RESULTS

SCGN is enriched in gonadotropes and lactotropes in the mouse anterior pituitary gland

By integrating publicly available scRNA‐seq data of the pituitary gland from C57BL/6 strain mice, we annotated and clustered cell populations based on the expression signatures of key transcription factors, categorizing them into major types including lactotropes, gonadotropes, somatotropes, thyrotropes, and corticotropes (Figure 1A). Enrichment analysis of SCGN transcript expression revealed that SCGN is primarily enriched in lactotropes and gonadotropes, with minor distribution observed in somatotropes, thyrotropes, and corticotropes (Figure 1B,C). We further quantified the proportion of SCGN‐expressing cells within each anterior pituitary cell type in female and male mice. The proportions were similar between sexes, and the majority of SCGN‐positive cells were gonadotropes and lactotropes (Figure 1D).

FIGURE 1: scRNA‐seq data analysis on the expression of SCGN in the anterior pituitary gland of mice (n = 5). A: Anterior pituitary gland cell clusters identified by genetic markers and visualized with UMAP. B: SCGN in the anterior pituitary gland of mice at the cell level. C: Expression of SCGN in 9 types of mice anterior pituitary gland cells. D: SCGN positive cells of male and female mice (2 female, 3 male). Circle represents the cell number percentage.

Considering the potential role of SCGN in regulating the secretion of pituitary hormones, we performed double immunofluorescence staining to analyze the colocalization of SCGN with specific hormone markers (e.g., LH, FSH, GH, PRL, ACTH, TSH) in the anterior pituitary gland tissues from 2‐month‐old female and male C57BL/6 mice (Figure 2A–L and Figure 3A–L). In the anterior pituitary gland, SCGN is expressed in the cytoplasm of adenohypophyseal cells. The various subtypes of pituitary hormone‐secreting cells display distinct distribution patterns. Quantitative analysis of SCGN colocalization with various anterior pituitary hormones was conducted to evaluate its expression distribution. In males, SCGN showed the highest colocalization with LH (37.293% ± 11.830%), FSH (25.808% ± 5.322%), moderate rates were observed with PRL (21.176% ± 5.647%) and ACTH (15.762% ± 3.914%), while colocalization was minimal with GH (4.618% ± 1.747%) and TSH (4.135% ± 2.928%) (Figure 2M). In females, SCGN frequently colocalized with LH (39.922% ± 8.655%), FSH (50.346% ± 11.917%), and PRL (43.367% ± 4.336%). Lower colocalization rates were found with ACTH (15.381% ± 3.763%), GH (19.747% ± 2.859%), and TSH (4.897% ± 2.337%) (Figure 3M). The immunofluorescence colocalization results indicated that SCGN was mainly distributed in gonadotropes and lactotropes in the mouse anterior pituitary gland, which are highly consistent with the scRNA‐seq results.

FIGURE 2: Immunofluorescence colocalization assays of SCGN in the anterior pituitary gland of 2‐month‐old male C57BL/6 mice (n = 5). A‐B: Colocalization of SCGN (red) and LH (green) in the anterior lobe (A), with a higher‐magnification view of the boxed area showing colocalization (arrows) (B). C‐D: Colocalization of SCGN (red) and FSH (green) in anterior lobe (C), with a higher‐magnification view of the boxed area showing colocalization (arrows) (D). E,F: Colocalization of SCGN (red) and ACTH (green) in anterior lobe (E), with a higher‐magnification view of the boxed area showing colocalization (arrows) (F). G‐H: Localization of SCGN (red) and TSH (green) in anterior lobe (G), with a higher‐magnification view of the boxed area showing the detailed localization (H). I‐J: Colocalization of SCGN (red) and PRL (green) in anterior lobe (I), with a higher‐magnification view of the boxed area showing colocalization (arrows) (J). K,L: Localization of SCGN (red) and GH (green) in anterior lobe (K), with a higher‐magnification view of the boxed area showing the detailed localization (L). Scale bars: 500 μm (A, C, E, G, I, K), 50 μm (B, D, F, H, J, L). Strong SCGN immunoreactivity observed in the posterior lobe.M: Quantitative analysis of colocalization rates between SCGN and individual anterior pituitary gland hormones in male mice. Data in the figure are mean ± SEM. Different letters above bars indicate significant differences (p < .05), bars sharing the same letter are not significantly different. One‐way ANOVA revealed a significant main effect of hormone type on colocalization rate (F (5, 24) = 27.75, p < .0001).

