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Protein phosphorylation is a rapid, reversible post-translational modification that plays a central role in mammalian spermatogenesis and sperm functional maturation. This review integrates evidence from phosphoproteomics, genetics, and functional studies to summarize how phosphorylation networks govern key steps of germ-cell development. We outline how major kinases and phosphatases – including the testis-specific serine/threonine kinases (TSSKs), Polo-like kinases (PLKs), IGF1R tyrosine kinase, metabolic kinases (AMPK, AK9, GK2), tyrosine kinases (C-kit, TAM family: TYRO3, AXL, MER), and the PP2A/PP1 phosphatases – coordinate spermatogonial proliferation and differentiation, meiotic chromosome dynamics, spermiogenesis, and post-epididymal capacitation. Metabolic kinases bridge phosphorylation signaling with energy metabolism to support sperm motility, while C-kit and TAM family kinases regulate germ cell maturation and Sertoli cell homeostasis (e.g., blood-testis barrier integrity, phagocytosis of apoptotic germ cells). We also highlight extensive crosstalk between phosphorylation and other regulatory layers, particularly histone modifications and ubiquitination, which together form an integrated network required for normal sperm development and function. Recent advances in mass spectrometry–based phosphoproteomics – especially DIA/SWATH-MS and emerging single-cell phosphoproteomic approaches – have enabled stage-resolved, system-level maps of phosphorylation dynamics, revealing critical sites and pathways that define specific developmental transitions. Importantly, disruption of these networks (including aberrant activity of metabolic kinases and tyrosine kinases) is closely associated with male infertility phenotypes, including oligozoospermia, asthenozoospermia, teratozoospermia, and increased sperm DNA fragmentation, supporting the use of phosphorylation signatures and kinase/phosphatase expression patterns as candidate diagnostic biomarkers. Finally, we propose a “mechanism–technology–clinical” research framework that combines spatiotemporally resolved phosphoproteomics, epigenetic interaction analyses, and AI-assisted network inference to define causal phosphorylation circuits. Prioritizing key regulatory nodes – such as the TSSK family, AKAP proteins, metabolic kinases, and TAM/C-kit tyrosine kinases – may accelerate the development of phosphorylation-based diagnostics and enable non-hormonal strategies for male contraception and fertility restoration. Overall, this review provides a unified view of how phosphorylation, integrating metabolic and tyrosine kinase signaling, shapes sperm development and function and offers direction for translational research in male reproductive health.
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
Kaili Zhou
College of Life Science Xinyang Normal University, Xinyang, China
Kejin Ren
College of Life Science Xinyang Normal University, Xinyang, China
Yijia An
College of Life Science Xinyang Normal University, Xinyang, China
Tiantian Meng
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
Xiaofang Cheng
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
Cencen Li
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
Pengpeng Zhang
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
Yongjie Xu
*
College of Life Science Xinyang Normal University, Xinyang, China
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain Xinyang Normal University, Xinyang, China
*Address all correspondence to: xyj@xynu.edu.cn
1. Introduction
Every cell in the body relies on a vast “switchboard” to decide when to grow, divide, move, or change its behavior. One of the fastest and most widely used switches is protein phosphorylation – a small chemical tag that cells attach to proteins to turn their activity up or down, change where they operate in the cell, or determine how long they survive. This tag is added and removed by two opposing groups of enzymes: protein kinases (which add the phosphate) and protein phosphatases (which remove it). Because the tag can be reversed within seconds or minutes, phosphorylation allows cells to react quickly to changing conditions. Large-scale studies have estimated that phosphorylation influences a substantial fraction of the proteome and sits at the heart of cellular communication, metabolism, gene regulation, and the cell cycle [1, 2]. Most phosphorylation occurs on serine, threonine, and tyrosine residues, but newer work has expanded the “phosphorylation landscape” to include less conventional sites, suggesting that phosphorylation-based regulation is broader than previously appreciated.
Few biological processes demand more precise control than spermatogenesis, the step-by-step transformation of stem cells in the testis into mature sperm [3]. This process unfolds over weeks and proceeds through three major phases: (1) repeated cell divisions that expand the germ-cell pool, (2) meiosis, which halves the chromosome number and reshuffles genetic information, and (3) spermiogenesis, a striking remodeling stage in which round cells are sculpted into streamlined sperm with a compact head and a propulsive tail. Each phase requires thousands of proteins to act at the right time and place – making spermatogenesis especially dependent on rapid, reversible control systems like phosphorylation [4, 5]. Research has shown that phosphorylation helps guide germ-cell proliferation and differentiation, coordinates the choreography of chromosome pairing and separation during meiosis, and directs key remodeling events during spermiogenesis, including chromatin packaging, acrosome formation, and flagellum assembly. After leaving the testis, sperm continue to mature and acquire fertilizing ability through capacitation, a process strongly marked by changes in protein phosphorylation – particularly tyrosine phosphorylation – on the sperm tail and signaling scaffolds that shape motility and fertilization competence [6–9].
Importantly, when this phosphorylation “switchboard” malfunctions, fertility can suffer. A growing body of evidence links abnormal phosphorylation patterns to common forms of male infertility, including low sperm count, poor motility, and abnormal morphology. Studies comparing fertile and infertile men have repeatedly identified altered phosphorylation signals in pathways that control motility, energy use, and capacitation, suggesting that phosphorylation signatures could serve as sensitive indicators of sperm quality and functional potential [10, 11]. At the same time, technological breakthroughs – especially mass spectrometry–based phosphoproteomics – now allow researchers to measure thousands of phosphorylation sites in a single experiment, across developmental stages or clinical samples. These tools have begun to reveal how phosphorylation networks change from spermatogonia to sperm and how specific kinases, phosphatases, and phosphorylation events may drive key transitions in germ-cell development [12–14].
Despite these advances, a major gap remains: we still lack a clear, integrated view of which phosphorylation events matter most, when they occur, and how they connect into functional networks that determine sperm production and sperm quality. Many studies identify large lists of phosphorylation sites, but translating those lists into a coherent biological story – and into clinical value – remains challenging.
Against this background, the goals of this paper are threefold: (1) To synthesize current knowledge on how phosphorylation regulates the major stages of spermatogenesis and sperm maturation. (2) To highlight key phosphorylation-controlled pathways and representative regulators that have emerged from genetic, biochemical, and phosphoproteomic studies. (3) And to discuss clinical relevance, including how phosphorylation changes may contribute to male infertility and how phosphoproteomics could support improved diagnosis and future therapies.
By connecting fundamental biology with emerging technology and clinical needs, this work aims to provide a clearer roadmap for understanding how a reversible chemical “tag” helps build one of the most specialized cells in the body – and how decoding that tag may open new possibilities for protecting and restoring male reproductive health.
2. Coregulation and molecular mechanisms of phosphorylation in spermatogenesis
Protein phosphorylation is governed by two antagonistic enzyme classes: protein kinases, which catalyze phosphate transfer to target residues, and protein phosphatases, which catalyze phosphate removal. During spermatogenesis, the stage-specific and subcellularly restricted expression and activity of these enzymes establishes a highly coordinated signaling architecture that regulates germ-cell proliferation, meiotic progression, spermiogenesis, and acquisition of fertilizing capacity. Testis-enriched regulators – including testis-specific serine/threonine kinases (TSSKs), Polo-like kinases (PLKs), and major serine/threonine phosphatases such as protein phosphatase 2A (PP2A) – modulate sperm development by altering the phosphorylation states of substrates that control chromatin remodeling, cytoskeletal dynamics, flagellar biogenesis, motility, and capacitation. This broad regulatory principle is consistent with the established view that reversible phosphorylation functions as a central mechanism for rapid and context-dependent control of protein function [1, 2].
2.1 Key kinases and their roles in spermatogenesis
Kinases constitute the principal catalytic drivers of phosphorylation-dependent regulation. In the testis, multiple kinase families operate in parallel and frequently converge on shared cellular modules, including chromatin, microtubules, and flagellar scaffolds. The TSSK family is especially critical during late spermatogenesis, whereas Fused (FU), IGF1R, and canonical cell-cycle/stress-responsive kinases (e.g., CDKs and MAPKs) contribute to spermatid remodeling and post-testicular sperm activation.
2.1.1 Testis-specific serine/threonine kinase (TSSK) family
TSSKs are predominantly expressed in testicular tissue and are evolutionarily conserved among mammals [15, 16]. The TSSK family, including multiple members such as TSSK1, TSSK2, TSSK3, TSSK4, and TSSK6, plays unique and important roles in different stages of spermatogenesis. Their activity is most prominent during spermiogenesis, where they regulate sperm morphogenesis and functional maturation; genetic disruption of individual TSSKs frequently results in subfertility or sterility.
