Given the broad physiological response scale of purinoceptors, purinergic signaling has been proposed to play a role in controlling germ cell maturation at different developmental stages. In mice, twelve such stages are sequentially transitioned to complete one seminiferous epithelial cycle. Accordingly, immunohistochemical investigation of cell type- and stage-dependent protein expression has been notoriously difficult. Early work described immunoreactivity for several P2X receptor subtypes in the rat testis (30). Various germ cell types throughout different stages of the seminiferous epithelial cycle were found immunopositive for P2X2, P2X3, and P2X5 receptors. By contrast, P2X4 and P2X6 receptors appeared absent from rat testis samples – a finding that was later contradicted by Ko and coworkers (31). P2X1 receptors were exclusively detected in blood vessels and P2X7 antibody staining was restricted to Sertoli cells (30). Notably, P2X2 and P2X3 isoforms, which frequently form functional heteromers in the nervous system (32), were usually observed in the same cell types and stages (30).

Recently, we combined gene expression analysis, immuno- and bioanalytical chemistry, protein knockdown, and single-cell electrophysiology to gather functional evidence for purinergic signaling in male germ cells (33). We identified a multidimensional ATP response pathway consisting of both P2X4 and P2X7 receptors and downstream Ca²⁺-activated large conductance (BK) K⁺ channels in prepubescent mouse spermatogonia ( Figure 2A_III ). P2X4 and P2X7 receptors display distinct ATP affinities, and their activation triggers transmembrane currents with characteristic kinetics that enable unequivocal electrophysiological isoform identification. Cooperatively activated by concurrent membrane depolarization and increased cytoplasmic Ca²⁺, hyperpolarizing BK channels provide a negative feedback mechanism that counteracts the effects of P2X receptor activation and ensures swift repolarization of the spermatogonial membrane potential (33).

While some of the apparent discrepancies between the above studies (30, 31, 33) likely result from species [mouse (33) versus rat (30, 31)] and/or age [juvenile (33) versus adult (30, 31)] differences, they also highlight the limitations of unidirectional (i.e., immunochemistry-only) protein expression analysis. Electrophysiological recordings from postmeiotic germ cells in acute seminiferous tubule slices of adult mice are technically challenging. Our own unpublished data nonetheless indicate functional expression of a fast activating and slowly desensitizing ATP-activated channel in postmeiotic spermatocytes and/or round spermatids ( Figure 2A_IV ). The molecular identity of this putative P2X receptor remains to be identified.

Given the emerging role of extracellular ATP in numerous physiological signaling processes, it is tempting to speculate that spermatozoa might be exposed to varying concentrations of extracellular ATP in the testis, epididymis, and/or female reproductive tract. ATP might, therefore, play a role in modulating sperm fertilizing capacity. In humans, extracellular ATP has been reported to increase the fertilizing potential of sperm and, accordingly, sperm exposure to ATP during IVF treatment has been suggested (36). Early studies report that extracellular ATP triggers acrosome exocytosis in human sperm via P2X-dependent Na⁺ influx (37, 38). In rat spermatozoa, P2X7 has been proposed to mediate the ATP-triggered acrosome reaction (39). While the acrosomal membrane is as yet inaccessible to electrophysiological recordings, acrosomal P2X receptor currents remain to be verified. A different mechanism was found in bovine spermatozoa, where extracellular ATP appears to activate P2Y receptors. The resulting elevation in cytoplasmic Ca²⁺ activates PKCα, which triggers acrosomal exocytosis (40). In 2007, Edwards et al. quantified the effects of extracellular ATP on acrosomal exocytosis, protein tyrosine phosphorylation, and sperm motility parameters in human sperm (41). In healthy and asthenozoospermic donors, ATP had no impact on acrosome exocytosis or tyrosine phosphorylation. However, it significantly altered sperm motility, increasing curvilinear velocity and percentage of hyperactivation. This observation might explain the previously described benefits of ATP supplement during IVF treatment.

Navarro et al. reported a nonselective cation current in the midpiece of mouse spermatozoa that is activated by external ATP exposure ( Figure 2A_V ) (34). This current matches the kinetics and pharmacological profile reported for recombinant P2X2 and, importantly, is absent in P2X2^-/- mice. Despite the loss of this ATP-gated current, P2X2^-/- spermatozoa show unaltered motility and acrosome reaction. However, P2X2^-/- males are subfertile when given the chance to mate at high frequencies, indicating that P2X2 adds a selective advantage under frequent mating conditions. The authors hypothesize that increased intracellular Ca²⁺ through P2X2 energizes sperm mitochondria in the midpiece, presumably as a consequence of Ca²⁺-dependent potentiation of enzymes in the Kreb’s cycle (42).