FIGURE 3: Immunofluorescence colocalization assays of SCGN in the anterior pituitary gland of 2‐month‐old female C57BL/6 mice (n = 5). A,B: Colocalization of SCGN (red) and LH (green) in the anterior lobe (A), with a higher‐magnification view of the boxed area showing colocalization (arrows) (B). C,D: Colocalization of SCGN (red) and FSH (green) in anterior lobe (C), with a higher‐magnification view of the boxed area showing colocalization (arrows) (D). E,F: Colocalization of SCGN (red) and ACTH (green) in anterior lobe (E), with a higher‐magnification view of the boxed area showing colocalization (arrows) (F). G,H: Localization of SCGN (red) and TSH (green) in anterior lobe (G), with a higher‐magnification view of the boxed area showing the detailed localization (H). I,J: Colocalization of SCGN (red) and PRL (green) in anterior lobe (I), with a higher‐magnification view of the boxed area showing colocalization (arrows) (J). K,L: Colocalization of SCGN (red) and GH (green) in anterior lobe (K), with a higher‐magnification view of the boxed area showing colocalization (arrows) (L). Scale bars: 500 μm (A, C, E, G, I, K), 50 μm (B, D, F, H, J, L). Strong SCGN immunoreactivity observed in the posterior lobe. M: Quantitative analysis of colocalization rates between SCGN and individual anterior pituitary gland hormones in female mice. Data in the figure are mean ± SEM. Different letters above bars indicate significant differences (p < .05), bars sharing the same letter are not significantly different. One‐way ANOVA revealed a significant main effect of hormone type on colocalization rate (F (5, 24) = 46.66, p < .0001).

SCGN is predominantly expressed in corticotropes in the male porcine anterior pituitary gland

We performed scRNA‐seq on pituitary gland from 3‐month‐old normal male pigs. Cell populations were annotated and clustered based on the expression signatures of key transcription factors, identifying 10 distinct cell types in the anterior pituitary, including corticotropes, gonadotropes, lactotropes, somatotropes, and thyrotropes (Figure 4A). Enrichment analysis of SCGN transcript expression revealed that SCGN transcripts were primarily enriched in corticotropes and gonadotropes, with lower expression levels in other cell types (Figure 4B,C). Furthermore, quantification of the proportion of SCGN‐expressing cells across various anterior pituitary gland cell types in male pigs demonstrated that among SCGN‐positive cells, the proportions classified as corticotropes and gonadotropes were significantly higher than those of other cell types (Figure 4D), suggesting a specific expression trend of SCGN in TPIT‐lineage corticotropes and SF‐1‐lineage gonadotropes.

FIGURE 4: scRNA‐seq data analysis on the expression of SCGN in the anterior pituitary gland of male pig (n = 3). A: Anterior pituitary gland cell clusters identified by genetic markers and visualized with UMAP. B: SCGN in the anterior pituitary gland of pig at the cell level. C: Expression of SCGN in 10 types of male porcine anterior pituitary cells. D: SCGN positive cells of male pig. Circle represents the cell number percentage.

To validate the expression distribution of SCGN at the protein level, we performed double immunofluorescence staining to analyze the colocalization of SCGN with six specific hormone markers in the anterior pituitary tissues of 3‐month‐old male pigs (Figure 5). Quantitative analysis of fluorescence colocalization revealed that SCGN had the highest colocalization rate with ACTH (63.581% ± 4.253%), followed by considerably lower rates with GH (10.759% ± 6.434%) and LH (14.688% ± 7.696%). In contrast, the colocalization with FSH (2.243% ± 1.992%), TSH (0.132% ± 0.118%), and PRL (0.200% ± 0.073%) was almost negligible (Figure 5M). These results indicate that SCGN is primarily and specifically localized in corticotropes. Notably, unlike the scRNA‐seq findings, the colocalization rate of SCGN with gonadotropes was relatively low. Collectively, integrating both single‐cell transcriptomic data and double immunofluorescence staining results, we confirmed that SCGN is primarily and specifically expressed in corticotropes in the anterior pituitary of male pigs.

