DISCUSSION

Through integrated analysis of scRNA‐seq data from mice, rats, pigs, and human pituitaries, combined with immunofluorescence and immunohistochemical validation, this study reveals the expression and distribution patterns of SCGN in the anterior pituitary across multiple species. The results demonstrate remarkable species‐specific SCGN expression: it is predominantly enriched in PIT1‐lineage lactotropes and SF‐1‐lineage gonadotropes in mice; primarily localized to TPIT‐lineage corticotropes in pigs; and highly expressed in PIT1‐lineage lactotropes and somatotropes in humans. Notably, no SCGN expression was detected in the rat anterior pituitary gland. These findings reveal that SCGN expression is species‐restricted rather than universally conserved. Importantly, the complete absence of SCGN in the rat anterior pituitary suggests that SCGN is not essential for fundamental anterior pituitary hormone secretion, at least in this species.

The species‐specific differential expression of SCGN in the pituitary gland is not an isolated phenomenon. Multiple studies have demonstrated that the expression and distribution of certain regulatory peptides and hormone receptors also vary across species in the pituitary. For instance, galanin is primarily expressed in lactotropes of female rats, where it promotes prolactin secretion, whereas in male rats, it is mainly found in somatotropes, thyrotropes, and corticotropes. 26 In adult male monkeys, galanin expression is more prominent in thyrotropes and gonadotropes, while in the normal human pituitary, it is almost exclusively restricted to corticotropes and exerts broader regulatory effects on multiple neuronal cell types. 27 , 28 Although mice and rats are both rodents, there are also other genes with similar species‐specific expression differences between the two. For example, the rat GnRHR promoter drives expression in the pituitary gland, hippocampus, and testes, while the mouse GnRHR is largely limited to the pituitary gland, and transgenic studies have shown that this difference is determined by the rat promoter itself. 29 Thus, the absence of SCGN in rat and its cell‐type‐specific enrichment in mice, pigs, and humans might reflect species‐specific transcriptional regulation rather than coding sequence differences.

