Mol. Hum. Reprod. Advance Access originally published online on June 13, 2007
Molecular Human Reproduction 2007 13(8):549-556; doi:10.1093/molehr/gam038
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
APEX/Ref-1 (apurinic/apyrimidic endonuclease DNA-repair gene) expression in human and ascidian (Ciona intestinalis) gametes and embryos *
1 Laboratoire Marcel Mérieux, Avenue Tony Garnier, Lyon, France 2 Stazione Zoologica ‘Anton Dohrn’, 80121 Napoli, Italy 3 National Research Council, 83100 Avellino, Italy 4 Laboratoire d'Eylau/Unilabs, 55 Rue Saint Didier, 75116, Paris
5 Correspondence address. Laboratoire d'Eylau/Unilabs, 55 Rue Saint Didier, 75116 Paris. Tel: +33-6-28-25-25-74; Fax: +33-1-53-70-64-94; E-mail: yves.menezo{at}eylau.fr, yves.menezo{at}club-internet.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
In recent years, the impact of sperm DNA damage on fertility has become an important issue. The different technologies developed to check sperm DNA fragmentation lead to the same conclusion: DNA damage negatively impacts upon reproductive processes. Oocyte DNA repair capacity is one of the cues to understanding embryo developmental arrest. APEX/Ref-1 (apurinic/apyrimidic endonuclease) is an enzyme involved in the DNA base excision repair pathway removing the abasic sites, the most common DNA decays. In humans, APEX has a multifunctional role, including the control of the redox status of transcription factors. RT–PCR allowed us to detect human APEX transcripts in oocytes, spermatozoa and preimplantation blocked embryos. In parallel, a comparative study on sea squirt Ciona intestinalis (ascidian) indicated that APEX transcripts are clearly detectable in oocytes and embryos until the larva stage, but not in spermatozoa, suggesting the appearance of the paternal contribution to DNA repair during development having arisen only late in Vertebrate evolution. Of additional phylogenetic significance is the observation that sea squirt APEX appears to lack redox transcriptional activity.
Key words: human/Ciona/APEX/Ref1/sperm/oocyte
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
The impact of male infertility is still a matter of debate. For many years male fertility has been defined as the ability of sperm to fertilize oocytes and obtain early cleavage stage embryos. In human in vitro fertilization (IVF), the generation of two to four cell embryos was considered a test for sperm fertilizing ability, assuming that all resulting embryos had the same developmental potential, irrespective of sperm and oocyte quality (Menezo, 2006; Menezo et al., 2006). Now, it is well established that differences in male fertility are not simply related to sperm penetration failure (Ron-el et al., 1991). In some cases, ICSI allows a better embryo development; however, this technique presents potential harmful aspects (Morozumi and Yanagimachi, 2005). More recently, careful attention has been paid to the quality of sperm DNA, and new methods have been developed to evaluate paternal DNA integrity, which can be totally independent of all semen parameters, including sperm morphology, concentration and motility (Evenson et al., 2002; Oger et al., 2003). Different techniques reached the same conclusions: sperm DNA fragmentation and increased reactive oxygen species (ROS) concentration are responsible for reduced fertility (Henkel et al., 2004). ROS have a heavily deleterious impact on sperm DNA (Lopes et al., 1998a,b), since they cause the formation of apurinic/apyrimidic (AP) sites (Xu et al., 1998; Hsieh et al., 2001), that, if left un-repaired, have profound effects on sperm physiology post fertilisation.
All mammalian cells possess DNA repair systems (Bessho et al., 1993; Wood et al., 2001). In oocytes and zygotes, DNA repair is probably one of the most important processes to be performed at the time of fertilization and immediately after, in order to allow complete embryonic development. The product of the APEX/Ref-1 (AP excision/Redox factor-1) gene is an enzyme capable of initiating the repair of AP sites, the most common decay in damaged DNA (Demple et al., 1991; Hsieh et al., 2001; Kelley and Parsons, 2001). APEX/Ref-1 is known by different names (e.g. APE; APX; APE1; APEN; HAP1; REF1; REF-1) and encodes the major AP endonuclease in human cells. Splice variants have been found for this gene: all encode the same protein. Variant-1 contains the full-length first exon and is the longest transcript (Fritz, 2000). In human cells, APEX/Ref-1 not only initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5' and adjacent to an AP site, but also possesses a Ref-1 activity, i.e. the ability to control the redox status of a number of transcription factors including Fos, Jun and p53 (Xanthoudakis and Curran, 1992; Jayaraman et al., 1997).
