Undifferentiated and Differentiated Spermatogonial Stem Cells

Abstract

Spermatogenesis is initiated and sustained by a rare population of singular spermatogonial stem cells (SSCs). These SSCs are connected to the basement membrane of the seminiferous tubules and possess distinctive morphological characteristics. They serve as a vital foundation for a robust stem cell system within the testis, crucial for spermatogenesis and reproductive processes. The isolation and cultivation of human SSCs would significantly enhance our understanding of germ and stem cell biology in humans. Although a challenging endeavor, the recent advancements in enriching and propagating spermatogonia carrying the male genome offer a significant stride toward future transplantation and the restoration of fertility in clinical settings.

1. Introduction

Male reproductive systems are characterized by spermatogenesis, which is a genital process. It is the spermatogonial stem cells (SSCs) that play the most important role in this system [1]. Somatic cells and Sertoli cells support SSCs in the basement membrane of seminiferous tubules. Seminiferous tubules support spermatogenesis by containing Sertoli cells (sustentacular cells) [2]. By secreting growth factors during spermatogenesis, these somatic cells support the fate of SSCs.

In spermatogenesis, genetic information is transmitted to the next generation. This process results in spermatogonia differing and proliferating into spermatozoa [3]. They can be subdivided into single spermatogonia (As), paired spermatogonia (Apr), or aligned spermatogonia (Aal) based on their topological organization [4]. Undifferentiated spermatogonia are called spermatogonia collectively. After proliferating throughout the seminiferous epithelium cycle, these cells become quiescent before they differentiate into A1 spermatogonia. An important subsequent division (A2-B) leads to the separation of differentiated SSCs into primary and secondary spermatocytes. As spermatozoa elongate following several meiotic divisions, secondary spermatocytes become round spermatids. Differentiated spermatogonia A1 through B are collectively referred to as spermatogonia A1 through B [5].

Unipotent stem cells called spermatogonial stem cells (SSC) produce sperm throughout the male’s lifetime. In the development, signaling pathway, growth regulation, and differentiation of cells, zinc finger, and BTB domain containing 16 (ZBTB16/PLZF) genes play several important roles. Sperm cells, embryonic stem cells, and pluripotent embryonic stem cells express PLZF [6]. Our study examined the expression of PLZF in testis, stem cells, pluripotent embryonic stem cells, and ES-like cells (Figure 1).

Figure 1.

Spermatogenesis can be divided into five successive stages of germ cell development: (1) spermatogonia, (2) primary spermatocytes, (3) secondary spermatocytes, (4) spermatids, and (5) spermatozoa.

There are two types of activities shown by spermatogonia. To maintain the primary pool of stem cells, self-renewal by mitotic divisions is necessary, followed by spermatogenesis, defined as the process of dividing undifferentiated spermatogonia into differentiated ones [7]. As a result of cytoskeletal activity, germ cell movements are also regulated in size and shape. Actin, microtubules, and intermediate filaments are part of the cytoskeleton, which governs the activities of those cells. Vimentin is crucial to these spermatogenic processes because it plays a critical role in the cytoskeleton [8].

2. Some gene expression between pluripotent stem cells and testicular germ cells

2.1 PLZF

The PLZF protein, as detected by immunohistochemistry (IMH), was localized in the differentiated tubular cells of the testis tubule center, as well as in the basal compartment of the seminiferous tubules of the undifferentiated testis [9]. A significant difference was found in PLZF IMH-positive cells in adult testis compared to neonates when positive cells were counted in sections of seminiferous tubules from undifferentiated and differentiated testes (P < 0.05). PLZF germ cell marker was strongly ICC-positive for SSC colonies in vitro but negative for ES cells and ES-like cells.

In pluripotent germ cells, PLZF is downregulated, but it is a transcription factor associated with testicular germ cell proliferation. As a result, in vitro and in vivo analysis of germ cell development can be supported (Figure 2).

Figure 2.

Testis section with PLZF positive cells. Sections of spermatogonial stem cells from undifferentiated and differentiated spermatogonials (A) and from in vitro spermatogonials (B) were analyzed for PLZF positive cells. There was a greater number of PLZF positive cells in undifferentiated testes compared to differentiated testes and in vitro testes.