2.1.1.1 TSSK1/2: Essential for spermiogenesis and chromatin remodeling
Murine studies demonstrate that Tssk1/Tssk2 double deletion causes complete male sterility, accompanied by profound sperm structural abnormalities and fertilization failure [17]. Work in a Drosophila model further supports an evolutionarily conserved role for TSSK homologs in regulating the histone-to-protamine transition, a prerequisite for sperm chromatin condensation [5]. Loss of TSSK activity compromises chromatin compaction and is associated with broad spermatid defects, including aberrant head morphogenesis and disorganization of the flagellum. Notably, expression of human TSSK rescues major phenotypes in this model, underscoring functional conservation and indicating that TSSK catalytic activity is required for male fertility.
2.1.1.2 TSSK3/4/6: Maintaining sperm motility and morphology
Other TSSK members exhibit functional specialization. TSSK3 is expressed in elongating sperm and localizes to the sperm tail [18]. The absence of TSSK3 results in disorganization of all stages of the testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology. Tssk4-null mice show subfertility, largely attributable to markedly reduced sperm motility, implicating TSSK4-mediated phosphorylation in the regulation of proteins required for effective flagellar beating [19]. TSSK6 is essential for the formation of γH2AX, which is closely related to histone-protamine conversion and male fertility. Knockout of the Tssk6 gene leads to severe defects in histone-protamine conversion in sperm cells, resulting in excessive retention of histones in sperm and affecting the normal concentration of chromatin [20]. Phosphoproteomic analyses further suggest that TSSKs engage broad substrate repertoires enriched in pathways related to microtubule organization, chromatin packaging, and flagellar assembly, supporting a model in which individual TSSKs act on partially overlapping yet functionally distinct substrate sets.
2.1.1.3 TSSKs and chromatin compaction: Phosphorylation of transition proteins and protamines
A hallmark of spermiogenesis is chromatin remodeling, in which histones are sequentially replaced by transition proteins (TNPs) and protamines (PRM1/PRM2) to achieve extreme DNA compaction. Experimental evidence indicates that TSSKs phosphorylate transition proteins and protamines in vivo and in vitro, providing a direct mechanistic link between kinase activity and chromatin packaging [16]. In human sperm, protamines contain multiple phosphosites (e.g., PRM1 S11 and PRM2 S59) that are implicated in regulating DNA binding and higher-order chromatin organization [14]. Although the temporal regulation of individual sites remains under investigation, aberrant protamine phosphorylation is plausibly associated with defective packaging and increased DNA fragmentation – features commonly correlated with male infertility.
2.1.2 Other kinases in the spermatogenic network
2.1.2.1 Fused (FU) kinase: Sperm head shaping and flagellar accessory structures
FU is a serine/threonine kinase required for normal sperm morphogenesis. Germ cell–specific Fu knockout mice are infertile, showing reduced sperm count, abnormal head shape, head–neck separation, and impaired motility [21]. While the axoneme can remain largely intact, the flagellar accessory structures, particularly the outer dense fibers (ODFs), are disorganized, consistent with motility defects. FU localizes to key microtubule-rich structures involved in shaping the sperm head and assembling the tail (e.g., the manchette and acrosome-associated complexes) and interacts with structural components such as ODF1 and kinesin-associated proteins, supporting a role in coordinating cytoskeletal remodeling.
2.1.2.2 IGF1R: A tyrosine-kinase pathway in human sperm capacitation
Capacitation is characterized by a robust increase in protein tyrosine phosphorylation, a widely used molecular indicator of sperm activation [22]. Phosphoproteomic and computational studies have identified IGF1R as a principal upstream tyrosine kinase in human sperm capacitation [23]. IGF1R activation increases phosphorylation of downstream targets, including AKAP3 and AKAP4, key scaffolds of the fibrous sheath. Multiple tyrosine residues on AKAP3 and AKAP4 become phosphorylated during capacitation, and this pathway is functionally linked to hyperactivated motility and acquisition of fertilization competence, supporting a direct connection between growth factor signaling and sperm functional maturation [24, 25].
2.1.2.3 CDKs and MAPKs: Coordinating proliferation, meiosis, and capacitation signaling
Cyclin-dependent kinases (CDKs) and mitogen-activated protein kinases (MAPKs) are canonical serine/threonine kinases that regulate cell-cycle transitions and stress-responsive signaling and are highly active in the testis [26, 27]. Large-scale phosphoproteomic surveys indicate that testicular phosphorylation is extensive and is dominated by serine and threonine phosphorylation, consistent with major inputs from kinase families such as CDKs, MAPKs, PAKs, PLKs, and TSSKs. These kinases collectively regulate spermatogonial proliferation, meiotic progression, and spermatid remodeling. For example, CDK7 plays an important role in various key aspects of spermatogenesis [28, 29]. During the spermatogonial stage, CDK7 affects cell proliferation and differentiation by regulating the expression of c-KIT mediated by retinoic acid. During meiosis, CDK7 affects meiosis initiation, DNA repair, and synapse complex formation by regulating the retinoic acid-mediated STRA8 and REC8 signaling pathways. During the stage of sperm formation, CDK7 activates STAT3 to affect germ cell apoptosis and sperm motility. STAT3 further regulates Coractin expression to affect sperm nuclear elongation, chromatin condensation, and acrosome formation. In mature sperm, MAPK signaling has also been implicated in capacitation-associated outcomes, including motility modulation and the acrosome reaction [30].
2.1.2.4 Metabolic and sperm-specific kinases linking phosphorylation to energy metabolism and motility
Phosphorylation-dependent regulation of spermatogenesis and sperm function is tightly coupled to energy metabolism: sperm motility, capacitation, and fertilization require precise coordination of ATP production and utilization. Three key kinases – AMP-activated protein kinase (AMPK), adenylate kinase 9 (AK9), and glycerol kinase 2 (GK2) – mediate this crosstalk by regulating ATP-generating pathways and, in the case of AMPK and AK9, by phosphorylating metabolic enzymes and structural proteins, thereby bridging signaling networks with metabolic homeostasis.
As a master regulator of cellular energy balance, AMPK is a heterotrimeric serine/threonine kinase (α/β/γ subunits) highly expressed in mammalian testes and mature sperm [31, 32]. Its activation depends on the phosphorylation of Thr172 in the α subunit, which is synergistically regulated by upstream signals, including the cAMP/PKA pathway, intracellular Ca2+, and Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα/β) [31, 33]. In spermatogenesis, AMPK phosphorylates key metabolic enzymes (e.g., ACC1/2, GSK3β) to shift cellular metabolism from anabolic to catabolic, thereby supporting spermatogonial proliferation and meiotic progression [32]. In mature sperm, AMPK localizes to the flagellum and midpiece (the mitochondrial-rich region), where it phosphorylates flagellar structural proteins (e.g., tubulin, AKAP3) and metabolic enzymes (e.g., PFKFB3) to regulate ATP production and flagellar beating [31]. Clinically, reduced Thr172 phosphorylation of AMPK is associated with asthenozoospermia, and impaired AMPK-dependent energy sensing is thought to contribute to defective hyperactivated motility during capacitation.
AK9 is a sperm-specific adenylate kinase predominantly expressed in post-meiotic spermatids and mature spermatozoa, where it localizes to the flagellum and acrosome [34]. AK9 catalyzes the reversible reaction ATP + AMP ↔ 2 ADP, thereby maintaining nucleotide homeostasis and supporting efficient ATP turnover. In addition, AK9 undergoes autophosphorylation and can phosphorylate downstream targets such as flagellar dynein light chain, which may modulate flagellar function in a phosphorylation-dependent manner [35]. A landmark clinical study in Chinese patients with idiopathic asthenozoospermia identified biallelic AK9 mutations that were associated with significantly decreased sperm ATP levels, suppressed glycolysis, and impaired sperm motility. Consistent with the clinical findings, Ak9-knockout mice generated by CRISPR-Cas9 exhibit typical features of male subfertility, including reduced sperm progressive motility and abnormal flagellar bending [35]. These results confirm that AK9 function is essential for sperm energy metabolism and motility.
GK2 is a testis-enriched metabolic kinase that phosphorylates glycerol to generate glycerol-3-phosphate (G3P), a critical intermediate linking glycolysis, lipid metabolism, and oxidative phosphorylation [36, 37]. Unlike the somatic isoform GK1, GK2 is specifically expressed in spermatocytes, spermatids, and mature sperm, and localizes to the sperm midpiece (mitochondrial sheath) and flagellum [38]. Its catalytic activity is positively regulated by PKA-mediated phosphorylation at Ser26, which enhances GK2’s ability to promote glycolysis-dependent ATP production – an essential energy source for sperm motility in the female reproductive tract, where oxidative phosphorylation is relatively limited [39]. Notably, GK2 can interact with voltage-dependent anion channel (VDAC) proteins in the mitochondrial outer membrane, thereby regulating ADP/ATP exchange between the cytosol and mitochondria [36]. Gk2−/− mice display reduced sperm motility, abnormal mitochondrial morphology, and male infertility [38]. In humans, decreased GK2 expression and impaired Ser26 phosphorylation are associated with asthenozoospermia, suggesting that GK2 may serve as a candidate biomarker for male infertility.
