Mol. Hum. Reprod. Advance Access originally published online on April 20, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular Human Reproduction, Vol. 10, No. 7, pp. 535-541, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology 2004; all rights reserved
Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation
1UPRES EA 2608-USC INRA, University of Caen, France 2Department of Genetics and Reproduction, CHRU Clemenceau, Caen 14032, France 3Laboratory of Reproductive Biology, Hopital Nord, 42055 Saint-Etienne, France
4 To whom correspondence should be addressed at: UPRES EA 2608, USC INRA, Université IRBA, esplanade de la Paix, 14032 Caen cedex, France. Email: carreau{at}ibba.unicaen.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The existence of a complex population of mRNA in human sperm is well documented but their role is not yet elucidated. Using discontinuous density gradients, we have isolated high and low motile sperm from the same semen sample. The levels of different transcripts coding for molecules either involved in nuclear condensation (protamines 1 and 2) or in capacitation [endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and c-myc] were then assessed in the two populations using semi-quantitative RT-PCR. Sperm viability was estimated by eosin–nigrosin staining and by hypo-osmotic swelling test; apoptosis percentage was measured by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling technique. The contamination by somatic and germ cells was assessed by looking for specific molecular markers of these cells, respectively CD-45 and E-cadherin for somatic cells and c-kit for germ cells. The viability of sperm was unchanged in high and low motile fractions, as well as DNA fragmentation percentage. The amount of Prm-1 mRNA was significantly higher in low density motile than in the high motile fraction. In most of high motile sperm samples eNOS and nNOS transcripts were undetectable whereas they were present in the low motile sperm. In contrast, no significant variation was found in the c-myc/Prm-2 mRNA ratio between the two populations. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. Thus analysing mRNA profiles could be helpful as a diagnostic tool and prognosis value for fertilization.
Key words: capacitation/human/motility/RNA/sperm
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
During the last decade, mRNA have been discovered either by gene-specific RT-PCR or in situ hybridization in human ejaculated sperm. A non-exhaustive list of transcripts including c-myc, HLA class 1, protamines 1 and 2, heat shock proteins 70 and 90, ß-integrins, variants of phosphodiesterase, progesterone receptor and aromatase reveals a wide range of transcripts in mammalian sperm (Lambard et al., 2003
; reviewed in Miller, 2000; Wykes et al., 2000). However, their putative roles are unknown, but several hypotheses could be suggested to explain the presence of mRNA in sperm.
The most common idea is to consider that these transcripts represent remnants of stored mRNA from post-meiotically active genes. Spermatogenesis consists of a complex and dynamic process of proliferation and differentiation leading to the transition of diploid spermatogonia to haploid sperm. During this process, nucleosomal histones, present in spermatogonia, are gradually replaced by transition proteins (Tnp-1 and Tnp-2) in round spermatids, and subsequently by protamines (Prm-1 and Prm-2) in elongating spermatids and sperm. These proteins are responsible for a highly stable chromatin condensation leading to a sperm genome transcriptionally inactive (reviewed in Grootegoed et al., 2000). Haploid post-meiotic germ cells are then characterized by a temporal uncoupling of translation and transcription (see for reviews: Dadoune, 2003; Steger, 2003). 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., 1995). More recently, Naz (1998) 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., 1988; Siffroi and Dadoune, 2001).
