Microarray Studies Of Gene Expression Profile Of Adult Sperms

Current studies indicated that about 10% of married couples suffered from infertile related abnormalities, for which the sperm dysfunction of adult male was considered to be the main cause. Sperm dysfunction induced infertility has become one of the problems puzzling medical professionals.It is believed that many factors might contribute to the sperm dysfunction. Though it has been studied for a long time, the mechanism of pathogeny and pathogenesis remained unknown, resulting to the lack of effective therapeutics.Modern genetics studies have demonstrated that most of the diseases relate to variations in genetic level to certain extent. The exploration of the causes of sperm dysfunction in the genetic level is of most important to find efficient cure. Generally there were two distinct approaches for mechanism study. One is to study whether the genetic DNA has mutated in certain locous. The other one is to study the variations in gene expression. There are 300 million bases in human DNA, but only producing 30 thousands proteins, as obviously many bases in DNA were none-transcriptional or belonged to repeated sequences. So gene expression profile also play important roles keeping healthy.This study focuses upon the gene expression profile of normal vs abnormal sperms.For a long time it was thought that there was no gene expressed in sperm because it was at the terminational differentiation stage. Because of this, researches of the molecular biology of sperm has not been carried out systematically. Recently however, studies have learned from the reports of Kramer J and Miller D that in sperms there have many genes expressed, such as protamine-2, hyaluronidase, lactate dehydrogenase, etc. These findings inspired strong interest to further investigate the gene expressions in sperm.To study the gene expression in sperm, the first step is to extract the total RNA from sperm. For RNA extraction, the conventional guanidinium isothiocyanate protocols proved to be inefficient, though effective extraction from other type of cells were successful. Sperms were haploid cells and had less RNAs compared to somatic cells, which brought out some difficulties in extracting the RNA. We extracted the total RNAs from normal sperm samples with RNeasy Mini Kit (QIAGEN).To continue our experiments we must evaluate the quality of extracted RNA extracted. Because the quantities of the extracted sperm RNA were too low, the ribosomal 28S, 18S and 5S could not be observed in conventional method (gel electrophoresis). The lab-on-chip gel electrophoresis (Agilent 2100 Bioanalyzer) was used to examine the quality of the total RNAs. With 25 ng spermatozoal RNA loaded onto each well of the lab-on-chip, we found 3 ribosomal bands in gel electrophoresis graph, but the location did not coincide with that of the normal somatic cells. On the basis of repeated results (13 times), we propose certain standardization of spermatozoal RNA, examined with Agilent 2100 lab-on-chip.Qualified spermartozoal total RNAs were reverse-transcribed into cDNA. With Restriction Display technique, 560 spermatozoal cDNA fragments were isolated and cloned, the clones are stored refrigiated for further analysis.The specific cDNA fragments mentioned above were amplified and purified, then the microarray on glass slides were fabricated, with GAPDH being the positivecontrol, HIV fragment being the negative control and 50% DMSO being blank control.Two sets of studies were carried out with our lab-made microarray, one was to detect the accuracy and reliability of the cDNA probes collected, the other was to do some basic research of spermatozoal gene expression. The results verified the probes collected were in good qualities, and genes expressed in sperm were studied. For example, some genes associated with replication, transcription, translation and regulation basically belonged to non-differentiate expression or down-regulated expression, whereas the genes involved in the spermatogenesis and spermatozoal particular antigens are in up-regulated expression, such as Homo sapiens spermatogenesis associated 2 (SPATA2), Homo sapiens sperm associated antigen 4 (SPAG4), et al. In addition, some genes linked with glycolysis are up-regulated, oxidative phosphorylation are down-regulated, which is consistent with inactivated sperms. In addition we found some new ESTs which have been submitted to GeneBank database.To further study the expression profile of the spermatozoal genes, Agilent Human IB microarray were adopted. Expressed gene differentiation between normal testis tissues and ejaculated sperm samples, and between normal and abnormal ejaculated sperm samples were studied systematically.Agilent Human IB microarray composed of 60mer oligos of 22153 spots, including 162 negative control genes and 918 reference genes, which are used to ensure the accuracy of the hybridization results.Considering about the low quantities of sperm RNA, conventionally used reverse-transcribed labeling method was not chosen. cRNA linear amplification technique was applied. This technique could amplify the genes in a linear fashion, not changing the numbers of genes in the primary situation, therefore more accurateand reliable hybridization results could be guaranteed.With Agilent Human IB microarray, normal spermatozoal gene expression profile was investigated. Of the 21073 genes spoted on the micoarray, 2157 were identified by the sperm cDNA in different 2 experiments with a common hybridization signal, including 22 known genes to be associated with spermatogenesis and asthenospermia. The hybridization between normal testis tissues and ejaculated sperm samples showed in ejaculated sperm, 67 genes were up-regulated, including 16 genes associated with development, 14 with spermatogenesis, mature and asthenospermia. Hybridization results between normal and abnormal sperm samples revealed that in abnormal sperms samples, 69 genes were up-regulated, 116 were down-regulated, which were involved in asthenospermia.Our experiments applying RNAs extraction, cDNA probes collection, microarray studies of spermatozoal gene expression profile, to study the normal sperm samples, testis tissues and abnormal sperm samples, it found that plenty of genes expressed in sperms. Genes found in the gene expression profile including some genes known closely associated with spermatogenesis, mature and fertility. Taking into consideration of our experimental results, certain groups of the genes were found to be associated with sperm maturity, while others were found to be associated with the inactivity of the abnormal sperms. These results will provide help assistance in establishing the normal gene expression fingerprints of the human sperms, ensuring more detailed study of the factors influencing the sperms quality. It is concluded that gene expression profile studies will be contributing to the functional genomics study in human sperms, leading to the discovery of novel therapeutics against male infertility.