The mouse GC-1 spg germ cell line (ATCC CRL-2053) was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 μg/ml penicillin-streptomycin, and was maintained in 5% CO₂ and a 95% humidified air atmosphere at 37°C.

Transfections were performed with Lipofectamine 2000 (Invitrogen). Cells were plated to 50–70% confluent culture in each well of a 6-well plate or a 60-mm dish 24 h before transfection. According to the manufacturer’s instructions, the GC-1 spg cells were transfected with 4 μg pRevTRE plasmid or pRevTRE-SPATA12 plasmid, respectively.

Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, digested by RNase-free DNase (Fermentas), dissolved in diethyl pyrocarbonate-treated water, and stored at −80°C prior to use. For quality control, RNA purity and integrity were evaluated by agrose gel electrophoresis and the OD260/OD280 ratio.

32K Mouse Genome Array (CapitolBio Corporation, Beijing, China), covering 99% of the current assembly of the mouse genome and including 32,256 Oligo DNA with 70 mer length, was applied to investigate the possible changes in the mRNA level in GC-1 spg cells following SPATA12 gene transfection by comparison to control GC-1 spg cells. Total RNA was extracted using TRIzol reagent (Invitrogen) and then applied to synthesize Cy3- or Cy5-conjugated dUTP-labeled cDNA probe using the RNA Fluorescence Labeling Core kit (M-MLV version 2.0; Takara, Dalian, China) and following the manufacturer’s instructions. Hybridization, scanning, and data extraction were conducted at CapitalBio Corporation. The number of genes affected by SPATA12 was determined using the scatter plots of control GC-1 spg cells vs. GC-1 spg cells transfected with SPATA12 (named GC-1 spg-SPATA12 cells). In addition, identified genes were also categorized specifically using biological process ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways using the software Molecule Annotation System 2.0 (http//:bioinfo.capitalbio.com/MAS/) (CapitolBio Corporation).

For cDNA synthesis, 2 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega). Then PCR was performed in a 10 μl reaction volume containing 5.7 μl of nuclease-free water, 0.1 μl of Takara Taq (5 U/μl), 1 μl of 10X PCR buffer, 0.8 μl of dNTP mixture (2.5 mM), 2 μl of cDNA and 0.2 μl each of the 20 μM gene-specific primers. After initial denaturation for 10 min at 94°C, 27–30 cycles of PCR were performed. Each cycle consisted of a denaturing period (30 sec at 94°C), an annealing phase (55–60°C), and an extension period (60 sec at 72°C). After the last cycle, all samples were incubated for an additional 10 min at 72°C. PCR products were separated by 1.5% agarose gel electrophoresis, and the DNA bands were stained with ethidium bromide. RT-PCR signals were normalized to the signals of the murine glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene.

For quantitative RT-PCR, a SYBR-Green real-time RT-PCR protocol (Invitrogen) was applied using an MX3000 (Stratagene, USA) instrument. PCR was performed in a 10 μl reaction volume containing 2.2 μl of nuclease-free water, 5 μl of Mix, 2 μl of cDNA and 0.4 μl each of the 2.5 μM gene-specific primers. The PCR profile was 95°C for 5 min followed by 94°C for 30 sec, 58°C for 20 sec, and 72°C for 20 sec for 40 cycles, with a final extension at 72°C for 10 min and storage at 4°C. The level of gene mRNA was evaluated in automated analysis by MxPro. All the primers for RT-PCR are shown in Table I.

Statistical analysis was carried out using the Student’s t-test. P-values ≤0.05 were considered to indicate statistically significant results.

Global gene expression levels induced by SPATA12 in GC-1 spg cells from the microarray analysis are represented in Fig. 1. The differentially expressed genes with fold changes of ≥2 or ≤0.5 (P≤0.05) were analyzed using t-test and P-value and clustered with the software package Cluster 3.0. Our data showed that the expression of 286 out of 32,256 genes was altered after SPATA12 was expressed. Of these, 182 genes were upregulated and 104 genes were downregulated specifically.

To test the reliability of the microarray data, the expression patterns of 10 selected genes (including 6 upregulated genes and 4 downregulated genes) (Table II) were examined by quantitative real-time RT-PCR. The overall profile of gene expression by real-time RT-PCR analysis was similar to that revealed by the microarray data for all the selected genes (Figs. 2 and 3).

To gain insight into the potential functional consequences of the SPATA12-induced expression in GC-1 spg cells, GO analysis was applied to distribute genes into groups according to biological process, molecular function and cellular component, respectively.

Functional analysis using GO of the biological process group revealed that genes related to immune response, antigen processing and presentation, positive regulation of T cell-mediated cytotoxicity, transcription regulation, and defense response to bacterium were upregulated in the SPATA12-transfected GC-1 spg cells (Fig. 4A and B). This suggests that based on the GO terms these upregulated genes combined with SPATA12 may play important roles in the physiology of the immune response. In contrast, genes related to carbohydrate metabolism, lipid metabolism, cholesterol biosynthesis and glycolysis were downregulated in the SPATA12-transfected cells, which indicates that based on these GO terms these genes affected by SPATA12 may be involved in metabolic processes.

In the cellular component group (Fig. 4C and D), GO analysis showed that the major differentially expressed genes, including 61 upregulated and 22 downregulated genes, were located in the nucleus. These data indicate that SPATA12 may play roles in the nucleus through interacting with genes associated in these GO categories. This observation was consistent with our previous bioinformatics analysis report, which predicted that SPATA12 probably functions as a testis-specific nuclear protein involved in spermatogenesis (21).

We also noted that these genes (molecular function group) (Fig. 4E and F), either upregulated or downregulated in SPATA12-transfected cells, were mostly involved in protein binding, ATP/GTP binding, zinc ion binding or DNA/RNA binding, indicating that the function of SPATA12 may be related with the activity of ‘binding’.

The analysis of the differential expression of genes led us to wonder whether these genes may represent related pathways in which SPATA12 is involved. Thus, biological pathway analysis was applied to characterize the unique gene networks associated with SPATA12 in germ cells. Most genes associated with immune-related pathways such as antigen processing and presentation, cell adhesion, T cell receptor signaling pathway, and development-related pathways such as MAPK, Jak-STAT and Wnt, were significantly overexpressed which provides evidence that both immune responses and developmental processes may be associated with various functions of SPATA12 (Table III). Changes in the expression of Wnt signaling-related genes such as β-catenin supported this possibility. Semi-quantitative RT-PCR results showed that the expression of β-catenin was obviously downregulated (Fig. 5) and its downstream target cell cycle gene cyclin E1 was strongly decreased (Fig. 6) in GC-1 spg cells transfected with SPATA12.