Indeed, as also reported for the PAF receptor mRNA (Roudebush et al., 2000), we have recently shown a significant decrease of aromatase mRNA level in low motile sperm compared to high motile sperm isolated from the same semen sample of normospermic patients (Lambard et al., 2003). 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., 1991; Herrero et al., 2003] between low and high motile sperm isolated from the same sample.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Human sample preparation
Sperm samples (n=41; mean age: 33.4±4.1 years) were obtained from healthy donors by masturbation after 3 days of sexual abstinence and allowed to liquefy for 30–60 min at room temperature before processing. 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 (1999)
guidelines. Infectious sperm and samples with >1 x 106 round cells/ml were excluded. 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 47.5%. After centrifugation (20 min at 300 g, 25°C), high motile (>90%) and low motile sperm (<30%) were isolated respectively from the 95% layer and from the interface 76–57%. Sperm obtained from the interface 76–57% showing motility >30% were excluded. These two fractions were then washed twice with Earle's medium (Eurobio, France) before being used. A microscopic examination of the enriched sperm fractions (95 and 76–57%) was performed to control the quality of cell preparations. No remaining round cells and cytoplasmic droplets were observed as described by Lambard et al. (2003). Human granulosa cells (positive control for eNOS and c-myc) were obtained by collection of human follicular fluid from pre-ovulatory follicles in the IVF centre (CHRU Clemenceau). 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., 2004). E-cadherin, a positive marker for epithelial cells, was also included in the quality control of our sperm cell preparations (Andersson et al., 1994).
Evaluation of sperm viability
Methods for evaluation of the sperm plasmalemma integrity in motile and immotile fractions included both EN vital stain and hypo-osmotic swelling test (HOST). For the EN test, smears were prepared by mixing 20 µl of sperm fraction and an equal volume of EN solution (4 g eosin plus 5 g nigrosin per litre of distilled water; Sigma Chemical Co., France; BDH Chemical Ltd, UK) (Blom, 1950). The percentage of membrane-intact sperm identified by EN was determined by counting 200 sperm under magnification (x400) with bright-field microscopy. Unstained sperm were viable whereas sperm showing partial or complete purple staining were considered as dead cells.
The HOST was performed by incubating 100 µl of sperm fraction with 100 µl of a hypo-osmotic solution (7.35 g sodium citrate plus 13.5 g fructose per litre of distilled water; Sigma) at 37°C for 60 min. Two hundred sperm were evaluated under magnification (x400) with bright-field microscopy. Sperm without swollen or coiled tails were considered as non-viable cells.
TdT (terminal deoxynucleotidyl transferase)-mediated dUDPnick-end labelling (TUNEL) assay
The DNA fragmentation was assessed using the Apo BrdU Kit (Phoenix Flow Systems, USA). Sperm diluted to 5 x 106 cell/ml were centrifuged at 600 g for 5 min, fixed and permeabilized in 70% ethanol at –20°C for >12 h. After two washes with 1 ml of phosphate-buffered saline (PBS), the elongation reaction was performed by incubating sperm in 50 µl of labelling solution containing the TdT enzyme and dUTP for 1 h at 37°C. 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. To perform the labelling reaction, the fluorescein-PRB1 antibody, providing high specificity for dUTP, was incubated with sperm for 30 min at room temperature in the dark. Before flow cytometry analysis, sperm was washed twice with PBS, labelled with 150 µmol/l of propidium iodide, and filtrated. Positive controls were prepared as described before but with an additional treatment with 10 IU DNAse I for 1 h at 37°C before the labelling reaction.
Analysis was performed using the cell-sorter FACS Vantage SE (BD Biosciences, USA). Fluorochromes were excited with the 488 nm line of the Enterprise laser (Coherent, USA). Green and red fluorescences were detected using FL1 and FL3 detectors respectively through a BP 530/30 nm and a BP 695/40 nm filters.
For each sample, 15 000 events were recorded at a flow rate stabilized at 200–300 cells/s. Cell doublet and debris were excluded using a FL3-A versus a FL3-w gate. Analysis of DNA fragmentation was performed using a FL3-A versus FL1-H cytogram. FL1 and FL3 fluorescence signals were respectively recorded before logarithmic and linear amplifications. Data were analysed with Cell Quest Pro Software (BD Biosciences).
Capacitation
Purified sperm were capacitated in Earle's medium supplemented with BSA (3 mg/ml; Roche, France) for 4 h at 37°C in 5% CO2 and 95% air.