In addition to the canonical signaling kinases TSSKs, PLKs, and IGF1R discussed above, these metabolic kinases integrate phosphorylation signaling with energy metabolism to form a more comprehensive regulatory network. Their stage-specific expression patterns and phosphorylation-dependent functions underscore the importance of metabolic-phosphorylation crosstalk in spermatogenesis and sperm functional maturation.
2.1.2.5 Tyrosine Kinases (TKs): C-kit and TAM family in germ cell maturation and sertoli cell function
Tyrosine kinases (TKs) mediate signal transduction through the reversible phosphorylation of tyrosine residues, acting as pivotal regulators of cell proliferation, differentiation, survival, and intercellular communication. In mammalian spermatogenesis, two evolutionarily conserved subgroups of receptor tyrosine kinases – c-Kit and the TAM family (TYRO3, AXL, and MER) – orchestrate germ cell development and Sertoli cell homeostasis, directly impacting sperm quantity, motility, and morphology [40, 41]. Their phosphorylation-dependent signaling is integrated with serine/threonine kinase pathways (e.g., CDKs and the PI3K–AKT axis) to form a coordinated regulatory network essential for male fertility.
c-Kit is a receptor tyrosine kinase highly expressed in differentiating type A spermatogonia and early spermatocytes, while its ligand, stem cell factor (SCF), is predominantly produced by Sertoli cells in the testis [42, 43]. Ligand-induced c-Kit dimerization triggers autophosphorylation of key tyrosine residues (Tyr568 and Tyr570 in the juxtamembrane domain; Tyr703 in the kinase domain), which creates docking sites for downstream signaling adaptors such as PI3K and GRB2 and activates cascades including PI3K–AKT and MAPK–ERK [44, 45]. During spermatogenesis, c-Kit phosphorylation governs two major processes: (1) spermatogonial fate determination – c-Kit activation promotes the transition from undifferentiated spermatogonia to differentiating type A spermatogonia, and retinoic acid-regulated c-Kit signaling helps balance spermatogonial self-renewal and differentiation [46]; and (2) meiotic progression – c-Kit-mediated AKT activation inhibits apoptotic signaling via BCL-2 family proteins, thereby protecting spermatocytes from premature cell death during meiosis [42]. Genetic disruption of c-Kit, as in W/Wv mice, leads to severe oligozoospermia or azoospermia due to depletion of differentiating germ cells and meiotic arrest [47]. Clinically, reduced Tyr568 phosphorylation of c-Kit has been detected in testicular tissue from patients with idiopathic oligozoospermia, and phospho-c-Kit (Tyr570) levels correlate with sperm concentration, suggesting that c-Kit phosphorylation status may serve as a biomarker of impaired spermatogenesis.
The TAM family comprises three receptor tyrosine kinases – TYRO3, AXL, and MER – that are expressed in germ cells and Sertoli cells. Their ligands, Gas6 and protein S, bind to the extracellular domain and induce autophosphorylation of intracellular tyrosine residues (e.g., Tyr789 in AXL, Tyr800 in MER, Tyr818 in TYRO3), thereby initiating downstream signaling [48]. In Sertoli cells, TYRO3 and AXL activation regulates proliferation, polarity, and blood–testis barrier (BTB) integrity via STAT3 and PI3K–AKT pathways [49]. MER deficiency impairs the phagocytosis of apoptotic germ cells by Sertoli cells, leading to seminiferous tubule disorganization and progressive germ cell loss. In spermatids, TYRO3 signaling promotes acrosome biogenesis and flagellar assembly and is associated with increased phosphorylation of tubulin and ODF1; accordingly, Tyro3-knockout mice display abnormal sperm head morphology (misshapen acrosomes) and reduced progressive motility [50]. AXL signaling, activated by Gas6 during epididymal maturation, enhances sperm motility in part by promoting phosphorylation of flagellar dynein light chain [51].
Notably, c-Kit and TAM kinases exhibit extensive crosstalk with other phosphorylation-dependent pathways to fine-tune spermatogenesis. For example, c-Kit–mediated ERK activation synergizes with CDK7 to regulate retinoic acid (RA)-dependent spermatogonial differentiation [52]. These interactions underscore the complexity of TK-driven phosphorylation networks in coordinating germ cell development and Sertoli cell function.
2.2 Key phosphatases and their roles in spermatogenesis
Phosphatases function as essential counter-regulators that terminate or tune phosphorylation-dependent signaling. By opposing kinase activity, they maintain phosphorylation homeostasis and ensure appropriate signaling kinetics. Accordingly, phosphatase dysregulation can perturb spermatogenesis and contribute to infertility [53].
2.2.1 PP2A
Protein phosphatase 2A (PP2A) is one of the most important serine/threonine phosphatases in eukaryotic cells, responsible for the dephosphorylation of most serine/threonine residues within the cell [54, 55]. In male reproductive cells, the expression and function of PP2A are strictly regulated, which is crucial for spermatogenesis and functional maturation.
2.2.1.1 PP2A in meiotic timing and progression
Meiotic transitions are strongly phosphorylation-dependent [56]. Classic studies using okadaic acid (OA) – an inhibitor of PP1/PP2A – showed that robust phosphatase inhibition induces premature meiotic entry in mammalian spermatocytes, demonstrating that serine/threonine phosphatases are required to preserve correct meiotic timing [57]. Although these experiments did not fully dissociate PP1 from PP2A contributions, they established a core principle: phosphatase activity prevents inappropriate or premature meiotic progression, consistent with a model in which PP2A dephosphorylates key cell-cycle substrates to enable orderly stage transitions [58, 59].
2.2.1.2 PP2A in sperm maturation and capacitation
After exiting the testis, sperm undergo epididymal maturation and capacitation, both of which require stringent control of phosphorylation dynamics. Partial PP2A inhibition in bovine and hamster sperm increases motility and the proportion of hyperactivated sperm, consistent with PP2A acting to restrain premature hyperactivation via dephosphorylation of motility-related substrates [60–62]. PP2A also modulates the acrosome reaction: selective inhibition can rapidly induce acrosome exocytosis in human sperm, indicating context-dependent roles in preventing premature activation while permitting fertilization-associated signaling at the appropriate time [60, 63].
2.2.2 PP1
Protein phosphatase 1 (PP1) is another major serine/threonine phosphatase that, together with PP2A, is responsible for most of the intracellular dephosphorylation events [64, 65]. Research has shown that PP1 is equally crucial for male fertility, especially during the spermatogenesis stage. PP1 participates in the morphogenesis and functional maturation of sperm cells by regulating specific substrate proteins [66]. Although its mechanism of action has not been fully elucidated, like PP2A, existing evidence suggests that dysfunction of PP1 can severely affect sperm development and lead to male infertility. In sperm capacitation studies, the use of inhibitors that can simultaneously inhibit PP1 and PP2A (such as okadaic acid and phellomycin A) to treat sperm can increase protein phosphorylation levels and motility in sperm from various mammals (including mice, cows, pigs, humans, and monkeys), indirectly indicating the role of PP1 in maintaining basal phosphorylation levels and motility status of sperm.
2.2.3 Other phosphatases
Human sperm expresses additional phosphatase classes that likely fine-tune capacitation signaling.
2.2.3.1 Protein Tyrosine Phosphatases (PTPs)
Unlike serine/threonine phosphorylation, tyrosine phosphorylation shows a significant increasing trend during sperm capacitation and is one of the markers of sperm functional activation [67]. Therefore, the role of protein tyrosine phosphatases (PTPs) has also attracted much attention. Research has found that, in the process of sperm capacitation, it is necessary to inhibit the activity of serine/threonine phosphatases (such as PPP1CC2) while maintaining the activity of tyrosine phosphatases in order to obtain normally functioning sperm. This indicates that tyrosine phosphatase plays a positive role in regulating signaling pathways related to sperm capacitation [68]. For example, PTP1D (also known as SHP-2) is expressed in sperm and may be involved in regulating signal transduction related to capacitation [69]. In addition, PTP1C (also known as SHP-1) has been identified for the first time in human sperm. Although its specific function in sperm remains to be elucidated, given its critical regulatory role in cell proliferation and differentiation signaling pathways in hematopoietic cells, it is likely to play an important role in spermatogenesis as well.
Dual-specificity phosphatases (DSPs) are capable of dephosphorylating both tyrosine and serine/threonine residues and play important negative regulatory roles in cell signaling [70]. Among them, MAPK phosphatase 1 (MKP1) was first identified in human spermatozoa [71]. The main function of MKP1 is to dephosphorylate and inhibit the activity of its target MAPKs, such as JNK and p38MAPK. In immune cells, such as macrophages, up-regulation of MKP1 expression correlates with a decrease in JNK and p38MAPK activity, acting as a negative feedback mechanism to control the inflammatory response [72]. A similar phenomenon was observed in human spermatozoa, suggesting that MKP1 may regulate certain functions of spermatozoa, such as the stress response or apoptotic processes, by inhibiting the MAPK pathway [73]. Given the important role of the MAPK pathway in sperm capacitation, MKP1 may be involved in maintaining the homeostasis of sperm function by finely regulating MAPK activity.
