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Epigenetics 6:11, 1354-1361; November 2011; © 2011 Landes Bioscience

RESEARCH PAPER

1354 Epigenetics Volume 6 Issue 11

*Correspondence to: Joana Marques; Email: cmarques@med.up.ptSubmitted: 06/27/11; Accepted: 09/06/11DOI: 10.4161/epi.6.11.17993

Introduction

Genomic imprinting regulates the expression of a subset of genes, leading to mono-allelic parental-dependent expression of imprinted genes.1 Parental alleles are differentially marked by epigenetic modifications such as DNA methylation at CpG dinucleotides located in the differentially methylated regions (DMRs) of imprinted genes. DNA methylation is postulated to be the primary imprinting mark, established in the gametes and maintained in the zygote, resisting the wave of DNA demethyl-ation occurring in both genomes in early embryo development.2

H19 and MEST/PEG1 (Mesodermal Specific Transcript/Paternally Expressed Gene 1) are two oppositely imprinted genes. H19 is methylated on the paternal allele and maternally tran-scribed into a non-coding RNA3,4 and is physically and function-ally linked to the reciprocally imprinted gene Igf2 (Insulin-like growth factor 2) that is expressed from the paternal allele. Transcription is controlled by common enhancers, located 3' of the H19 gene,5 and by DMR methylation of the H19 gene. This DMR, located 5' of the H19 gene, has several binding sites for the insulator protein CTCF (CCCTC-binding factor), which binds

Paternal imprinting marks were shown to be erased in the mouse primordial germ cells and progressively re-established throughout the male germ line development, starting in fetal prospermatogonia and continuing post-natally through the onset of meiosis. We here evaluated imprinting marks in human adult spermatogenic cells and analyzed mRNA and protein expression of DNA Methyltransferases (DNMTs). Spermatogonia A, primary and secondary spermatocytes, round spermatids and elongated spermatids/spermatozoa were isolated by micromanipulation from testicular biopsies of men with normal spermatogenesis. DNA methylation at two imprinted genes, H19 and MEST/PEG1, was analyzed using bisulphite genomic sequencing and DNMTs expression was determined by qRT-PCR and immunofluorescence. H19 was completely methylated at the spermatogonia stage in the analyzed individuals and MEST/PEG1 was completely demethylated, with the exception of few CpGs. The analysis of DNMT1, DNMT3A and 3B expression showed peaks of mRNA transcripts in primary spermatocytes and in mature ejaculated spermatozoa, with DNMT1 transcript level being the most abundant in all cell stages. Immunolocalization showed that DNMT proteins are present throughout the spermatogenic cycle, with stage-specific shuttling between the nucleus and cytoplasm. We conclude that, in humans, methylation imprints are established in spermatogonia A and are maintained in subsequent stages up to elongated spermatid/spermatozoa. Additionally, DNA methyltransferases are expressed throughout human spermatogenesis, possibly maintaining the methylation patterns in order to avoid the transmission of imprinting errors by the male gamete.

DNA methylation imprinting marks and DNA methyltransferase expression in human

spermatogenic cell stagesC. Joana Marques,1,* Maria João Pinho,1 Filipa Carvalho,1 Ivan Bièche,2 Alberto Barros1,3 and Mário Sousa1,3,4

1Department of Genetics; Faculty of Medicine; 4Department of Microscopy; Laboratory of Cell Biology; UMIB; ICBAS; University of Porto; Porto, Portugal; 3Centre for Reproductive Genetics Alberto Barros; Porto, Portugal; 2Laboratoire d’Oncogénétique; Institut Curie-Hôpital René Huguenin; St. Cloud, France

Key words: genomic imprinting, human spermatogenesis, DNA methyltransferases, H19, MEST/PEG1

to the unmethylated maternal allele, thereby establishing a chro-matin structure that prevents the Igf2 promoters from accessing the enhancers, and activating only the H19 promoter.6,7 On the paternal allele, DMR methylation prevents the binding of CTCF and allows expression of Igf2 by interaction with downstream enhancers. MEST is unmethylated on the paternal allele and expressed.8,9 Mest deficiency was shown to cause general embryo and placental growth retardation, as well as disturbed maternal behavior towards newborns.10

