Mol. Hum. Reprod. Advance Access originally published online on December 10, 2004
Molecular Human Reproduction 2005 11(2):133-140; doi:10.1093/molehr/gah137
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Identification of transcripts by macroarrays, RT–PCR and in situ hybridization in human ejaculate spermatozoa
1Laboratoire de Cytologie et Histologie, Centre Universitaire des Saints-Pères, 75270, Paris, 2EA 1533 Génétique de la Reproduction Humaine, Hôpital Tenon (AP-HP), 75970, Paris and 3Unité INSERM 581, Hôpital Henri-Mondor, 94010 Créteil, Paris, France
4 To whom correspondence should be addressed at: Laboratoire de Cytologie et Histologie, Centre Universitaire des Saints-Pères, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France. Email: jean-pierre.dadoune{at}univ-paris5.fr
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Abstract |
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Round spermatids contain high levels of extremely varied mRNAs that are synthesized either throughout early spermatogenesis or during spermiogenesis from the haploid genome. Concomitantly, with major changes in the chromatin organization, arrest of transcription occurs at midspermiogenesis. However, previous investigations using RT–PCR have revealed the persistence of numerous and different transcripts in ejaculated spermatozoa. In the present study, a step-by-step analysis by means of macroarray hybridization, RT–PCR and in situ hybridization was performed to identify more accurately the different mRNA species found in the human ejaculated spermatozoa. The data showed an extended pattern of various transcripts encoding a diverse range of proteins involved in signal transduction and cell proliferation. For the first time, they demonstrated that mRNAs coding for the transcription factors NF
B, HOX2A, ICSBP, protein kinase JNK2, growth factor HBEGF and receptors RXRß and ErbB3 accumulate within the sperm nucleus. The origin and fate of the sperm transcripts remain subject to discussion. Key words: haploid genome/messenger RNAs/spermatozoa
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Introduction |
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In mammals, round spermatids contain a number of transcripts that are produced either throughout early spermatogenesis (Eddy, 2002
) or during spermiogenesis from the haploid genes coding for sperm-specific proteins such as transition proteins and protamines (Steger, 1999) or sperm tail cytoskeletal proteins implied in the molecular make-up of the outer dense fibres (Petersen et al., 1999) and fibrous sheath (Eddy et al., 2003). The arrest of transcription that is concomitant with major changes in the chromatin organization occurs during midspermiogenesis (Dadoune, 2003). However, the presence of extremely varied transcripts in mature sperm cells has been described in both rodents (Pessot et al., 1989; Passananti et al., 1995; Levallet et al., 1998) and man (Kumar et al., 1993; Chiang et al., 1994; Miller et al., 1994; Rohwedder et al., 1996; Kramer and Krawetz, 1997; Wykes et al., 1997; Miller et al., 1999; Richter et al., 1999; Goodwin et al., 2000a,b; Miller, 2000; Sachdeva et al., 2000; Carreau et al., 2001; Siffroi and Dadoune, 2001; Aquila et al., 2002; Lin et al., 2002; Luconi et al., 2002; Ostermeier et al., 2002; Wong et al., 2002; Com et al., 2003; Park et al., 2003; Lambard et al., 2004). Most investigations on RNA identification in mature spermatozoa have been performed with techniques based on the detection of specific or particular sets of RNAs by means of PCR after reverse transcription (RT–PCR). Attempts, using differential RNA fingerprinting following anchor-primed RT–PCR, have previously been made to characterize the diverse transcripts present in human spermatozoa (Miller et al., 1994). More recently, microarray technology has been employed for a large-scale analysis of complementary DNAs prepared from spermatozoan RNA by RT–PCR. This method has allowed the rapid detection of thousands of transcripts in human sperm cells (Ostermeier et al., 2002). Nevertheless, RNA extracted from ejaculated spermatozoa may be exposed to low levels of somatic contaminants, despite purification of semen samples. Consequently, the verification of PCR results by in situ hybridization (ISH), using either riboprobes (Kumar et al., 1993; Wykes et al., 1997) or labelled antisense oligonucleotides (Siffroi and Dadoune, 2001), is recommended to establish the unquestionable spermatic origin of the transcripts detected by PCR. ISH also permits bypassing another problem raised by the high sensitivity of PCR. Indeed, nested RT–PCR of RNAs from a single spermatozoon has shown apparently aberrant transcripts in human sperm cells like those coding for synapsin 1, immunoglobulins or T cell receptor 
(Kimoto, 1998). Such a phenomenon, named illegitimate transcription, has been defined as a very low-level transcription of any gene in any cell type (Chelly et al., 1989). In this respect, some transcripts accumulated in the sperm nucleus would not have any functional meaning in the male germ cell line. Thus, the questions raised by the sensitivity of detection methods prompted us to further identify the different mRNA species found in human ejaculated spermatozoa by carrying out a step-by-step analysis with macroarray hybridization, RT–PCR and ISH. The results showed an extended pattern of various transcripts encoding factors essential for cellular functioning and, for the first time, demonstrated the presence of the NFB, HOX2A, ICSBP, JNK2, HBEGF, RXRß and ErbB3 transcripts in human sperm nuclei. |