2.3 Functional phosphosites and protein interaction networks
Ultimately, phosphorylation matters because it changes how specific proteins behave and interact. In spermatogenesis, many key proteins carry stage-specific phosphosites that alter their DNA binding, stability, localization, or ability to form complexes – thereby shaping sperm structure and function.
2.3.1 Protamine phosphorylation
Protamines are the ultimate executors of highly compressed sperm chromatin, and their post-translational modifications, especially phosphorylation, are crucial for the proper packaging of chromatin. In human sperm, multiple sites of protamines PRM1 and PRM2 have been confirmed to undergo phosphorylation, including the serine 11 site (PRM1S11) of PRM1, the serine 59 site (PRM2S59) of PRM2, and the serine 9 site (PRM1S9) of PRM1 [74–76]. Although the precise regulatory mechanisms and functions of these phosphorylation sites are still under further investigation, it is widely believed that they play a critical role in the binding of fish sperm proteins to DNA and the interactions between fish sperm proteins. Phosphorylation of protamines may be a dynamic process, and in the early stages of sperm formation, phosphorylation may contribute to the correct folding of protamines and their binding to DNA [77]. In the late stage of sperm maturation, with the formation of disulfide bonds and zinc bridges, phosphate groups may be removed, making the chromatin structure more stable and dense. TSSK kinase has been shown to phosphorylate fish sperm proteins, which directly links kinase activity to chromatin remodeling. The phosphorylation abnormalities of these key sites may lead to chromatin packaging defects, which are closely related to male infertility, especially an increase in the sperm DNA fragmentation rate.
2.3.2 Phosphorylation of flagellar structural proteins
Sperm flagella are the executive organs of sperm movement, and their complex structure and function are precisely regulated by phosphorylation modifications. This intricate regulation is crucial for the successful navigation of spermatozoa through the female reproductive tract to achieve fertilization. The CatSper channel, a sperm-specific calcium channel, plays a pivotal role in this process by organizing signaling proteins along the flagella into distinct calcium domains, which, in turn, orchestrate tyrosine phosphorylation and motility [78]. This spatial and temporal organization of phosphorylation events is essential for sperm to acquire the capacity to fertilize, highlighting the importance of phosphorylation in regulating sperm motility.
2.3.2.1 AKAP3/4 tyrosine phosphorylation in motility and capacitation
A-kinase-anchoring proteins (AKAPs) serve as critical structural components of the sperm fibrous sheath, facilitating the spatial organization of signaling molecules, notably protein kinase A (PKA), thereby enabling localized regulation of downstream substrate phosphorylation [79]. During the process of human sperm capacitation, AKAP3 and AKAP4 undergo substantial tyrosine phosphorylation. This phosphorylation event has been demonstrated to be regulated by an insulin-like growth factor 1 receptor (IGF1R)-mediated signaling pathway [23]. The tyrosine phosphorylation of AKAP3 and AKAP4 may induce alterations in the fibrous sheath structure or facilitate interactions with other proteins, thereby promoting the hyperactivated motility of spermatozoa, which is essential for penetrating the egg’s outer structure [80, 81]. Consequently, the phosphorylation of AKAPs not only serves as a hallmark of sperm capacitation but also provides a molecular foundation for its functional maturation.
2.3.2.2 ODF1 and flagellar integrity
Outer dense fibers (ODFs) constitute the primary structural elements within the mid-segment of the sperm flagellum, serving to provide essential rigidity [21]. The protein ODF1 is identified as the predominant constituent of ODFs. Recent findings indicate an interaction between the Fused (Fu) kinase and the ODF1 protein [82]. Although direct phosphorylation of ODF1 by Fu was not demonstrated in this study, the kinase activity of Fu and its co-localization with ODF1 suggest a potential regulatory role. It is hypothesized that Fu may influence the assembly or stability of ODFs through the phosphorylation of ODF1. Notably, Odf1 knockout mice display phenotypes characterized by sperm head and neck detachment, alongside male infertility, highlighting the critical role of ODF1 in preserving spermatozoa structural integrity [83]. Consequently, the phosphorylation regulation of flagellar structural proteins such as ODF1 by Fu kinase may represent a pivotal mechanism in ensuring proper sperm flagellar formation and function.
2.3.3 Phosphorylation as a driver of network remodeling
Phosphorylation is a critical post-translational modification (PTM) that plays a pivotal role in regulating protein function and cellular processes. However, it often does not act in isolation but works synergistically with other PTMs to regulate sperm development and function. This synergy is evident in the complex interplay between phosphorylation and other PTMs, such as ubiquitination, acetylation, and SUMOylation, which collectively influence sperm capacitation, motility, and fertility potential.
The role of phosphorylation in sperm function is underscored by its involvement in sperm capacitation, a process essential for acquiring fertilization competence. During capacitation, phosphorylation events are closely linked with oxidative PTMs, such as S-nitrosylation and S-glutathionylation, which modulate protein function and sperm motility. The interplay between these modifications is crucial for maintaining redox balance and ensuring proper protein phosphorylation, as demonstrated by the regulation of zona-pellucida binding proteins and flagellar proteins during capacitation [84]. This crosstalk between phosphorylation and oxidative modifications highlights the integrated nature of PTMs in sperm function.
Moreover, the human sperm proteome reveals that various PTMs, including phosphorylation, acetylation, and glycosylation, are intricately involved in regulating sperm proteins. These modifications are not only essential for basic cellular functions but also play a direct role in sperm-specific processes, such as motility and fertilization. The functional enrichment of these PTMs suggests that they may serve as potential biomarkers for fertility-associated traits, emphasizing the importance of PTM crosstalk in sperm biology [85]. This is further supported by studies showing that PTMs like SUMOylation and ubiquitination are involved in the regulation of protein stability and function, which are critical for spermatogenesis and sperm function [86, 87]. A recent investigation into sperm maturation in buffaloes elucidated a synergistic regulatory network involving these modifications by integrating data from phosphorylation and ubiquitination proteomics [88]. The study identified numerous proteins subjected to both phosphorylation and ubiquitination within two critical pathways: the proteasome and glycolysis. Specifically, in the proteasome pathway, eight proteins, including PSMC1 and PSMA4, exhibited both modifications, while in the glycolysis pathway, five proteins, such as GAPDH and ALDOC, were similarly co-modified. This phenomenon of co-modification suggests that one modification may influence the occurrence of the other or collaboratively determine the ultimate fate of the protein, affecting aspects such as activity regulation, subcellular localization, or degradation. These complex interactions between modifications offer novel insights into the molecular mechanisms underlying sperm maturation.
The dynamic nature of PTM interactions is also evident in the regulation of deubiquitinating enzymes (DUBs), where PTMs such as phosphorylation, acetylation, and SUMOylation modulate enzyme activity and substrate specificity. This regulation is crucial for maintaining the ubiquitin-proteasome system, which is vital for protein turnover and cellular homeostasis in sperm cells [89]. Additionally, the crosstalk between phosphorylation and O-GlcNAcylation, where these modifications target similar amino acids, further illustrates the complexity of PTM interactions in regulating protein function and cellular processes [90].
In conclusion, phosphorylation modification in sperm development and function is part of a broader network of PTMs that collectively regulate protein activity and cellular processes. The synergistic interactions between phosphorylation and other PTMs, such as ubiquitination, acetylation, and oxidative modifications, underscore the complexity and precision of regulatory mechanisms in sperm biology. Understanding these interactions provides valuable insights into the molecular underpinnings of sperm function and fertility, with potential implications for therapeutic interventions in male infertility.
3. Roles of phosphorylation in key biological processes of spermatogenesis
Protein phosphorylation regulates virtually every major transition in spermatogenesis – from the proliferation and differentiation of spermatogonia, meiotic recombination and chromosome segregation, to spermatid remodeling (spermiogenesis), and finally to post-testicular sperm maturation and capacitation. By modulating protein activity, localization, stability, and interactions, phosphorylation provides the timing and specificity required for orderly germ-cell development (Figure 1).
Figure 1.
Key phosphorylation regulatory networks in spermatogenesis and sperm maturation. The integrated flow diagram illustrates the sequential stages of spermatogenesis, highlighting stage-specific roles of key kinases and phosphatases in orchestrating this process through dynamic protein phosphorylation. From left to right, the developmental timeline encompasses: (1) spermatogonial proliferation and differentiation, regulated by the balanced action of kinases (e.g., IGF1R tyrosine kinase) and phosphatases (e.g., PP1/PP2A), controlling cell cycle progression and stem cell renewal at the seminiferous tubule basement membrane; (2) meiosis and chromosome dynamics, in spermatocytes, kinases such as polo-like kinases (plks) and phosphatases (PP1/PP2A) coordinate essential events including chromosome pairing, recombination, and segregation; (3) spermiogenesis, the morphological transformation of spermatids is driven by a distinct kinase repertoire (e.g., TSSK family, TTBK2, CSNK1G1), which regulates critical processes such as acrosome formation, flagellar assembly, and histone-to-protamine transition for chromatin condensation; (4) epididymal maturation and post-epididymal capacitation, final functional maturation, including motility acquisition and hyperactivation, is governed by kinase cascades (e.g., PKA, tyrosine kinases), with phosphorylation levels markedly increasing during capacitation. Integrated regulation and PTM crosstalk (bottom panel): phosphorylation events are functionally integrated with other post-translational modifications (ptms) – such as ubiquitination, acetylation, and oxidative modifications – through synergistic or competitive crosstalk, collectively fine-tuning protein function, localization, and stability throughout spermatogenesis.