In mice, methylation imprints are erased in primordial germ cells (PGCs) after their entry into the genital ridge, at 12.5–13.5 dpc,11 and later re-established according to the sex of the germ line. Paternal H19 re-methylation starts in prospermatogonia (14.5 dpc) and is described to be differentially established in the two parental alleles, with complete methylation of the paternal allele occurring prior to the spermatogonia A stage whereas the maternal allele became hypermethylated after completion of mei-osis I.12 This suggests that parental alleles are still recognizable after global DNA demethylation in PGCs, possibly by another epigenetic mark or by residual methylation still present in the paternal allele, favoring a more rapid re-methylation.11,13-15

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RESEARCH PAPER RESEARCH PAPER

DNA methyltransferases are expressed throughout the sper-matogenic cycle, with stage-specific mRNA levels and protein compartmentalization.

Results

H19 and MEST/PEG1 methylation imprinting marks. Methylation patterns of H19 and MEST/PEG1 DMRs were analyzed in all cell stages of human spermatogenesis, namely in mitotically dividing cells (spermatogonia A-SGA), pre-meiotic (primary spermatocytes-ST1) and post-meiotic divisions (sec-ondary spermatocytes-ST2 and round spermatids-Sa) and in differentiating cells (elongated spermatids-Sd) isolated by micro-manipulation from testicular biopsies (T) of individuals with normal spermatogenesis (Fig. 1).

Spermatogonia A presented almost complete methylation of the H19 gene in all the individuals (Fig. 2) and this high level of methylation was maintained in all subsequent cell stages, with the number of unmethylated CpGs varying from only 1 to 3 (excluding CpG 7 since it’s a polymorphic site between a cyto-sine and a thymine). Based on an informative polymorphic site between CpG 10 and 11 (between a cytosine and an adenine), it was possible to discriminate both alleles in individuals A and C, since they were heterozygous. We confirmed that both alleles, of paternal and maternal origin, were amplified in most of the stages analyzed with the exceptions of SGA and Sd in patient A and in ST2 and Sd in patient C, possibly due to the technical limitations inherent to analyzing small numbers of cells (varying between 100 and 300).30 For MEST gene, we observed almost complete demethylation (93.1–100%) in all cell stages analyzed, with the number of methylated CpGs varying from 1–4 (Fig. 3).

Expression of DNA methyltransferases. To investigate if DNMTs are present in human spermatogenic cells, we deter-mined, by quantitative real-time RT-PCR (qPCR), the relative expression of DNMT1, DNMT3A and DNMT3B in all spermato-genic cell stages from individuals with normal spermatogenesis (Fig. 4A). Using spermatogonia (SGA) as the calibrator sample (expression = 1), we observed that the expression of DNMT1 is relatively constant with a potential decrease in round spermatids (Sa). We did not detect expression of any DNMT in elongated spermatids (Sd), despite the amplification of the reference genes (RPLP0, UBB and ACTB). Expression of DNMT3A was also held relatively constant with a possible decrease in secondary spermatocytes (ST2) and DNMT3B was detected in spermato-gonia (SG), primary spermatocytes (ST1) and round sperma-tids (Sa). In all cell stages, DNMT1 expression was higher than DNMT3A and DNMT3B (Table 1; normalization of DNMT1 against DNMT3A and DNMT3B within each cell stage). In purified ejaculated sperm (Sz) from an individual with normal spermiogram parameters, we observed high levels of DNMT1 and DNMT3A transcripts. Whole testicular samples (T1 and T2), from the individuals studied and from a commercial source, were used as positive controls for the amplification of the three DNMTs. DNMT3L mRNA expression was not observed in any of the cell stages, albeit its positive detection in whole testicular tissue (T1 and T2) used as controls (data not shown).