Material and methods |
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Preparation of spermatozoa
Semen samples were collected from 20 healthy voluntary donors, after 3–5 days of sexual abstinence. A complete semen analysis was performed on each sample using the World Health Organization guidelines (1999
). The sperm concentration was >20 x 106/ml, with motility >65% and containing <1% immature germ cells and lymphocytes. At least, 30% of the spermatozoa had a normal morphological appearance. Samples from 3 to 5 donors were pooled for further treatments. Following liquefaction at 37°C, motile spermatozoa were selected by density gradient centrifugation. Two millilitres of semen were layered on top of a 45–90% discontinuous gradient of isotonic PureSperm 100 (Nidacon, Gothenburg, Sweden) diluted with FertiCult IVF mediumTM (FertiPro N.V., Beernem, Belgium). After centrifugation (20 min at 400 g), the pellets were resuspended in 0.25 ml of BM1 medium (Eurobio, Les Ulis, France) and the purity of the selected spermatozoa population was checked under a light microscope. For some samples, the sperm heads were separated from the sperm tails by sonication (4 x 6 min) at the maximal power of a Labsonic U sonicator (Biotech. International, Melsungen, Germany). Sperm heads were recovered by centrifugation at 400 g and resuspended in BM1 medium. Sperm smears were prepared with 20 µl of the cell suspension per slide using VectabondTM silane-coated slides (Vector, Burlingame, CA, USA). The smears were fixed in methanol–acetic acid (3/1) for 5 min, rinsed briefly in distilled sterile water, air-dried and then stored at –20°C until used.
RNA extraction
Total RNA was extracted from the entire spermatozoa and the sperm heads using the TRIZol solutions (Invitrogen SARL, France), following the manufacturer's instructions. The final RNA concentration was measured on a GeneQuant II spectrophotometer (Pharmacia biotech, UK).
Macroarray hybridization
Two micrograms of RNA were treated for 30 min at 37°C with 10 units of RQ1-DNase, RNase Free (Promega, WI, USA) in the presence of 40 units of RNasin (Promega, WI, USA). The RNA was then extracted using phenol/chloroform and precipitated by ethanol. The pellet was washed with 75% ethanol and dried. The dried RNA was resuspended in 4 µl of sterile water. Two microlitres of RQ1-DNase treated RNA were used to synthesize the 32PdATP labelled probe following the supplier's instructions (BD Biosciences Clontech, CA, USA). The AtlasTM Human cDNA Expression Array membranes (BD Biosciences Clontech, CA, USA) were hybridized overnight and washed as described in the user's manual. After the last washing step, the membranes were sealed in Saran wraps and exposed for 1 week to a detection screen (Molecular Dynamics, CA, USA). The screen was then analysed using a Storm 840 phosphorImager and the ImageQuant program (Molecular Dynamics, CA, USA). The complete list of the cDNAs and controls immobilized on the membrane is available on Clontech's website (www.bdbiosciences.com/clontech/atlas/genelists/index.shtml).
RT–PCR analysis
Two micrograms of RQ1-DNase treated spermatozoa RNA were used in a final volume of 50 µl for reverse transcriptase reactions. Briefly, the RNA was incubated for 10 min at 70°C in 20 µl of sterile water containing 0.4 mM of oligodT18–22. The RNA/oligodT solution was shielded on ice before the addition of AMV reverse transcriptase buffer (50 mM tris–HCl pH 8.3, 6 mM MgCl2, 40 mM KCl, 4 mM DDT), dNTPs (0.2 mM of each), RNasine (20 units) and 40 units of AMV reverse transcriptase (Finnzyme, Finland). The reaction mixture was incubated for 1 h at 48°C, extracted using phenol/chloroform and precipitated by ethanol. The cDNA pellet was washed with 75% ethanol, dried and resuspended in water at a final concentration of 10 ng/µl. The equivalent of 50 ng of cDNA were used in the PCR reaction under the following conditions: 5' and 3'specific oligonucleotide primers (0.3 µmoles of each), DyNAzymeTM EXT optimized buffer (1x final containing 1.5 mM MgCl2), dNTP (200 µM final each), dimethylsulphoxide (5% final), and 1 unit of enzyme DyNAzymeTM EXT DNA polymerase (Finnzyme). The PCR protocol consisted of a 2 min denaturation step at 94°C followed by 40 cycles of a denaturation step at 94°C for 30 s, an annealing step at 57°C for 30 s. and an elongation step at 72°C for 1 min. The reaction was ended by a 10 min step at 68°C and the samples were stored at 4°C until analysis. For analysis, the products of the reaction, separated by electrophoresis on a 1.5% agarose gel, were blotted on a Hybond N+ membrane (Amersham Biosciences, France), which was hybridized with [32P] labelled oligonucleotides (2x107 cpm) specific for each selected RNA. The sequence of the specific PCR primers and the corresponding detection primers used in this study are listed in Table I.
