Microenvironment of spermatogonial stem cells: a key factor in the regulation of spermatogenesis

Abstract

Spermatogonial stem cells (SSCs) play a crucial role in the male reproductive system, responsible for maintaining continuous spermatogenesis. The microenvironment or niche of SSCs is a key factor in regulating their self-renewal, differentiation and spermatogenesis. This microenvironment consists of multiple cell types, extracellular matrix, growth factors, hormones and other molecular signals that interact to form a complex regulatory network. This review aims to provide an overview of the main components of the SSCs microenvironment, explore how they regulate the fate decisions of SSCs, and discuss the potential impact of microenvironmental abnormalities on male reproductive health.

Graphical Abstract

Introduction

Definition and significance of SSCs

Normal spermatogenesis in mammals is fundamental to the maintenance of male fertility, and spermatogenesis is a complex and highly efficient process involving a series of mitotic, meiotic, and morphological changes in the cells [1]. Spermatogenesis is the process of formation of mature spermatozoa in the testis, which is divided into three main stages: mitosis of spermatogonia, two meiotic divisions of spermatocytes, and metamorphosis and maturation of spermatocytes [1]. The classification of spermatogonia in mammals is different in primates and rodents [2] (as shown in Fig. 1). In primates, there are two main types of spermatogonia, type A and type B. Type A spermatogonia are stem cells with the ability to self-renewal and proliferate, and type A spermatogonia are subdivided into Adark and Apale cells. Type B spermatogonia are the precursor cells that differentiate into spermatocytes, and they undergo a series of cell divisions and differentiation processes that lead to the formation of mature spermatids [3, 4]. Adark spermatogonia are usually considered true SSCs, while Apale spermatogonia proliferate and differentiate as male germline progenitors into further germ cells [2, 5]. In contrast, spermatogonia in rodents are mainly classified into Asingle, Apaired, and Aaligned types. Aaligned spermatogonia differentiate into type A1 spermatogonia, which subsequently differentiate into type A1–A4, intermediate, and type B spermatogonia. Asingle spermatogonia are considered the sole spermatogonial type with a self-renewal capacity [6]. The balance and transformation between type A and type B spermatogonia are essential for the maintenance of normal spermatogenesis. Through the self-renewal and differentiation of SSCs, the male can continuously produce new spermatozoa and maintain the normal function of the reproductive system. Primary spermatocytes are cells formed by mitosis of spermatogonia. Primary spermatocytes undergo meiosis I to form two secondary spermatocytes, the two secondary spermatocytes undergo meiosis II to form four round spermatids, and the round spermatids undergo a series of morphological and functional changes to form mature spermatozoa with complete structures and functions [1]. This process is regulated by various factors, including the influence of hormones, growth factors and cytokines, among which neurotrophic factors such as Glial cell line-derived neurotrophic factor (GDNF) play an important role in regulating spermatogenesis [7].

Fig. 1

Schematic diagram of testicular spermatogenesis. The formation of spermatogonia differs between mouse and human spermatogonia, with type A spermatogonia in mice mainly divided into Asingle, Aparied, Aaligned, and A1–A4 spermatogonia; whereas human type A spermatogonia are divided into Adark and Apale spermatogonia

SSCs are a key cell type in the male reproductive system and they play a crucial role in reproduction and fertility maintenance. Firstly, studying the molecular regulatory network of SSCs helps us understand the molecular mechanisms of male infertility and may identify new targets for its treatment [8]. Secondly, SSCs are the only adult stem cells that can transmit genetic information to offspring. Therefore, gene editing of SSCs may offer new possibilities for treating genetic disorders [9]. By gene editing SSCs in vitro (e.g. using CRISPR/Cas9 technology), defective genes can be repaired or replaced. Then these repaired cells can be transplanted back into the body, making it possible to treat genetic disorders in germline cells [10]. Thirdly, cryopreservation and transplantation of SSCs can be used to preserve fertility in childhood cancer patients [11], where SSCs are extracted from the testes of cancer patients for cryopreservation, and thawed SSCs can be used for in vitro culture to become functional spermatozoa to be used for in vitro fertilization (IVF). Fourthly, the in vitro differentiation potential of SSCs offers potential applications in regenerative medicine. In particular, SSCs could potentially be used to repair or replace damaged tissues and organs in treating neurodegenerative diseases or heart disease [12]. Multipotent SSCs (mSSCs), like embryonic stem cells (ESCs), have self-renewal and differentiation properties and are capable of differentiating into vascular endothelial cells (VECs) and smooth muscle cells in vitro [13]. SSCs, as germ cell initiating cells, are the cornerstone of male reproductive health, and they maintain sperm production through self-renewal and differentiation. In turn, the proper performance of these functions of SSCs relies on their stable microenvironment, a complex network of multiple biological signals that ensures the correct behavior of SSCs. Understanding the composition and function of this microenvironment is important for unravelling the underlying mechanisms of male infertility and developing new therapeutic approaches.

SSCs microenvironment

The concept of the "niche" was first proposed in the context of hematopoietic stem cells (HSCs) [14]. Niche refers to the specific microenvironment or cell population in which stem cells are found in a tissue or organ, as well as the niche-specific ECM [15]. The niche of SSCs, also known as the reproductive ecosystem or the microenvironment within the seminiferous tubules, is a complex system comprising a variety of cell types and molecular signals that work together to maintain the self-renewal and differentiation of SSCs. The microenvironment of SSCs consists mainly of cellular fractions of Sertoli cells, Leydig cells, peritubular myoid cells (PMCs), macrophages, and VECs, as well as the ECM [16, 17].

