Spermatogonial stem cells, a distinct cellular entity within the testes of male mammals, demonstrate extraordinary self-renewal and pluripotential differentiation capabilities (Lin et al., 2022; Qin et al., 2023). These cells are not only pivotal in tissue regeneration and immunomodulation but are also increasingly recognized for their prospective contributions to regenerative medicine, emerging as a novel source for stem cell therapies (Hashemi Karoii and Azizi, 2023). Recent research has evolved from focusing on the in vivo colonization, survival, and differentiation of spermatogonial stem cells post-transplantation to examining their interactions with the microenvironment and immunomodulatory roles (Hashemi Karoii and Azizi, 2023). Specifically, the regulatory dynamics between spermatogonial stem cells and their target cells within signaling pathways have garnered substantial research interest. In the realm of tissue engineering, spermatogonial stem cells are integral to tissue repair and organogenesis. Their interactions with cellular signaling pathways elucidate their functional mechanisms. Notably, the JAK-STAT (Janus kinase signal transducer and activator of transcription) signaling pathway, which is instrumental in vital cellular processes such as proliferation, maturation, and differentiation, exerts a profound influence on the functions of spermatogonial stem cells and their target cells (Shields et al., 2014; Zhang et al., 2015). This review aims to summarize the advancements in research on the JAK/STAT signaling pathway and the multifaceted cellular functions of spermatogonial stem cells, encompassing immunoregulation, tissue differentiation, homing, and microenvironmental adaptation. The findings discussed herein are intended to offer a critical reference for elucidating the signaling pathway mechanisms underlying functional changes in spermatogonial stem cells and to provide theoretical underpinnings for related preclinical studies and cellular engineering research.

The term “stem cell” was first coined in 1901 by Regaud, who, in his study of spermatogonia in the testes, postulated the existence of a stem cell system to replenish the plethora of differentiated cells (Regaud, 1901). Presently, it is acknowledged that multiple stem cell types exist, with adult stem cells being a prominent category. An adult stem cell, located in specific tissues, possesses the dual capacity to both proliferate, generating new stem cells, and differentiate, yielding more progenitor cells (Dym et al., 2009). Spermatogonial stem cells, a subset of adult stem cells from the testes, have the unique ability to self-renew, sustaining their population, and differentiate, giving rise to a multitude of daughter cells (de Rooij and Russell, 2000). These spermatogonia are situated at the basement membrane of the seminiferous tubules and initially exist as single cells (As). Upon division, these cells can either independently form two as spermatogonia or, by connecting through cell bridges, form two pairs of spermatogonia (Apr). Subsequently, Apr spermatogonia divide to yield pairs (Apr), which then divide to form chains of 4, 8, or 16 cells (Aal). Following approximately 6-7 mitotic divisions, these cells develop into spermatocytes, progressively migrating away from the basement membrane towards the tubular lumen, undergoing meiosis to transform into spermatids, and ultimately entering the tubular lumen. Collectively, As, Apr, and Aal spermatogonia are categorized as undifferentiated spermatogonia. Given the lack of 100% purified SSCs, the SSCs under investigation are also referred to as undifferentiated spermatogonial stem cells (de Rooij and Russell, 2000). The existence of SSCs has been acknowledged since the 1950s, although research progress was initially sluggish (Nakagawa et al., 2007). A primary reason for this was the reliance on traditional research methodologies that focused predominantly on the morphology of SSCs, rather than exploring molecular mechanisms (de Rooij and Russell, 2000). However, around 1990-2000, there was a significant paradigm shift, marking the onset of a new era in SSC research. This change was catalyzed by two pivotal technological breakthroughs: the first being the development of the testis transplantation technique by Brinster and Avarbock (1994), which spurred unprecedented advancements in SSC research (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). The second milestone was achieved in 2003 when Kanatsu-Shinohara and his team established a protocol for the long-term in vitro culture and proliferation of SSCs (Kanatsu-Shinohara et al., 2003; Kanatsu-Shinohara et al., 2005; Kubota and Brinster, 2006). These methodologies have enabled the transformation of SSCs into pluripotent stem cells and their genetic manipulation in vitro. Recent studies report that pluripotent stem cells derived from neonatal mice, adult mice, and human testicular tissue can, in vitro, differentiate into various cell lines capable of forming teratomas upon injection into immunodeficient mice (Kanatsu-Shinohara et al., 2004; Guan et al., 2006; Conrad et al., 2008). Additionally, gene targeting and homologous recombination techniques in SSCs have led to the successful creation of knockout mice, and while knockout rats have not yet been produced, successful homologous recombination in the rat genome has been achieved (Kanatsu-Shinohara et al., 2006; Kanatsu-Shinohara et al., 2011; He et al., 2015). For the cell culture aspect of SSCs, there are quite a few experiences to learn. SSCs were isolated from the testes of C57BL/6J mice or SD rats on the 7th day after birth. Subsequently, after removal of the tunica albuginea and epididymal curvature, the seminiferous tubules were excised and flushed with Hanks balanced salt solution (HBSS). They were physically sheared and digested with a solution of DnaseI, hyaluronidase, collagenase, and trypsin using a two-step enzymatic digestion method in which the digestive enzymes included DnaseI, hyaluronidase, collagenase, and trypsin. The dissociated single cell suspension was resuspended and cultured.

