Screening Of Subtractive CDNA Libraries Of Bovine X-/Y- Spermatozoa And Analysis Of Positive Differentially Expressed Genes

Male mammals can generate both X- and Y- spermatozoa simultaneously. Researches on transcripts in these two kinds of spermatozoa would have significant theoretical meanings, and be important to sex control study of livestock. It's considered traditionally that the products of gene expression in spermatozoa with the identical origination were shared via the intercellular bridges, thus the detection of differences between two kinds of spermatozoa had no meanings. To research the different gene expressions between two kinds of spermatozoa, subtractive cDNA libraries of bovine X- and Y-spermatozoa had been constructed in our laboratory. In this study, the libraries were screened and analysed using microarray analysis combined with sequence homology analysis, and the results of microarray analysis were validated using real-time RT-PCR.1. Screening for subtractive cDNA libraries of bovine X- and Y-spermatozoa.Two hundreds and one clones representing all unigenes in the forward and reverse libraries were selected to be used in cDNA microarray preparation according to informations from libraries. Total RNAs of X-/Y- spermatozoa were extracted, and reverse transcriptions and fluorescence labelings were performed after two rounds of mRNA linear amplification respectively. Then the prepared microarrays were hybridized with labled cDNAs. SAM software was used to analyse the microarray data for three biological repetitions. The analysis result showed that thirty one unigenes were considered as positive differentially expressed genes, four of which were up-regulated in Y-spermatozoa while twenty seven of which were in X-spermatozoa.2. Sequence homology analysis on differentially expressed genes.The matching results using Blastx software displayed that 12.9% of unigenes (four contigs) had high homology with the annotated proteins in SwissProt database (E-value≤1×10-10), one of which was up-regulated in Y-spermatozoa while the other three were highly expressed in X-spermatozoa. The matching results using Blastn software for the left differentially expressed genes showed that 32.26% of unigenes had high homology with sequences in bovine EST database (E-value≤1×10-10), one of which was up-regulated in Y-spermatozoa while nine of which were in X-spermatozoa. 54.84% of unigenes havd no homologous sequences in any database (E-value≥1×10-10) were considered as novel genes.3. Detection of two differentially expressed genes using real-time RT-PCR.The detection results showed that the cytochrome b gene (CYTB) and iron-sulfur cluster scaffold gene (ISCU) expressed as 1.731 fold and 5.125 fold highly in X- spermatozoa respectively as expressed in Y- spermatozoa. The real-time RT-PCR results validated the accuracy of microarray analysis. The results of this study prove that differential gene expression products do exist between X- and Y- spermatozoa even though the existing of the intercellular bridge among the spermatid. It provides the theoretical basis for researches on gene expressions in spermatozoa development, and impulses the researches about sex control from spermatozoa level.