Sertoli Cells are one of the most important cell types in the SSCs niche [18]. They bind tightly to SSCs, provide physical support and secrete a variety of growth factors and cytokines, such as GDNF, fibroblast growth factor 2 (FGF2), and colony stimulating factor 1 (CSF1), which are essential for the self-renewal and differentiation of SSCs [19, 20]. Leydig cells are mainly responsible for the production of testosterone, an important sex hormone. Testosterone has a major influence on the differentiation of SSCs and the entire spermatogenesis process [21]. PMCs surround the seminiferous tubules and not only provide structural support but may also regulate the function of SSCs by secreting growth factors or cytokines [22]. Macrophages play an immunosurveillance role in the niche of SSCs and may influence the functions of SSCs by secreting cytokines [23]. VECs constitute a vascular network within the testis that provides oxygen and nutrients to SSCs, and VECs can act as feeder layer cells to maintain the proliferative and self-renewal capacity of SSCs while retaining their stemness properties [24]. Together, these cell types form a complex network that interacts through direct signaling exchanges and works together to maintain the homeostasis and proper function of SSCs [25]. Clinical studies suggest that idiopathic male infertility may be associated with disturbed signaling communication in the testicular microenvironment [26]. Imbalances in the microenvironment may lead to abnormalities in spermatogenesis, thereby affecting male fertility. Therefore, understanding how these cells interact is important for revealing the regulatory mechanisms of SSCs and developing new strategies for treating male infertility.

Impact of the microenvironment on SSCs

Influence of microenvironmental cellular fractions on SSC

The role of Sertoli cells

During spermatogenesis, Sertoli cells provide structural support, form the blood-testis barrier (BTB), supply nutrients, and regulate metabolism. They also participate in complex intercellular communication through secreted signaling molecules, which are essential for maintaining the stem cell state of SSCs and regulating their differentiation into mature spermatozoa [18]. At the same time, Sertoli cells can also participate in maintaining the number of Leydig cells with the normal functioning of PMCs [27]. Here, we focus on the function of signals secreted by Sertoli cells in regulating SSC self-renewal as well as differentiation through paracrine manners (Table 1).

Table 1 Sertoli cells regulate SSC self-renewal and differentiation

Sertoli cells regulate the self-renewal of SSCs

GDNF

Signaling molecules such as GDNF, FGF, Ets variant 5 (ETV5/ERM), WNT, NOTCH, etc., which are expressed in Sertoli cells, play important roles in the regulation of SSC self-renewal (e.g., Fig. 2). GDNF is an important neurotrophic factor with protective and survival-promoting effects on many types of neurons [28]. In 2000, Meng et al. demonstrated that GDNF is involved in the self-renewal of the SSC [29]. In mammalian testis, GDNF is mainly secreted by Sertoli cells but is also produced by PMCs stimulated by testosterone [22]. GDNF binds to its high-affinity receptor, GDNF family receptor alpha (GFRα). This binding is typically followed by an interaction with the tyrosine kinase receptor RET, which initiates downstream signaling pathways. The GDNF-RET-GFRα signaling axis is essential for the self-renewal of SSCs [19, 30, 31]. Additionally, GDNF can bind to GFRα independently of RET to mediate signaling [32]. In SSCs, GDNF signaling secreted by Sertoli cells is particularly important for maintaining the self-renewal of SSCs and preventing their premature differentiation. The testes of Gdnf-deficient mice are morphologically normal until adulthood, whereas the testes of older Gdnf ^± mice have only Sertoli cells and no spermatogonia, and show the Sertoli cell-only syndrome (SCOS) phenotype [29]. The high expression of Gdnf inhibits spermatogonia differentiation, leading to the overproliferation of undifferentiated spermatogonia, leading to seminoma [33]. This result was also validated in human testicular tissues, where mRNA and protein expression of GDNF was significantly lower in SCOS tissues than in normal [34]. Restoration of GDNF expression in mice deficient in GDNF signaling and a small amount of SSCs in the tubules can reconstitute the SSC pool [35], we hope that this research will enable SCOS patients to bear their own children.

Fig. 2

Regulatory Network of SSC Self-Renewal by Sertoli Cells. Sertoli cells regulate SSC self-renewal through key signaling pathways such as GDNF, which is essential for SSC maintenance, and FGF2, which synergizes with GDNF to enhance SSC proliferation. ETV5 aids in the transcriptional regulation of SSC self-renewal genes. Other signaling molecules and pathways also contribute to maintaining the balance between SSC self-renewal and differentiation

GDNF is a key factor in the maintenance of self-renewal of SSCs, and multiple signaling molecules in Sertoli cells mediate SSC self-renewal by regulating GDNF expression [7]. Thus, GDNF and its receptor system play a central role in regulating the fate of SSCs and spermatogenesis. Both Sertoli cells-derived GDNF and recombinant GDNF induce spermatogonia migration in vitro, and GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRα1 and upregulates the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) in a dose-dependent manner, which in turn affects cell migration [36]. Similarly, macrophage migration inhibitory factor (MIF) is expressed in Sertoli cells, whereas its receptor MIFR is expressed on spermatogonia, Leydig cells and Sertoli cells. MIF is involved in spermatogonia migration and MIF was found to synergize with GDNF to increase spermatogonia migration [37]. Many signaling molecules in Sertoli cells are involved in the maintenance of SSC self-renewal through the regulation of GDNF and thus the maintenance of SSCs. Cell division control protein 42 (Cdc42) is a major regulator of the actin cytoskeleton [38], which maintains the SSC pool by mitogen-activated protein kinase 1/3 (MAPK1/3)-dependent secretion of GDNF, and Sertoli cells-specific knockout of Cdc42 downregulates GDNF, leading to loss of SSCs [39]. AT-rich interaction domain 4B (ARID4B) is involved in maintaining the balance between self-renewal capacity and differentiation of HSCs [40]. SSCs from Sertoli cell-specific Arid4b knockout mice cannot maintain self-renewal, and the Arid4b knockout leads to a reduced expression of GDNF, which in turn affects the self-renewal of SSCs [41]. Follicle-stimulating hormone (FSH) has been shown to regulate GDNF expression, and FSH stimulates the expression of nuclear orphan receptor Nur77 in Sertoli cells during prenatal and early postnatal, whereas Nur77 binds to the promoter of GDNF to promote GDNF expression. The FSH/Nur77/GDNF pathway affects SSC proliferation [42].