Spermatogonial stem cells also exhibit several distinctive biological properties. SSCs are characterized by their inherent potential for self-replication and differentiation. De Rooij (1988) identified as cells as the spermatogenic stem cells in various animals, including rats, mice, Chinese hamsters, and sheep. Dym (1994) categorized undifferentiated spermatogonia into five subpopulations (A0—A4) based on morphological characteristics. A0 cells divide slowly, are in a dormant state, and can reintegrate into the spermatogonial lineage, while A1—A4 cells are proliferative, with A4 cells being true stem cells that not only generate differentiated In and B cells but also new A2 cells, thus maintaining the stem cell count within their population. Under conditions such as X-ray irradiation or treatment with the alkylating agent leucovorin, as cells exhibit a degree of damage tolerance, whereas Apr and subsequent spermatogonial stages succumb or are depleted. Upon removal of the damaging conditions, the surviving As cells can regenerate the entire spermatogenic cell lineage through self-replication and differentiation (Huckins, 1971). SSCs possess a unique trait of immortality. While other adult tissue stem cells are capable of self-renewal, they do not contribute genetic information to offspring, thus not influencing the genetic makeup of the progeny. In contrast, oocytes transmit genetic information but lose self-replicative ability post-birth (Brinster and Zimmermann, 1994). SSCs uniquely embody both aforementioned capabilities, making them the only replicating population of diploid cells in the adult body, hence considered immortal (Brinster and Avarbock, 1994) (Figure 1) (Images in this article were generated by Midjourney).

Janus Kinase (JAK) is a family of non-receptor tyrosine protein kinases comprising four members: Janus Kinase 1, Janus Kinase 2, Janus Kinase 3, and Tyrosine Kinase 2. Cytokine activation triggers the opening of the intracellular structural domain of the receptor, facilitating its binding to JAK. This interaction leads to the phosphorylation of JAK, forming p-JAK, which in turn recruits intracellular STAT transcription factors. The phosphorylated STAT transcription factors then dimerize and polymerize, enabling them to recognize and bind to specific regions of the DNA, thus modulating gene expression within the target cell. This cascade of events delineates the JAK/STAT signaling pathway (Stark and Darnell, 2012).