Work from multiple laboratories suggests that extracellular ATP triggers a rapid and transient increase in the cytosolic Ca²⁺ concentration of Sertoli cells, albeit with partly conflicting propositions for the underlying purinoceptor isoforms (31, 33, 43–47).

Endocrine control of spermatogenesis along the hypothalamic–pituitary–testicular axis converges on Sertoli cells (48). Sertoli cell function is centrally regulated by gonadotropins, either directly by follicle stimulating hormone (FSH) or indirectly by luteinizing hormone-dependent generation of dihydrotestosterone. FSH surges trigger cAMP production and mobilization of cytosolic Ca²⁺ in Sertoli cells (48). Interestingly, both ATP and its uridine derivative UTP inhibit FSH-dependent cAMP accumulation by 70% in rat Sertoli cells, suggesting that P2Y2 or P2Y4 receptors are involved (43). Moreover, rapid IP₃ accumulation was observed upon ATP exposure in primary cultures of rat and mouse Sertoli cells, in line with P2Y2 or P2Y4 receptor activation (49). In rat Sertoli cells, extracellular ATP evoked 17β-estradiol production/secretion. This effect depended on both membrane depolarization via Na⁺ influx (implicating P2X receptors) and Ca²⁺ release from internal stores (suggesting a concurrent role of P2Y receptors) (47).

Both receptor identification and direct functional characterization of purinergic signaling in mouse Sertoli cells were performed by Veitinger et al. in 2011 as well as Fleck et al. in 2016 (33, 45). P2X2 and P2Y2 are the prevailing purinoceptors ( Figure 2A_II ) with confirmatory results obtained from both Sertoli cell–germ cell co-cultures (45) and acute seminiferous tubule sections (33). These (electro-)physiological observations are in accordance with early findings by Foresta et al. in rat Sertoli cells. Here, the authors claimed that ATP exposure generates both an increase in cytosolic Ca²⁺ by release from intracellular stores (P2Y receptors) and a depolarizing Na⁺ influx consistent with P2X receptor activation (43). Notably, Veitinger and coworkers establish that mitochondria serve as essential regulatory components of Sertoli cell purinergic Ca²⁺ signaling (45).

Spermatogenesis completes with the release of still immotile spermatozoa from the seminiferous epithelium into the lumen of the seminiferous tubule. After detachment from Sertoli cells, sperm must therefore be transported towards the rete testis and epididymis for further maturation. Accordingly, precisely regulated tubular transport mechanisms are imperative for reproduction.

Early on, observations of minute motions of seminiferous tubule segments (50, 51) have sparked speculation about a critical role for smooth muscle-like TPCs (52, 53) in male (in)fertility through mediating contractile tubule movements (54, 55). However, direct experimental in vivo evidence for paracrine control of TPC contractions has been lacking (56) and quantitative live-cell measurements of seminiferous tubule contractions are rare and controversial (57–60). Somewhat surprisingly, early work explicitly excluded extracellular ATP as an activator of TPCs (61). By contrast, we recently reported both ATP-dependent Ca²⁺ signals and adenosine-dependent proinflammatory actions in human TPCs in vitro (62, 63). Notably, we also identified purinergic signaling pathways as physiological triggers of tubular contractions both in vitro and in vivo. By acting on ionotropic (P2X2 and/or P2X4) and metabotropic (P2Y2) purinoceptors ( Figure 2 ), extracellular ATP elevates cytosolic Ca²⁺ ( Figure 2B ), activates TPC contractions, and triggers stage-dependent directional sperm movement within the mouse seminiferous tubules (3). Combining recordings from primary mouse and human TPC cultures as well as acute mouse seminiferous tubule slices with intravital multiphoton imaging of intact tubules, we provide direct and quantitative evidence for purinergic TPC signaling that triggers robust peristaltic movement of luminal sperm (3). Electrophysiological and Ca²⁺ imaging data suggest that, while metabotropic P2Y signaling is sufficient to induce ATP-dependent contractions, influx of extracellular Ca²⁺ through ionotropic P2X receptors enhances TPC contractions. While the full picture is admittedly still incomplete, current data support a concept of Ca²⁺-induced Ca²⁺ release mechanisms that amplify ATP-dependent excitation-contraction coupling.

Being under androgen control, expression of TPC contractility proteins initiates with puberty and, notably, TPC-selective androgen receptor knock-out renders mice infertile (64). Both findings underscore a potential role of TPC contractions in male (in)fertility. Accordingly, pharmacological targeting of purinergic signaling pathways to (re)gain control of TPC contractility represents an attractive approach for male infertility treatment or contraceptive development (3). Still, translation of TPC contractions and their putative role(s) from mice to humans awaits further physiological investigation.