DNA methyltransferases (DNMTs) are responsible for add-ing methyl groups to the C5 (carbon 5) position of cytosines located at CpG dinucleotides. DNMT1 is considered to be the maintenance methyltransferase, due to its catalytic preference for hemi-methylated DNA16,17 and its association to S-phase repli-cation foci,18 and was shown to cooperate with DNMT3A and DNMT3B in maintenance and establishment of methylation.19,20 Dnmt1 was also described to have substantial de novo methyla-tion activity, about 5–20% of the activity on hemi-methylated DNA,16 being capable of spreading methylation in a partially methylated region,21,22 even in the absence of Dnmt3a and Dnmt3b.23 It has a male germ line specific mRNA transcript, Dnmt1p, whose transcription is restricted to pachytene sper-matocytes, but without detectable levels of protein.24,25 Dnmt3a and Dnmt3b are essential de novo methyltransferases.26 Dnmt3a establishes both maternal and paternal imprints and regulates normal spermatogenesis, with conditional mutant mice show-ing maturation arrest at the spermatogonia stage.27 Dnmt3L (Dnmt3-Like) protein shares regions of homology to Dnmt3a and Dnmt3b. Although lacking enzymatic activity due to the absence of conserved catalytic motifs,28 Dnmt3L was shown to be essential for normal spermatogenesis and for the establishment of maternal imprints during oogenesis.29

To better understand the mechanisms underlying the estab-lishment and maintenance of paternal imprints in human spermatogenesis, we here analyzed H19 and MEST methyla-tion imprinting marks and DNA methyltransferases (DNMTs) mRNA and protein expression in purified human spermato-genic cell stages, isolated by micromanipulation from testicular biopsies of individuals with normal spermatogenesis. We show that paternal imprinting marks are already established in adult spermatogonia and are maintained across meiotic divisions and differentiation steps. Additionally, de novo and maintenance

Figure 1. Spermatogenic cells isolated by micromanipulation from a testicular biopsy (T), namely spermatogonia A (SGA), primary spermato-cytes (ST1), secondary spermatocytes (ST2), round spermatids (Sa) and elongated spermatids (Sd). Magnification 400x.

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Discussion

In the present study, we analyzed DNA methylation imprint-ing marks and DNA methyltransferases expression in all human spermatogenic cell stages isolated by micromanipulation from testicular biopsies of individuals with normal spermatogenesis. Our results indicate that paternal imprints are already established in human adult spermatogonia, indicating that H19 re-methyl-ation and MEST erasure of methylation occurred prior to this stage. Additionally, we describe for the first time that de novo and maintenance DNA methyltransferases are expressed in human spermatogenesis, during proliferation, meiosis, cell divisions, spermatid differentiation and in mature ejaculated spermatozoa.

Previous studies have addressed the timing of imprints estab-lishment in murine spermatogenesis, with H19 active methyla-tion being shown to start on both alleles in fetal (14.5–18.5 dpc) prospermatogonia,15,31 and to be completed on the paternally inherited allele in primitive type A spermatogonia and on the maternally inherited allele at meiosis I.12 However, other stud-ies in mice have shown that both paternal and maternal alleles

DNMT1, DNMT3A and DNMT3B proteins were detected by immunofluorescence in all spermatogenic cell stages (Fig. 4B), although a fainter staining was observed in round spermatids (Sa) for DNMT3A and in elongated spermatids (Sd) for DNMT3A and DNMT3B. DNMT1 was localized in the nucleus and cytoplasm of spermatogonia A (SGA), zygotene (ZGT) and pachytene (PCT) spermatocytes, being translocated to the nucleus of leptotene (LPT) spermatocytes, secondary sper-matocytes (ST2) and late spermatids (Sd), and to the cytoplasm during cell divisions (telophases Tel1 and Tel2) and in round sper-matids (Sa). DNMT3A was mainly confined to the cytoplasm, shuttling to the nucleus of pachytene spermatocyte (PCT) and secondary spermatocytes (ST2), whereas DNMT3B was mainly confined to the nucleus. Another finding of particular interest was observed in purified ejaculated sperm, with DNMT1 local-izing in the equatorial region, DNMT3A in the mid-piece and DNMT3B in the anterior half of the sperm head (Figs. S1–S3). While the presence and localization of DNMT1 and DNMT3A proteins was uniform in all spermatozoa analyzed, DNMT3B was present in the majority, but not all of the cells.