The JAK/STAT pathway is a common conduit utilized by many cytokines, capable of transducing diverse extracellular signals into specific cellular responses (Cao et al., 2015; Amano et al., 2016; Ahmad et al., 2017; Browning et al., 2018). STAT molecules, central to this pathway, can form various structures including homodimers, heterodimers, or tetramers, and subsequently translocate to the nucleus or mitochondria to bind DNA and regulate the transcription of target proteins (Brinster and Zimmermann, 1994; Wei et al., 2010; Begitt et al., 2014; Meier and Larner, 2014; Villarino et al., 2015). Moreover, the JAK/STAT pathway is subject to negative feedback regulation, crucial for maintaining intracellular homeostasis. Post-activation, suppressors of cytokine signaling (SOCS) are released, inhibiting JAK phosphorylation and thereby modulating the expression of target proteins (Brinster and Zimmermann, 1994; Wei et al., 2010; Begitt et al., 2014; Meier and Larner, 2014; Villarino et al., 2015). This negative regulation, exemplified by SOCS, plays a vital role in cellular equilibrium (Tamiya et al., 2011).

The influence of the JAK/STAT pathway on gene expression is both specific and extensive (Stark and Darnell, 2012; Villarino et al., 2015). Regarding specificity, different cytokines may activate identical STAT molecules, such as interleukin 6, interleukin 27, and interleukin 10 activating STAT3, yet their effects on cellular functions vary. For instance, interleukin 10 exerts a pain-inhibitory effect and promotes neural repair via STAT3 activation, whereas interleukin 6, through the same pathway, impedes neural repair and induces nociceptive sensitization (Meka et al., 2015; Zhu et al., 2017; Uciechowski and Dempke, 2020). This specificity stems from varying mechanisms within the signaling pathways, where distinct cytokine stimuli lead to diverse cellular responses through the activation of other signaling cascades, ultimately affecting gene transcription and translation, and resulting in specific cellular functions.

In terms of broader impact, STAT molecules can bind to multiple sites on the DNA, influencing gene expression by attaching to promoter or enhancer regions. For example, interleukin 4 activates STAT6 molecules in T cells, which then bind to enhancer regions in T cell nuclei, prompting phenotypic changes and differentiation into helper T 2 cells (Xu et al., 1996; Kanno et al., 2012; Vahedi et al., 2012) (Figure 2).

Recent developments have led scholars to transition from perceiving spermatogonial stem cells (SSCs) as monopotential to recognizing them as multipotential stem cells. In 2004, Kanatsu-Shinohara et al. (2004) first cultured multipotential embryonic-like stem cells from neonatal mouse SSCs and successfully induced their transdifferentiation into various adult cell types. A parallel study by Guan et al. (2006), akin to that of Kanatsu-Shinohara et al. (Conrad et al., 2008), yielded similar results, producing multipotent adult male germline stem cells (maGSCs) derived from adult mouse testis. In 2007, GPR125 positive germline progenitor cells from adult mice were isolated and cultured, yielding embryonic-like stem cells that were further transdifferentiated into cells of other embryonic lineages, even contributing to chimeric formations (Seandel et al., 2007). Both induction systems generated multipotent embryonic-like stem cells, yet the origin of these cells and the mechanisms underlying their dedifferentiation remained elusive.

In 2010, to gain insight into the dedifferentiation process of SSCs, researchers delineated it into three phases: the SSC stage, intermediate-type SSCs (iSSCs), and the embryonic-like stem cell stage (Kim et al., 2010). Takashima et al. (2013) suggested that the autonomous reprogramming of SSCs might be influenced by unstable DNA methylation and the Dmrt1-Sox2 cascade reaction, critical in regulating SSC multipotency. Spermatogonial stem cells have been shown to differentiate into specific cell types suitable for transplantation in vitro, such as vascular endothelial and smooth muscle cells (Im et al., 2012), renal parenchymal cells (Wu et al., 2008; Heer et al., 2013; De Chiara et al., 2014), omphalocele-like cells (Wang et al., 2012), neuroepithelial-like cells (Nazm Bojnordi et al., 2013), neuronal-like cells (Liu et al., 2012), and hepatic stem cell-like cells (Zhang et al., 2013). However, studies have consistently reported that transplanted SSCs exhibit low in vivo colonization rates and brief survival times, implying alternative mechanisms of action.

