Comparative expression profiling of testis-enriched genes regulated during the development of spermatogonial cells

Abstract

The testis has been identified as the organ in which a large number of tissue-enriched genes are present. However, a large portion of transcripts related to each stage or cell type in the testis still remains unknown. In this study, databases combined with confirmatory measurements were used to investigate testis-enriched genes, localization in the testis, developmental regulation, gene expression profiles of testicular disease, and signaling pathways. Our comparative analysis of GEO DataSets showed that 24 genes are predominantly expressed in testis. Cellular locations of 15 testis-enriched proteins in human testis have been identified and most of them were located in spermatocytes and round spermatids. Real-time PCR revealed that expressions of these 15 genes are significantly increased during testis development. Also, an analysis of GEO DataSets indicated that expressions of these 15 genes were significantly decreased in teratozoospermic patients and polyubiquitin knockout mice, suggesting their involvement in normal testis development. Pathway analysis revealed that most of those 15 genes are implicated in various sperm-related cell processes and disease conditions. This approach provides effective strategies for discovering novel testis-enriched genes and their expression patterns, paving the way for future characterization of their functions regarding infertility and providing new biomarkers for specific stages of spematogenesis.

Fig 1. RT-PCR of mouse and human testis-enriched genes.

Expression of mouse and human testis-enriched genes in various tissues are presented. Te: testis, M: muscle, Li: liver, Br: brain, Lu: lung, K: kidney, F: fat, H: heart, Sp: spleen, Int: intestine, Thy: thymus. Murine cyclophilin (Cyc) and human cyclophilin (CYC) genes were used as loading controls for an equal amount of cDNA.

Fig 2. Immuno-localization of testis-specific genes in human testis based on figures obtained from the Human Protein Atlas (www.proteinatlas.org).

Among genes that have been published regarding their localization in mouse testis, but not in human testis, proteins expressed in both pachytene spermatocytes and round spermatids (A) and in round spermatids (B) are described. Genes that have not been published regarding their localization in both human and mouse testis are displayed for their expression in both pachytene spermatocytes and round spermatids (C) and in round spermatids (D). In addition, proteins that are expressed in early spermatogenic cells (type A and B spermotogonia) are presented as non-testis-specific controls (E). A: type A spermatogonia, B: type B spermatogonia, PC: pachytene spermatocytes, RS: round spermatids, ES: elongating spermatids, AG: acrosomal granule, SZ: spermatozoa, ST: sertoli cells, LD: Leydig cells.

Fig 3. Real-time PCR analysis of developmental expression patterns of testis-specific genes in mouse testis.

Quantitative real-time PCR (qPCR) results are presented for testis-related genes which are described in Fig 2 at 10, 21 and 91 days postpartum (dpp). According to categories of Fig 2, genes expressed in both pachytene spermatocytes and round spermatids (A) and in round spermatids (B) are presented for expression patterns. Among genes that have not been published for localization, genes expressed in both pachytene spermatocytes and round spermatids (C) and in round spermatids (D) are examined for expression patterns. In addition, non-testis-specific controls that are expressed in early spermatogenic cells (type A and B spermotogonia) are presented (E). The Y-axis represents relative expression value using cyclophilin (Cyc) as a housekeeping control. Each bar represents mean ± SEM. To compare means, one-way ANOVA was followed by Tukey’s post hoc test. Different letters above the bars indicate significant differences between developmental time points.

Fig 4. Signaling pathways associated with testis-enriched proteins.

The pathway analysis was conducted using Pathway Studio (v 11.2.5.9) following a database search based on PubMed/Medline hyperlink. Pathway inhibition is indicated with flat-headed lines, and activation with arrow-headed lines.