RNA extraction
Total RNA from purified sperm fractions and granulosa cells were extracted using the guanidium thiocyanate-derived method (Chomczynski and Sacchi, 1987). Briefly, after centrifugation the cell pellets were homogenized on ice in a 1 mol/l Tris buffer containing guanidinium thiocyanate 4 mol/l. The RNA was then isolated with a phenol–chloroform–isoamylic acid solution and was precipitated from the aqueous phase with isopropanol. The RNA was again re-extracted and precipitated with isopropanol, washed with 75% ethanol, dryed on speed-vac and dissolved in Diethyl pyrocarbonate (DEPC)-treated water and then stored at –80°C. The purity of RNA samples was checked spectrophotometrically at 260 and 280 nm.
RT-PCR assay
Four hundred nanograms of total RNA were reverse-transcribed to first strand cDNA as follows: 1 h at 37°C with 200 IU M-MLV-RT (Promega, France), 500 µmol/l dNTP, 0.2 µg oligo-dT (12–18 mers) and 24 IU RNasin in a final volume of 10 µl, then 5 min at 94°C. The DNA were further amplified by PCR using selected oligonucleotides. PCR were performed in the presence of 1.5 mmol/l MgCl2, 200 µmol/l dNTP, 1.5 IU Taq polymerase and 50 pmol of the forward and reverse primers (Life Technology, France) in a final volume of 50 µl. The applied PCR primers and the expected lengths of the resulting PCR products for eNOS, nNOS, c-myc, Prm-1 and Prm-2 are shown in Table I. 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 1.5% agarose gel stained with ethidium bromide and visualized under UV transillumination.
|
|
Semi-quantitative RT-PCR
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 PCR amplifications were carried out for 26, 22 and 37 cycles for Prm-1, Prm-2 and c-myc respectively (Figure 1
). The resulting PCR products were analysed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Gels were photographed using Polaroid film under UV light and analysed using AGFA Snap-Scan 1200U Scanner, Photoexpress software and the NIH image computer program (http://rsb.info.nih.gov/nih-image). The intra- and inter-assay coefficients of variation were <12%.
|
Statistical analysis
Results are means±SEM. Statistical analyses were determined by two-way analysis of variance (ANOVA). P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Characterization of the high and low motile sperm fractions
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. (2003)
. 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 viability of the low motile fraction has been estimated both by EN test and HOST. The percentage of live cells was >80% in low motile sperm and was not significantly different with respect to the method used (EN: 76.7±4.3% versus HOST: 79.8±4.4%, n=10) (Figure 3A
).
|
The percentages of non-apoptotic and live cells, respectively estimated by TUNEL technique and EN staining, were similar whatever the technique used (85.7±3.1 versus 86.6±1.9%) (Figure 3B
). The percentage of non-apoptotic cells in motile and low motile sperm was 92.7±1.8% in the high motile fraction versus 85.7±3.1% in the low motile fraction (P<0.005; n=6) whereas the motility was >90 and <30% respectively (data not shown).
Protamine mRNA status
The quantification of Prm-1 and -2 mRNA has been performed by a semi-quantitative RT-PCR. 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. Thus, the low motile population showed a significant increase (43%) of the Prm-1/Prm-2 mRNA ratio compared to the high motile fraction.
|
Expression of NOS mRNA
The presence of eNOS and nNOS proteins has been demonstrated in human sperm (Herrero et al., 1996
), but there is no report about the presence of eNOS and/or nNOS transcripts. Our RT-PCR results demonstrated a large difference in eNOS and nNOS mRNA distribution between the two fractions of sperm (Figure 5). All sperm samples analysed presented a similar and intensive signal at the expected size for eNOS (477 bp) and nNOS (471 bp) in the low motile population of sperm. In the high motile fraction, the expression of eNOS and nNOS mRNA was more variable: no signal was observed for eNOS and nNOS in respectively seven and eight different samples, whereas a weak signal was detected in the others (Figure 5).