FGF2

In addition to GDNF, which primarily regulates SSC self-renewal, researchers have identified the FGF2 signaling pathway as perhaps a novel mode of self-renewal regulation independent of GDNF [43]. FGF2 is expressed in Sertoli cells in mouse testis [44], and its receptor, fibroblast growth factor receptor 3 (FGFR3), is expressed in A-type spermatogonia [45]. Without GDNF signaling, the expansion of SSC cultured in vitro can be maintained by adding FGF2 [43]. In the presence of testosterone, FGF2 secreted by Sertoli cells causes activation of the Extracellular signal-regulated kinase (ERK) signaling pathway by interacting with receptors in germ cells, where the transcription factor cAMP-response element binding protein (CREB) binds to the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) promoter and initiates transcription [46]. Previous studies indicate that FGF2 regulates the self-renewal of SSCs. However, recent research suggests that FGF2 might also promote spermatogonia differentiation. Additionally, FGF2 has been found to inhibit GDNF production and retinoic acid (RA) metabolism, potentially favoring differentiation. Therefore, the precise spatiotemporal role of FGF2 warrants further investigation [47]. FGF2 also stimulates the expression of ETV5 along with GDNF in Sertoli cells through the MAPK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling cascade response, but unlike GDNF, the expression of ETV5 is increased in response to epidermal growth factor (EGF) [48]. The transcription factor Etv5 is expressed in Sertoli cells, and loss of Etv5 prevents SSCs from maintaining self-renewal but does not affect the differentiation process. This ultimately results in progressive germ cell loss, leading to SCOS [49]. Furthermore, Etv5 does not affect the maintenance of SSCs through the modulation of GDNF and consequently SSCs [50]. In addition, microtubule-associated serine/threonine kinase 4 (MAST4) in Sertoli cells has been found to regulate SSC self-renewal via the FGF2-ETV5 pathway, and Mast4 knockout testes have a similar phenotype to SCOS, with germ cell loss [51]. Further exploration revealed that MAST4 was able to regulate the cell cycle by inducing cyclin-dependent kinase 2 (CDK2) to activate promyelocytic leukemia zinc-finger (PLZF), and the activated PLZF inhibited the transcription of p21, p53, and cyclin-A2 (Ccna2) to keep SSCs in the stem cell state [52]. Unlike Fgf2, Fgf9 is predominantly expressed in mouse Leydig cells, and Fgf9 promotes the proliferation of SSCs [53]. By activating its signaling cascade within the SSCs, it induces the phosphorylation of p38 MAPK, upregulates Etv5 and increases B-cell CLL/lymphoma 6 member B protein (Bcl6b) expression, ultimately promoting SSC proliferation [54]. Berberine (BBR), an antioxidant and anti-inflammatory agent, enhances the self-renewal capacity of SSCs by strengthening the reduced Sertoli cells and Leydig cells in the testis, which in turn upregulates the expression of Etv5, Gdnf, and Bcl6b [55], perhaps BBR could be applied in the future long-term in vitro culture of SSCs. Phosphoglycerate mutase 1 (Pgam1) in Sertoli cells increases the expression of Fgf2 and Gdnf through the glycolytic pathway, which in turn promotes self-renewal of SSCs [56]. Thus, FGF2 plays an important role in the balance of SSC self-renewal and differentiation.

GATA4

GATA Binding Protein 4 (GATA4) is also a key regulator in Sertoli cells involved in stem cell pool maintenance. Rubicon, a negative regulator of autophagy in Sertoli cells, maintains the normal function of Sertoli cells by preventing autophagic degradation of the transcription factor Gata4, thereby maintaining the normal function of the SSC pool [57]. Silencing of Gata4 in Sertoli cells affects germ cell development, leading to loss of the SSC pool and also allowing increased permeability of the BTB [58, 59]. GATA4 is also expressed in Leydig cells, and defects in Gata4 in Leydig cells lead to reduced expression of genes related to androgen synthesis and cause an increase in apoptotic cells, suggesting that Gata4 also acts as a critical transcription factor in Leydig cells [60].

WNT, NOTCH

The WNT and NOTCH signaling pathways in Sertoli cells are involved in regulating SSC self-renewal. The specific activation of beta-catenin 1 (CTNNB1) in Sertoli cells reduces the activity of endogenous SSCs through Wnt4 signaling, which results in germ cell apoptosis and failure of spermatogenesis [61]. Sertoli cells-secreted Wnt5a maintains SSC self-renewal by promoting SSC survival [62], whereas luteinizing hormone (LH) negatively regulates SSC self-renewal by inhibiting Wnt5a expression via testosterone [63]. The cell-specific secretion of Wnt6 supports the activation of Wnt/β-catenin signaling and the expression of the Wnt target gene Axin2, which promotes SSC proliferation but does not affect SSC self-renewal [64]. Deletion of Collaborator of ARF (Carf) in Sertoli cells downregulates Wnt signaling and Gdnf expression, which in turn impairs self-renewal of SSCs and proliferation of undifferentiated spermatogonia, leading to SCOS [65]. Wilms' tumor 1 (WT1) also regulates spermatogenesis through the WNT signaling pathway. WT1 is specifically expressed in Sertoli cells. When Wt1 is defective, undifferentiated spermatogonia accumulates in the testis, and germ cells fail to undergo normal meiosis [66]. This defect leads to a progressive loss of developing germ cells and Sertoli cells, ultimately destroying the spermatogenic tubule structure [67]. Defects in Wt1 result in decreased expression of Wnt4 and Wnt11. This downregulation of Wnt4 subsequently reduces the expression of cell polarity-related genes, such as Par6b and E-cadherin, which in turn disrupts spermatogenesis [68]. The heterogeneous nuclear ribonucleoprotein U (HNRNPU) in Sertoli cells interacts with WT1 and SOX9 and binds directly to the promoter regions of Sox8 and Sox9, which in turn regulates the development of both Sertoli cells and germ cells. Deficiency in Hnrnpu impairs spermatogonia proliferation and differentiation [69]. Leydig cells can be classified as adult Leydig cells (ALCs) and fetal Leydig cells (FLCs) [70, 71]. Deletion of Wt1 downregulates NOTCH signaling via a paracrine pathway, disrupting the differentiation of FLCs from PMCs. Additionally, Wt1 may play a role in maintaining the balance between FLCs and ALCs differentiation [72]. Deletion of Wilms tumor 1-associated protein (Wtap) in Sertoli cells can lead to defects in SSC self-renewal and proliferation, leading to a depletion of the SSC pool. However, the absence of Wtap does not affect meiosis [73]. In contrast to the WNT signaling pathway, NOTCH signaling in Sertoli cells acts as a negative regulator, modulating GDNF and controlling SSC self-renewal [74]. Overexpression of NOTCH signaling can lead to premature differentiation and progressive apoptosis of germ cells [75]. Transcription factor Rbpj is a key factor of NOTCH signaling, and the absence of Rbpj in Sertoli cells does not affect the development of the Sertoli cells themselves, but leads to an increase in the number of SSCs and enhances the expression of key factors that maintain undifferentiated spermatogonia [76]. This indicates that NOTCH signaling plays a crucial role in maintaining the balance between SSC self-renewal and differentiation.