FIGURE 5: Immunofluorescence colocalization assays of SCGN in the anterior pituitary gland of 3‐month‐old male pig (n = 5). A,B: Colocalization of SCGN (red) and LH (green) in the anterior lobe (A), with a higher‐magnification view of the boxed area showing colocalization (arrows) (B). C,D: Localization of SCGN (red) and FSH (green) in anterior lobe (C), with a higher‐magnification view of the boxed area showing the detailed localization (D). E,F: Colocalization of SCGN (red) and ACTH (green) in anterior lobe (E), with a higher‐magnification view of the boxed area showing colocalization (arrows) (F). G,H: Localization of SCGN (red) and TSH (green) in anterior lobe (G), with a higher‐magnification view of the boxed area showing the detailed localization (H). I,J: Localization of SCGN (red) and PRL (green) in anterior lobe (I), with a higher‐magnification view of the boxed area showing the detailed localization (J). K,L: Colocalization of SCGN (red) and GH (green) in anterior lobe (K), with a higher‐magnification view of the boxed area showing colocalization (arrows) (L). Scale bars: 1.25 mm (A, C, E, G, I, K), 50 μm (B, D, F, H, J, L). Strong SCGN immunoreactivity observed in the posterior lobe. M: Quantitative analysis of colocalization rates between SCGN and individual anterior pituitary gland hormones in male pig. Data in the figure are mean ± SEM. Different letters above bars indicate significant differences (p < .05), bars sharing the same letter are not significantly different. One‐way ANOVA (arcsine square‐root transformed): F(5, 24) = 107.1, p < .0001. Welch’ s ANOVA (heteroscedasticity‐robust): W(5, 10.13) = 318.9, p < .0001.

SCGN expression is enriched in lactotropes, somatotropes, and thyrotropes in human anterior pituitary gland

To investigate the expression pattern of SCGN in the human anterior pituitary gland, we first performed immunohistochemical staining on human anterior pituitary gland tissue sections. The IHC results confirmed SCGN expression at the histological level, with positive signals detected in the cytoplasm of anterior pituitary gland cells (Figure 6A).

FIGURE 6: Immunohistochemistry and scRNA‐seq results of SCGN expression in the human anterior pituitary gland. A: Immunohistochemistry validation confirms SCGN expression in the human anterior pituitary gland (n = 3). B: Anterior pituitary gland cell clusters identified by genetic markers and visualized with UMAP. C: SCGN in the anterior pituitary gland of human at the cell level. D: SCGN positive cells of male and female (4 male, 3 female). Circle represents the cell number percentage. E: Expression of SCGN in 10 types of human anterior pituitary gland cells. Scale bars: 50 μm.

Subsequently, we integrated and analyzed scRNA‐seq data from 4 male and 3 female human pituitary gland samples, which enabled annotation and clustering of cell populations based on key transcription factor expression signatures, identifying 10 major cell types including lactotropes, gonadotropes, somatotropes, thyrotropes, and corticotropes (Figure 6B). Subsequent enrichment analysis of SCGN transcript expression revealed significant enrichment in lactotropes and somatotropes, with minimal distribution observed in thyrotropes, gonadotropes, and corticotropes (Figure 6C–E). Further quantification of SCGN‐expressing cells across anterior pituitary cell types demonstrated similar proportional trends between sexes, with higher rates predominantly observed in lactotropes, somatotropes, thyrotropes, and gonadotropes (Figure 6D). Collectively, the scRNA‐seq data suggest that SCGN exhibits a specific expression trend within the human anterior pituitary, particularly in PIT1‐lineage lactotropes and somatotropes.

Absence of SCGN expression in the rat anterior pituitary gland

By integrating publicly available scRNA‐seq data of the pituitary gland from SD rats, we annotated and clustered cell populations based on the expression signatures of key transcription factors, categorizing them into major types including lactotropes, gonadotropes, somatotropes, thyrotropes, and corticotropes (Figure 7A). Enrichment analysis of SCGN transcript expression revealed no significant enrichment of SCGN in any of these classified cell populations (Figure 7B). Meanwhile, we selected the pancreas and hypothalamus of rats as positive controls, as the presence of SCGN expression has been reported in these tissues. 24 , 25 The RT‐qPCR results showed that SCGN mRNA was detected at high transcription levels in the pancreas and hypothalamus of both male and female rats, but almost none was detected in the anterior pituitary gland (Figure 7C). Similarly, positive cytoplasmic immunostaining signals of SCGN were detected in the pancreas and hypothalamus of rats by immunofluorescent experiments, but no immunostaining signal was observed in the anterior pituitary region (Figure 7D,E).

FIGURE 7: scRNA‐seq data and immunofluorescence analysis of SCGN expression in the anterior pituitary gland of rats. A: Anterior pituitary gland cell clusters identified by genetic markers and visualized with UMAP. B: SCGN in the anterior pituitary gland of rat at the cell level (n = 4). C: Relative SCGN mRNA expression in pancreas, hypothalamus and anterior pituitary gland of male and female rats measured by RT‐qPCR (n = 3). D: Immunofluorescent staining of SCGN (red) in male rat pancreas, hypothalamus and anterior pituitary gland. E: Immunofluorescent staining of SCGN (red) in female rat pancreas, hypothalamus and anterior pituitary gland. Scale bars: 50 μm.