Figure 2. Methylation patterns of H19 in human spermatogenic cell stages—spermatogonia A (SGA), primary spermatocytes (ST1), secondary sper-matocytes (ST2), round spermatids (Sa) and elongated spermatids (Sd)—isolated by micromanipulation from testicular biopsies of three individuals (A–C). A hemi-nested PCR was performed in the samples where only 18 CpGs were analyzed. Methylated CpG (blue), unmethylated CpG (yellow), CpG7 is a polymorphism C/T (grey), number of clones (C) presenting the same pattern of methylation.

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non-imprinted genes.39 Consistent with this hypothesis, main-tenance (DNMT1) and de novo (DNMT3A and DNMT3B) methyltransferases were here found to be expressed at the mRNA and protein level in all germ cell stages, with DNMT1 mRNA levels being higher than DNMT3A and DNMT3B. Therefore, it is interesting to note that DNMT1 is present in non-replicat-ing spermatogenic cells and DNMT3A and 3B are present after the establishment of paternal imprints, supporting the hypoth-esis that de novo methylation is required for the maintenance of methylation patterns after their establishment.40

Quantitative RT-PCR analysis of stage-specific male germ cells in mice showed increased DNMTs expression in spermato-gonia A, leptotene/zygotene spermatocytes and round sper-matids, and then becoming almost undetectable in elongated spermatids.41 Similarly to mice, we observed increased transcrip-tion in human primary spermatocytes (DNMT1, DNMT3B) and round spermatids (DNMT3B), with an absence of expression in late spermatids. This could have been due to a limited number of cells or to absence or very low DNMT mRNA synthesis in this final step of spermiogenesis. In contrast, high levels of DNMT1 and DNMT3A, but not of DNMT3B mRNA, were observed in purified ejaculated sperm, indicating a post-testicular upregu-lation of DNMT1 and DNMT3A. This observation might be related to re-methylation events previously described to occur in the epididymis42 and might reveal methylation repair events that occur before the release of male gametes. We have also confirmed

are almost fully methylated by the newborn gonocyte stage.14,15 In humans, a previous study described that spermatogonia still present 25% of the H19 clones completely unmethylated and that H19 imprints are fully established at the primary spermatocyte stage.32 In our case, using purified cells by micromanipulation from fresh testicular biopsies and analyzing a high number of cells and clones, we were not able to detect unmethylated H19 clones in spermatogonia. Additionally, the present data confirms previous observations, in mice and human, showing that H19 methylation is completed by the elongated spermatid/spermato-zoa stage.12,13,32-36 This is an important observation concerning the therapeutic safety of using elongated spermatids, for elon-gated spermatid injection (ELSI), in terms of their imprinted status.37

Interestingly, the present analysis at the single CpG level suggests that the few sites with an altered methylation status (unmethylated for H19 and methylated for MEST ) might undergo positional changes from one germ cell stage to the subsequent one, indicating that both demethylation of previous methylated CpGs and re-methylation of previously unmethylated CpGs might occur in human male germ cells during stage transition. DNA re-methylation and demethylation events were previously observed in pachytene spermatocytes in mice, in three testis-specific genes—transition protein 1 (Tnp1), protamine 1 (Prm1) and protamine 2 (Prm2),38 and from spermatogonia to pachy-tene spermatocytes in several other sites, including imprinted and

Figure 3. Methylation patterns of MEST in human spermatogenic cell stages isolated by micromanipulation from testicular biopsies of three individu-als (A, B and D)—spermatogonia A (SGA), primary spermatocytes (ST1), secondary spermatocytes (ST2), round spermatids (Sa) and elongated sperma-tids (Sd). Methylated CpG (blue), unmethylated CpG (yellow), number of clones (C) presenting the same pattern of methylation.

