on PLLA). In the present study, colonies were composed of cells that expressed and stained positive for the mitosis markers PLZF and α6 integrin. Moreover, we used alkaline phosphatase staining to evaluate the colonies of SSCs for alkaline phosphatase activity. It is well known that PLZF and α6 integrin are markers for spermatogonial stem/progenitor cells in many species [14, 16, 45].
After the fourth week of culture, we analyzed ID-4, Plzf, and c-kit gene expression levels. Our data clearly show that melatonin supplementation can increase the proliferation rate of SSCs. The level of PLZF and ID-4 (undifferentiated genes) expression in the melatonin group were significantly higher than in the control group, whereas the level of c-kit (differentiated gene) expression decreased in the melatonin group. In support of our results, Aliakbari et al. also reported a decrease in c-kit expression after they cultured post-thawed SSCs treated with antioxidants in both control and treated groups . Our findings are in line with the results of other previous studies [14, 16, 58].
Results of this research suggest several practical applications. We emphasize the importance of adding melatonin to the culture medium as an antioxidant.