CXCL12/CXCR4

The CXC motif chemokine ligand-12/CXC motif chemokine receptor-4 (CXCL12/CXCR4) signaling regulates SSC migration and ensures they settle in specific locations within the seminiferous tubules to maintain their stem cell properties. Both CXCL12 and CXCR4 are crucial for maintaining the SSC pool. CXCL12 is expressed by postnatal mouse testicular Sertoli cells, while CXCR4 is expressed by undifferentiated spermatogonia [77]. Overexpression of Cxcl12 in Sertoli cells increased SSC colonization in vivo [78]. Sin3a is required for the establishment and maintenance of germline stem cells. The absence of Sin3a in Sertoli cells affects the expression of stem cell markers and results in the absence of Cxcl12/Cxcr4 for the maintenance of undifferentiated spermatogonia [77]. Consequently, this inhibition of Cxcl12/Cxcr4 leads to a loss of SSCs [79]. The expression of CXCL12 in Sertoli cells and CXCR4 in germ cells has also been demonstrated in the adult testis [80]. However, recent single-cell RNA sequencing revealed that in human testis, CXCL12 is mainly expressed in Leydig cells, whereas CXCR4 is expressed in both macrophages and spermatogonia [17]. The interaction network of CXCR12 and CXCR4 needs to be further investigated. ASK-1 interacting protein (Aip1) is also involved in regulating germ cell migration, and Aip1 deficiency in Sertoli cells reduces SSC self-renewal but increases differentiated spermatogonia [81].

Other factors

Maintaining the SSC pool also depends on several essential paracrine factors secreted by Sertoli cells, including Insulin-like growth factor 1 (IGF1), Insulin Growth Factor Binding Protein 7 (IGFBP7), the Na+-K+-Cl-transporter isoform 1 (NKCC1), and protein-tyrosine phosphatase, non-receptor type 11 (PTPN11, also known as SHP2), all of which promote SSC stemness and proliferation [82,83,84]. IGF1 secreted by Sertoli cells, Leydig cells and PMCs stimulates mSSCs proliferation by promoting the G2/M phase of the cell cycle [85]. Previous studies have shown that combining IGF1 with GDNF enhances SSC activity in in vitro cultures [7]. And IGFBP7 secreted by Sertoli cells maintains SSC stemness by inhibiting the downstream PI3K signaling pathway through competition with IGF1 for binding to IGF1R [82]. NKCC1 is expressed in both germ cells and Sertoli cells, and deletion of NKCC1 leads to meiosis and spermatogenesis abnormalities [83]. Further investigation of its role in Sertoli cells revealed that NKCC1 is a downstream target gene of oxidative stress-responsive kinase 1 (OSR1) and SPS1-related proline/alanine-rich kinase (SPAK). Double knockout of Osr1 and Spak resulted in a decrease in Nkcc1 activity in Sertoli cells, resulting in loss of germ cells and finally a SCOS phenotype [86]. PTPN11 in Sertoli cells is crucial for balancing SSC self-renewal and differentiation. Although the absence of Ptpn11 does not affect Sertoli cell development, it leads to premature SSC differentiation, depleting the SSC pool, and ultimately resulting in the absence of germ cells in the seminiferous tubules [84]. PTPN11 has been reported to be expressed in Leydig cells and to participate in testosterone production. However, its potential role in regulating spermatogenesis warrants further investigation [87, 88].

Sertoli cells regulate the differentiation of SSCs

Sertoli cells are involved not only in regulating SSC self-renewal but also in the differentiation of SSCs by secreting a variety of growth factors and cytokines (as shown in Fig. 3).

Fig. 3

Regulatory network of SSC differentiation by Sertoli cells. VEGFA165B regulates SSC differentiation by binding to VEGFR, and both SCF and KITL are involved in SSC differentiation by binding to c-kit. Also LRH1, WT1, Cx43, Erbb4/Nrg1, and Rdh10 regulate SSC differentiation

Retinoic acid (RA) has an important effect on SSC differentiation in the reproductive system. RA is considered one of the important signaling molecules to promote the differentiation of spermatogonia into spermatozoa [89]. It has been shown that RA induces SSCs into a differentiated state and promotes the progression of spermatogonia to the spermatocyte lineage [90]. RA affects the differentiation process of spermatogonia by binding to the retinoic acid receptor (RAR), regulating the expression of downstream genes and promoting spermatogonia differentiation and maturation [91]. Retinol dehydrogenase 10 (RDH10) is essential for RA biosynthesis [92]. The absence of Rdh10 in the Sertoli cells causes a defect in spermatogonia differentiation, resulting in only Sertoli cells with undifferentiated spermatogonia in the seminiferous tubules [93].

