Digital gene expression tag profiling analysis of the gene expression patterns regulating the early stage of mouse spermatogenesis

Abstract

Detailed characterization of the gene expression patterns in spermatogonia and primary spermatocytes is critical to understand the processes which occur prior to meiosis during normal spermatogenesis. The genome-wide expression profiles of mouse type B spermatogonia and primary spermatocytes were investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early spermatogenesis were systematically analyzed. Gene expression patterns vary significantly between mouse type B spermatogonia and primary spermatocytes. The functional analysis revealed that genes related to junction assembly, regulation of the actin cytoskeleton and pluripotency were most significantly differently expressed. Pathway analysis indicated that the Wnt non-canonical signaling pathway played a central role and interacted with the actin filament organization pathway during the development of spermatogonia. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of early spermatogenesis.

Figure 1. Scatter plot indicates the comparing results of log transformed gene expression levels and differentially expressed genes' distribution between GC-1spg and GC-2spd (ts).

Figure 2. Correlation between the expression fold change level of 15 randomly selected differently expressed genes between GC-1spg and GC-2spd (ts).

The selected genes cover different expression levels of both up- and down-regulated genes defined by digital gene expression, and the fold change level validated by qPCR. Gene expression levels are presented at the normalized gene expression levels in primary spermatocytes, relative to type B spermatogonia.

Figure 3. Differentially expressed genes involved in the focal adhesion signaling pathway during early spermatogenesis.

Genes up-regulated and down-regulated during the development process from GC-1spg to GC-2spd (ts) are marked in red and green, respectively.

Figure 4. Differentially expressed genes involved in Wnt signaling pathway during early spermatogenesis.

Genes up-regulated and down-regulated during the development process from GC-1spg to GC-2spd (ts) are marked in red and green, respectively.

Figure 5. Differentially expressed genes involved in MAPK signaling pathway during early spermatogenesis.

Genes up-regulated and down-regulated during the development process from GC-1spg to GC-2spd (ts) are marked in red and green, respectively.

Figure 6. Differentially expressed genes involved in the regulation of action cytoskeleton pathway during early spermatogenesis.

Genes up-regulated and down-regulated during the development process from GC-1spg to GC-2spd (ts) are marked in red and green, respectively.

Figure 7. Protein levels of FGF13, SFRP2, RAC, WNT10A and FGF7.

(A) Protein levels of FGF13, SFRP2 and RAC in GC-1spg and GC-2spd (ts) cells were detected by western blotting respectively. β-Actin were used to normalize the individual expression levels. (B) Protein level of WNT10A and FGF7 in the medium supernatants from GC-1spg and GC-2spd (ts) cells were determined by ELISA. The data are presented as the mean plus SEM from a representative of three independent experiments, with each performed in triplicate. Statistically significant differences between GC-1spg and GC-2spd (ts) cells are indicated above the bars: * means P<0.05.