The interaction between the JAK/STAT signaling pathway and spermatogonial stem cells is twofold: ① The JAK/STAT pathway in target cells modulates the biological properties of SSCs, exemplified by support cells signaling to SSCs via the JAK-STAT pathway, thereby shaping the microenvironment conducive to SSC self-renewal (Tulina and Matunis, 2001). ② The JAK/STAT pathway can influence the biological attributes of SSCs themselves; for instance, Brawley and Matunis (2004) demonstrated that mitotically active spermatogonia could repopulate their niche and revert to a stem cell state when JAK-STAT signaling was conditionally altered (Brawley and Matunis, 2004). Therefore, the exploration of the relationship between the biological characteristics of SSCs and the JAK/STAT pathway can be approached from two perspectives: firstly, examining the impact of the target cell’s JAK/STAT pathway on SSCs (Tulina and Matunis, 2001); and secondly, investigating the influence of the SSC’s own JAK/STAT pathway on its biological properties (Brawley and Matunis, 2004).

The influence of the JAK/STAT pathway in target cells on spermatogonial stem cells (SSCs) primarily manifests in aspects such as stem cell self-renewal, dedifferentiation, proliferation, development, and the immune microenvironment. Sheng et al. (2009a) discovered that a subset of mesenchymal stromal cells, termed “hubs” in Drosophila testis, activate the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in neighboring germ cells and somatic stem cells. This activation is crucial for maintaining germline stem cells (GSCs), creating a microenvironment favorable for GSC maintenance. Under certain conditions, spermatogonia can revert to GSCs when the JAK/STAT pathway is active. Conversely, when this pathway is inhibited, spermatogonia lose their dedifferentiation capability. STAT92E, a critical protein in the JAK/STAT pathway, is enriched in GSCs and progenitor cells’ vesicles, with increased activation in spermatogonia near the testis apex, indicating local upregulation of JAK/STAT signaling during dedifferentiation. Unpaired (Upd), a ligand of the JAK/STAT pathway, does not vary in mRNA levels and distribution during dedifferentiation, suggesting its role in activating the pathway. The JAK/STAT pathway also contributes to cell motility, potentially related to cell rearrangements during spermatogonia’s reversion to GSCs.

Wawersik et al. (2005) provided direct evidence of the JAK/STAT pathway mediating key signals from somatic gonads that regulate sexual development in the male germ line, highlighting its role in sex determination and development of SSCs. Brawley and Matunis (2004) found that the restoration of JAK/STAT signaling in the testis can regenerate GSCs, even from a complete absence. These regenerated GSCs were functional and could segregate from adjacent spermatogonia, reverting to stem cells. They demonstrated that spermatogonia, even after undergoing limited mitotic divisions, could reoccupy their niche and revert to stem cells under appropriate microenvironmental conditions. This finding implies that transit-amplifying cells can dedifferentiate into functional stem cells for tissue regeneration. Additionally, GSC differentiation occurs when the function of stat92E (Drosophila STAT homologue) is lost, but its restoration can revert differentiated spermatogonia back to stem cells. This process does not occur through symmetric division of remaining GSCs but through the restoration of already differentiated spermatogonia to stem cell identity, challenging previous theories on GSC regeneration.

Sheng et al. (2009b) showed that the JAK/STAT pathway activation in a subset of primordial germ cells (PGCs) linked to newly formed embryonic hubs leads to these PGCs expressing GSC markers and functioning as GSCs. In contrast, PGCs not linked to the hub began to differentiate, indicating the pathway’s importance in establishing GSCs. Tulina and Matunis (2001) concluded that support cells activate the JAK-STAT pathway to signal neighboring stem cells, defining an ecological niche for stem cell self-renewal, emphasizing the pathway’s critical role in maintaining SSC self-renewal and homeostasis.

