The Exploratory Study Of The Characterization And Potential Function Of Cell-free MRNA Present In Semen And Epididymal Fluid

Part 1:Detection of Cell-free Seminal RNA (cfsRNA) in Healthy Individuals:to Set Up Method of cfsRNA Extraction, Quantity and Microarray AnalysisObjection:We have detected cell-free seminal RNA (cfsRNA). To find whether it can become a promising non-invasive biomarker for male reproductive system and germline epigenetics, we first set up method of cfsRNA extraction and analyzed its quantities and species through microarray technology.Methods:A modified cfRNA isolation protocol was performed using Trizol LS reagent (Invitrogen) followed by purifying using an RNeasy minicolumn (RNeasy kit. Qiagen).cfsRNA quantity was assessed by real time RT-PCR targeting the 5'region ofβ-actin (ACTB) transcript. The species of cfsRNA was analyzed by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer, and its sources was analyzed through microarray technology using the Affymetrix Human Gene 1.0 ST array.Results:The cfRNA concentration in the seminal plasma of the 10 healthy individuals varied from 0.87 up to 3.64μg/mL with a mean±SD of 1.75±0.92μg/mL. The chromatogram of the size distribution of cfsRNA showed three distinct peak signals, which correspond to the three predominant RNA classes (28S,18S rRNA and "small RNAs "such as 5S rRNA, tRNAs and microRNA), and a broad distribution of molecular weights with relatively weak signals. Microarray analysis showed 20 106 different transcripts were detected in the mixed cfsRNA of 12 healthy participants, including various male reproductive organ specific transcripts.Conclusion:cfsRNA in healthy individuals, with abundant quantities and species, could meet the need of almost all routine RNA analysis techniques, including microarray, northern blot, electrophoresis and RT-PCR. Therefore, such abundant quantities and species of cfsRNA can provide comprehensively cell-and tissue-specific information. A non-invasive transcriptome profiling of male reproductive organs may be carried out via analyzing cfsRNA. Part 2:Characterization of Human Cell-free Seminal mRNA: Implications for non-invasive Disease Diagnosis and Gene Expression Study of Male Reproductive SystemExperiment 1:Integrity and Stability of Cell-free Seminal mRNA In Healthy IndividualsObjection:we aimed to investigate whether the characteristics of cfsRNA can meet the basic requirements of molecular diagnostic assays. For this purpose, we mainly report the characterization of integrity and stability of cfsRNA in healthy individuals, and presumed the possible mechanisms for its protection against seminal ribonuclease activity.Methods:The integrity of cfsRNA was analyzed amplification of full-length ACTB and DEAD(Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) transcript, as well as the relative amount of different regions of ACTB and DDX4. The stability of cfsRNA was measured by a time-course analysis of different regions of ACTB and DDX4. To find out whether cfsRNA was protected by complex forms, the semen plasma was processed by two means:filtration through pores of different sizes and Triton X-100 treatment before RNA recovery.Results:Most cfsRNA was present in partially degraded forms, with less of the middle and 3' gene amplicons compared to the 5', compared with the 5'region of the ACTB mRNA, we detected 71.0% of the middle region (P＜0.05), whereas 24.2% of the 3'region amplicon (P<0.001). Analysis of DDX4 mRNA showed strongly less of the middle amplicon (middle-to-5'ratio=0.484, P＜0.001) as well as the 3'amplicon (3'-to-5'ratio=0.02, P＜0.001) compared with the 5'amplicon. These data suggested preferential degradation of 3'region of cfsRNAs, so that further researches or clinical usages of cfsRNAs would likely focus on the 5'region.No significant degradation of each region of ACTB mRNA was observed among these serial time points (P>0.05). To DDX4 transcripts, despite the 5'region was still stable up to 24 hr, the amount of the middle region was slightly reduced at 90min and reduced to 25.8% after 24 hr incubation (P<0.01), whereas the amount of the 3' region was 72.6% reduced at 20 min and thereafter degraded completely by 90 min incubation (P<0.001), compared with the amount at time 0, respectively. As a control, exogenous rat RNA added to seminal plasma was immediately degraded:6.6×1012 copies/mL of rat-ACTB mRNA was detected at the beginning, whereas absent after only 15s incubation, which confirmed the highly active ribonuclease in semen.No significant change in seminal ACTB mRNA concentration followed by 5μM filtration, suggesting these mRNA transcripts were not contained within intact cells. In comparison with 5μm-filtered samples, only 51.9% of ACTB mRNA was detected in samples filtered through 0.45μm filters (P<0.05), and decreased to 23.3% when samples were filtered through 0.22μm filters (P＜0.01). These data showed 0.45 and 0.22μm filtration had clearly reductive effects on seminal ACTB mRNA concentration, indicating more than three-quarters of cfsRNA were associated with macromolecules, and the remainder might exist in some other forms.The dramatic degradation of cfsRNA was observed after treatment with Triton-X 100. After 10 min and 20 min,6.1% and 2.3% of the 5'region of ACTB mRNA could be detected respectively (Fig.4B, P＜0.001) and no DDX4 mRNA