3.1 Phosphorylation regulation in proliferation and differentiation of spermatogonia
The initiation phase of spermatogenesis is characterized by the proliferation and differentiation of spermatogonia, a process intricately regulated by various signaling pathways, with phosphorylation modifications playing a pivotal role. Cell cycle protein-dependent kinase 7 (CDK7) is a crucial kinase involved in the regulation of spermatogonia proliferation and differentiation [26, 28]. CDK7 influences spermatogonia development by modulating retinoic acid (RA)-mediated c-KIT expression. Inhibition of CDK7 activity results in impaired proliferation and abnormal differentiation of spermatogonia, indicating that CDK7 is essential for maintaining normal spermatogonial development. The mammalian target of rapamycin protein complex 1 (mTORC1) is integral to the transition from spermatogonial stem cells to progenitor cells [91, 92]. Research has demonstrated that mTORC1 facilitates the formation of progenitor clones from activated spermatogonial stem cells by regulating transcriptional and insulin-like growth factor (IGF) signaling pathways. Inhibition of mTORC1 leads to the accumulation of activated spermatogonial stem cells, thereby hindering their differentiation into progenitor cells. Retinoic acid (RA) is a key signaling molecule that initiates spermatogonia differentiation [93]. It has been found that there are differences in the responsiveness of different types of spermatogonia to RA, and this difference is partly determined by the intracellular phosphorylation status. Cytochrome P450 family 26 enzyme (CYP26) prevents progenitor spermatogonia from responding prematurely to RA by degrading RA, whereas a small fraction of undifferentiated spermatogonia enriched for spermatogonial stem cells exhibit catabolism-independent insensitivity to RA [94, 95].
In addition to CDK7 and mTORC1, the C-kit tyrosine kinase plays a pivotal role in spermatogonial regulation. SCF-induced C-kit phosphorylation (Tyr568/Tyr570) promotes the transition of undifferentiated spermatogonia to differentiated progenitors, while its sustained activity – modulated by retinoic acid and CDK7-mediated signaling – maintains SSC self-renewal [43, 96]. Dysregulation of this pathway leads to SSC depletion and oligozoospermia, highlighting the interdependence of tyrosine and serine/threonine kinase networks in spermatogonial homeostasis.
3.2 Meiosis
Meiosis is a germ cell–specific division that halves chromosome number and promotes genetic diversity through homologous recombination. Its progression depends on precisely coordinated phosphorylation events. The role of phosphorylation in meiosis is underscored by the involvement of cyclin-dependent kinases (CDKs) and Dbf4-dependent kinase (DDK), which are crucial for genome duplication and maintenance during both mitotic and meiotic divisions. These kinases orchestrate diverse processes during cellular reproduction, with meiosis-specific adaptations in their regulation and functions in DNA metabolism [97]. The phosphorylation of key proteins, such as FZR1/CDH1 by CDKs, is essential for the temporal regulation of the Anaphase Promoting Complex/Cyclosome (APC/C) activity, which is critical for meiosis II entry and the maintenance of spermatogonia [98].
3.2.1 Chromosome pairing, double-strand break repair, and recombination
During prophase of meiosis, the precise pairing of homologous chromosomes (association) and the exchange of genetic material (recombination) are critical processes. These processes are facilitated by a complex array of protein machinery, whose functions are modulated by phosphorylation. For instance, the histone methyltransferase PRDM9 orchestrates the formation of DNA double-strand breaks (DSBs) – the initial step of homologous recombination – by catalyzing the deposition of H3K4me3 at specific genomic loci [99]. Conversely, the deposition of H3K9me2/3, catalyzed by Suv39h1/2, is crucial for maintaining genomic integrity [100]. Both these histone modifications and the proteins that recognize them may have their activity or localization regulated by phosphorylation. For example, knockout mice lacking the Zcwpw1 gene experience meiotic arrest due to inefficient DSB repair, underscoring the significance of modifications such as phosphorylation in the DSB repair pathway [101]. Furthermore, the protein phosphatase PP2A has been identified as a key regulator of meiotic progression, with dysregulation of its activity leading to disruptions in meiotic timing [102].
3.2.2 Spindle assembly and chromosome segregation
Both meiotic divisions necessitate accurate spindle assembly and precise chromosome segregation. The spindle apparatus is primarily composed of microtubules, whose dynamic instability is modulated by various kinases and phosphatases. Members of the Polo-like kinase (PLK) family, including Plk1 and Plk4, are integral to centromere replication and spindle assembly [103, 104]. Research indicates that transient deletion of Plk1 results in aberrant sperm flagellar structure, while disruption of Plk4 leads to the complete absence of flagella, thereby underscoring the critical role of these kinases in the regulation of microtubule-associated structures. During meiosis, these kinases modulate spindle morphology and dynamics by phosphorylating substrates such as microtubule-binding proteins, thereby ensuring the equitable distribution of chromosomes into daughter cells. Conversely, phosphatases such as PP1 and PP2A maintain phosphorylation homeostasis by counteracting kinase activity, thus facilitating accurate chromosome segregation [105–107].
3.3 Spermatid morphogenesis (spermiogenesis)
Spermiogenesis converts round spermatids into streamlined, motile spermatozoa through coordinated organelle remodeling, tail assembly, and chromatin compaction. Phosphorylation is a central regulator of these transformations.
3.3.1 Acrosome biogenesis
The acrosome is a lysosome-like organelle essential for fertilization. Its formation depends on Golgi-derived vesicle trafficking and membrane fusion events that are regulated by kinase and phosphatase signaling [108]. For example, Fused (FU) kinase localizes to acrosomal structures, and its loss disrupts sperm head shaping, consistent with impaired acrosome development [21]. Acrosome biogenesis is also influenced by epigenetic control of acrosomal genes; SETD2-mediated H3K36me3, for instance, regulates the expression of acrosome-associated factors such as Acrbp1 [109]. Together, these findings suggest coordinated regulation between phosphorylation signaling and chromatin-based mechanisms during acrosome formation.
3.3.2 Flagellum formation and axoneme assembly
The sperm flagellum, required for motility, is built around a “9 + 2” axonemal microtubule core and associated accessory structures (outer dense fibers, fibrous sheath). Multiple kinases contribute to flagellar assembly and maintenance. TSSK family kinases are essential: TSSK1/2 double-knockout mice display severe flagellar defects and infertility [110]. FU kinase also regulates flagellar architecture, including the organization of accessory structures [21]. In addition, PLK1 and PLK4 have been implicated in normal flagellum formation, consistent with kinase control of microtubule organization and associated protein networks [111]. Mechanistically, these kinases likely act by phosphorylating tubulins, dynein components, and structural proteins to coordinate assembly, stability, and movement.
3.3.3 Chromatin remodeling and the histone-to-protamine transition
A defining feature of spermiogenesis is extreme chromatin condensation, driven by histone replacement with transition proteins (TNP1/2) and protamines (PRM1/2). This process is governed by coordinated post-translational modifications. Histone hyperacetylation (e.g., H4 hyperacetylation) promotes chromatin relaxation and facilitates histone eviction [112]. TSSK kinases can phosphorylate transition proteins and protamines, and these phosphorylation events are important for proper DNA binding and orderly packaging [20]. Protamines also undergo dynamic phosphorylation: early phosphorylation may promote folding and deposition onto DNA, whereas later dephosphorylation may stabilize the final condensed state. Epigenetic regulators, such as JMJD1A (JHDM2A), which removes H3K9me2 at promoters of Tnp1 and Prm1, are required for correct expression of packaging factors; defects in this pathway impair spermiogenesis and cause infertility [113, 114]. Overall, spermatid chromatin remodeling emerges from tightly coupled phosphorylation, acetylation, and methylation networks.
3.4 Sperm capacitation
Capacitation is the functional maturation process that occurs in the female reproductive tract (or in vitro) and enables fertilization. It is characterized by the acquisition of hyperactivated motility and the competence to undergo the acrosome reaction. Phosphorylation – particularly tyrosine phosphorylation of flagellar proteins and serine/threonine phosphorylation during membrane remodeling – is a core molecular driver of this process.