A marker of spermatogonial differentiation, kit, is directly repressed by PLZF, according to Filipponi et al. In the testis niche, PLZF plays an important role in maintaining the self-renewal and maintenance of the SSC. In undifferentiated spermatogonia, PLZF is co-expressed with Oct4 [9]. PLZF loss leads to a limited number of normal spermatozoa and, after birth, a lack of respected germline due to the progressive loss of spermatozoa. The expression of genes regulating limb and axial skeletal development is regulated by PLZF during embryogenesis. There is a genetic relationship between PLZF and Gli3 and Hox5 genes during limb development [10]. Testis and SSCs expressed PLZF, making it a SSC marker according to previous studies. PLZF expression in neonate and adult testicular sections, isolated SSCs, ES cells, and ES-like cells of mouse testicular culture was examined to determine if PLZF expression is the same in both testicular germ cells and pluripotent stem cells. According to the results, plunging stem cells do not express PLZF [9].

2.2 Vimentin

There are two types of activity in spermatogonia. To maintain the primary pool of stem cells, self-renewal occurs through mitotic division, followed by spermatogenesis, which refers to the differentiation of undifferentiated spermatogonia into differentiated spermatogonia. Associated with these events are widespread adjustments in germ cell movements in relation to cytoskeletal activity. Microtubules, actin, intermediate filaments, and the cytoskeleton make up the cytoskeleton, which governs the activities of those cells [10, 11, 12]. During these spermatogenic processes, vimentin plays a critical role in cytoskeleton function. Spermatogenesis begins with the expression of vimentin, an intermediate filament. The filamentous intermediate filament of vimentin connects the tubulin and actin cytoskeleton to the nuclear periphery [13]. As spermatogenesis progresses, vimentin functions primarily to ensure cellular stiffness, to maintain actin position, to facilitate cell migration, to divide cells, and to organize organelles. Additionally, vimentin has a number of essential roles, including determining cell shape, differentiation, motility, maintaining cell junctions, contributing to the maintenance of ordinary spermatogonia morphology, and anchoring germ cells to the seminiferous epithelium to anchor them [14].

It has been suggested that vimentin plays an important role in the differentiation of SSCs. However, it has been unclear whether the vimentin intermediate filament is necessary during differentiation in vitro. Finally, vimentin is expressed in male germ cells in a few studies. Separating germ cells in the adluminal and luminal compartments of seminiferous tubules expressed high levels of vimentin, while undifferentiated cells located in the basal compartment expressed low levels of vimentin. Immunohistochemical analysis indicated that vimentin expression was associated with Sertoli cells near the basal membrane. Afterward, we differentiated germ cells from Sertoli cells using SOX9 specific markers. According to IMH analysis, Sertoli cells expressed SOX9 cytoplasmically and differentiating germ cells expressed SOX9 negatively (Figures 3 and 4). Growth factors were added to isolated cells after enzyme digestion. In a previous study, we characterized isolated testicular cells. Immunocytochemistry was used to examine vimentin expression in primary and secondary spermatocytes, round spermatids, and undifferentiated spermatogonia [11].

Figure 3.

Characterization of vimentin intermediate filaments in adult mouse seminiferous tubules by immunohistochemistry. Differentiating germ cells located in the middle compartment of seminiferous tubules expressed high levels of vimentin, whereas undifferentiated cells located in the basal compartment expressed low levels. Merged image with DAPI. (A) Vimentin, red, (B) DAPI, blue (scale bar: 15 μm), and merge (C).

Figure 4.

DAZL and vimentin immunocytochemistry. In (A), DAZL expression is marked by green fluorescence; in (B), vimentin expression is marked by red fluorescence; in (C), DAPI is shown merged with the green fluorescence. The white arrow represents undifferentiated spermatogonia, and the yellow arrow represents germ cells that are differentiating. DAZL is green; vimentin is red; and DAPI is blue (scale bar: 15 μm), and (D) bright field. (scale bar: 15 μm) (get this figure from our recent article [11]).