The importance of metabolic pathways involving APEX/Ref-1 activity, led us to check the presence of APEX/Ref-1 mRNA in human oocytes and preimplantation embryos, in order to clarify the role of the enzyme in these models in response to oxidative stress. Accordingly to recent estimations, spermatozoa can carry up to 5000 mRNAs (Ostermeier et al., 2002; Miller et al., 2005); therefore, we tested the expression of APEX/Ref-1 also in spermatozoa.
To investigate how evolutionarily conserved was the expression of APEX/Ref-1 mRNA during the early stages of development, we extended the study to oocytes and spermatozoa of the ascidian Ciona intestinalis (sea squirt), an organism intensively studied in developmental biology (Satoh, 2003) and, more recently, proposed as a model to study meiotic regulation (Russo et al., 1996; Nixon et al., 2000; Russo et al., 2004). From an evolutionary point of view, Tunicates (appendicularians, salps and sea squirts) have very recently been re-evaluated as the closest relatives of vertebrates, more than cephalochordates, like amphioxus (Delsuc et al., 2006). This important discovery has been made possible since the advent of genomics that actually provide the opportunity for phylogenetics to resolve a number of outstanding evolutionary questions (Delsuc et al., 2005). In this respect, the draft copy of the C. intestinalis genome became publicly available, providing new insights into origin and evolution of chordates (Dehal et al., 2002; Ciona Genome Group, 2003). Based on this evidence, we used C. intestinalis gametes and embryos as comparative models to study the conservation of APEX/Ref-1 functions during evolution.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Collection of human sperm, oocytes and embryo
Human oocytes were collected from an IVF centre (IRH/Laboratoire Marcel Mérieux, Centre de FIV du Val d'Ouest, chemin de la Vernique, 69130 Ecully). Hormonal stimulation was performed according to classical protocols involving a semi-long treatment with gonadotrophin-releasing hormone (GnRH) agonists (decapeptyl or buserelin) followed by ovarian stimulation with urinary or recombinant follicle stimulating hormone (FSH). In the absence of fertilization, metaphase II (MII) oocytes were collected 48 h after insemination. All the fertilization and embryo culture procedures were performed under oil to allow better developmental potential and to avoid oxidative stress. Germinal vesicle (GV) oocytes were collected from intracytoplasmic sperm injection (ICSI) patients, when maturation was not completed. MII oocytes (one pool of eight, and two pools of five) were gathered when, in two cases, the husband was not able to produce sperm at the time of IVF/ICSI). As the access for GV was not restricted, most of our controls (ß-actin) were performed on pools of 10 GV oocytes. Tests were also performed on individual GV oocytes. Care was taken to ensure the total absence of corona cells on the oocytes and to avoid any extra cellular contamination by observation under an inverted microscope at x200 magnification. Arrested or lysed embryos (after freezing and thawing) were used for the determination of APEX according to the French bioethical law. Analyses were performed on two pools of five and one pool of two 2–3 cell blocked embryos, two pools of eight, and one pool of four early blocked morula (eight to 16 cell stages), of two blocked late morulas and five blocked/lysed blastocysts. No four-cell blocked or lysed embryos were available. This protocol was performed in accordance with an agreement of the CNMBRDP (Commission Nationale de Medecine, Biologie de la Reproduction et Diagnostic Preimplantatoire). This agreement allows research only on embryos that were unsuitable for transfer. Six sperm samples, with characteristics largely over WHO parameters were treated twice with 45/90% Percoll (Suprasperm, Medicult France) gradient. We especially took care, in the crude samples, of the absence of round cells and blood cells. After treatment the samples were checked under an inverted microscope to be absolutely sure of the total absence of foreign cells.
RNA extraction, reverse transcription (RT) and polymerase chain reaction (PCR) from human samples
Total RNA was extracted from human sperm (5 x 105 higly selected spermatozoa) using Trizol protocol (Invitrogen, Milano, Italy). For human oocytes and blocked embryos, a direct thermolysis was performed. Briefly, samples were placed in PCR tubes in 2 µl of sterile diethylpyrocarbonate (DEPC)-treated water and covered with one drop of mineral oil. Samples underwent thermolysis for 1 min at 100°C in order to release nucleic acids.