3.4.1 Tyrosine phosphorylation and hyperactivated motility
The most prominent functional manifestation of sperm capacitation is a transition in motility pattern, from symmetrical progressive movement to vigorous, asymmetric, hyperactivated motion. This shift is closely linked to a marked increase in tyrosine phosphorylation of proteins within the sperm tail [41]. Several sperm tail components, notably the fibrous sheath proteins AKAP3 and AKAP4, undergo tyrosine phosphorylation during capacitation-associated changes in ATP production [79]. This post-translational modification is thought to induce subtle alterations in flagellar architecture and/or modulate the activity of dynein motor proteins, thereby facilitating the emergence of hyperactivated motility. In addition, α- and β-tubulin have been identified as substrates for tyrosine phosphorylation in capacitated sperm [115]. Collectively, these findings support tyrosine phosphorylation as a crucial signaling mechanism that drives hyperactivated motility and promotes efficient penetration of the oocyte’s surrounding barriers, including the cumulus matrix and zona pellucida.
3.4.2 Serine/threonine phosphorylation and membrane remodeling
In addition to tyrosine phosphorylation, serine/threonine phosphorylation contributes to capacitation, particularly in the context of plasma membrane remodeling. In human sperm, increases in serine/threonine phosphorylation have been observed, which are independent of the classical bicarbonate/cAMP axis and instead linked to albumin-driven cholesterol efflux. Because cholesterol loss is an early capacitation event that increases membrane fluidity and primes downstream signaling and the acrosome reaction, these phosphorylation changes are thought to regulate membrane-associated proteins and help establish a capacitation-competent membrane state. In addition to PKA- and IGF1R-mediated phosphorylation, sperm capacitation is also tightly regulated by metabolic kinases. Specifically, AMPK activation (Thr172 phosphorylation) can be promoted by the cAMP/PKA pathway and intracellular Ca2+, enhancing glycolytic ATP production to support hyperactivated motility. In parallel, PKA-mediated phosphorylation of GK2 at Ser26 strengthens glycerol metabolism, which is critical for maintaining fertilization competence in the largely glycolysis-dependent microenvironment of the female reproductive tract [36].
3.4.3 cAMP/PKA signaling and pathway integration
The bicarbonate–cAMP–PKA pathway is a canonical initiator of capacitation. Bicarbonate activates soluble adenylyl cyclase (sAC), elevating cAMP and activating PKA, which phosphorylates downstream targets to trigger capacitation-associated changes. AKAP proteins spatially organize this signaling by anchoring PKA to specific subcellular domains (notably the flagellum), enabling localized phosphorylation of motility regulators. Capacitation signaling also intersects with nitric oxide (NO) pathways: NO can modulate sperm proteins through S-nitrosylation and phosphorylation, influencing chaperones (e.g., HSPA2) and metabolic enzymes (e.g., PKM2), thereby affecting motility and sperm–oocyte interactions. Collectively, these interconnected pathways form a multilayered phosphorylation network that coordinates capacitation.
4. Association of phosphorylation with male infertility
Aberrant protein phosphorylation is closely associated with multiple forms of male infertility. Because phosphorylation regulates spermatogenesis, sperm maturation, and fertilization, disruption of this network can impair sperm number, morphology, or function, and ultimately reduce fertility.
4.1 Abnormal phosphorylation as diagnostic biomarkers
Conventional semen analysis evaluates sperm concentration, motility, and morphology, but it often fails to explain idiopathic infertility. Phosphoproteomics provides an additional molecular layer of information that can improve diagnosis and patient stratification (Figure 2).
Figure 2.
Pathological mechanisms of phosphorylation dysregulation in male infertility. The diagram contrasts the functional consequences of a precisely regulated versus a malfunctioning phosphorylation “switchboard” in the context of male fertility, highlighting its role as a key determinant of sperm quality. Left panel – balanced phosphorylation network (fertile): illustrates a state of kinase-phosphatase equilibrium, where correct phosphorylation of key effector proteins governs essential sperm functions. Examples include: proper phosphorylation of flagellar motor proteins enabling normal motility; accurate modification of chromatin condensation factors ensuring compact chromatin and genomic stability; and regulated phosphorylation of capacitation regulators leading to successful capacitation. This coordinated signaling network culminates in the production of functionally competent sperm and fertility. Right panel – dysregulated phosphorylation network (infertile): depicts a pathological state characterized by network imbalance (e.g., hyperactive kinases or suppressed phosphatases), resulting in aberrant phosphorylation signatures. This dysregulation directly underpins common infertility phenotypes: faulty phosphorylation of motor proteins causes poor motility/asthenozoospermia; incorrect modification of chromatin factors leads to abnormal chromatin compaction and increased DNA fragmentation; and dysregulated signaling through key spermatogenesis regulators can result in meiotic arrest or oligozoospermia. The collective outcome is infertility or subfertility. Bottom – diagnostic potential: the contrasting “normal” and “altered” phosphorylation signatures, visualized as distinct mass spectrometry profiles, underscore the utility of phosphoproteomic signatures as sensitive, molecular-level indicators of sperm quality and diagnostic biomarkers for male infertility.
4.1.1 Sperm tyrosine phosphorylation patterns and sperm quality
The extent of tyrosine phosphorylation during sperm capacitation serves as a critical indicator of sperm functional potential. The pattern of sperm tyrosine phosphorylation has been shown to be closely associated with the sperm’s capacity to bind to the egg’s zona pellucida [116, 117]. Abnormal tyrosine phosphorylation is frequently observed in the sperm of men with idiopathic infertility. For example, a study in patients with teratospermia, characterized by morphologically abnormal spermatozoa, demonstrated that the extent of tyrosine phosphorylation under capacitating (“energizing”) conditions was significantly reduced in abnormal sperm compared with normal sperm [118]. This reduction may impair capacitation-associated energy production and thereby decrease fertilization success. Another study corroborated these findings by showing that tyrosine phosphorylation levels in the neck and principal piece of abnormal spermatozoa were significantly lower than those in normal spermatozoa following incubation in capacitating medium. Together, these observations suggest that the levels and patterns of sperm tyrosine phosphorylation could serve as potential biomarkers for evaluating sperm functional quality and diagnosing male infertility.
4.1.2 Phosphorylation changes in specific proteins
In addition to global phosphorylation changes, abnormalities in specific phosphorylated proteins have been implicated in male infertility. Heat shock protein family A member 2 (HSPA2) is a critical chaperone expressed in spermatozoa and enriched at the sperm surface; reduced HSPA2 expression is correlated with diminished sperm binding to the zona pellucida and may contribute to male infertility [119]. Experimental data indicate that the phosphorylation state of HSPA2 is modulated by nitric oxide (NO), suggesting that its function may be regulated through phosphorylation-dependent mechanisms [120]. Interestingly, proteomic analyses of spermatozoa from men with asthenozoospermia have reported upregulation of HSPA2 expression, accompanied by downregulation of other proteins such as KLK2, SORD, and ANXA2 [121]. These findings suggest that dysregulation of HSPA2 – including altered expression and phosphorylation status – together with changes in other phosphoproteins, may reflect disruptions in sperm energy metabolism and function.
AKAP3 and AKAP4 are pivotal scaffolding components of the sperm fibrous sheath that organize PKA signaling and regulate flagellar function; their phosphorylation status is integral to ATP production during capacitation and motility [79]. Abnormal phosphorylation of HSPA2, AKAP3, AKAP4 and related proteins may therefore contribute to defective sperm function and could provide informative molecular biomarkers for male infertility.
Abnormal phosphorylation of tyrosine kinases further contributes to infertility: reduced C-kit Tyr568 phosphorylation is associated with oligozoospermia due to SSC depletion [42], while impaired TYRO3/AXL phosphorylation correlates with asthenoteratozoospermia – attributed to disrupted Sertoli-germ cell communication and flagellar assembly defects [50]. These phosphorylation signatures, detectable in testicular tissues or seminal plasma, expand the pool of potential molecular biomarkers for idiopathic male infertility.
4.1.3 Phosphatase expression profiles as diagnostic indicators
Alterations in the expression and activity of phosphatases, which act as the “erasers” of phosphorylation marks, can also significantly affect sperm function. A study examining phosphatase expression profiles in human spermatozoa identified distinct differences between normal and abnormal samples [69]. Specifically, serine/threonine phosphatases such as PPP1CC2, PPP1CB, PPP4C, and PPP6C, together with tyrosine phosphatases like PTP1C and the dual-specificity phosphatase MKP1, exhibited differential expression and subcellular localization in spermatozoa. These variations in phosphatase expression profiles may reflect disruptions in the phosphorylation regulatory network during spermatogenesis, suggesting their potential utility as molecular biomarkers for the diagnosis and possibly the prognosis of male infertility. In the future, diagnostic platforms integrating phosphatase expression and activity, either independently or in conjunction with traditional semen analysis, may offer more precise and mechanistically informed diagnostic insights for clinical practice.
In line with the metabolic kinases discussed above, dysregulated phosphorylation of these enzymes further expands the pool of potential infertility biomarkers. Reduced Thr172 phosphorylation of AMPK (impairing energy sensing) and hypophosphorylation of AK9 at Ser115 (disrupting nucleotide homeostasis) have both been reported in men with asthenozoospermia, while impaired Ser26 phosphorylation of GK2 may serve as a specific indicator of motility disorders linked to mitochondrial energy metabolism defects [31, 32].