|
Expression of c-myc transcripts
C-myc transcripts were the first mRNA described in human ejaculated sperm (Kumar et al., 1993
). No significant variation of c-myc mRNA levels was observed between the two populations of sperm. Therefore c-myc mRNA:Prm-2 mRNA ratio was not different between high motile sperm and low motile fraction (Figure 6).
|
Expression of c-myc and Prm-2 transcripts in capacitated 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. After capacitation for 4 h, the expression of Prm-2 transcripts remained constant whereas the signal intensity corresponding to the c-myc mRNA decreased in capacitated sperm compared to uncapacitated sperm cells (Figure 7
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
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., 2002
; May-Panloup et al., 2003; Mengual et al., 2003; O'Connell et al., 2003) and is commonly utilized in assisted reproduction protocols. 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. It is of note that only N-cadherin mRNA is present in mature sperm whereas E-cadherin mRNA is absent (Goodwin et al., 2000). 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, 1999; Gil-Guzman et al., 2001). 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.
In our study, differences in the levels of Prm-1 and Prm-2 mRNA have been observed between low and high motile sperm (increase of Prm-1 mRNA in low motile population). 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., 2003). Data from Steger et al. (2003) and our group reported the absence of modifications of Prm-2 transcripts, suggesting that Prm-1 is one of the main factors that could be studied in male infertility. Contrary to our work and that of Steger et al. (2003), it has been assumed that both Prm-2 protein and mRNA could be other markers of male fertility since a complete absence of Prm-2 mRNA in round spermatids (Ziyyat et al., 1999) or P2 protein (deYebra et al., 1993) has been noted. No significant differences in the P1:P2 ratio in the different populations of sperm isolated from Percoll fractions have been observed (Mengual et al., 2003) but these data are in disagreement with other studies (Colleu et al., 1996; de Yebra et al., 1998).
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. The motility of human sperm is increased by low NO concentrations (Hellström et al., 1994) but inhibited in the presence of high NO levels (Rosselli et al., 1995). 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., 1996). The administration of high amounts of the NOS substrate, L-arginine, induces infertility in male rats (Ratnasooriya and Dharmasiri, 2002). 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., 1995).
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. mRNA coding for c-myc was first described in human sperm by Kumar et al. (1993). The c-myc protein could play a potential role in capacitation and/or acrosome reaction, and in oocyte fertilization (Kumar et al., 1993). It could also modify characteristics of sperm movement such as head lateral amplitude and velocity (Naz et al., 1991).
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., 2001). The mechanisms of translational control ensuring the absence of mRNA translation until several days after gene expression, involve either protein repressors binding specific sequences located in the 3'-UTR (untranslated region) or to 160–180 residues counting the poly-A tail of mRNA (Steger, 1999; Grootegoed et al., 2000). Transgenic male mice lacking the normal Prm-1 3'-UTR are infertile, resulting in a premature translation of protamine mRNA and therefore in an arrest of transcription (Lee et al., 1995). At first, most of the translationally silent mRNA stored as polyadenylated forms in messenger ribonucleoprotein particles (Schmidt et al., 1999) become translationally active when the poly-A tails are shortened by a partial deanylation (Domenjoud et al., 1991).
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., 1999).
Protein phosphorylation, transcription and translation steps have been observed in human sperm (Naz, 1998, Sakkas et al., 2003). Indeed, the histone-to-protamine exchange process seems to be incomplete in human spermatids compared to rat or mouse spermatids (Tanphaichitr et al., 1978; Gatewood et al., 1987) which therefore suggest a putative transcription and/or translation in sperm. 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. The existence of a 70 kDa polysome-associated protein binding specifically to a coding region of c-myc mRNA and serving to protect it from endonucleolytic action, thus regulating mRNA stability, has been reported (Prokipcak et al., 1994; Ross, 1995). Recently, it has been shown that mouse sperm contain an endogenous reverse transcriptase (Giordano et al., 2000), which can reverse-transcribe RNA into cDNA that are transmitted to offspring upon fertilization (Sciamanna et al., 2003). 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., 1997) that could reduce RNA carriage in capacitating sperm.