The reverse transcription reaction was performed in a final volume of 20 µl containing RT buffer 1 x, 10 mM dithiothreitol (Sigma, Milan, Italy), 0.5 mM of each dNTP (Invitrogen), 0.5 µg oligo(dT)12–18 (Invitrogen), 10 IU RNase inhibitor (Promega, Chrabonnières, France) and 200 IU SuperScript reverse transcriptase (Invitrogen). For each sample, 18 µl of the RT mix was added. RT reaction was carried out at 42°C for 50 min followed by heating to 70°C for 15 min to inactivate the reaction.
PCR analyses were carried out in a final volume of 50 µl containing the cDNA (half of the RT product) from 10 oocytes and a minimum of five blocked embryos, or from 5 x 105 spermatozoa, 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 0.2 mM each of dNTP, 0.4 µM of each primer (MWG biotech, France) and 2 IU of Taq DNA polymerase (Perkin Elmer Cetus, Courtaboeuf, France). To avoid any risk of genomic contamination during PCR, the forward APEX-F and the reverse APEX-R were designed on exons three and five of human APEX gene, respectively (Accession Number M80261, Table 1). Internal positive control of PCR was performed on the same samples using human ß-actin specific primers (Table 1, Accession Number E00829 [GenBank] ). A positive control was also performed on 1 µg of total RNA from human amniotic cells, which we knew to express human APEX, under the same conditions described above. After an initial denaturation step of 1 min at 94°C, 35 amplification cycles were performed. Each cycle included denaturation at 94°C for 45 s, annealing at 56°C for 1 min and extension at 72°C for 1 min. A final extension step of 10 min at 72°C was performed in order to complete the PCR reaction. The size of APEX external product was 499 base pairs (bp), while the couple of nested primers generated an internal amplicon of 292 bp. To confirm the identity of the human APEX transcripts, each RT–PCR product was cleaved with AluI restriction enzyme. PCR products and AluI reaction mixtures were separated by electrophoresis on 2% agarose gel, stained by ethidium bromide and visualized under UV.
|
DNA/RNA extraction from C. intestinalis gametes and embryos
Ciona intestinalis individuals were supplied from Stazione Zoologica of Naples ‘A. Dohrn’. Unfertilized oocytes and spermatozoa were collected as reported (Russo et al., 1996). Genomic DNA was prepared from gametes and embryos using a commercially available kit (Wizard, Promega, Milano, Italy). Typically, each single sperm preparation started from 50–100 µl of dry sperm (4–6 x 1010 spermatozoa/ml). Oocytes were collected from the ovary of three to five samples, depending on their size, and fertilized to obtain embryos at different stages. RNA extraction from sperm, unfertilized oocytes and selected embryos was performed using Trizol reagent (Invitrogen), quantitated by spectrophotometric reading and analysed on denaturing agarose gel as reported (Sambrook and Russell, 2001). The total RNA extracted was subjected to extensive treatment with DNAse–RNAse/free (BD-Clontech, Milano, Italy), in order to eliminate any trace of contaminating genomic DNA, before cDNA synthesis. The amount of unfertilized oocytes and embryos collected was enough to isolate 1–3 µg of total RNA.
RT–PCR and real-time qPCR on C. intestinalis oocytes and embryos
The protocols for reverse-transcriptase PCR, opportunely optimized, were carried out on approximately 2 µg of RNA total employing the One-Step and RT-Omniscript kits (Qiagen, Milano, Italy), following the manufacturer's instructions. Synthesized cDNA was amplified using the following protocol: 5 µl cDNA, 10 x PCR buffer (100 mM Tris/HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2; Roche, Mannheim, Germany) 2.5 mM dNTP; 25 pmol of specific primer; 2.5 U Taq Polimerase enzyme (Biogem; supplied by Stazione Zoologica of Naples); water to a final volume of 50 µl. PCR primers were generated using ‘Primer3’ software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesized by the Molecular Biology Core Facility at Stazione Zoologica of Naples. Their sequences are reported in Table 1. PCR reactions were placed in a Thermocycler Ptc-100 (M.J. Research, Inc., 590 Lincoln Street, Waltham, MA 02451, USA) using the following parameters: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min; annealing at 52–60°C for 1 min; extension at 72°C for 1 min; final extension at 72°C for 10 min. Only when genomic DNA was amplified, cycle numbers were lowered to 25. Amplified products were separated on 2% (w/v) agarose gel and visualized by ethidium bromide staining. The images were acquired and digitized using the Gel-Doc 2000 equipment (Bio-Rad, 3 boulevard Raymond Poincaré, 92430 Marne La Coquette, France).