Because phosphorylation is mechanistically linked to sperm development and function, components of phosphorylation circuits represent potential therapeutic targets for infertility treatment – and, conversely, for non-hormonal contraception.
4.2.1 Targeting IGF1R signaling to improve capacitation and motility
The IGF1R-mediated tyrosine phosphorylation pathway is essential for human sperm capacitation and hyperactivated motility [23]. Consequently, modulating IGF1R activity through pharmacological or biological agents may represent a therapeutic strategy to enhance sperm function. For instance, in patients experiencing impaired sperm capacitation due to diminished IGF1R signaling, the use of IGF1R agonists could be explored to augment this pathway, thereby improving sperm motility and fertilization potential. Conversely, for individuals seeking contraception, the development of specific IGF1R inhibitors could offer a novel approach to male contraception.
4.2.2 Modulating key kinases and phosphatases
TSSK kinase and PP2A phosphatase are integral to the processes of spermatogenesis and functional maturation [15, 60]. Consequently, they represent promising therapeutic targets. In cases where patients exhibit defective sperm chromatin packaging attributable to aberrant TSSK function, future pharmacological interventions could potentially involve the development of agents that mimic or enhance TSSK activity. Regarding PP2A, its bifunctional role in sperm capacitation – both in preventing hyperactivation and facilitating the acrosome reaction – indicates that its activity must be precisely regulated. The creation of drugs capable of selectively modulating PP2A activity within specific substrates or cellular compartments may offer novel therapeutic strategies for addressing sperm motility disorders or abnormalities in the acrosome reaction.
4.2.3 Site-specific intervention strategies
A more comprehensive understanding of the roles of critical phosphorylation sites could enhance the precision of future therapeutic strategies, enabling direct targeting of specific phosphorylation events. For instance, identifying key phosphorylation sites associated with sperm dysfunction, such as those on A-kinase anchoring proteins (AKAPs) or specific sites on piscine sperm proteins, could facilitate the development of small molecules or peptides designed to selectively modulate phosphorylation at these sites. Such targeted intervention strategies are anticipated to reduce side effects while achieving precise regulation of sperm function. Nonetheless, achieving this objective necessitates more in-depth and systematic investigations into the phosphorylation regulatory network.
5. Applications of emerging phosphoproteomics technologies in spermatogenesis research
Recent advances in mass spectrometry–based phosphoproteomics have substantially deepened our understanding of phosphorylation networks in spermatogenesis. These high-throughput, sensitive methods enable systematic identification and quantification of phosphoproteins and phosphosites in germ cells and sperm, providing a mechanistic foundation for studying development, maturation, and infertility (Figure 3).
Figure 3.
Phosphoproteomics in spermatogenesis: from discovery to clinical translation. The schematic illustrates a transformative three-phase workflow that transitions from foundational discovery to clinical impact, enabled by advanced phosphoproteomic technologies and integrative analysis. Discovery and mapping (left): this initial phase utilizes high-resolution mass spectrometry-based phosphoproteomics (e.g., DIA/SWATH-MS, single-cell approaches) to analyze testicular tissues (across developmental stages) and sperm samples from fertile and infertile individuals. The output is a high-definition, stage-resolved map of phosphorylation dynamics, identifying thousands of phosphorylation sites and pinpointing key regulatory kinases and phosphatases (e.g., TSSK2, CSNK1G1). Integrated “mechanism-technology-clinical” analysis (center): the core of the framework integrates multidimensional data: (1) spatiotemporally resolved phosphoproteomics, (2) epigenetic interaction analyses to decipher crosstalk between phosphorylation and other ptms, and (3) AI-assisted network inference. This synthesis defines causal phosphorylation circuits and identifies master regulatory nodes (e.g., TSSK kinase family, AKAP scaffolds) within the spermatogenic regulatory network. Clinical and translational applications (right): the identified regulatory nodes drive three key translational avenues: (1) Novel diagnostic biomarkers: phosphorylation signatures for non-invasive assessment of conditions like oligozoospermia and sperm DNA fragmentation. (2) Non-hormonal male contraceptives: development of targeted inhibitors against essential spermatogenic kinases (e.g., TSSK-specific compounds). (3) Fertility restoration and assisted reproduction: application of phosphorylation state knowledge to refine sperm selection for ICSI and optimize in vitro culture conditions, potentially improving IVF outcomes. This integrated framework represents a paradigm shift toward mechanism-driven diagnostics and therapeutics in andrology.
5.1 Advances in phosphoproteomics technologies
A typical phosphoproteomics workflow includes (i) phosphopeptide enrichment, (ii) LC–MS/MS identification and quantification, and (iii) computational analysis [122]. Progress across all three steps has markedly increased coverage, precision, and reproducibility.
5.1.1 Quantitative MS-based phosphoproteomics
Phosphorylated proteins are typically present in lower abundance within the total protein pool, necessitating their enrichment prior to mass spectrometry analysis. Techniques such as immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) chromatography are frequently employed for the enrichment of phosphorylated peptides [123, 124]. When combined with high-performance liquid chromatography (LC) separation and tandem mass spectrometry (MS/MS) identification, these methods enable large-scale identification and quantification of phosphorylated proteins. For instance, one study utilized IMAC-TiO2 enrichment coupled with LC–MS/MS to compare the phosphorylated proteome of human spermatozoa before and after capacitation, successfully identifying key phosphorylated proteins, including AKAP3 and AKAP4 [11]. Another study employed quantitative phosphoproteomics analysis to elucidate a 56-protein interaction network during sperm capacitation. The implementation of these techniques offers a robust approach for mapping the dynamic phosphorylation events associated with spermatogenesis.
5.1.2 Data-independent acquisition (DIA) and high-resolution mass spectrometry
Traditional Data-Dependent Acquisition (DDA) methodologies encounter challenges in detecting low-abundance phosphorylated peptides. In contrast, Data-Independent Acquisition (DIA) techniques, exemplified by SWATH-MS, facilitate the acquisition of more comprehensive and reproducible quantitative data through the systematic fragmentation of all peptides [125]. When integrated with high-resolution, high-sensitivity mass spectrometers, such as the Orbitrap Astral, DIA technology allows for enhanced proteomic coverage and more precise quantification of the sperm proteome. The deployment of these advanced technologies empowers researchers to more reliably identify critical proteins and pathways associated with sperm function, discover biomarkers linked to male infertility, and establish a foundation for targeted therapeutic interventions. Astral-DIA technology is acknowledged as a significant conduit between fundamental research and clinical applications, owing to its superior accuracy and reproducibility [126].
5.1.3 Multi-omics integration to reconstruct regulatory networks
A singular histological technique typically elucidates only one aspect of biological activity. By integrating phosphoproteomics with other histological methodologies, such as genomics, transcriptomics, ubiquitination, and metabolomics, researchers can systematically elucidate the regulatory mechanisms governing spermatogenesis from multiple perspectives. For instance, the combination of phosphoproteomics with ubiquitination proteomics can uncover the interactions between these two critical post-translational modifications, as demonstrated in studies of buffalo sperm maturation [88]. Conversely, integrating phosphoproteomics with metabolomics can investigate the role of phosphorylation signaling in regulating sperm energy metabolism, which is crucial for understanding the mechanisms underlying sperm motility dysfunction, such as impaired spermatogenesis [127]. This integrated multi-omics analytical approach offers a more comprehensive and systematic understanding of spermatogenesis and male infertility.
5.2 Single-cell and spatial phosphoproteomics
Spermatogenesis involves substantial cellular heterogeneity across stages and lineages, making bulk measurements inherently averaging. Emerging single-cell and low-input phosphoproteomic methods address this limitation [128]. For example, SPARCE (Streamlined Phosphoproteomic Analysis of Rare Cells) enables multiplexed phosphoproteomic quantification from low cell numbers (on the order of ~ 1,000 sorted cells) by integrating streamlined processing, advanced isobaric labeling (e.g., TMT), and phosphopeptide enrichment [129]. Microfluidic platforms further miniaturize and integrate capture, lysis, labeling, and enrichment, improving throughput and sensitivity for rare populations [130]. In parallel, spatial approaches such as MALDI imaging mass spectrometry (often combined with shotgun proteomics) can localize molecular signatures within testis tissue architecture, providing spatial context for phosphorylation-associated processes and infertility-related alterations [131].
5.3 Advances in bioinformatics and network inference
Bioinformatics is essential for extracting biological meaning from phosphoproteomics. Tools such as iGPS (in vivo GPS) predict kinase–substrate relationships by combining sequence motif preferences with protein–protein interaction context to reduce false positives [132]. More recent approaches use machine learning (including node-embedding methods trained on curated resources such as PhosphoSitePlus) to improve kinase–substrate association accuracy [133]. Beyond site assignment, integrative pipelines map phosphoproteomic changes onto functional annotations and networks – using GO/KEGG enrichment, protein interaction networks, and pathway-based modeling – to reconstruct signaling circuits and identify stage-specific regulators in spermatogenesis [134].