The KIT ligand (KITL)-KIT signaling pathway is essential for spermatogenesis, and KITL is expressed in Sertoli cells, and its receptor c-kit is expressed in spermatogonia and Leydig cells [94]. Kitl mutant mice exhibit impaired sperm production. When testicular tissues from these mutant mice were cultured with recombinant KITL and CSF1, it was observed that CSF1 enhanced the effects of KITL and promoted spermatogenesis [95]. Further exploration revealed that low concentrations of KITL induced the differentiation of stem Leydig cells (SLCs), while high concentrations of KITL inhibited the differentiation of SLCs. Thus, KITL is an essential growth factor that regulates the development of SLCs [96]. Stem cell factor (SCF) also binds to c-kit to regulate spermatogonia differentiation and apoptosis. While SCF is broadly expressed in various testicular cell types, the absence of Scf specifically in Sertoli cells impairs spermatogonia differentiation, leading to male infertility. The lack of SCF in Sertoli cells results in the loss of most c-kit⁺ spermatogonia and subsequent germ cells, underscoring the critical role of Sertoli cell-specific SCF production in spermatogenesis [97]. In vitro experiments demonstrated that β-estradiol induced an increase in the expression level of SCF in human fetal Sertoli cells, which in turn promoted the proliferation and inhibited the apoptosis of SSCs [98]. Neuregulin-1 (Nrg1) is expressed in Sertoli cells, and Nrg1^Ser−/− leads to spermatogonia cell death and inhibits spermatogonia proliferation and differentiation into spermatocytes [99]. NRG1 and SCF may interact to promote the proliferation of type A spermatogonia, while the differentiation of spermatogonia into spermatocytes may depend on the NRG1/ERBB4 signaling pathway. Deletion of Erbb4 in Sertoli cells leads to apoptosis and impaired spermatogenesis. Additionally, dysregulation of ERBB4 affects the formation of the BTB, disrupts Sertoli and Leydig cell function, and ultimately compromises male fertility [100].

Connexin 43 (Cx43, also known as GJA1) is regulated in Sertoli cells by androgens through WT1 [101]. Knockout of Cx43 in Sertoli cells prevents spermatogenesis [102], leading to male sterility by impeding spermatogonia differentiation [103, 104]. Cx43 is a major gap junction protein in the BTB, and its knockout also disrupts the integrity of the BTB [105], and the overexpression of Cx43 rescues the function of the BTB and re-initiates meiosis [106]. Knockout of Cx43 in Sertoli cells adversely impacts both germ cell development and the integrity of the BTB, while also affecting the Sertoli cells’ maturation [107].

Vascular endothelial growth factor A (VEGFA) is a key molecule that is highly expressed on Sertoli cells to regulate the balance between SSC self-renewal and differentiation [108], and multiple isoforms of VEGFA act by binding to vascular endothelial growth factor receptor (VEGFR) expressed on SSCs. VEGFA164 promotes SSC self-renewal, while VEGFA165b promotes SSC differentiation [109, 110]. Liver receptor homologue-1 (Lrh1) is also expressed in Leydig cells, Sertoli cells and germ cells. Lrh1 deficiency in Leydig cells has no significant effect on spermatogenesis. However, Lrh1 deficiency in Sertoli cells results in the dysfunction of Sertoli cells, and unable to maintain spermatogenesis, and Lrh1 in germ cells is mainly involved in the maintenance and differentiation of SSCs [111]. The lack of RING finger protein 20 (Rnf20) in Sertoli cells leads to the loss of H2BK120ub, which further reduces the expression of the Claudin11 (Cldn11) in Sertoli cells, disrupting cell adhesion and leading to apoptosis of spermatogonia and spermatocytes, resulting in a SCOS phenotype [112].

The normal formation of the BTB is essential for spermatogenesis, and deletion of F-Box and WD Repeat Domain Containing 7 (Fbxw7) in Sertoli cells leads to excessive germ cell loss and spermatogenesis blockage, age-dependent tubular atrophy, and subsequent infertility in mice. Knockout of Fbxw7 in Sertoli cells disrupts the integrity of the BTB and causes reduced testosterone secretion by Leydig cells [113]. In contrast, in a previous study, Fbxw7 in SSCs was found to negatively regulate its self-renewal by degrading MYC [114]. Thus, Fbxw7 may play different roles in different cells, and the exact molecular mechanisms remain to be explored. Alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) is expressed in Leydig cells, Sertoli cells and germ cells [115], and depletion of Alkbh5 in Sertoli cells affects the stability of N- cadherin (Cdh2) mRNA stability which disrupts BTB integrity [116].

Contribution of Leydig cells

Leydig cells are endocrine cells located in the testicular interstitium that synthesize and secrete testosterone, which is essential for the development and function of the male reproductive system (as shown in Fig. 4). Leydig cells and Sertoli cells may originate from the same progenitor cells. Knockout of the Wt1 gene reprogrammed testicular Sertoli cells into Leydig cells [117]. Nes-GFP⁺ cells were identified as SLCs, and Nes-GFP⁺ cells transplanted into Leydig cells functionally damaged or senescent testes were able to increase testosterone and restore germ cell meiosis [118]. In our previous study, human testicular SLCs were isolated and cultured in vitro, and we found that WNT5A promotes proliferation, DNA synthesis and stemness maintenance of human testicular SLCs through activation of the β-catenin signaling pathway [119]. Future studies may investigate whether WNT5A in SLCs affects human SSC function through co-culture. Leydig cells indirectly influence the function of SSCs and the spermatogenesis process mainly through secreted testosterone, which is secreted by ALCs originating from the proliferation and differentiation of SLCs [120]. Testosterone is essential for promoting the differentiation of SSCs to mature spermatocytes during spermatogenesis. It affects the microenvironment of SSCs through endocrine pathways, thereby regulating the proliferation and differentiation of SSCs. Testosterone can affect the function of Sertoli cells, including the levels of growth factors and cytokines they secrete, which in turn influence the behavior of SSCs. Specifically, Testosterone regulates the levels of GDNF and FGF2 secreted by Sertoli cells, which are key factors in maintaining the self-renewal of SSCs [121, 122]. Testosterone regulates the pituitary–gonadal axis through an endocrine feedback mechanism, affecting the release of gonadotropins, such as FSH and LH, which further influence the proliferation and differentiation of SSCs [123].