Real-time PCR (qPCR) was performed using an ABI PRISM 7000 sequence detector system (Applied Biosystems, 25 Avenue de la Baltique BP96, 91943 Courtaboeuf Cedex, France) and SybrGreen (SYBR) chemistry as a double strand DNA-specific fluorescent (Invitrogen). Specific primer sets for each gene were designed on the basis of the recently sequenced C. intestinalis genome (http://genome.jgi-psf.org) (Dehal et al., 2002) using the Primer3 software. Primers for housekeeping (ribosomal protein S-27; RP-S27) (Olinski et al., 2006) and CiAPEX genes used in qPCR are reported in Table 1.
Four different sample preparations were carried out for each time point analysed: unfertilized oocytes, two-, four-, 16-cell stages, larvae (legend of Fig. 1). Each reaction was performed at least in triplicates in optical strip tubes in two different runs, one for each sample preparation. The qPCR mixture, in the final volume of 25 µl, contained 2 µl of cDNA (dilution 1 : 2 of starting material); 12.5 µl of Platinum SYBR Green qPCR super mix-UDG with Rox (Invitrogen) and 10.5 µl of specific primer pairs (7.5 pmol). The following experimental run protocol was used: denaturation program (95°C for 10 min), 40 cycles of amplification (15 s at 95°C; 1 min at 60°C and 1 min at 72°C). Specificity of every amplification reaction was verified by melting curve analysis. For all experiments, raw data output were internally normalized against RP-S27 mRNA (the endogenous control), whose expression levels remain constant during all the developmental stages examined (data not shown). Analyses were performed using the standard curve method. The relative standard curve method uses a set of relative standards from which unknown samples are quantitated. The quantity is expressed relative to some base sample, such as the calibrator (e.g. unfertilized oocytes). A calibrator is a sample used as the basis for comparing results. For all experimental samples, target quantity is determined by interpolating from the standard curve and then dividing by the target quantity of the calibrator. The calibrator becomes the 1 x fold and all other quantities are expressed as an n-fold difference relative to the calibrator. qPCR results have been reported as fold increase/decrease compared to the expression level of CiAPEX determined in unfertilized oocytes (Fig. 1).
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Expression of APEX in human gametes and early embryos
In order to verify the expression of APEX (HsAPEX) in human oocytes and preimplantation embryos, a RT–PCR was performed employing the primers listed in Table 1. Fig. 2A shows that the expected 499 bp product was clearly detectable in GV oocytes, two cell embryos, morula (Mo) and blastocyst (BL). No clear signal was evidenced in MII oocytes (Fig. 2A). An internal positive control was represented by the amplification of human ß-actin transcripts starting from all cDNA preparation showed in Fig. 2 (data not shown). A subsequent nested-PCR led to the amplification of a 292 bp fragment, reinforcing the presence of HsAPEX transcripts in GV and preimplantation embryos. In addition, the increased sensitivity due to the nested-PCR allowed us to detect a clear transcript in MII oocytes (Fig. 2B), suggesting that the negative result shown in Fig. 2A was probably due to a limited amount of HsAPEX mRNA available for the RT–PCR reaction. The careful design of primers employed in the primary PCR and in the following nested-PCR (Table 1) excluded any possible contaminating amplification due to the presence of genomic DNA in the cDNA preparation.
|
The same 499 bp amplicon, corresponding to HsAPEX transcript, was also observed in human sperm cells (Fig. 2C). No nested-PCR was necessary on these samples, confirming the high expression of HsAPEX in human sperm. As a further characterization of HsAPEX transcript in the sperm, an AluI restriction profile was performed. The enzyme generated the two expected fragments of 307 and 147 bp (Fig. 2D), confirming the identification of HsAPEX transcript in human sperm.