Using the DAZL specific marker, we distinguished primary and secondary spermatogonia as well as spermatids from undifferentiated spermatogonia. Undifferentiated spermatogonia expressed DAZL at a high level, while differentiated germ cells did not. Differentiating germ cells express high levels of vimentin, while undifferentiated spermatogonia display low levels. Last time, spermatogonia were differentiated from undifferentiated spermatogonia by utilizing the Ki67 specific marker. As expected, differentiated germ cells express a high level of Ki67, while undifferentiated spermatogonia display a low level of Ki67 expression [11, 14].

2.3 POU5F1

With the advent of SSC culture techniques and genetic analysis, important genes were identified that maintain the stem-cell function of SSCs. As, Apr, and Aal spermatogonia express POU5F1 (POU domain, class 5, transcription factor 1), one of the molecular markers of undifferentiated spermatogonia. The POU5F1 gene encodes a transcription factor that plays an essential role in controlling embryonic development and maintaining pluripotency and self-renewal. POU5F1A, POU5F1B, and POU5F1B1 are produced during alternative splicing, but only POU5F1A maintains stemness.

Developing and optimizing treatment methods for male infertility requires understanding how SSCs differentiate and how genes involved in spermatogenesis are expressed at different stages of SSC differentiation. Since male fertility depends on accurate spermatogenesis and the population of SSCs in the testis, understanding the mechanisms behind SSC differentiation is essential. The POU5F1 protein localization in neonate and adult mice testis did not distinguish the populations of SSCs in our previous study using three antibodies [15]. A comparison between this study and the previous study in this article reveals differences in POU5F1 expression between the two populations of spermatogonia. Since SSCs play an important role in regenerative medicine, it is important to understand the differences between two different populations of SSCs in order to utilize each one appropriately. Our research has helped the scientific community gain a better understanding of what POU5F1 actually does during spermatogenesis [16].

In mouse seminiferous tubules, we analyzed the expression pattern of POU5F1 using immunohistochemistry. Using POU5F1 Proprietary Antibody, we identified SSCs using immunohistochemistry. After merging and staining mouse seminiferous tubules with DAPI, it was found that they express this marker. Seminiferous tubule basal spermatogonial cells exhibit the highest POU5F1 expression, as seen in fluorescent microscope images [17].

Images obtained from the bright field microscope demonstrated the difference between differentiated and undifferentiated spermatogonia when cells were extracted from two spermatogonial populations and cultured on their respective media. Spermatogonial cells that were undifferentiated did not grow much after culturing; they were also small and tended not to form specific shapes. Unlike undifferentiated spermatogonial cells, differentiated spermatogonial cells expand during culturing and tend to form clusters. Thus, spermatogonial cells can be diagnosed based on their morphology [17].

The comparison of two SSC populations shown that there are differentiated and undifferentiated. A bright field of spermatogonia cells during differentiation indicates that undifferentiated and differentiated spermatogonial cells differ morphologically. In the subsequent study, we examined POU5F1 expression in differentiated and undifferentiated spermatogonia by ICC. In two spermatogonia populations, DAPI staining was used to determine SSCs and to stain for the POU5F1 marker. According to ICC analysis of images obtained with a scanning UV laser microscope, undifferentiated cells expressed more POU5F1 than differentiated cells. According to IMH analysis, basement membrane cells expressed POU5F1 at high levels and differentiated cells expressed it at low levels (Figure 5).

Figure 5.

In the seminiferous tubules, immunohistochemistry (IMH) analysis revealed the expression pattern of POU5F1. POU5F1 (A), DAPI (B), and 4′,6-diamidino-2-phenylindole (POU5F1) (C) show the mixed images and sharp expression of POU5F1 in the basal membrane (scale bar: 15 μm).

2.4 VASA

It was discovered that the VASA gene plays a crucial role in the development of female germ stem cells (GSCs) in Drosophila [18]. The VASA gene is eliminated in mice with a systematic genetic deficiency that results in a loss of sperm production in the males. During meiosis phases, GSCs in males appear to die at the zygotene stage, but the ovary appears to function normally [19]. On embryonic day 12.5 and subsequent to entry into the gonadal anlage, mice show localization of VASA in PGCs. PLZF has been implicated in direct repression of Kit transcription, a spermatogonial differentiation marker, in previous studies. The loss of the PLZF gene also causes limited numbers of normal spermatozoa to be produced, resulting in an impaired germline after birth. In embryogenesis, PLZF regulates gene expression during the patterning of the limbs and axial skeleton. Two types of cell populations present in seminiferous tubules were analyzed for co-expression of PLZF and Oct4.