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In situ hybridization
Specific oligonucleotides for each selected RNA were labelled using terminal transferase and DIG-dUTP as described by the supplier (Roche Applied Science, Germany). The probes were quantified and the labelling efficiency estimated using the DIG Teststrips (Roche Applied Science, Germany). Finally, the probes were kept at –20°C until use.
Before hybridization, sperm head decondensation was performed by treating slides with 0.1 M Tris–HCl pH 8, 10 mM dithiothreitol (Sigma–Aldrich Chimie SARL, France) for 30 min at room temperature followed by a 3 h incubation in 0.1 M Tris–HCl pH 8, 10 mM 3,5-diiodosalicylic acid (LIS, Sigma–Aldrich Chimie SARL, France). The slides were then washed twice in 2 x SSC (1 x SSC: 150 mM NaCl, 15 mM sodium citrate pH 7.0), dried and prehybridized for 2 h at 37°C in a solution of 1 x Denhardt (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 1.5 x SSC and 4% deionized formamide containing 110 µg/ml tRNA and 20 µg/ml heat denatured Salmon Sperm DNA (SSDna). After a two-step wash, 1 h at 37°C in 2 x SSC and 1 h in 1 x SSC at room temperature, the slides were treated for immunodetection using the DIG Nucleic Acid Detection Kit (Roche Applied Science, Germany) following the manufacturer's instructions.
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Results |
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To enrich for motile spermatozoa and remove somatic contaminants, the pooled samples were purified by centrifugation through a discontinuous gradient of PureSperm. As shown in Figure 1a, no somatic contamination became visible in the pellets resuspended in the BM1 medium. All the selected spermatozoa exhibited normal morphological features. After DTT treatment, which allows a better accessibility to the probes used for ISH, sperm cells display moderately uncondensed nuclei (Figure 1b).
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Two identical expression array membranes contained in the kit were hybridized with two independently prepared sperm-specific probes. A representative result obtained on a membrane is presented in Figure 2aFigure 2a. The signals obtained on the two membranes were similar in strength (Figure 2b). Analysis of the hybridization signals indicated the presence of a large variety of RNAs in the spermatozoa. The D region of the membrane, in which the sequences corresponding to the transcription factors and the regulators are located, exhibits signals stronger than those from the other regions. The region with the lowest signal is the A region corresponding to the oncogenes and the tumour suppressor sequences. The 20 strongest signals classified by decreasing signal strengths are listed in Table II. From this list, the three most abundant signals correspond, respectively, to the 90 kDa heat-shock protein A (Hsp86), the heparin-binding epidermal growth factor-like growth factor (HBEGF) and the interferon consensus sequence-binding protein (ICSBP). Similar results were obtained from entire spermatozoa and sperm heads collected after sonication (data not shown).