Analysis of C. intestinalis APEX gene and protein
To verify if the presence of APEX transcripts in gametes and preimplantation embryos was an event that is evolutionarily conserved, we measured its expression in the ascidian C. intestinalis. The rationale of this choice is based on the observation that sea squirts are closest relatives of vertebrates, more than cephalochordates (Delsuc et al., 2006). Therefore, using the Smith–Waterman algorithm into the Joint Genome Institute (JGI) browser, we found clone fgenesh3_pm.C_chr_12p000020 (CiAPEX), protein ID: 203216, located on chromosome 12p:962878–963822 of C. intestinalis and homolog to HsAPEX (CiAPEX). Fig. 3A reports the gene sequence downloaded from the JGI website (http://genome.jgi-psf.org/ciona4/ciona4.home.html). Different from HuAPEX, CiAPEX does not present introns. In accordance with the evolutionary position of C. intestinalis, at the diverging point between invertebrates and chordates, similarity, for both mRNAs and proteins, between CiAPEX and its vertebrate homologs (human, mouse, frog and fish) was generally higher compared to invertebrate species, such as insects and protozoa (Table 2). Clustal alignment method (Higgins et al., 1996) for both DNA and amino acid sequences between HsAPEX and CiAPEX showed a 33.5 and 44.3% identity, respectively, with several functional regions perfectly conserved between them (Fig. 3B). The four acidic residues, namely Asp-90, Glu-96, Asp-219, Asp-308 of human protein, essential for DNA repair activity are conserved in CiAPEX, including Asp-219 (Asp-215 in C. intestinalis), whose mutation causes loss of both enzyme functions: DNA binding and AP-endonuclease activity (Fig. 3B). Conversely, residues Cys-65 and Cys-93, forming a disulphide bond in HsAPEX, are not conserved in CiAPEX. The absence of these two cysteine residues in CiAPEX suggests that the ascidian enzyme may lack redox transcriptional activity (Fig. 3b). This conclusion is also supported by the absence of PKC phosphorylation sites in CiAPEX, which are thought to be an important regulatory element for the human enzyme increasing its binding activity to transcription factors such as Fos, Jun and AP-1 (Hsieh et al., 2001). It is worthwhile to note that among the alignments shown in Table 2, acidic residues Asp-90, Glu-96, Asp-219, Asp-308 of the human protein are perfectly conserved in all species reported, from mouse to protozoa, in accordance with the primary role of APEX as a DNA repair gene. However, APEX Cys-65 and Cys-93 (numbers refer to human residues) are not conserved among invertebrates, from C. intestinalis to D. discoideum, suggesting the later evolution of Ref-1 activity (Table 2).
|
|
Expression of APEX in ascidian gametes and early embryos
Before determining if CiAPEX was expressed in unfertilized oocytes, a ß-tubulin control was routinely performed to exclude the presence of contaminating genomic DNA in RNA preparations. Fig. 4A shows amplification products obtained from genomic DNA (lane 1), or cDNA synthesized from total RNA isolated from C. intestinalis oocytes (lane 2) using ß-tubulin specific primers (CiTu-F/CiTu-R; Table 1). Similar to humans, C. intestinalis oocytes presented a clearly detectable expression of CiAPEX mRNA (Fig. 4B, Ov), that persisted during the early phase of embryo development (two to 16 cells stage; Fig. 4B) and remained detectable during larva stages (Fig. 4C). Nested PCR and sequence analyses confirmed the identity of the 618 bp band shown in Fig. 4B–C as CiAPEX transcript (data not shown), while PCR amplifications using primers specific for genomic CiAPEX (AXG-F/AXG-R in Table 1 and Fig. 5), or C. intestinalis ß-tubulin (Fig. 4C, + lane) excluded the possible amplification of genomic DNA contaminating sequences.
|
|
To investigate if a variable level of expression of CiAPEX was present during development, a qPCR was performed on the same samples reported in Fig. 4. Data were normalized using as endogenous reference the gene encoding for the ribosomal protein S-27 (Table 1), whose expression remains constant in all the developmental stages examined (data not shown). CiAPEX was present as maternal transcript at relatively low levels. Its expression significantly changed during early divisions of the embryos, reaching its maximal expression at four-cells stage (about eight-fold compared to the expression in oocytes). At larva stage, CiAPEX mRNA expression decreased reaching a value lower compared to that observed in unfertilized oocytes (Fig. 1).
