Haploid post-meiotic germ cells are then characterized by a temporal uncoupling of translation and transcription see for reviews: Dadoune, ; Steger, Translation of some mRNA into proteins could then occur for up to several days after the cessation of transcription before the completion of spermiogenesis.
Thus, the mRNA present in mature sperm seem to be roughly similar to those found in testis and could reflect the accurate development of spermatogenesis. It has also been reported that there is the persistence of a low but detectable level of transcription in mature sperm cells Miteva et al. More recently, Naz has described the existence of transcriptional and translational activities in human sperm during capacitation and acrosome reaction, which could also explain the presence of mRNA in mature sperm.
Finally, one can argue that these stored mRNA may be used during the first steps of fertilization, which are not sensitive to transcriptional inhibitors, and then contribue to the paternal printing Braude et al. These results suggest that the establishment of sperm mRNA profiles could be used as a genetic fingerprint of normal fertile men. We have therefore tested the hypothesis that sperm quality could be related to its mRNA distribution by comparing the levels of different transcripts coding for molecules either involved in nuclear condensation protamines 1 and 2 or in sperm function [endothelial nitric oxide synthase eNOS , nNOS and c-myc respectively; Naz et al.
Informed patient consent was obtained for the use of sperm samples in this study. The selected specimens had normal semen parameters according to the World Health Organization guidelines. A spermocytogram was performed to eliminate samples with cytoplasmic droplets. To rule out the possibility of any contamination by residual cells germ cells or polynuclear cells , the liquefied semen samples were fractionated on a discontinuous Puresperm gradient JCD, France consisting of four successive layers with the following densities: 95, 76, 57 and These two fractions were then washed twice with Earle's medium Eurobio, France before being used.
No remaining round cells and cytoplasmic droplets were observed as described by Lambard et al. Human sperm samples contaminated by either testicular germ cells or leukocytes were used as positive controls respectively for c-kit and CD45 Lambard et al. E-cadherin, a positive marker for epithelial cells, was also included in the quality control of our sperm cell preparations Andersson et al.
Methods for evaluation of the sperm plasmalemma integrity in motile and immotile fractions included both EN vital stain and hypo-osmotic swelling test HOST.
Unstained sperm were viable whereas sperm showing partial or complete purple staining were considered as dead cells. Sperm without swollen or coiled tails were considered as non-viable cells. For each experimental set, a negative control was prepared by omitting TdT from the reaction mixture. Two subsequent washes were performed to stop the reaction.
FL1 and FL3 fluorescence signals were respectively recorded before logarithmic and linear amplifications. Total RNA from purified sperm fractions and granulosa cells were extracted using the guanidium thiocyanate-derived method Chomczynski and Sacchi, The RNA was then isolated with a phenol—chloroform—isoamylic acid solution and was precipitated from the aqueous phase with isopropanol. PCR were performed in the presence of 1. All primers have been chosen in different exons to eliminate a possible contamination by genomic DNA.
The different cycle profiles are summarized in Table II. For all PCR amplifications, negative control only water and positive control were included. All cDNA fragments were run on a 1.
In order to quantify different transcripts Prm-1, Prm-2 and c-myc in human sperm samples, we have determined the optimal conditions for the RT-PCR. The resulting PCR products were analysed by electrophoresis on a 1. We have checked the purity of the two fractions by controlling for the absence of round cells and cytoplasmic droplets in the two selected populations as described by Lambard et al.
Moreover, we have been unable to amplify either CD45, c-kit or E-cadherin mRNA which are positive markers for leukocytes, testicular germ cells and epithelial cells respectively, thus demonstrating the purity of our preparations Figure 2. The percentages of non-apoptotic and live cells, respectively estimated by TUNEL technique and EN staining, were similar whatever the technique used The percentage of non-apoptotic cells in motile and low motile sperm was Prm-1 mRNA levels were significantly higher in the low motile fraction compared to the high motile fraction Figure 4.
No significant variation was observed between the two groups for Prm-2 mRNA levels. C-myc transcripts were the first mRNA described in human ejaculated sperm Kumar et al. No significant variation of c-myc mRNA levels was observed between the two populations of sperm. Because c-myc mRNA has a short half-life in a great number of cell types, we have analysed its expression during capacitation, together with that of Prm-2 mRNA. In this study, semen samples from normospermic patients were fractionated on a four layer discontinuous Puresperm gradient in order to separate high motile from low motile sperm from the same sample.
This technique has already been used by several authors Weng et al. Herein we have observed a differential distribution of some mRNA between high and low motile sperm, such as Prm-1, eNOS and nNOS, whereas no variation was observed for other transcripts such as Prm-2 or c-myc. One explanation for the discrepancies observed between the two populations of sperm might lie in the presence of round cells germ cells, leukocytes and epithelial cells , which contain much more RNA than sperm.
In order to avoid this, we have used a four layer gradient, and in the various sperm preparations obtained, the absence of specific molecular marker of leukocytes, germ cells and epithelial cells respectively CD45, c-kit and E-cadherin has been demonstrated. Moreover, the viability of the high and low motile fractions was estimated and the percentage of apoptotic cells measured to control for the integrity of the sperm cells. The discrepancies observed in this study between low and high motile sperm could be due to the presence of immature sperm with residual cytoplasm in the low fraction.