Using a microarray analysis, Ostermeier et al. (2002) have reported concordant mRNA profiles from testes and sperm of normospermic patients, suggesting that the study of sperm could reflect past events of spermatogenesis and/or spermiogenesis. 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 Prm-1-Prm-2-TNP-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, 1999). As proposed by Ostermeier et al. (2002), the establishment of sperm mRNA profiles could be used as a genetic fingerprint of normal fertile men. But the presence of transcripts such as eNOS and nNOS in low motile sperm, but not in motile sperm cells, must be taken into account. Motility was thus found to be an important physiological marker, especially for ICSI procedures. The development of new investigation methods such as the analysis of mRNA profiles in high and low motile sperm and the understanding of the transcripts significance could be helpful as additional diagnostic tools and be of prognostic value to fertilization and pregnancy.
|
Acknowledgements |
|---|
S.L. was supported by a fellowship from the French Ministry of Education and Research.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Andersson AM, Edvardsen K and Skakkebaek NE (1994) Expression and localization of N- and E-cadherin in the human testis and epididymis. Int J Androl 17, 174–180.[ISI][Medline]
Braude P, Bolton V and Moore S (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332, 459–461.[CrossRef][Medline]
Chen MJ and Bongso A (1999) Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum Reprod 14, 759–764.
Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB and Eddy EM (2001) Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nature Genet 28, 82–86.[CrossRef][ISI][Medline]
Chomczynski P and Sacchi N (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156–159.[ISI][Medline]
Colleu D, Lescoat D and Gouranton J (1996) Nuclear maturity of human spermatozoa selected by swim-up or by Percoll gradient centrifugation procedures. Fertil Steril 65, 160–164.[ISI][Medline]
Dadoune JP (2003) Expression of mammalian spermatozoal nucleoproteins. Microsc Res Tech 61, 56–75.[CrossRef][ISI][Medline]
de Yebra L, Ballesca JL, Vanrell JA, Bassas L and Oliva R (1993) Complete selective absence of protamine P2 in humans. J Biol Chem 268, 10553–10557.
de Yebra L, Ballesca JL, Vanrell JA, Corzett M, Balhorn R and Oliva R (1998) Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Fertil Steril 69, 755–759.[CrossRef][ISI][Medline]
Domenjoud L, Kremling H and Burfeind P (1991) On the expression of protamine genes in the testis of man and other mammals. Andrologia 23, 333–337.[ISI][Medline]
Gatewood JM, Cook GR, Balhorn R, Bradbury EM and Schmid C (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science 236, 962–964.
Gil-Guzman E, Ollero M, Lopez MC, Sharma RK, Alvarez JG, Thomas AJ and Agarwal A (2001) Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Reprod 16, 1922–1930.
Giordano R, Magnano AR, Zaccagnini G, Pittoggi C, Moscufo N, Lorenzini R and Spadafora C (2000) Reverse transcriptase activity in mature spermatozoa of mouse. J Cell Biol 148, 1107–1113.
Goodwin LO, Karabinus DS and Pergolizzi RG (2000) Presence of N-cadherin transcripts in mature spermatozoa. Mol Hum Reprod 6, 487–497.
Grootegoed JA, Siep M and Baarends WM (2000) Molecular and cellular mechanisms in spermatogenesis. Baillière's Clin Endocrinol Metab 1, 331–343.
Hellström WJG, Bell M, Wang R and Sikka SC (1994) Effect of sodium nitroprusside on sperm motility, viability and lipid peroxidation. Fertil Steril 61, 1117–1122.[ISI][Medline]
Herrero MB, Perez Martinez S, Viggiano JM, Polak JM and Gimeno MF (1996) Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa. Reprod Fertil Dev 8, 931–934.[CrossRef][Medline]
Herrero MB, de Lamirande E and Gagnon C (2003) Nitric oxide is a signaling molecule in spermatozoa. Curr. Pharmaceut Des 9, 419–425.[CrossRef][ISI][Medline]
Kumar G, Patel D and Naz RK (1993) c-myc mRNA is present in human sperm cells. Cell Mol Biol Res 39, 111–117.[ISI][Medline]
Lambard S, Galeraud-Denis I, Bouraima H, Bourguiba S, Chocat A and Carreau S (2003) Expression of aromatase in human ejaculated spermatozoa: a putative marker of motility. Mol Hum Reprod 9, 117–124.