In some studies, it has been shown (Morimoto et al., 2009; Marega et al., 2021) that Kit, a cell surface receptor in the JAK/STAT signaling pathway, and its downstream signaling pathway can activate STAT3, and the phosphorylated STAT forms a dimer and then enters the nucleus, which causes Kit to be significantly downregulated in certain phenotypes, thus affecting the behavior of cells. Some studies have shown (Morimoto et al., 2009) that in germline stem cells, Kit binding to SCF not only promotes cell survival and proliferation, but may also maintain their undifferentiated state by activating the JAK/STAT pathway. Although in vivo SSCs do not express the c-kit tyrosine kinase receptor (Kit), Kit expression is upregulated when cultured on laminin in vitro. Both Kit-positive and Kit-negative cells showed considerable levels of SSC activity after germ cell transplantation. Unlike differentiated spermatogonia, which depend on Kit for survival and proliferation, Kit on SSCs does not play any role in SSC self-renewal. Kit expression changes dynamically when SSCs begin to proliferate after germ cell transplantation. This implies that SSCs may upregulate Kit expression during active proliferation. Despite the limited role of Kit in SSC self-renewal, the regulation of Kit activation is crucial during subsequent differentiation. This may be related to the role of Kit in differentiated spermatogonia, which depend on Kit for survival and proliferation.

Hasan et al. (2015) demonstrated that the anti-apoptotic gene Drosophila inhibitor of apoptosis 1 (DIAP1), enriched in GSCs and CySCs and required for their survival, is a target of the JAK-STAT pathway. Enhanced JAK-STAT signaling upregulates DIAP1 in stem cells and their progeny, whereas decreased signaling increases their sensitivity to stress-induced apoptosis due to lower DIAP1 levels. Non-autonomous overexpression of DIAP1 in somatic cells rescued spermatogonia from stress-induced apoptosis, suggesting that microenvironmental signaling can promote stem cell survival by upregulating anti-apoptotic proteins, a potential general strategy for stem cell apoptosis resistance.

This paper synthesizes previous research on the JAK/STAT signaling pathway and spermatogonial stem cells (SSCs), highlighting that these studies predominantly concentrate on the protein expression and genetic material content of JAK/STAT signaling pathway molecules. The role of the JAK/STAT signaling pathway in SSCs and their target cells has been elucidated in two principal areas:

First, the impact of the JAK/STAT pathway in target cells on SSCs, which is instrumental in regulating microenvironmental imbalances and has potential applications in disease treatment and adjuvant therapy. Second, the differentiation, immunomodulation, adaptive changes, and homing of SSCs are all linked to the JAK/STAT signaling pathway:1.Differentiation: Cytokine-activated JAK/STAT signaling contributes to SSC differentiation. 2. Immunomodulation: SSCs foster immune cell proliferation through specific surface antigen expression and cytokine release via the JAK/STAT pathway. 3.Adaptive Changes: SSCs exhibit enhanced survival rates in both in vivo and in vitro environments when the JAK/STAT pathway is activated. 4.Homing: The JAK/STAT pathway guides SSC migration towards chemokine-releasing cells.

The main stem cell sources for studying the biological properties and JAK/STAT pathway include Drosophila, rats, and sheep, with human and large mammalian studies being relatively rare. There is a need to develop a comprehensive theoretical framework for the biological properties and JAK/STAT signaling pathway of SSCs across different species.

SSCs hold considerable promise for clinical applications, particularly in adjuvant therapy and disease treatment. Current tissue engineering research focuses on the differentiation function of SSCs to replace damaged or destroyed cells. However, foundational research on preclinical applications of SSCs is closely linked to the exploration of cellular signaling pathways. This paper contributes to the understanding of SSCs by examining their relationship with the JAK/STAT signaling pathway, offering vital insights into the signaling mechanism and providing theoretical support for further preclinical studies.