Spermatogenesis defects are often responsible for infertility in humans. It is essential to understand normal spermatogenesis in order to develop subfertility and infertility in humans. RNA-binding proteins play a crucial role in the formation of germ cells. In addition to rhesus macaques, goats, cattle, pigs, and other animals, VASA is expressed in germ cells [20]. ATP-dependent RNA helicases and RNA-binding proteins are encoded by the VASA gene. Spherical spermatids, spermatogonia, and spermatocytes can be identified in human testicular tissues based on the expression of the VASA protein. Human spermatogenesis might be better understood by understanding how these proteins are expressed in different germ cells at different stages [21, 22].

Drosophila cells dispersed VASA protein evenly throughout their cytoplasm. VASA proteins function as RNA chaperones and are connected to chromatoid bodies. Various studies have shown that VASA functions as the mRNA transcript and CB in spermatozoa when the genome is inactive. In addition to spermatogenesis, VASA is essential for the differentiation of embryonic stem cells into primordial germ cells (Figure 6).

Figure 6.

Immunohistochemy image VASA and vimentin in vitro. (A) Immunohistochemy of VASA, (B) Immunohistochemy image of vimentin, (C) DAPI, and (D) merge (scale bar: 100 μm).

2.5 SOX2

In stem cells and progenitor cells, Sox2 plays an important role in maintaining pluripotency and differentiation. Furthermore, it plays a role in cell reprogramming in the inner cell mass (ICM) and ectoderm of blastocysts (10). As well as regulating Sall4, Plzf, Gfra1, Oct4, Klf4, Foxm1, Cux1, Zfp143, Trp53, E2f4, Esrrb, Nfyb, and c-Myc, Sox2 can also convert somatic cells to pluripotent stem cells. Reprogramming and pluripotency are dependent on each other for the production of induced pluripotent stem cells (iPS) [23]. Mice’s primordial germ cells also expressed Nanog and Sox2. Human primordial germ cells may still contain Sox2, but further research is needed to confirm this. The number of pluripotent cells decreases when Sox2 expression is reduced, and cell differentiation begins. There have also been reports of high levels of Sox2 expression in brain cancers that can cause pituitary tumors and decreased levels of Sox2 expression in patients with ocular abnormalities [24]. Breast cancer, colorectal cancer, and glioblastoma have been linked to increased Sox2 expression, while gastric cancer is associated with its decrease. Different cell types and tissues express Sox2 differently in humans. Bone marrow, endometrium, heart, kidney, liver, and pancreas have lower expression levels than the lung, prostate gland, stomach, testis, and fallopian tube. Sox2 gene interaction with spermatogenesis genes was the goal of this study. We investigated and compared gene expression in differentiated and undifferentiated spermatogonial stem cells. Moreover, this gene was examined in differentiated and undifferentiated spermatogonia to determine its quantity and mode of expression. To improve male infertility treatment, this research aimed to understand the mechanisms involved in sperm generation [25].

The interaction network between Sox2 protein and some other proteins involved in spermatogenesis was analyzed in this experiment, as shown in Figures 1 and 2. Using STRING and Cytoscape databases, key genes were identified that are not connected to Sox2, including Sim2 and Rfx4. These two figures illustrate the origins of these genes, the sources of measurement and connection, as well as where each gene is expressed in each testicle and its biological function. A greater or lesser degree of relationship was also detected between genes. Oct4, Nanog, and Klf4 are strongly connected with Sox2, but Smad1, Gdnf, Egr2, and Stra8 are poor connections. In addition to Pou5fA, Stra8 and Gdnf, Sox2 appears to be related to Pou5f1, Stra8, Klf4, and Bmp8b. In addition, Sox2 is connected with Pou5f1, Klf4, Kit, and Nanog in stem cell population maintenance. The expression of Sox2 was examined by immunohistochemical analysis of cross sections of seminiferous tubules. According to immunohistochemistry analysis by confocal scanning UV laser microscope, Sox2 nuclear expression increased during spermatogenesis in vivo over time (Figure 3). In this figure, single undifferentiated cells can be seen along with a group of spermatogonial stem cells. Different expressions of Sox2 were observed in isolated spermatogonia that had been cultured and differentiated in vitro (Figure 4). The expression of Sox2 in differentiated cells exceeded expectations when considered as a pluripotency factor. A Fluidigm PCR analysis of spermatogonia grown in vitro showed that differentiated spermatogonia expressed much more Sox2 than undifferentiated spermatogonia. A high level of Sox2 expression was observed in differentiated cells, and a significant difference in Sox2 expression between differentiated and undifferentiated cells was also observed (p < 0.05). Undifferentiated spermatogonia under in vitro conditions also expressed high levels of Sox2. However, Sox2 was highly expressed in differentiated spermatogonia, similar to in vivo conditions (Figure 7). The study’s most interesting finding was that undifferentiated cells expressed high levels of Sox2 cytoplasmically and differentiated cells expressed high levels of Sox2 [24].