An increase in intracellular calcium concentration serves as the primary intracellular signal triggering the fusion of secretory vesicles with the plasma membrane for the release of hormones and neurotransmitters. Consequently, the presence of SCGN as a calcium‐sensing protein in different hormone‐secreting cell types suggests a potentially conserved mechanism: SCGN may sense intracellular calcium signals triggered by hypothalamic releasing hormones and interact with cytoskeletal and vesicle trafficking‐related proteins, thereby facilitating the directed transport and priming of secretory vesicles toward the plasma membrane. 10 , 30 , 31 , 32 The secretion of LH/FSH is mainly promoted by the binding of GnRH and its receptors in the hypothalamus, which facilitates the influx of extracellular Ca2+ and activates G protein, initiating downstream typical signaling pathways and regulating the biosynthesis and secretion of gonadotropins. 33 , 34 , 35 , 36 SCGN has been identified as a multifunctional calcium sensor that alternately binds SNAP‐25 and Syntaxin‐4 via its hydrophobic groove, acting as a temporal regulator during vesicle recruitment, docking, and release, while competitively inhibiting premature SNARE complex assembly. 37 , 38 , 39 Therefore, in gonadotropes, SCGN may bind SNAP‐25 under resting conditions to prevent excessive spontaneous vesicle fusion. Upon signal activation, Ca2+ influx may induce SCGN dissociation from SNAP‐25 or a conformational change, permitting efficient SNARE complex assembly and thus enabling precise, pulsatile release of LH and FSH. ACTH release is regulated by hypothalamic corticotropin‐releasing hormone (CRH) and occurs via Ca2+‐dependent exocytosis. 40 , 41 We thus speculate that SCGN may also participate in ACTH secretion by modulating the Ca2+‐SNAP‐25 axis. CRH induces Ca2+ influx, SCGN binds to Ca2+, undergoes conformational changes, and releases SNAP‐25, allowing for effective SNARE complex assembly and Ca2+‐triggered ACTH secretion. This mechanism is similar to the process of SCGN in insulin secretion at the molecular level, suggesting that SCGN could act as a Ca2+‐regulated exocytosis‐dependent promoter in cell types where it is expressed. Furthermore, studies have shown that SCGN is involved in CRH secretion regulation within neurons and participates in the systemic stress response pathway, 12 , 42 , 43 suggesting that SCGN's role may extend beyond local pituitary regulation, potentially acting as a component coordinating the hypothalamic–pituitary–adrenal axis at multiple levels to modulate ACTH release. The expression of SCGN in PIT1‐lineage cells, particularly lactotropes, in both humans and mice, suggests its potential involvement in a fundamental secretion mechanism shared across this lineage. Beyond regulation by hypothalamic inhibiting and releasing factors, PRL synthesis and secretion are stimulated by various agents including dopamine, TRH, estradiol, and olfactory marker protein. 44 , 45 , 46 Unlike gonadotropes, a defining secretory characteristic of lactotropes is their capacity for high basal hormone release. This is attributed to spontaneous plateau‐bursting action potentials that drive sustained, efficient voltage‐gated calcium influx, thereby continuously activating Ca2+‐dependent hormone exocytosis. 45 , 47 In this context, SCGN may maintain a readily releasable pool of secretory vesicles by stabilizing SNAP‐25. Additionally, by sensing the persistent calcium influx during action potential plateaus, SCGN could fine‐tune vesicle priming and release probability, supporting the high basal secretory activity of these cells. Meanwhile, the marked species‐specific distribution of SCGN suggests that it is not a universal or essential exocytotic factor. In species where it is expressed, SCGN could act as a cell‐type‐specific auxiliary modulator (or “gain control”) that fine‐tunes Ca2+‐triggered hormone release according to local physiological demands. SCGN enrichment in mouse gonadotropes may support rapid reproductive cycling; in pig corticotropes, it may underpin robust stress‐axis reactivity; in human lactotropes/somatotropes, it may align with sustained lactation and linear growth.

Regarding expression profile similarities, SCGN in the human pituitary gland is predominantly enriched in PIT1‐lineage‐derived lactotropes and somatotropes. Correspondingly, SCGN in the mouse pituitary gland is also distinctly enriched in PIT1‐lineage lactotropes. This key commonality suggests that mice and humans may share similar conserved SCGN‐dependent mechanisms in regulating hormone pathways related to growth, metabolism, and lactation. In contrast, in the porcine model, SCGN is specifically localized to TPIT‐lineage corticotropes. Given the highly overlapping PIT1‐lineage expression profiles between mice and humans, the mouse may serve as a useful model for studying SCGN expression and regulation. In species where SCGN is expressed, it could play a modulatory role in exocytosis. However, the absence of SCGN in the rat anterior pituitary argues against a critical, non‐redundant function, and any speculation about SCGN dysfunction leading to secretory deficiencies must be considered with caution. The specific expression of SCGN in pituitary cells across different species suggests that its expression may be regulated by specific transcription factors (e.g., PIT1, TPIT, SF‐1). Aberrant expression of these transcription factors themselves serves as a core marker for the molecular subtyping of pituitary adenomas. Therefore, SCGN may function as a downstream effector, mediating the tumor proliferation or secretory phenotypes driven by specific transcription factors.

In summary, SCGN exhibits species‐specific expression patterns in the anterior pituitary gland and is completely absent in the rat, suggesting SCGN may not be a universal core component in the exocytosis mechanism of the anterior pituitary. The expression of SCGN in the anterior pituitary gland seems to be evolutionarily different—existing in some mammalian lineages but unnecessary in others. Whether other calcium‐binding proteins compensate for the absence of SCGN in the rat pituitary, or whether SCGN is truly functionally redundant, remains an open question for future investigation. These findings challenge the assumption that SCGN plays a conserved role in endocrine function across mammals and underscore the importance of species‐specific considerations when extrapolating findings from animal models to human physiology.