Lambard S, Galeraud-Denis I, Saunders PTK and Carreau S (2004) Human immature germ cells and ejaculated spermatozoa contain aromatase and estrogen receptors. J Mol Endocrinol 32, 279–289.[Abstract]
Lee K, Haugen HS, Clegg CH and Braun RE (1995) Premature translation of protamine I mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proc Natl Acad Sci, USA 92, 12451–12455.
Maione B, Pittogi C, Achene L, Lorenzini R and Spadafora C (1997) Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol 16, 1087–1097.[ISI][Medline]
May-Panloup P, Chrétien MF, Savagner F, Vasseur C, Jean M, Malthiery Y and Reynier P (2003) Increased sperm mitochondrial DNA content in male infertility. Hum Reprod 18, 550–556.
Mengual L, Ballesca JL, Ascaso C and Oliva O (2003) Marked differences in protamine content and P1/P2 ratios in sperm cells from Percoll fractions between patients and controls. J Androl 24, 438–447.
Miller D (2000) Analysis and significance of messenger RNA in human ejaculated spermatozoa. Mol Reprod Dev 56, 259–264.[CrossRef][ISI][Medline]
Miller D, Briggs D, Snowden H, Hamlington J, Rollinson S, Lilford R and Krawetz SA (1999) A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gene 237, 385–392.[CrossRef][ISI][Medline]
Miteva K, Valkov N, Goncharova-Peinoval J, Kovachev K, Zlantarev S, Pironcheva G and Russev G (1995) Electron microscopic data for the presence of post-meiotic gene expression in isolated ram sperm chromatin. Cytobios 88, 7–10.
Naz RK (1998) Effect of actinomycine D and cycloheximide on human sperm function. Arch Androl 41, 135–142.[ISI][Medline]
Naz RK, Ahmad K and Kumar G (1991) Presence and role of c-myc proto-oncogene product in mammalian sperm cell function. Biol Reprod 44, 842–850.[Abstract]
O'Connell M, McClure N, Powell LA, Steele EK and Lewis SEM (2003) Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril 79, 754–762.
Ostermeier GC, Dix DJ, Miller D, Khatri P and Krawetz SA (2002) Spermatozoal RNA profiles of normal fertile men. Lancet 360, 772–777.[CrossRef][ISI][Medline]
Perera DM, Katz M, Heenbanda SR and Marchant S (1996) Nitric oxide synthase inhibitor NG-monomethyl-L-arginine preserves sperm motility after swim-up. Fertil Steril 66, 830–838.[ISI][Medline]
Prokipcak RD, Herrick DJ and Ross J (1994) Purification and properties of a protein that binds to the C-terminal coding region of human c-myc RNA. J Biol Chem 269, 9261–9269.
Ratnasootiya WD and Dharmasiri M (2002) L-arginine, the substrate of nitric oxide synthase, inhibits fertility of male rats. Asian J Androl 3, 97–103.
Ross J (1995) mRNA stability in mammalian cells. Microbiol Rev 59, 423–450.
Rosselli M, Dubey RK, Imthurn B, Macas E and Keller PJ (1995) Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity. Hum Reprod 10, 1786–1790.
Roudebush WE, Wild MD and Maguire EH (2000) Expression of the platelet-activating factor in human spermatozoa: differences in messenger ribonucleic acid content and protein distribution between normal and abnormal spermatozoa. Fertil Steril 73, 967–971.[CrossRef][ISI][Medline]
Sakkas D, Leppens-Luisier G, Lucas H, Chardonnens D, Campana A, Franken DR and Urner F (2003) Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding. Biol Reprod 68, 1463–1469.