Figure 7.

The pattern of Sox2 expression. (A) the expression of Sox2 in seminiferous tubules, (B) the DAPI (blue) staining shows the nuclear cells, and (C) merged images (scale bar 50 μm).

Sox2 expression is essential for stem cells and SSCs to remain pluripotent, and it also plays an important role in maintaining, increasing, and specializing SSCs and PGCs. Additionally, both in vivo and in vitro spermatogonia expressed Sox2. The nucleus of differentiated spermatogonia expressed more Sox2 than the cytoplasm of undifferentiated spermatogonia in in vitro experiments. Differentiated spermatogonia expressed more Sox2 in in vivo experiments than undifferentiated spermatogonia. Still, this gene expression played a critical role in maintaining stem cell pluripotency, which is crucial for spermatogenesis. By studying Sox2 expression in spermatogenesis, we may be able to improve male infertility treatments.

3. Gene expression profiling of SSCs seems to be age dependent

An embryonic germ layer can be differentiated into ectodermal, mesodermal, and endodermal cells using pluripotent stem cells (PSCs). PSCs have been generated using several different approaches, including ESCs obtained from embryonic blastocysts after fertilization. The so-called induced pluripotent stem cells (iPSCs) were also obtained by enforcing the expression of pluripotency genes in somatic cells; SSCs have proven to be a promising method for establishing PSCs in a more natural and ethically acceptable manner, especially for therapeutic approaches in medicine [26]. It is possible to isolate and expand SSCs in vitro, as they are found in small numbers in the testis. They are unipotent stem cells under the control of their stem cell niches, but under specific culture conditions outside the niche and without exogenous pluripotency genes, they are capable of converting into ESC-like cells at various times after culture or isolation of SSCs [27].

In neonatal and adult SSCs obtained from 7- and 12-week-old mice, real-time PCR was used to quantify and analyze the expression of important germ cell-enriched genes (LHX1, Stella, VASA, DAZL, CD9, EPCAM, GPR125, GDF3, THY1, STRA8, GFRa1, 1ITGB1, KIT, ETV5, and BCL6B) and pluripotency associated genes (Oct4, Nanog, Sox2, TDGF4, KLF4, MYC, LIN28, SALL4, and DPPA3).

Figure 7 shows how neonatal SSCs and adult SSCs were grouped according to hierarchical clustering (dendrograms) and principal component analysis (PCA). There was a significant difference between neonatal and adult SSC clusters in the heat map analysis of pluripotency and germ cell genes [28].

The Oct4, NANOG, TDGF1, and Sox2 expression levels of neonatal SSCs were significantly higher than those of adult SSCs. MYC, NODAL, LHX1, GDF3, GPR125, CD9, ITGB1, VASA, TAF4b, EPCAM, BCL2L2, ETV5, DAZL, KLF4, RET, and THY1 were significantly higher expressed in adult SSCs (fold change >2 and -test) than in neonatal SSCs [28].

These differences became even more apparent when neonatal SSCs were compared to SSCs obtained from 12-week-old mice (see Supplementary Tables). In addition, 7-week-old mice have significantly higher expression levels of pluripotency genes than 12-week-old mice (Figure 8).

Figure 8.