Overall, these results suggest that the expression of the APEX gene in oocytes and early embryos might have an important functional role, since it is conserved during evolution from Urochordata to humans.
To verify if also in the male gametes the expression of APEX was conserved during evolution, total RNA was isolated from C. intestinalis spermatozoa and employed to synthesize the corresponding cDNA. The procedure was carefully carried out in order to exclude the presence of genomic DNA in the preparation, a problem often encountered when cDNA is synthesized from sperm. Fig. 5A shows that AXG-F/AXG-R primers, specific for genomic CiAPEX (lane G), did not amplify any transcript among the population of cDNA synthesized from C. intestinalis sperm (lane 1). However, any combination of primers specific for CiAPEX mRNA used in both, direct or nested-PCR, failed to detect any amplification product (data not shown). The good quality of C. intestinalis sperm cDNA preparation was confirmed by the positive control showed in Fig. 5B. A search in the Ciona Genome website led us to select the C. intestinalis homolog of human hexosaminidase A (Cioin2/chr_04q:1503589–1509804, protein ID 201985), a gene specifically expressed in testis (http://hoya.zool.kyoto-u.ac.jp/cgi-bin/gbrowse/ci). We designed the TS1-F/TF1-R PCR primers that were able to selectively amplify a 175 bp fragment (Fig. 5B, lane 2). The size of the fragment was exactly that expected based on the sequence of hexosaminidase A transcript reported on the JGI website, and its identity was also confirmed by sequence analysis (data not shown).
These data indicated the putative absence of CiAPEX transcript in ascidian spermatozoa, different from the observation reported for human sperm (Fig. 2).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Even under physiological conditions, the genome is continuously subjected to aggressions. In human IVF/ICSI, in vitro manipulations are supposed to increase these assaults by generating ROS (Pabon et al., 1989; Nasr-Esfahani et al., 1990). Abasic sites (AP) are the most common DNA decays, especially those induced by ROS, which can form oxidized AP (Sung and Demple, 2006). In human cells, APEX/Ref-1 is the major enzyme involved in the removal of baseless sites. Since persistence of AP sites results in a DNA replication block, cytotoxic mutations and genetic instability, we hypothesized a functional role for APEX/Ref-1 in human oocytes, supported by the presence of the corresponding mRNA. In sheep oocytes, completion of DNA repair involves upregulation of polymerase beta by estrogens and of DNA ligase (Murdoch and Van Kirk, 2001). In addition to its excision activity, APEX/Ref-1 is a multifunctional protein, controlling the redox status of transcription factors such as Fos, Jun, hypoxia inducible factor-1-alpha, and p53 maintaining them in an active reduced state (Kelley and Parsons, 2001). The in vitro redox activity of APEX/Ref-1 is activated by PKC phosphorylation (Flaherty et al., 2001; Pines et al., 2005). In addition, APEX/Ref-1 is associated with thioredoxin (Trx) in the up-regulation of the redox potential (Hedley et al., 2004). Elevated levels of both APEX/Ref-1 and Trx increases cell growth and resistance to programmed cell death (Powis et al., 2000). Using Affimetrix chips, we are characterizing the presence of more than 10 000 human transcripts in different samples of pooled oocytes. Preliminarily, we identified mRNAs coding for Trx, Trx reductase and APEX/Ref-1. The transcripts for six members of the peroxiredoxin family, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase mRNAs were also detected. The pentose pathway generating NADPH necessary for Trx reductase is actively used in the human embryo (Menezo et al., manuscript in preparation).
Many studies reported the presence of mRNA species in ejaculate spermatozoa, suggesting that these transcripts are delivered to the oocyte during the fertilization process (Ostermeier et al., 2004). The function and utility of sperm mRNAs remains essentially unexplored, although circumstantial evidence suggests an involvement of sperm mRNAs in early embryonic development (Ostermeier et al., 2004, 2005). In addition, it has been hypothesized that some of these mRNA species might represent a good marker for sperm quality (Ostermeier et al., 2005). In this context, HsAPEX expression might be considered a valuable indicator of DNA repair capacity in preimplantation embryos.