5.4 Applications of phosphoproteomics in spermatogenesis research
Phosphoproteomics has become a core approach for dissecting phosphorylation-dependent regulation during spermatogenesis and sperm function, enabling systematic discovery of stage-specific signaling events, key regulators, and clinically relevant biomarkers.
5.4.1 Mapping the dynamic phosphorylation landscape of spermatogenesis
By profiling phosphoproteomes across developmental stages – spermatogonia, spermatocytes, round spermatids, and mature sperm – researchers can reconstruct the dynamic phosphorylation program that accompanies germ-cell differentiation. For example, a large-scale mouse study identified 735 testis-specific, highly expressed phosphoproteins enriched in pathways related to histone regulation, chromosome organization, and ciliogenesis [11]. Such stage-resolved maps capture spatiotemporal phosphorylation dynamics and generate prioritized candidates for downstream functional validation.
Guo et al. conducted a comprehensive mapping of the dynamic phosphorylation events occurring during mouse spermatogenesis by utilizing phosphorylation modification histology technology [13]. The researchers successfully isolated spermatocytes from four critical developmental stages of mouse spermatogenesis and identified 3,681 differentially expressed proteins along with 5,119 differentially phosphorylated sites, 2,059 of which were novel discoveries. These phosphorylated proteins were found to play significant roles in acrosomogenesis, mRNA processing, flagellar assembly, and chromatin remodeling, exhibiting distinct regulatory patterns across different stages. Through cluster analysis and the integration of a kinase substrate annotation database, CSNK1G1 and TTBK2 were identified as key kinases. In vivo experiments further demonstrated that CSNK1G1 is involved in the regulation of acrosome biosynthesis and sperm motility, whereas TTBK2 contributes to the stability of IFT88 through phosphorylation, thereby maintaining flagellum formation and sperm morphology.
In 2025, researchers from Nanjing Medical University and Southeast University conducted a comprehensive analysis of the phosphorylation profile associated with mouse epididymal sperm maturation, utilizing phosphoproteomics technology [13]. Their study identified 1,407 phosphorylated proteins and 6,447 phosphorylated sites across sperm from the head, body, and tail regions of the mouse epididymis. The differentially phosphorylated proteins were significantly enriched in pathways pertinent to sperm motility, energy metabolism, sperm capacitation, and fertilization. Subsequent investigations revealed that TSSK2 plays a crucial regulatory role in the acquisition of sperm motility, with its inhibition markedly reducing sperm motility in the tail region of the mouse epididymis.
5.4.2 Identifying key phosphoproteins and pathways underlying sperm function
Comparative phosphoproteomics is particularly effective for pinpointing proteins and signaling pathways that control sperm motility, capacitation, and the acrosome reaction [11]. For instance, phosphoproteome comparisons before and after capacitation have highlighted regulators such as AKAP3/4 and have implicated IGF1R-dependent tyrosine phosphorylation as a central pathway in human sperm capacitation [23]. Likewise, contrasts between normal and dysfunctional sperm can reveal phosphorylation changes linked to impaired function, helping to define pathogenic mechanisms.
5.4.3 Discovering phosphorylation biomarkers for male infertility
Reliable biomarkers are essential for improving the diagnosis and stratification of male infertility, and phosphoproteomics offers a high-throughput route to identify them. By comparing sperm or seminal plasma from fertile controls with patients with asthenozoospermia, teratozoospermia, or azoospermia, studies can nominate phosphorylation sites or phosphoproteins with diagnostic potential [11, 77]. For example, altered phosphorylation of HSPA2 and AKAP family proteins has been associated with reduced sperm quality [119]. While clinical translation requires rigorous validation in large, diverse cohorts and standardized assays, phosphoproteomics provides a strong foundation for developing more precise diagnostic tools.
6.1 Untangling the complexity of phosphorylation networks
Although phosphorylation research has advanced rapidly, phosphorylation regulatory networks in spermatogenesis remain incompletely defined. Future work should integrate multi-omics datasets (genomics, transcriptomics, proteomics, and metabolomics) to build a layered, systems-level map of phosphorylation control across spermatogenesis. Such frameworks will clarify how phosphorylation interfaces with other regulatory mechanisms to generate stage-specific and cell-type–specific outcomes. Because spermatogenesis is highly ordered in space and time, key priorities include technologies that capture spatiotemporal dynamics – such as real-time monitoring and spatially resolved phosphoproteomics. Another unresolved challenge is functional redundancy: multiple sites or pathways may converge on the same process, masking causal relationships. Defining when redundancy is protective, compensatory, or biologically essential will be critical for therapeutic strategies.
6.2 Building dynamic, spatiotemporally resolved phosphorylation maps
A major goal is to generate high-resolution, dynamic phosphorylation atlases across developmental stages and cell types. Advances in mass spectrometry imaging and single-cell approaches make it feasible to map phosphorylation with spatial and cellular precision, enabling direct links between phosphorylation states and specific spermatogenic transitions. Complementary strategies – such as pulse labeling and time-resolved profiling – are needed to capture phosphorylation kinetics and identify causal, transient events. Given marked cellular heterogeneity during spermatogenesis, improved single-cell phosphoproteomic methods will be essential, particularly to characterize rare or short-lived populations.
6.3 Phosphorylation in epigenetic regulation
The interplay between phosphorylation and epigenetic control is a key emerging frontier. Site-specific demethylation events during spermatogenesis appear to predefine nucleosome retention in sperm. Future studies should determine how phosphorylation regulates methylation/demethylation machinery and how these processes jointly shape gene expression programs. In addition, phosphorylation likely cooperates with histone methylation, acetylation, and ubiquitination through coordinated “histone code” mechanisms. For example, DOT1L recognition of methylated H3K79 and ubiquitinated H2B suggests combinatorial regulation [135]. Systematic mapping of these interactions will be necessary to establish mechanistic rules. Epigenetic information carried by sperm can influence offspring phenotypes. Whether phosphorylation contributes directly to such inheritance – and how environmental exposures modulate these marks – remains largely unexplored and could reshape our understanding of paternal effects.
6.4 From mechanism to clinical application
A central objective is to translate basic discoveries into tools for reproductive medicine. Large-scale phosphoproteomics should enable robust phosphorylation-based biomarkers for male infertility, supported by standardized assays and clinically validated thresholds. In addition, because phosphorylation defects may arise from distinct genes and pathways across patients, precision strategies should combine genotype, pathway signatures, and phosphorylation profiles to guide targeted treatment, including emerging gene- and RNA-based approaches where appropriate.
Because phosphorylation is essential for spermatogenesis and sperm function, selective inhibitors of key regulatory kinases (e.g., TSSK family members) are promising candidates for non-hormonal male contraceptives, provided specificity and safety can be established. Mechanistic insights may refine IVF/ICSI protocols by identifying phosphorylation states linked to fertilization competence and embryo quality, enabling improved sperm selection and culture conditions.
Protein phosphorylation is a core regulatory mechanism across mammalian spermatogenesis, governing spermatogonial proliferation and differentiation, meiotic progression, spermiogenesis, and epididymal maturation. Recent advances in high-resolution mass spectrometry, single-cell methods, and computational analysis have greatly expanded the male germline phosphoproteome. Large-scale studies now report thousands of phosphorylation sites and identify stage-specific regulators – such as CSNK1G1, TTBK2, and TSSK2 – with defined roles during spermatogenic progression. Clinically, phosphorylation abnormalities are increasingly associated with male infertility, supporting the development of phosphorylation-based biomarkers and targeted interventions. Continued innovation in single-cell phosphoproteomics and mass spectrometry imaging should further accelerate mechanistic discovery.
Despite this progress, the phosphorylation circuitry of spermatogenesis remains incompletely defined. Key priorities include: (i) constructing integrated regulatory maps that capture crosstalk among phosphorylation events and with other post-translational and epigenetic modifications; (ii) developing technologies that resolve phosphorylation dynamics with high spatiotemporal precision; (iii) determining how phosphorylation contributes to epigenetic programming and potential transgenerational effects; and (iv) strengthening translational pipelines to deliver validated diagnostics, personalized therapies, and non-hormonal contraceptive strategies. Clarifying how phosphorylation coordinates sperm development will advance reproductive biology and improve the diagnosis and treatment of male infertility.
This research was funded by the National Natural Science Foundation of China (31972537), the Department of Science and Technology in Henan Province (242102111015), the Key Research Projects of Higher Education Institutions in Henan Province (24A230015), the Postgraduate Education Reform and Quality Improvement Project of Henan Province (YJS2025KC33), and the Nanhu Scholars Program of Xinyang Normal University.
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Written By
Haixia Xu, Kaili Zhou, Kejin Ren, Yijia An, Tiantian Meng,
Xiaofang Cheng, Cencen Li, Pengpeng Zhang and Yongjie Xu
Submitted: 31 December 2025Reviewed: 13 January 2026Published: 23 February 2026
Open access peer-reviewed chapter - ONLINE FIRST