In fact, it has been shown that the morphology of sperm recovered from the bottom of the gradient is improved compared to the lower density layers Chen and Bongso, ; Gil-Guzman et al. In order to rule out this hypothesis, a spermocytogram was systematically performed to eliminate sperm with anormal morphology or with an excess of cytoplasmic droplets.
An important decrease of Prm-1 gene expression is also observed in testicular biopsies from non-obstructive azoospermia compared to obstructive azoospermia, associated with a normal spermatogenesis Steger et al.
Data from Steger et al. Contrary to our work and that of Steger et al. No significant differences in the P1:P2 ratio in the different populations of sperm isolated from Percoll fractions have been observed Mengual et al. We have also noted an almost complete absence of eNOS and nNOS transcripts in motile sperm whereas for the first time an intensive signal for these two mRNA is observed in the low motile fraction.
Nitric oxide synthesized by NOS is a potential modulator of sperm function, especially in the acquisition of motility and capacitation. In the presence of NOS inhibitors, the motility is increased in low motile sperm initially characterized by a very elevated NO production Perera et al. The administration of high amounts of the NOS substrate, L-arginine, induces infertility in male rats Ratnasooriya and Dharmasiri, The high levels of eNOS and nNOS transcripts in low motile sperm can be related to the excessive production of NO responsible for an inhibition of sperm motility Rosselli et al.
Contrary to the previous tested mRNA analysed herein Prm-1, eNOS and nNOS , no significant difference in c-myc transcripts has been observed between high and low motile sperm fractions.
It could also modify characteristics of sperm movement such as head lateral amplitude and velocity Naz et al. The presence of a complex population of mRNA in human mature sperm is well documented, but their roles are unknown.
It is generally accepted that sperm are in an inactive transcriptional state. The histone-to-protamine exchange and, consequently, the resulting chromatin condensation finally leads to the cessation of transcription. Recently, protamine haplo-insufficient mice have been shown to be infertile by preventing chromatin assembly Cho et al. At first, most of the translationally silent mRNA stored as polyadenylated forms in messenger ribonucleoprotein particles Schmidt et al.
As evoked by the persistence of mRNA coding for transition proteins and protamines, the reported transcripts could be considered as untranslated stored remnants and as a fingerprint of spermatogenesis quality Miller et al. Protein phosphorylation, transcription and translation steps have been observed in human sperm Naz, , Sakkas et al.
Indeed, the histone-to-protamine exchange process seems to be incomplete in human spermatids compared to rat or mouse spermatids Tanphaichitr et al. However, until now there has been no evidence for the presence of ribosomal RNA in human sperm. Our results observed during capacitation could be explained by a modification of the c-myc half-life leading to the disappearance of c-myc transcripts.
Recently, it has been shown that mouse sperm contain an endogenous reverse transcriptase Giordano et al. This observation could also explain the diminution of c-myc mRNA level in the capacitated sperm. Sperm nuclei also have endogenous endonuclease activity Maione et al. Using a microarray analysis, Ostermeier et al. However, some mRNA distinct from those found in oocytes but similar to those observed in zygotes seem to be testis- and sperm-specific and essential to the early embryo development.
The identification of SMARS sperm-specific nuclear matrix attachment regions on the PrmPrmTNP-1 domain associated with the nuclear matrix and the DNAse I sensitivity are in favour of an alternative chromatin structure, and can represent the initiation point for replacing protamines by histones during the male pronucleus formation Wyke and Krawetz, As proposed by Ostermeier et al. No tRNAs recognize these codons. Thus, in the place of these tRNAs, one of several proteins, called release factors, binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.
The translation process is very similar in prokaryotes and eukaryotes. Although different elongation, initiation, and termination factors are used, the genetic code is generally identical. As previously noted, in bacteria, transcription and translation take place simultaneously, and mRNAs are relatively short-lived. In eukaryotes, however, mRNAs have highly variable half-lives, are subject to modifications, and must exit the nucleus to be translated; these multiple steps offer additional opportunities to regulate levels of protein production, and thereby fine-tune gene expression.
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European Journal of Biochemistry , — Grunberger, D. Codon recognition by enzymatically mischarged valine transfer ribonucleic acid. Science , — doi Kozak, M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature , — doi Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
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Copy Number Variation. Copy Number Variation and Genetic Disease. Copy Number Variation and Human Disease. Tandem Repeats and Morphological Variation. Chemical Structure of RNA. Eukaryotic Genome Complexity. RNA Functions. Citation: Clancy, S. Nature Education 1 1 How does the cell convert DNA into working proteins? The process of translation can be seen as the decoding of instructions for making proteins, involving mRNA in transcription as well as tRNA.
Aa Aa Aa. Figure Detail. Where Translation Occurs. Figure 3: A DNA transcription unit. A DNA transcription unit is composed, from its 3' to 5' end, of an RNA-coding region pink rectangle flanked by a promoter region green rectangle and a terminator region black rectangle. Genetics: A Conceptual Approach , 2nd ed. All rights reserved. The Elongation Phase. Figure 6. At Moderna, we are leveraging the fundamental role that mRNA plays in protein synthesis.
We have developed proprietary technologies and methods to create mRNA sequences that cells recognize as if they were produced in the body. Using mRNA as a drug opens up a breadth of opportunities to treat and prevent disease. We have the potential to treat or prevent diseases that today are not addressable — potentially improving human health and impacting lives around the world.
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