Neonatal SSCs (colored dark blue), adult SSCs (colored light blue), and adult SSCs (colored blue-green) express pluripotency and germ cell genes differently. The red arrows indicate genes downregulated in adult SSCs, while the purple arrows indicate genes upregulated (more than twofold). Note the downregulation of Oct4, Nanog, and Sox2 in 12-week-old mice’s SSCs) (get this figure from our recent article [29]).

4. An analysis of haGSCs with predefined gene sets related to germline, pluripotency, fibroblasts, and mesenchymal stem cells

There has been some evidence that human adult germ stem cells (haGSCs) derived from highly enriched spermatogonia isolated from adult human testicular tissue are highly versatile and share some similarities with human embryonic stem cells (hESCs). They can be differentiated in vitro into a variety of cell lineages comprising the three germ layers and express genes associated with pluripotent cells. Based on some studies, mesenchymal stem cells or cells similar to MSCs may have been the source of cells expressing markers of pluripotency. HaGSCs may also be low-differentiated testicular fibroblasts, according to some studies. The stem cells from human testis biopsy, on the other hand, were derived from both germ and mesenchyme and could differentiate into cells from all three germ layers. Other research has shown that haGSCs can produce small teratomas similar to hESCs. Each of these studies raised new questions regarding the true nature of pluripotency in haGSCs. Generally, the activation of a transcriptional regulatory network is required for the pluripotency of cells, which has been observed in ex vivo cultures of early embryonic cells, as well as germ cells. Members of the pluripotency network are normally active in these cells, including morula and blastocyst-stage (inner cell mass) embryonic cells, epiblasts, primordial germ cells (PGCs), and germline stem cells.

Fluidigm real-time PCR analysis was performed on the following germ cell- and pluripotency-associated genes based on microarray results in addition to the initial panel of germ cell- and pluripotency-associated genes: L1TD1, SALL4, JARID2, HOOK1, EPCAM, PROM1, SALL2, IGFR2BP3, REX1, and GATA4. A similar pattern of gene expression was observed in haGSCs derived from two additional patients, with VASA, DAZL, and PLZF predominant. VASA, DAZL, and PLZF expression in haGSCs was significantly lower than in hSSCs. While STELLA and GFR1 were strongly expressed in haGSCs, the other two germ cell-specific genes were not. As compared to hSSCs, haGSCs expressed REX1, LIFR, and NANOS in similar ranges, while CD9 expressed at a higher level. In hFibs, neither DAZL nor LIFR were expressed. Compared to hFibs, hSSCs and haGSCs showed significantly higher expression of germ cell-associated genes. Similar to hESCs, haGSCs possess a rudimentary gene expression profile associated with germ cells. There were higher levels of CD9 and GFR1 expression in haGSCs than in hESCs (Figure 9).

Figure 9.

Based on microarray data, haGSCs are upregulated by pluripotency-associated genes compared to hFibs.

Cell culture produces haGSCs from spermatogonia and MACS enriched in CD49f but never from negatively selected fractions or from patients without spermatogonia. A central cluster of haGSCs with outgrowing “epithelial”-like cells characterized these colonies from hFibs. The expression of germ- and pluripotency-related genes was quite different in haGSCs compared to hFibs based on single-cell Fluidigm analysis. The majority of outliner hESCs and haGSCs did not share any similarities with hFibs. In addition, it became clear that haGSC colonies were heterogeneous, displaying similar characteristics to pluripotent states. Moreover, different haGSC colonies showed a relatively heterogeneous expression of germ- and pluripotency-associated genes in the microarray study. In comparison with hESCs and hFibs, the haGSC transcriptome and high variance genes showed a distinct separation from hFibs.

5. Spermatogonia stem cell gene ontology and signaling pathway bioinformatics analysis

Statistic and bioinformatics analyses are the main bottleneck in transcriptomic studies. Candidates were usually identified using widely accepted statistical criteria (such as P values and fold changes). In order to translate the gene list into biomedical significance, automatic functional annotation was performed using knowledge bases, such as Gene Ontology (GO) and KEGG pathways. We have recently proposed a framework for revising candidate protein lists and identifying novel proteins based on reanalysis of published proteomics data. We also believe that reanalyzing transcriptomes using optimized bioinformatics methods would help us interpret the results better.