could be amplified, suggesting that this RNA-macromolecule interaction is important for cfsRNA stability.Conclusion:We characterized seminal cfRNAs as partially degraded, fairly stable and particle-associated. Consequently, cfsRNA is a potential non-invasive biomarker for male reproductive system and germline epigenetics; however, very existence and the latent effects of cfsRNA in male reproduction deserve further research.Experiment 2:A Substantial Proportion of Cell-Free Seminal mRNA Is Highly Protected Within Seminal Microparticles (SMPs) for Healthy Individuals and Severe Oligzoospermic PatientsObjection:cell-free seminal RNA may be still considered as an unreliable diagnostic biomarker because excellular RNA is labile in abstracto. This reflects our inadequate understanding of the very existence of cfsRNA, and may hinder further research and clinical applications. Since cfsRNA is readily detected in seminal plasma, moreover. Seminal Microparticles (SMPs) have been identified as stable and abundant fragments in semen, we hypothesized cfsRNAs would be protected within SMPs that released from male reproductive organs.Methods:In this study, we first determined whether cfsRNA would exist in the SMPs of healthy participants, by extracting RNA from SMPs (smpRNA) and amplifyingβ-actin (ACTB) mRNA using PCR. And if so, other 5 male reproductive organ-specific transcripts were amplified as models for a universal demonstration. In particular, they were chosen from our previous microarray analysis, including DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4), Protamine 2 (PRM2), P-defensin 129 (DEFB129), serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5 (SERPINA5) and transglutaminase 4 (TGM4). Also, we quantitatively analyzed proportions of cfsRNA that included within SMPs, by comparing the concentrations of smpRNA and cfsRNA using RT-qPCR targeting these 6 transcripts, respectively. As SMPs was likely to be heterogeneous both in size and origin, the SMPs-associated nature of cfsRNA was further investigated by filtration through pores of different sizes. Lastly, we analyzed cfsRNA of oligzoospermic patients to determine whether these results could be generalized.Results:To validate whether these transcripts could be derived from different male reproductive organs, we analyzed the extracted RNA from 10 healthy participants by using the respective real-time RT-PCR assays to detect 6 mRNAs derived from different male reproductive organs. These genes were ACTB, DDX4, PRM2, DEFB129, SERPINA5 and TGM4. In these 10 participants, we were able to amplify all 6 mRNAs with 100% detectivity in their seminal plasma.Meanwhile, we isolated SMPs from the same participants and successfully detected PCR amplicons for all these 6 mRNAs. As shown in Fig.1A, we randomly selected 3 cases from these 10 healthy participants and showed their PCR amplicons of cfsRNA in seminal plasma and in the form of SMPs. These data confirm that cell-free seminal mRNAs are indeed released from different male reproductive organs, and they appear to be at least partially present in the form of SMPs.No significant change in ACTB, DDX4, PRM2, DEFB129, SERPINA5 and TGM4 mRNA levels between cfsRNA and smpRNA for healthy individuals both severe oligozoospermic patients. Our data therefore suggested that the quantity of SMPs-sequestered cfsRNA varied depending on the size of SMPs and characteristic of concentration distribution may relate to their specific cell origin. Conclusion:For healthy participants and severe oligozoospermic patients, cfsRNA could be derived from different male reproductive organs, and the predominant cfsRNA is contained in SMPs to protect from degradation. it might open a new area of research for study of cfsRNA biology might be helpful to explore more effective methods of cfsRNA enrichment and search for noninvasive biomarker of the male reproductive system and of germline epigenetics.Part 3:Epididymal Cell-Free mRNAs is Associated with Epididymosomes and Acquired by SpermObjection:It has been demonstrated that SMPs released from epididymal epithelium called epididymosome could transfer into sperm and play a role in sperm epididymal maturation by providing sperm surface proteins that required for the binding to the surface of the egg. As this transfer might also include transmission of epididymal mRNAs, our laboratory has actively investigated whether the event of RNA delivery happened in epididymal segment and ultimately contributed to the epigenetic properties of spermatozoa maturation.Methods:We used RT-PCR, in situ hybridization, electron microscopy, to find that mouse cauda epididymal cell-free Crispl mRNAs is associated within cauda epididymosome secreted by the epididymal epithelium and binds to its caput sperm.Results:we show that cauda epididymal cell-free Crispl mRNAs secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Crispl mRNAs relocated from epididymal cells to caput sperm directly through cauda epididymosomes. It mainly located in mouse sperm head, mid piece and principal piece of the sperm tail, as well as mitochondrial sheath.Conclusion:This surprising phenomenon of transmission of epididymal cell-free mRNAs relocated from epididymal cells to its caput sperm directly through cauda epididymosomes might provide a possible mechanism for sperm maturation and epigenetic changes.