Schmidt EE, Hanson ES and Capecchi MR (1999) Sequence-independent assembly of spermatid mRNAs into messenger ribonucleoprotein particles. Mol Cell Biol 19, 3904–3915.
Sciamanna I, Barberi L, Martire A, Pittoggi C, Beraldi R, Giordano R, Magnano AR, Hogdson C and Spadafora C (2003) Sperm endogenous reverse transcriptase as mediator of new genetic inforamtion. Biochem Biophys Res Commun 312, 1039–1046.[CrossRef][ISI][Medline]
Siffroi JP and Dadoune JP (2001) Accumulation of transcripts in the mature human sperm nucleus: implication of the haploid genome in a functional role. It J Anat Embryol 106, 189–197.[Medline]
Steger K (1999) Transcriptional and translational regulation of gene expression in haploid spermatids. Anat Embryol 199, 471–487.[CrossRef][Medline]
Steger K (2003) Possible predictive factors for ICSI? Molecular biology techniques in combination with therapeutic testicular biopsies. Andrologia 35, 200–208.[CrossRef][ISI][Medline]
Steger K, Fink L, Failing K, Bohle RM, Kliesch S, Weidner W and Bergmann M (2003) Decreased protamine-1 transcript levels in testes from infertile men. Mol Hum Reprod 9, 331–336.
Tanphaichitr N, Sohbon P, Taluppeth N and Chalermisarachai P (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp Cell Res 117, 347–350.[CrossRef][ISI][Medline]
Weng SL, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S and Oehninger S (2002) Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod 8, 984–991.
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th edn. Cambridge University Press, UK.
Wyke SM and Krawetz SA (1999) Gene potentiation: forming long-range open chromatin structures. Gene Ther Mol Biol 4, 303–312.
Ziyyat A, Lasalle B, Testart J, Briot P, Amar E, Finaz C and Lefevre A (1999) Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients. Hum Reprod 14, 379–387.
Submitted on February 17, 2004; resubmitted on March 24, 2004; accepted on March 30, 2004.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Lalancette, C. Thibault, I. Bachand, N. Caron, and N. Bissonnette Transcriptome Analysis of Bull Semen with Extreme Nonreturn Rate: Use of Suppression-Subtractive Hybridization to Identify Functional Markers for Fertility Biol Reprod, April 1, 2008; 78(4): 618 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gilbert, N. Bissonnette, G. Boissonneault, M. Vallee, and C. Robert A molecular analysis of the population of mRNA in bovine spermatozoa Reproduction, June 1, 2007; 133(6): 1073 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-G. Li, X.-F. Ding, A.-H. Liao, X.-B. Kong, and C.-L. Xiong Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility Mol. Hum. Reprod., May 1, 2007; 13(5): 299 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Platts, D. J. Dix, H. E. Chemes, K. E. Thompson, R. Goodrich, J. C. Rockett, V. Y. Rawe, S. Quintana, M. P. Diamond, L. F. Strader, et al. Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs Hum. Mol. Genet., April 1, 2007; 16(7): 763 - 773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Miller and G.C. Ostermeier Towards a better understanding of RNA carriage by ejaculate spermatozoa Hum. Reprod. Update, November 1, 2006; 12(6): 757 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-M. Cai, Y.-T. Gui, X. Guo, J. Yu, L.-D. Guo, L.-B. Zhang, H. Wang, and J. Yu Low Expression of Glycoprotein Subunit 130 in Ejaculated Spermatozoa from Asthenozoospermic Men J Androl, September 1, 2006; 27(5): 645 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Oliva Protamines and male infertility Hum. Reprod. Update, July 1, 2006; 12(4): 417 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.P. Dadoune, A. Pawlak, M.F. Alfonsi, and J.P. Siffroi Identification of transcripts by macroarrays, RT-PCR and in situ hybridization in human ejaculate spermatozoa Mol. Hum. Reprod., February 1, 2005; 11(2): 133 - 140. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||