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For RT–PCR analysis, we choose 16 RNA sequences including the three previously mentioned strongest signals, two negative hybridization signals for the RNAs coding for deoxyribonuclease I-like protein 1 (XIB) (coordinates C7n) and glucagon (coordinates F7f), respectively, and finally 11 randomly chosen sequences exhibiting various degrees of hybridization signal. These RNAs are listed in Table I. To check the quality of the reverse transcriptase reaction, we performed, at first, a PCR reaction using specific oligonucleotides for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as positive control (Lin et al., 2002
; Ohmura et al., 1999). The amplified products were separated on agarose gel and visualized by ethidium bromide staining under UV light (Figure 3a). Two of the five samples tested gave a low amount of amplified products for GAPDH. These samples were discarded from the study and we pooled the samples 1–3 for the PCR reactions with the selected RNA sequence-specific primers. Four additional pools of the samples were treated independently in the same conditions. A typical pattern of the amplification products detected by hybridization with specific labelled oligonucleotides is shown in Figure 3b. The strongest signals detected on each tested sample correspond to the GAPDH, HBEGF, HSP90 and c-Myc transcripts. Due to the heterogeneous quality of the extracted samples, RXRß, HOX2A, NFB1 and JNK2 amplification products were detected in three samples out of five whereas RAB4 was detected only in one sample. No specific signal was detected for all the other oligonucleotides tested. The correlation between macroarray hybridization signals and RT–PCR blots was good for most of the specific mRNAs tested except for ICSBP. The latter, in spite of a strong macroarray signal, did not show any specific RT–PCR signal while c-Myc showed a very low signal on macroarray membranes and a strong RT–PCR signal. The comparison of the ICSBP sequence with the EST database indicated that the 5' oligonucleotide designed for RT–PCR was located in a region that is deleted by splicing at least in Jurkat cells (human leukaemic T lymphocytes). Consequently, we designed a new 5' oligonucleotide that allowed the detection of a strong specific amplification product for ICSBP at the right size (164 bp) in all tested samples (Figure 3c).
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ISH analysis was made to assess whether the nine transcripts detected by RT–PCR with a wide range of hybridization signal strengths (Figure 3b) were present in mature spermatozoa. As shown in Figure 4, all spermatozoa display a similar distribution of the RXRß, JNK2, HOX2A, ErbB3, NF
B, c-Myc and HBEGF transcripts, localized specifically within the entire head region. The presence of ICSBP detected by RT–PCR using a new 5' oligonucleotide (Figure 3c) was confirmed by ISH results. The reaction intensity of the eight transcripts was comparable to that for GAPDH used as control.
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Discussion |
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The data provided by hybridization of the Atlas Human cDNA expression array membranes indicated that a large number of RNAs are present in ejaculated spermatozoa. In addition to observations based on the amplification of the targeted mRNA species (Table III), recent investigations using a conventional screening strategy (Miller et al., 1999
) or microarray technology (Ostermeier et al., 2002) have shown that most spermatozoan RNAs encode a diverse range of proteins participating in signal transduction, oncogenesis and cell proliferation. Our results are in accordance with previous findings, even when macroarray hybridization signals were not strong. For example, c-Myc and N-cadherin that give very low hybridization signals on the membrane were previously detected in spermatozoa by RT–PCR and/or in situ hybridization (Kumar et al., 1993; Goodwin et al., 2000a). However, it is noteworthy that the region of the membrane containing the transcription factors and regulators (region D) exhibits the largest number of signals. The significance of this finding remains to be elucidated. In addition, our macroarray hybridization data revealed that the three most represented mRNAs in human spermatozoa are HSP90, HBEGF and ICSBP. The well-detected HSP90 mRNA has already been identified in human spermatozoa (Miller et al., 1999; Miller, 2000). HSP90 is an abundant and highly conserved molecular chaperone with a large conformational flexibility that allows it to assist a wide range of substrates (Picard, 2002). As a component of the hetero-oligomeric structure of steroid receptors, it would act as a positive and negative modulator of the steroid receptor function (Catelli et al., 1999).
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The presence of the HBEGF transcript and the ICSBP transcript variant, described for the first time in human sperm cells, is demonstrated by our RT–PCR and ISH results. HBEGF is one of the major ligands for the epidermal growth factor receptor (EGFR) that belongs to the ErbB family of receptor tyrosine kinases (Anderson et al., 2004
). It is thought to be involved in preimplantation embryo development and implantation (Leach et al., 1999; Tamada et al., 1999; Armant et al., 2000). Until now, its production by the testis has not been established. The ICSBP mRNA detected in human spermatozoa is a spliced variant that is deleted at least at exon 7. ICSBP has been identified as a transcription factor of the interferon (IFN) regulatory factor family (IRF), which regulates the IFN-inducible gene expression and which would participate in the regulation proliferation and differentiation of the haematopoietic progenitor cells (Schmidt et al. 1998). Both type I and type II IFNs are expressed in the seminiferous tubules (Dejucq et al., 1995). Thus, it can be hypothesized that these cytokines act on IFN-inducible genes in male germ cells through a transcription factor which is a truncated protein translated from the ICSBP transcript variant.