Fig. 4

Regulation of SSCs by Leydig cells, PMCs, VECs, and macrophage. Leydig cells regulate SSCs mainly by secreting CSF1, ESR2, and IGF1. PMCs affect SSCs mainly by secreting AR, GDNF, CSF1, and pnliprp2. VECs affect SSCs by secreting GDNF, and macrophages regulate SSCs by secreting CSF1 and NR2C2

Testosterone activates FGF2 expression in spermatogonia through an internal ribosome entry site (IRES)-dependent mechanism in the adult testis, while FSH rather than testosterone is used to support spermatogenesis in the fetal testis [122]. Casein kinase 1α (Ck1α), tumor necrosis factor induced protein 3 (Tnfaip3), and Nrg1 regulate testosterone secretion in Leydig cells. CK1α regulates testosterone synthesis through the LH/PKA/EGFR/ERK1/2 signaling pathway, and Ck1α deficiency in Leydig cells results in fewer germ cells, aberrant spermatogenesis and reduced male fertility [124]. Tnfaip3 inhibits apoptosis in Leydig cells and promotes testosterone production by upregulating CCAAT/enhancer-binding protein β (Cebpb) expression [125]. Nrg1 is an LH target gene, and knockout of Nrg1 in Leydig cells significantly reduced the proliferation of infant testicular Leydig cells, leading to reduced testosterone production and causing abnormal spermatogenesis in mutant mice, but deletion of Nrg1 did not affect SSCs development [126]. D-aspartate (D-asp) may further promote the proliferation of GC-1 cells by stimulating the secretion of estradiol/estrogen receptor β (Esr2) from Leydig cells [127]. Csf1 is mainly expressed in Leydig cells and PMCs in the testis [128]. The absence of Leydig cells leads to insufficient secretion of CSF1, which affects the self-renewal ability of SSCs, whereas melatonin reduces apoptosis of Leydig cells, increases CSF1 secretion, and ameliorates the reduction of spermatogenesis induced by diabetes [129]. Unlike the knockout of Nuclear Receptor Subfamily 5 Group A Member 1 (Nr5a1) in Sertoli cells, which does not impact male fertility, the deletion of Nr5a1 in Leydig cells affects Leydig cell development but also does not impair fertility. However, a double knockout of Nr5a1 in both Sertoli and Leydig cells leads to abnormal Sertoli cell localization, morphological abnormalities in Leydig cells, and degeneration of spermatogenic tubules [130]. Similar to CSF1, IGF1 secreted by Leydig cells activates the PI3K/Akt signaling pathway by binding to IGF1 receptors on SSCs, thereby promoting SSC self-renewal [131].

Functions of macrophages, PMCs, and TECs in the microenvironment of SSCs

Macrophages, PMCs and TECs play complex and important roles in the microenvironment of SSCs. They maintain the stability of the microenvironment, support the function of SSCs and participate in the regulation of the spermatogenic process through cytokine secretion and intercellular signaling.

Macrophages in the testis are divided into a peritubular macrophage population and an interstitial macrophage population, which play different roles due to different localization [132]. The peritubular macrophage population can stimulate the differentiation of spermatogonia, whereas the interstitial macrophage is mainly involved in intercellular communication with Leydig cells or other surrounding somatic cells [133, 134]. It has been shown that a short-term absence of peritubular macrophages leads to a block in spermatogonia differentiation without affecting the maintenance of the SSC pool [23]. Moreover, macrophages secrete CSF1 and RA, which suggests they may promote SSC self-renewal in a manner similar to Leydig cells through CSF1 secretion, and regulate SSC differentiation through RA secretion [23]. However, experimental validation is required to confirm these proposed functions. Nuclear receptor subfamily 2 group C member 2 (NR2C2) is expressed in macrophages in the testis, and NR2C2 promotes the expression of Interleukin 1β (IL-1β) and IL-6 in macrophages through the NF-κB signaling pathway, exerting a pro-inflammatory effect, which in turn inhibits the proliferation of mouse spermatogonia [135].

GDNF production by PMCs is essential for developing undifferentiated spermatogonia in vivo [22, 136], and GDNF production by PMCs is regulated by testosterone secreted by Leydig cells [137]. PMCs also express the androgen receptor (AR), and progressive loss of spermatogonia occurs after AR knockout in PMCs [138]. However, knockout of AR in Sertoli cells does not affect the number of spermatogonia and mainly affects meiosis in germ cells [139]. Overexpression of AR in Sertoli cells also affects Sertoli cell/germ cell homeostasis and male fertility [140]. Pancreatic lipase-related protein 2 (Pnliprp2) is specifically expressed in testicular PMCs, and Pnliprp2^−/− leads to a disruption of homeostasis in undifferentiated spermatogonia that does not affect meiotic progression but results in a decrease in the number of SSCs [141]. Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is specifically expressed in PMCs, and the absence of Lgr4 may affect the balance between self-renewal and differentiation of SSCs, leading to an increase in undifferentiated spermatogonia and a decrease in differentiated germ cells [142].

VECs are also vital components of the niche. Co-culturing VECs with mouse SSC promoted SSC self-renewal, and the addition of GDNF, FGF2, and VECs-specific secretion of IGFBP2, CXCL12, and macrophage inflammatory protein 2 (MIP2) was found to be sufficient for long-term culture of human and mouse SSCs in the absence of feeder layer cells [143]. Additionally, FGF2 binds to FGFR1 on VECs, thereby promoting the secretion of GDNF from these cells [143]. Then, researchers also demonstrated that VECs enhanced the self-renewal capacity and preserved the stemness of rat SSCs [24].

