Expression and significance of Rap1A in testes of azoospermic subjects

Table 1. Gene classification

The total RNA was isolated using modified single-step extraction technique. Briefly, frozen tissues were crushed and homogenized in solution D and 1 % mercap-toethanol. The supernatant was extracted with phenol:chloroform (1:1), phenol:chloroform (5:1) and NaAc (pH 4.5) successively. The supernatant was precipitated in an equal volume of isopropanol at -20 ℃ for 1.5 hours and then precipitated in LiCl for purification. mRNAs were purified using the Oligotex mRNA Midi Kit (Quagen Inc., USA). Fluorescent cDNA probes were prepared by reverse transcription and then purification. The mRNA from normal testicular tissue was labeled with Cy3-dUTP and those from the azoospermic tissue, with Cy5-dUTP. Equal amounts of the two probes were mixed, precipitated by ethanol and resolved in 20 ×hybridization buffer (5×SSC + 0.4 % SDS, 50 % formamide, 5×Enhardt's solution).

Samples for cDNA hybridization were collected from one azoospermic and one normal testes. The samples were snap-frozen in liquid nitrogen within 15 min after biopsy and stored at -80 ℃. Microarray slides were pre-hybridized in hybridization buffer containing 0.5 mg/mL denatured clupeine DNA at 42 ℃ for 6 hours. After denaturation at 95 ℃ for 5 min, the probe mixture was added onto the pre-hybridized slides and sealed with cover glass. After hybridizing in the HybChamber (United Gene Holdings Ltd., China) at 60 ℃ for 15 hours, the slides were washed in solutions of 2×SSC + 0.2 % SDS, 0.1×SSC + 0.2 % SDS and 0.1×SSC successively for 10 min each and then dried at room temperature.

The slides were scanned with a ScanArray 3000 laser scanner (General Scanning Inc., USA) at two wavelengths to obtain fluorescence intensities for both dyes (Cy3 and Cy5). The original value of each spot was normalized by the values of the housekeeping genes. The fluorescence intensities of Cy3 and Cy5 were analyzed and the ratio was calculated with ImaGene 3.0 software (BioDiscovery Inc.). The intensities of the two fluorescent signals represent the quantities of the two tagged probes. The ratio of Cy5:Cy3 in certain spots on the slide represents the mRNA abundance of this gene expressed in the azoospermic versus the normal tissues.

The probe for hybridization was designed and synthesized by the Boster Co. (Wuhan, China) using the Primer 3 software with respect to the sequence released in NCBI. The specific oligonucleotide sequence of the probe is 5'-TTTGTTCAGGGAATTTTTGTTGAAAAATAT-3', 5'-GTATATTCTATTACAGCTCAGTCC-ACGTTT-3' and 5' TGTGCCTTTTTAGAATCTTCT-GCAAAGTCA-3'. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects according to the kit guideline. The probe was terminally labeled with digoxigenin-dUTP with a 1:75 working dilution. The sections were incubated with phosphatase-labeled anti-digoxigenin antibody (1:400) at 37 ℃ for 2 hours. To assess the specificity of labeling, control sections were co-incubated with a 100-fold excess of unlabeled probe and the corresponding DIG-labeled probe. No labeling above the background was detected in these sections. Positive staining shows a brown coloration appearing in the cytoplasm.

Hybridization was repeated at least for 3 times to eliminate false positive results. Only those genes with the Cy5-dUTP/Cy3-dUTP ratio more than 2.0 or less than 0.5 were selected.

The Chi-square test and SPSS 10.0 for Windows software were used to analyze the results.

Table 2. Up-regulated genes in azoospermic testes.

Table 3 Down-regulated genes in azoospermic testes

Figure 2. Scattered dot image in cDNA microarray. Points between two inclined lines: genes not changed; Points out of these 2 lines: genes changed.