A previously published dataset was used to extract the expression data for two cell types (primitive and differentiated type A spermatogonia) from a previously published study. In the next step, eight canonical markers were evaluated using RNA-Seq data. As well as the expression index, we proposed a new parameter for integrating absolute and relative expression abundances. Our statistical model used this parameter to dynamically select the best cutoff considering biological relevance. To understand and study the maintenance of SSCs, we constructed a refined network by combining information about physical interaction, expression change, biological function, and disease association.

Despite transcriptomics’ ability to profile gene expression and regulation, bioinformatics analysis is crucial for translating gene lists into functional biomedical applications. The two groups are usually screened using a one-size-fits-all cutoff using statistical inference. Considering both absolute abundance and relative change, we ranked genes using the expression index proposed in this study. By taking well-studied genes associated with SSC self-renewal as a positive reference, we developed a statistical model that dynamically screens for the best cutoff to prioritize candidate genes. Based on predicted genes involved in cell proliferation or differentiation, an optimal cutoff was determined for identifying functionally important genes [29].

SSCs are thought to be proliferating and surviving by activating and silencing various endogenous genes in response to exogenous factors. The mechanism of SSC self-renewal in vivo is still poorly understood despite the identification of a few key regulators and signaling pathways. Our transient model for self-renewal versus differentiation is based on SG-A cells (primitive versus differentiated). Based on the expert knowledge-guided and dynamic statistical model described above, we identified 1119 candidate genes with the best enrichment of canonical markers. By combining physical interactions, expression changes, cellular function, and disease associations with these genes, we finally created a refined network [30]. A high quality and relevance of gene prioritization can be seen in this network, which contains five of the eight canonical markers. As well as finding novel regulators of SSC self-renewal, we suggest the refined network could be used to identify target genes for male infertility and testicular cancer treatment [16, 29, 31, 32, 33].

The protein–protein interaction network with 945 genes was visualized using the STRING (v.11) database. Spermatogenesis was largely regulated by vimentin interaction and regulation, according to the study. There is a strong interaction between vimentin and Stat3, Mmp2, Trp53, Casp7, AURKB, Pik3r1, Ctnnb1, Lgals3, Cdkn1a, and Snai1. There was also a clear association between Trp53, Mmp2, Casp7, Stat3, and Pik3r1. Reactome and KEGG selected any spermatogenesis-related signaling pathway as the master regulator of the pathways involved in spermatogenesis. Figure 10 shows a strong correlation between the highlighted genes.

Figure 10.

In silico analysis in spermatogonial stem cell genes. (A) PPI network in spermatogonial stem cell genes, and (B) gene ontology in spermatogonial stem cell.

6. Conclusion

A large number of spermatogonia are produced during each epithelial cycle when undifferentiated spermatogonia proliferate. When these Aal spermatogonia are in quiescence, they do not divide and develop into the AJ spermatogonia, the first generation of differentiating spermatogonia. Testis undifferentiated regions and the basal section of the seminiferous tubule are strongly expressed with POU5F1, VASA, and PLZF factors, according to the investigation. A comparison of differentiated and undifferentiated populations of spermatogonial stem cells was also conducted. It was found that POU5F1, VASA, and PLZF levels decrease with differentiation, whereas vimentin and sox2 levels increase in differentiated spermatogonial stem cells. In light of the use of SSCs for clinical and therapeutic purposes, such as male infertility, the study of spermatogonial stem cells will provide better insight into the regulation of stem cells in the testis. Also, molecular research and analysis, as well as improved understanding of how genes such as these genes contribute to male infertility, can lead to new treatments or improvements to existing ones. The laboratory can also be used to treat Azoospermia and Oligospermia, abnormal sperm function, and blockages that prevent sperm delivery by investigating the differentiation process of spermatogonial stem cells and better understanding the methods of differentiation.

Acknowledgments

This study was funded by Centre for International Scientific Studies and Collaboration (CISSC), Tehran University and Amol University of special modern technologies.

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

SSCs	spermatogonial stem cells
ZBTB16/PLZF	zinc finger and BTB domain containing 16
ICC	immunocytochemical analysis
iPS	induced pluripotent stem
PSCs	pluripotent stem cells
GO	gene ontology