Int J Stem Cells.  2024 Aug;17(3):298-308. 10.15283/ijsc23093.

Cytoplasmatic Localization of Six1 in Male Testis and Spermatogonial Stem Cells

• Qin M
• Ma L
• Du W
• Chen D
• Luo G
• Liu Z

• KMID: 2558607
• DOI: http://doi.org/10.15283/ijsc23093

Abstract

Sine oculis homeobox 1 (Six1) is an important factor for embryonic development and carcinoma malignancy. However, the localization of Six1 varies due to protein size and cell types in different organs. In this study, we focus on the expression and localization of Six1 in male reproductive organ via bioinformatics analysis and immunofluorescent detection. The potential interacted proteins with Six1 were also predicted by protein-protein interactions (PPIs) and Enrichr analysis. Bioinformatic data from The Cancer Genome Atlas and Genotype-Tissue Expression project databases showed that SIX1 was highly expressed in normal human testis, but low expressed in the testicular germ cell tumor sample. Human Protein Atlas examination verified that SIX1 level was higher in normal than that in cancer samples. The sub-localization of SIX1 in different reproductive tissues varies but specifically in the cytoplasm and membrane in testicular cells. In mouse cells, single cell RNA-sequencing data analysis indicated that Six1 expression level was higher in mouse spermatogonial stem cells (mSSCs) and differentiating spermatogonial than in other somatic cells. Immunofluorescence staining showed the cytoplasmic localization of Six1 in mouse testis and mSSCs. Further PPIs and Enrichr examination showed the potential interaction of Six1 with bone morphogenetic protein 4 (Bmp4) and catenin Beta-1 (CtnnB1) and stem cell signal pathways. Cytoplasmic localization of Six1 in male testis and mSSCs was probably associated with stem cell related proteins Bmp4 and CtnnB1 for stem cell development.

Keyword

Subcellular localization; Six1; Testis; Mouse spermatogonial stem cells; Protein-protein interactions

Figure

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  Fig. 1 SIX1 expression in human cancers and localization in testicular germ cell tumor (TGCT) samples. (A) The expression levels of SIX1 in 33 human cancer samples based on GEPIA2 online examination compare with normal tissues. TGCT, breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), ovarian serous cystadenocarcinoma (OV), uterine corpus endometrial carcinoma (UCEC), and uterine carcinosarcoma (UCS). T: tumor, N: normal. Red bar represents tumor, blue bar represents normal. *p<0.05. (B) SIX1 localization in testis and tumor tissues by HPA001893 staining. Black arrows indicated the positive signal for staining and scale bar=10 μm.

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  Fig. 2 Sub-localization of SIX1 in cancer samples vs. the controls by HPA001893 staining. (A) SIX1 expression is undetectable in normal ovary. The localization of SIX1 in ovarian serous cystadenocarcinoma is in the nucleus. (B) SIX1 expression is undetectable in normal endometrium, but highly expressed in the uterine corpus endometrial carcinoma and localized in the nucleus. (C) SIX1 expression is undetectable in normal breast tissue, but highly expressed in the breast invasive carcinoma and localized in the nucleus. (D) SIX1 highly expressed in glandular cells of cervix samples, and localized in both cytoplasmic/membranous and nucleus, while SIX1 in cervical squamous cell carcinoma and endocervical adenocarcinoma was high expression in the nucleus. All scale bars=200 μm.

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  Fig. 3 Six1 expression and localization in mouse testis. (A) Dotplot illustration for gene expression based on single cell RNA-sequencing data analysis of mouse testis. L: leptotene, Z: zygotene, P: pachytene, D: diplotene, MII: meiosis II, RS: round sperm. (B) Triple immunofluorescent staining of 10-week adult mouse testis by Vasa (red), Gfra1 (green), and Six1 (purple). The nucleus was stained by DAPI (blue). Scale bar=10 μm.

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  Fig. 4 Six1 expression in mouse spermatogonial stem cells (mSSCs). (A) EGFP positive mSSCs were cultured and photographed with fluorescent microscope. Scale bar=20 μm. (B) Western blot analysis by Tubulin (55 kDa), Gfra1 (55 kDa), and Six1 (37 kDa) in mSSCs. (C) Immunofluore-scent examination of Vasa (red)/Gfra1 (green) and Vasa (red)/Six1 (green) colocalization in mSSCs. The nuclei were stained by DAPI (blue). All pictures were taken with 63× magni-fication. Scale bar=20 μm.

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  Fig. 5 Protein-protein interaction (PPI) examination of potential proteins that may interact with Six1. (A) Human SIX1 PPI network was analyzed and visualized by STRING and Cytoscape. (B) Mouse Six1 PPI network was analyzed and visualized by STRING and Cytoscape. (C) Gene ontology (GO) cell component term analysis of human SIX1 interacted proteins based on PPI analysis. (D) GO cell component term analysis of mouse Six1 interacted proteins based on PPI analysis. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of human Six1 interacted proteins based on PPI analysis. (F) KEGG analysis of mouse Six1 interacted proteins based on PPI analysis. Asterisk (*) indicates significant difference with p<0.01, and the blue-colored part indicates p<0.05.

Reference

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