Role of the ECM

The process of self-renewal and differentiation of SSCs requires the regulation of different signals in the microenvironment. The signals provided by the microenvironment change to facilitate the transition of SSCs into mature sperm. For example, changes in the composition of the ECM may affect cell migration and differentiation. The ECM modulates gene expression patterns by influencing the interaction between chromatin and the nuclear matrix and affecting the association between the cytoskeleton and mRNAs [144]. ECM is continuously degraded, remodeled and synthesized by cells in each tissue, rendering it tissue-specific [145]. The ECM of SSCs is a complex network of laminin, fibronectin, collagen and proteoglycans [146,147,148,149], which provide structural support and biochemical signals to SSCs and are essential for the processes of SSC self-renewal, differentiation and spermatogenesis (Table 2).

Table 2 The influence of extracellular matrix on SSCs

Collagen is the most abundant protein in the ECM and provides the structural framework for spermatogenic tubules [150]. In the ECM of SSCs, collagens contribute to a three-dimensional support network that maintains tubule morphology and mechanical stability [151]. It has been suggested that collagen may not only provide structural support but may be involved in the formation of tight junctions [152]. Collagen has been found to promote SSCs localization in in vitro culture [153]. Collagen type I alpha 1 chain (Col1a1) is also involved in SSC self-renewal, and Col1a1 deficiency inhibits SSC self-renewal and promoted spermatogonia differentiation [154]. In an in vitro culture of porcine SSCs, collagen type IV (Col IV) promotes SSCs growth and differentiation [155]. Laminins are crucial structural components in the testis, serving as key basement membrane elements that bind to integrin receptors and other cell surface receptors and are involved in cell adhesion, differentiation, and migration [152, 156,157,158]. Laminin alpha2 (Lama2) deficiency leads to abnormalities of the testicular basement membrane, which causes male infertility and can be rescued by overexpression of laminin alpha1 (Lama1) [159]. In vitro culture of testicular tissue reveals that Lama1 is a key component in maintaining the niche of the SSCs, and the loss of germ cells may be associated with a reduction of Lama1 in the culture medium [160]. Laminin deficiency and altered expression patterns of all ECM proteins in the testes of SCOS patients [161] and dysregulation of ECM genes in infertile patients were also identified by single-cell RNA sequencing [162]. Proteoglycans are molecules consisting of a protein core and one or more glycosylated polysaccharide chains. They act as fillers in the ECM and are involved in water retention and regulation of cell signaling. In the ECM of SSCs, proteoglycans such as hyaluronic acid and chondroitin sulphate are essential for maintaining the physical properties of the microenvironment and regulating cell behavior. It was found that the use of cell scaffolds containing hyaluronic acid (HA) promoted the proliferation and differentiation of SSCs more than the 2D culture system, and that the combination of silicone nanoparticles (SNs) with HA promoted spermatogenesis [163]. Integrins are cell surface receptors crucial for the adhesion and localization of SSCs, with their activity and expression patterns being vital for these processes. The absence of β1-integrin in Sertoli cells impairs SSC colonization in vivo and decreases SSC homing. Additionally, integrin binding to adhesion molecules has been shown to co-regulate SSC homing [21, 164]. α9β1-integrin is expressed in porcine SSCs and is essential for sustaining SSC self-renewal in pigs. [165]. Exposure of rat testes to petrol exhaust leads to abnormal spermatogenesis. Petrol exhaust impairs SSC self-renewal by down-regulating α6-integrin and β1-integrin [166].

Extracellular vesicles (EVs) in the testis are also part of the ECM [167]. EVs include exosomes and microvesicles, small membrane structures released by cells into the extracellular environment, which have a significant effect on the microenvironment of SSCs [168]. These vesicles can contain a variety of biomolecules such as proteins, lipids, RNA and DNA, and testicular EVs can cross the BTB and play a crucial role in intercellular communication. Extracellular vesicles from different sources serve distinct functions. miR-486-5p in extracellular vesicles secreted by Sertoli cells can promote SSCs differentiation by interacting with PTEN [169]. Similarly, miR-493-5p in Sertoli cells-derived exosomes can inhibit GDNF expression, thereby inhibiting self-renewal and promoting differentiation [170]. However, inhibition of exosomes released from Sertoli cells reduces spermatogonia proliferation [171], and undifferentiated A spermatogonia-derived EVs are also able to inhibit the proliferation of SSCs in vitro [172]. Recent studies have found that palmitoylation of VMP1 affects the secretion of EVs from Sertoli cells and thus the maintenance of SSCs-like cells (SSCLCs). Disruption of VMP1 palmitoylation leads to a decrease in EV release, inhibiting the self-renewal and proliferation of SSCLCs and increasing the number of apoptotic cells [173]. Exosomes can cross the BTB, and exosomes secreted by rat Sertoli cells deliver Ccl20 mRNA to Leydig cells, which promotes Leydig cell survival via the Akt pathway [174]. Consequently, exosomes are pivotal mediators of intercellular signaling. Future research should explore the molecular mechanisms by which EVs facilitate cellular communication within the testicular microenvironment.

Together, these ECM components form a dynamic network that not only provides physical support but also participates in cell fate regulation. The ECM, with its diverse and complex composition, ensures that SSCs can effectively self-renew and differentiate within the appropriate microenvironment. The removal of testicular cells from decellularized testes, leaving only the ECM components, produces what is known as the decellularized testicular matrix (DTM) [175]. DTM promotes SSC proliferation and differentiation in the in vitro culture of SSCs [176]. Studies have shown that a serum-free, feeder layer-free system using ECM extracted from homologous tissue can support the culture of hSSCs for a period. However, further research is needed to optimize this in vitro culture system for hSSCs [177].