Our comparative study suggests that the male contribution to DNA repair functions, at fertilization and during the early stage of development, may have been established only later in the Chordate evolution. In fact, CiAPEX does not appear to be expressed in sea squirt spermatozoa (Fig. 5; Table 2). This observation is particularly interesting considering the structural differences existing between the human and ascidian enzymes: different from CiAPEX, HsAPEX transcript is present in sperm and HsAPEX enzyme is potentially capable of Ref-1 activity since the residues essential for this function (i.e. Cys-65 and Cys-93) are conserved. This might suggest that the presence of HsAPEX in the male gametes could not only be associated to a DNA-repair function, but also to a transcriptional activity in fertilized oocytes. An interesting exception to this is D. melanogaster where both Cys-65 and Cys-93 are conserved (Table 2), although the presence of a redox activity has not been demonstrated. Future studies in low vertebrates (Fishes, Amphibians, Reptiles) will be devoted to clarify when the Ref-1 function of APEX/Ref-1 appeared during evolution. The constant publication of new genomes, including those of low vertebrates, will certainly facilitate this study.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
We want to thank the ARCEFAR (Association pour la recherche clinique et fondamentale appliqué à la reproduction) and the ORGANON laboratory (France) for their financial support. We are indebted to Dr Giuseppe Iacomino (National Research Council, Avellino, Italy) for their help in setting up qPCR. We are grateful to P. Cirino, A. Toscano (Marine Resources for Research Service) for providing and maintaining Ciona intestinalis. We thank Dr. E. Biffali (Molecular Biology Service) for oligonucleotide synthesis and DNA sequencing. Finally, we thank G. Gargiulo for his help in preparing figures.
|
Footnotes |
|---|
* A part of this work was presented (oral presentation) at the 2004 ESHRE meeting in Berlin.
These two authors equally contributed to the data presented.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Bessho T, Tano K, Kasai H, Ohtsuka E, Nishimura S. Evidence for two DNA repair enzymes for 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in human cells. J Biol Chem (1993) 268:19416–19421.
Ciona Genome Group. The Ciona genome issue. Dev Genes Evol (1993) 213:1–218.
Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, et al. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science (2002) 298:2157–2167.
Delsuc F, Brinkmann H, Philippe H. Phylogenomics and the reconstruction of the tree of life. Nat Rev Genet (2005) 6:361–375.[ISI][Medline]
Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature (2006) 439:965–968.[CrossRef][Medline]
Demple B, Herman T, Chen DS. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes. Proc Natl Acad Sci (1991) 88:11450–11454.
Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl (2002) 23:25–43.[ISI][Medline]
Flaherty DM, Monick MM, Hunninghake GW. AP endonucleases and the many functions of Ref-1. Am J Respir Cell Mol Biol (2001) 25:664–667.
Fritz G. Human APE/Ref-1 protein. Int J Biochem Cell Biol (2000) 32:925–929.[CrossRef][ISI][Medline]
Hedley D, Pintilie M, Woo J, Nicklee T, Morrison A, Birle D, Fyles A, Milosevic M, Hill R. Up-regulation of the redox mediators thioredoxin and apurinic/apyrimidinic excision (APE)/Ref-1 in hypoxic microregions of invasive cervical carcinomas, mapped using multispectral, wide-field fluorescence image analysis. Am J Pathol (2004) 164:557–565.
Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, Schill WB, Kruger TF. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril (2004) 81:965–972.[CrossRef][ISI][Medline]
Higgins DG, Thompson JD, Gibson TJ. Using CLUSTAL for multiple sequence alignments. Methods Enzymol (1996) 266:383–402.[ISI][Medline]
Hsieh MM, Hegde V, Kelley MR, Deutsch WA. Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation. Nucleic Acids Res (2001) 29:3116–3122.
Jayaraman L, Murthy KGK, Zhu C, Curran T, Xanthoudakis S, Prives C. Identification of redox:repair protein Ref-1 as a potent activator of p53. Genes Dev (1997) 11:558–570.
Kelley MR, Parsons SH. Redox regulation of the DNA repair function of the human AP endonuclease Ape1/ref-1. Antioxid Redox Signal (2001) 3:671–683.[CrossRef][ISI][Medline]
Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod (1998a) 13:896–900.
Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril (1998b) 69:528–532.[CrossRef][ISI][Medline]
Menezo Y Jr, Viville S, Veiga A. Epigenetics and assisted reproductive technology. Fertil Steril (2006) 85:269–270. author reply.[ISI][Medline]
Menezo YJ. Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment. Reprod Biomed Online (2006) 12:616–621.[ISI][Medline]
Miller D, Ostermeier GC, Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol Med (2005) 11:156–163.[CrossRef][ISI][Medline]
Morozumi K, Yanagimachi R. Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development. Proc Natl Acad Sci U S A (2005) 102:14209–14214.
Murdoch WJ, Van Kirk EA. Estrogenic upregulation of DNA polymerase beta in oocytes of preovulatory ovine follicles. Mol Reprod Dev (2001) 58:417–423.[CrossRef][ISI][Medline]
Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development (1990) 109:501–507.[Abstract]
Nixon VL, McDougall A, Jones KT. Ca2+ oscillations and the cell cycle at fertilisation of mammalian and ascidian eggs. Biol Cell (2000) 92:187–196.[CrossRef][ISI][Medline]
Oger I, Da Cruz C, Panteix G, Menezo Y. Evaluating human sperm DNA integrity: relationship between 8-hydroxydeoxyguanosine quantification and the sperm chromatin structure assay. Zygote (2003) 11:367–371.[CrossRef][ISI][Medline]
Olinski RP, Dahlberg C, Thorndyke M, Hallbook F. Three insulin-relaxin-like genes in Ciona intestinalis. Peptides (2006) 27:2535–2546.[CrossRef][ISI][Medline]
Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet (2002) 360:772–777.[CrossRef][ISI][Medline]
Ostermeier GC, Miller D, Huntriss JD, Diamond MP, Krawetz SA. Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature (2004) 429:154.[Medline]
Ostermeier GC, Goodrich RJ, Diamond MP, Dix DJ, Krawetz SA. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. Fertil Steril (2005) 83:1687–1694.[CrossRef][ISI][Medline]
Pabon JE Jr, Findley WE, Gibbons WE. The toxic effect of short exposures to the atmospheric oxygen concentration on early mouse embryonic development. Fertil Steril (1989) 51:896–900.[ISI][Medline]
Pines A, Perrone L, Bivi N, Romanello M, Damante G, Gulisano M, Kelley MR, Quadrifoglio F, Tell G. Activation of APE1/Ref-1 is dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP. Nucleic Acids Res (2005) 33:4379–4394.
Powis G, Mustacich D, Coon A. The role of the redox protein thioredoxin in cell growth and cancer. Free Radic Biol Med (2000) 29:312–322.[CrossRef][ISI][Medline]
Ron-el R, Nachum H, Herman A, Golan A, Caspi E, Soffer Y. Delayed fertilization and poor embryonic development associated with impaired semen quality. Fertil Steril (1991) 55:338–344.[ISI][Medline]
Russo GL, Kyozuka K, Antonazzo L, Tosti E, Dale B. Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II. Development (1996) 122:1995–2003.[Abstract]
Russo GL, Tosto M, Mupo A, Castellano I, Cuomo A, Tosti E. Biochemical and functional characterization of protein kinase CK2 in ascidian Ciona intestinalis oocytes at fertilization. Cloning and sequence analysis of cDNA for alpha and beta subunits. J Biol Chem (2004) 279:33012–33023.
Sambrook JF, Russell DW. Molecular Cloning: A Laboratory Manual (2001) 3rd edn. Cold Spring Harbor Laboratory (NY): Cold Spring Harbor Laboratory Press.
Satoh N. The ascidian tadpole larva: comparative molecular development and genomics. Nat Rev Genet (2003) 4:285–295.[ISI][Medline]
Sung JS, Demple B. Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA. FEBS J (2006) 273:1620–1629.[CrossRef][Medline]
Wood RD, Mitchell M, Sgouros J, Lindahl T. Human DNA repair genes. Science (2001) 291:1284–1289.
Xanthoudakis S, Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1DNA binding activity. EMBO J (1992) 11:653–665.[ISI][Medline]
Xu YJ, Kim EY, Demple B. Excision of C-4'-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta. J Biol Chem (1998) 273:28837–28844.
Submitted on April 7, 2007; accepted on April 19, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||