The Screening Of Differential Expression Genes Related With Adult Male Asthenozoospermia By Genome Wide Microarray

Clinical studies have shown that asthenozoospermia is the major factor leading to male infertility. We actually understand little about the molecular mechanisms or pathogenesis. Therefore, it is difficult to make a clear diagnosis of the aetiology of the disease and the effect of treatment is not satisfactory. Recent researches have found a few mRNAs in human sperm are associated with asthenozoospermia, such as Tektin-t, DNAI1, DNAH5, DNAH11. In addition to these, a few mRNAs, including Prml, Aromatase, ENOS and NNOS, have shown a significant expressed differences in low motile sperm compared to high motile sperm isolated from the same semen sample of normospermic patients. These studies tell us that a further understanding of sperm mRNA expression and molecular function has a important significance for elucidating the functions of sperm. Therefore, we planned to obtain gene expression profiles of spermatozoa from asthenozoospermia and normal controls by microarrays, screen differentially expressed genes in asthenozoospermic semen samples and explore their functions and significance. The following are the research methods, contents and conclusions of this study. Part 1 The selection and collection of adult male asthenozoospermic and normal samplesObjectiveThe aim was to collect semen samples of asthenozoospermia and normal controls. Single (50%) and two-layer (90% and 45%) Percoll density gradient centrifugation were separately used to separate and purify spermatozoa. We wanted to explore which separation method was more suitable for our research.Methods1. According to the World Health Organizaion, the patient samples corresponded to consecutive asthenozoospermic patients confirmed by the analysis of three different semen analyses. Controls were proven normozoospermic semen with normal sperm parameters. Sperm morphology was assessed after staining with Diff-Quick. None of the semen samples had significant numbers of round cells or leukocytospermia. In this study, patients and controls were selected, excluding those with teratozoospermia and leukocytospermia. Other exclusion criterions were abnormal semen liquefaction, urinary tract infection, history of cryptorchidism or orchitis and systemic diseases. Specialist medical examination were normal. The bioethics committee of our hospital approved this project and informed consent was obtained from the participants.2.10 semen specimens had been dealed with by single (50%) and two-layer (90% and 45%) density gradient centrifugation separately. Before and after the treatment, we used SCA (Sperm Class Analyzer) to analyse the sperm density,motilty,dynamic parameters and round cell density.Results1. We have separately collected 120 and 160 cases of semen samples from asthenozoospermia and normal controls. 2. After separation, Sperm recovery rate of single-layer mothod was (56.9±10.3)%, significantly higher than two-layer method (P= 0.004, P<0.01). A grade sperm percentage of single and two-layer method was significantly higher than before (P=0.016, P<0.05; P=0.001, P<0.01). However, C grade sperm percentage of single method was significantly higher than two-layer method(P=0.021, P<0.05). A and B grade sperm percentage of two-layer method was significantly higher than before(P=0.037, P<0.05), but A and B grade sperm percentage of single-layer method was no difference with before(P=0.677, P>0.05). The round cell density of single and two-layer method was significantly lower than before (P=0.002, P<0.01; P=0.000, P<0.01), but there is no difference between the two methods (P=0.414,P>0.05).Conclusions1. The quantity of samples we have collected could guarantee the need of the following experiments.2. Single-layer method has a higher recovery rate of sperm, and little changes the sperm motility. It can better reflect the level before the separation and have the advantage for research of sperm motility. Therefore, we used 50% Single-layer Percoll density gradient centrifugation to separate and purify the semen.Part 2 The extraction, detection and analysis of sperm RNAObjectiveWe used RNeasy Mini kit (Qiagen, Germany) to extract sperm RNA and Agilent 2100 bioanalyzer to analyse the sperm RNA. The aim was to establish the intact procedure of sperm RNA extraction and detection.Methods1. Sperm samples after serparation were stored in-80℃refrigerator. After thawing in 37℃water box, then every four spermatozoal pellets from asthenozoospermia or healthy donors were pooled. RNA was isolated from these solutions using an RNeasy Mini kit (Qiagen, Germany), according to the producers'manuals.2. RNAs quantity was measured spectrophotometrically by a Nanodrop spectrophotometer (Nanodrop technologies, USA). The quality of the RNAs was checked using an Agilent Bioanalyzer and 1.5% agarose gel electrophoresie.Results1. The total concentration of RNA was about 5ug and A260/A280 ratios were more than 2.0 by UV spectrophotometer. This indicated that extracted sperm RNA had high purity.2. We did not find 28S and 18S belts, but can see some flaky white areas by 1.5% agarose gel electrophoresie.3. The integrity of the total RNAs was evaluated by Agilent 2100 bioanalyzer. The spermatic RNA samples clearly showed an absence of 18S and 28S ribosomal subunits and the overall RNA profile, displayed a majority of short-length fragments, which is typical of this cell type.Conclusions1. The results show that we have established a complete set of sperm RNA extraction and detection methods.2. We found that the sperm contains a large number of small molecular weight RNA. The extracted sperm RNA had high purity and low density, but it could supply the RNA for the following studies.Part 3 The screening, validation and analysis of asthenozoospermia molecular markersObjective The aim was to screen the differently expressed genes in asthenozoospermia by Agilent human 4×44K microarray and preliminarily analyse through bioinformatics.Methods1. Total RNAs were used as a template for in vitro transcription using the Ambion MessageAmpII cRNA Amplification Kit to cRNA synthesis.The Cy3 or Cy5 (GE healthcare) was added chemically to the amino allyl-dUTPs groupings to label the cRNA according to the instructions. The cRNAs were hybridized on the Agilent human 4×44K slides(Agilent, USA) containing 41000 single-spotted human cDNA. After hybridization, each array was washed and scanned in an Agilent 2565BA scanner. The acquired images were analyzed using Feature Extraction software.2. Molecular functions and biological processes of differentially expressed genes were analyzed by the PANTHER online analytical tools.3. Real-time PCR was used to verify the microarray results.Results1. By applying the threshold value of 2 folds and p<0.05 (Student's t-test), we identified 1265 differently expressed genes in Agilent human 4×44K slides containing 41000 single-spotted human cDNA,307 genes up-regulation and 958 down-regulation in asthenozoospermia compared to normal controls.2. PANTHER online analyses found that mRNAs in sperm could have a set of cell functions and take part in a variety of biological activities. A subset of differentially expressed genes were known to be involved in spermatogenesis and sperm-motility, included ZFP90, PKDREJ, CRISP2, SEMG1 and SPAG6.3. Real-time PCR tests indicated similar mRNA expression levels of ZFP90, C14ORF140 and SEMG1 to the results on chips.Conclusions 1. The results tells us that the microarray date supported the view that the sperm-motility is related to the levels of mRNA expression of spermatozoa.2. Spermatozoa mRNAs could have a set of cell functions and take part in various biology process.3. Such well-characterized genes and many novel genes discovered by this endeavour could be candidates for potential diagnostic biomarkers and further functional studies might help us to explain the pathogenesis involved in asthenozoospermia.