Correlation between microenvironmental abnormalities and male infertility

Microenvironmental abnormalities are strongly correlated with male infertility. In non-obstructive azoospermia (NOA) patients, abnormalities in Sertoli cells, Leydig cells, and peritubular myoid cells (PMCs) have been observed. Disruptions in intercellular communication are likely to be the molecular mechanisms underlying the development of NOA [178]. The homozygous + 48845 G > T (TT allele) variant of ETV5 has been identified as a genetic risk factor for developing SCOS [179]. Abnormal expression of various genes in Sertoli cells may lead to male infertility, and the expression of GDNF, FGF8, BMP4, FGF9 and CX43 were significantly reduced in SCOS patients, suggesting that the abnormal expression of these genes may be associated with the development of SCOS [180,181,182]. In KS and SCOS patients, ECM proteins are abnormally expressed and laminin expression is notably absent compared to normal controls [183]. In cryptorchidism, the testicular ECM is also disrupted, with decreased expression of Col IV and laminin [184]. Disruption of homeostasis in Sertoli cells may accelerate testicular senescence, as revealed by single-cell transcriptome analyses [185]. Whole exome sequencing of 529 patients with non-obstructive azoospermia (NOA) identified six missense mutations in WT1 gene. Knockout of Wt1 revealed that most seminiferous tubules contained only Sertoli cells, with extensive germ cell apoptosis. This suggests that WT1 mutations may be a potential cause of NOA in humans [68]. Gene chip and bioinformatics analyses revealed abnormal expression of numerous genes, including members of the G-protein-coupled receptors (GPCR) superfamily, in Sertoli cells from NOA patients [186].

Researchers have attempted to treat male infertility caused by microenvironmental abnormality. Since abnormal Leydig cell function leads to insufficient testosterone production, which impairs spermatogenesis and results in infertility, Leydig cell transplantation has been investigated as a means to restore testosterone levels and address the issue. In a recent study, it was found that transplantation of CXCR4-SF1 bifunctional adipose-derived stem cells (CXCR4-SF1-ADSCS) into BPA-induced Leydig cells injury model mice using tail vein injection was able to differentiate into Leydig-like cells, increasing the expression of testosterone and restoring the function of Leydig cells, rescuing the defects in reproductive function in the future. CXCR4-SF1-ADSCs are expected to treat male infertility caused by Leydig cells dysfunction in the microenvironment [187, 188]. Lhcgr is essential for the maturation of Leydig cells. Deletion of Lhcgr in Leydig cells impairs their function, and mutations in Lhcgr lead to hypoplasia of Leydig cells [189]. AAV8-Lhcgr treatment restores testosterone in the testis and significantly promotes the formation of round and elongated spermatozoa, increases sperm count and viability, and rescues spermatogenesis [190]. Currently, these findings are based on animal studies, but it is hoped that they will eventually translate into clinical applications, offering viable solutions for male infertility patients.

Directions for future research

Although several genes involved in the self-renewal and differentiation of SSCs have been identified, understanding the complex regulatory mechanisms within the testicular microenvironment remains incomplete. Key challenges include the difficulty in obtaining human testicular tissue, which is both scarce and requires ethical approval. Additionally, the testicular microenvironment is dynamically influenced by hormonal changes, affecting cellular states and interactions. While in vitro models can partially simulate this environment, they may not fully replicate its complexity. Consequently, current studies fall short of fully elucidating the regulatory networks of the testicular microenvironment.

With the development of sequencing technology, the signaling communication between somatic cells and germ cells can be revealed using single-cell RNA sequencing [191]. Single-cell RNA sequencing analysis reveals that classical signaling networks, such as GDNF-GFRa1/Ret and FGFs-FGFRs, are involved in the interaction between Sertoli cells and spermatogonia. Additionally, new signaling networks were identified, such as INHA produced by Sertoli cells, with its receptors,  ACVR2B and TGFBR3, being expressed in spermatogonia and Leydig cells, respectively [192]. The specific molecular mechanisms of the interactions need to be demonstrated by in vivo and in vitro experiments in future work. Existing studies on the molecular mechanisms of microenvironmental regulation of SSCs have relied on the following three approaches (e.g., Fig. 5). The first is in vivo experiments, in which somatic cells are infected in vivo by injecting specific lentiviruses infecting somatic cells or constructing knockout mice [54, 193, 194]. The second is in vitro transplantation experiments, in which Sertoli cells, Leydig cells, etc., are modified in vitro and then transplanted back into the mouse testis [195]. The third is somatic cells and SSCs co-culture system [78]. A new option in the co-culture system is to construct a three-dimensional scaffold in vitro to study the functions played by the niche components of SSCs and their molecular mechanisms. A study used a gel scaffold composed of hyaluronic acid (HA), chitosan, and decellularized testicular matrix (DTM) for in vitro SSCs culture, and this scaffold was found to support SSC differentiation and proliferation in vitro [196]. With the development of technology, many researchers are culturing testicular organoids, which may be used in subsequent studies to investigate the specific regulatory mechanisms of the microenvironment on SSC self-renewal and differentiation [197]. The construction and application of testicular organoids can not only study the signal communication between the testicular microenvironment and germ cells in infertile patients, but perhaps can also be applied to the recovery of spermatogenesis in infertile patients [198].

Fig. 5

Four approaches to explore microenvironmental regulation of SSCs. The four research methods mainly include in vivo experiments (A), in vitro transplantation experiments (B), establishment of in vitro co-culture systems (C), and construction of testicular organoids (D)

The combination of testicular organoid culture technology and spatial proteomics to study the testicular microenvironment is a novel field of research that allows a deeper understanding of the complexity of the testicular microenvironment. Spatial proteomics is an emerging direction that can provide spatial information on proteins in tissue sections, revealing their localization and function in the testicular microenvironment [199]. Based on spatial proteomics, the composition and conditions of testicular organoid culture models can be optimized to more closely mimic the natural testicular microenvironment and improve the biological relevance of experimental results. Potential drugs or therapeutic regimens can be tested in organoid models to evaluate their effects on the testicular microenvironment and SSC function [200].

Conclusion

In this article, we detail the mechanisms by which Sertoli cells, Leydig cells, PMCs, macrophages, and VECs in the microenvironment regulate SSCs, summarize the role of ECM in the microenvironment such as collagen, laminin, and EVs on SSCs, and finally also explore the correlation between microenvironmental abnormalities and male infertility. The microenvironment of SSCs is a complex network, and normal signaling communication in the microenvironment is essential for the self-renewal and differentiation of SSCs. Future studies could further reveal the specific roles of the various components of this microenvironment and how they interact under different conditions through advanced sequencing techniques such as spatial proteomics, which will help develop new therapeutic strategies for male infertility.