Two other new mRNAs encoding transcription factors have been found in the sperm nucleus. The HOX2A mRNA is transcribed from the HOX2A gene (human homeobox HOXB5) that is located on the human chromosome 17 (Galang and Hauser, 1992). HOX homeobox genes display a pattern of expression in the testis, suggesting a role in spermatogenesis (Lindsey and Wilkinson, 1996). The NFB mRNA codes for a NFB protein that belongs to the Rel/nuclear factor (NF)-kappa B family of transcription factors. Nuclear NFB activity has been detected in murine spermatocytes and spermatids, leading to the hypothesis that NFB factors may play a role during the development of sperm cells (Delfino and Walker, 1998; Lilienbaum et al., 2000). In addition to transcription factor mRNAs, transcription factor proteins such as Oct-1, Oct-4, Ets-1, CEBP and TBP have been localized by immunofluorescence in the chromatin of mouse spermatozoa, questioning the role of these proteins after fertilization (Pittoggi et al., 2001).
Finally, altogether, our RT–PCR and ISH data have shown the localization of the JNK2, RXRß, and ErbB3 transcripts within the human sperm head. JNK2 (c-Jun N-terminal kinase 2) is a member of the MAP kinase group. After activation by dual phosphorylation on tyrosine and threonine residues, it phosphorylates the transcription factors c-Jun, ATF-2 and Elk-1 (Gupta et al., 1996). It is well known that the nuclear transcription factor c-Jun is expressed during the premeiotic and the meiotic steps of spermatogenesis (Muller et al., 1982). The RXRß and ErbB3 mRNAs code for the nuclear retinoid X receptor ß (RXRß) and for a member of the epidermal growth factor receptor (EGF-R or ErbB) family, respectively. RXRß was immunolocalized in A spermatogonia, pachytene spermatocytes and spermatids in adult mice (Gaemers et al., 1998) and in rat gonocytes during a short neonatal period (Boulogne et al., 1999), suggesting that retinoids act on the male germ cell development. The epidermal growth factor superfamily of peptide growth factors (EGF-GFs) is implicated in the spermatogenic events and RT–PCR analysis has revealed that the EGF receptors ErbB1, ErbB2, ErbB3 and ErbB4 are expressed in all the stages of spermatogenesis (Wahab-Wahlgren et al., 2003).
The origin and the fate of the mRNAs found in the human sperm nucleus are yet to be discussed. The question is raised as to whether these RNAs are stored in the sperm nucleus after being transcribed in early spermatids or are eventually synthesized in the mature spermatozoa, in spite of the apparent transcriptionally inert state of the nucleus (Kierszenbaum and Tres, 1975). Interestingly, the presence of residual DNA and RNA polymerase activity within the sperm chromatin has been formerly reported (Hecht, 1974; Witkin et al., 1975; Chevaillier and Philippe, 1976; Miteva et al., 1995). Later on, complementary investigations have indicated that in spite of a high degree of DNA packaging within the human sperm head, chromatin retains some features of active chromatin, mainly acetylated histones (Gatewood et al., 1990) and the arrangement of certain chromatin domains into the nucleosomes (Gineitis et al., 2000; Zalenskaya et al., 2000). Therefore, it is tempting to speculate that DNA regions linked to histones may represent sites of active transcription. However, transcription within the sperm genome is yet to be conclusively demonstrated in vivo. In fact, persistence of transcripts in mature spermatozoa is more likely to be the result of an overexpression process in the spermatid genome, as illustrated by the presence of transcripts for transition proteins and protamines whose synthesis and deposition are known to be achieved at the end of spermiogenesis (Dadoune, 2003).
With a few exceptions concerning, for example, c-Myc (Naz et al., 1991; Kumar et al., 1993), transcription factor Stat 4 (Herrada and Wolgemuth, 1997; Siffroi and Dadoune, 2001), angiotensin converting enzyme (Kohn et al., 1998; Siffroi and Dadoune, 2001), N-cadherin (Goodwin et al., 2000a) and P450 aromatase (Carreau et al., 2001), there is no evidence that the proteins corresponding to the majority of mRNAs found in the mature spermatozoa are also present. Therefore, to date, the possible role of these transcripts in the function of spermatozoa cannot be inferred. However, if the mRNAs accumulated in the sperm nucleus are not residual non-functional materials, they might be viewed as the male gametes contribution to early embryogenesis. Delivering spermatozoan RNA to the ovocyte has been demonstrated in mice (Hayashi et al., 2003) and humans (Ostermeier et al., 2004) as well. Some sperm transcripts encoding proteins known to participate in fertilization and embryonic development have been specifically detected in early embryos after in vitro fertilization failure, while they have not been found in the ovocyte (Ostermeier et al., 2002). Thus, human spermatozoa could act not only as genome carriers but also as providers of specific transcripts necessary for zygotic viability and development before activation of the embryonic genome.
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Submitted on September 28, 2004; resubmitted on November 10, 2004; accepted on November 17, 2004.
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