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detection, an absent to residual germ cell expression or, alterna-tively, expression by somatic (Sertoli and/or Leydig) testicular cells.

the presence of DNMT3L mRNA in human testicular tissue43 but transcripts were not detected in isolated cells, suggesting that the limited amount of cells analyzed could be limiting the

Figure 4. DNA methyltransferases expression in human adult spermatogenesis. (A) Relative expression of DNMT1, DNMT3A and DNMT3B in sper-matogonia (SGA-calibrator sample, expression = 1), primary spermatocytes (ST1), secondary spermatocytes (ST2), round spermatids (Sa), elongated spermatids (Sd), ejaculated spermatozoa (Sz), whole testicular biopsy (T1), Clontech RNA from human testis (T2). mRNA levels for each gene were de-termined in five replicates and are represented as Mean ± SD. One of three biological replicates is represented. (B) Detection, by immunofluorescence, of DNMT1, DNMT3A and DNMT3B in spermatogonia (SGA), primary spermatocytes in leptotene (LPT), zygotene (ZGT) and pachytene (PCT) stages, telophase 1 (Tel1), secondary spermatocytes (ST2), telophase 2 (Tel2), round spermatids (Sa), elongated spermatids (Sd) and ejaculated spermatozoa (Sz). Nucleus is stained with DAPI (blue) and DNMTs with Alexa Fluor 568 (red). Magnification 1,000x.

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Materials and Methods

Isolation of spermatogenic cells from testicular biopsies. Experiments were conducted after informed consent of the patients and according to the principles expressed in the Declaration of Helsinki. Spermatogenic cells were isolated from testicular biopsies of patients with normal, conserved spermatogenesis, undergoing ICSI-TESE (Intracytoplasmic Sperm Injection after Testicular Sperm Extraction) mainly due to spinal cord injuries. Elongated type A spermatogonia, primary spermatocytes, second-ary spermatocytes, round spermatids and elongated spermatids/spermatozoa were isolated by micromanipulation as described before in reference 45 and 46. Briefly, each fragment of testi-cle biopsy was collected in SPM (Sperm Preparation Medium, Medicult, Copenhagen, Denmark), digested for 1 hour at 37°C in SPM containing 25 μg/ml of crude DNase and 1,000 U/ml of collagenase-IV (Sigma, Barcelona, Spain), washed, resuspended in IVF medium (Medicult) and incubated at 32°C, 5% CO

2

until use. Cells were individually selected in an inverted Nikon microscope, equipped with Hoffman optics and a heated stage (32°C), using Narishige micromanipulators and micropipettes of 15–20 μm in diameter (Swemed, Frolunda, Sweden). About 100–300 cells were collected for methylation analysis by sodium bisulphite treatment and 3,000 cells for gene expression analysis by quantitative real-time RT-PCR and kept at -80°C until use. Additionally, ejaculated spermatozoa were isolated from semen of individuals with normal spermiogram parameters by gradi-ent centrifugation followed by swim-up technique, as described before in reference 35.

Bisulphite genomic sequencing. DNA extraction and bisul-phite modification were performed in isolated spermatogenic cells, as previously described in reference 47. Briefly, DNA was isolated with a phenol-chloroform method, re-suspended in 3 μl of sterile water, digested with Pst1 restriction enzyme (New England Biolabs, Ipswich, MA), embedded in an agarose bead and treated with sodium bisulphite for 4 hours (30 min in ice and 3 h 30 min at 50°C).

H19 and MEST differentially methylated regions (DMRs) were amplified by PCR.32 Patients A and B were analyzed for both H19 and MEST genes; patient C was only analyzed for H19 and patient D only for MEST gene (due to the limiting amount of DNA). For H19, 21 CpGs were analyzed within a sequence of 323 bp (GenBank Accession n° AF087017: nt 6,006–6,328) that includes the sixth CTCF-binding-site (CpG 4–8) and two polymorphisms, one at CpG7 (Genbank acces-sion n° AF125183, nt 7,966, C/T) and another between CpG 10 and 11 (Genbank accession n° AF125183, nt 8,008, C/A). For MEST, a sequence with 290 bp was amplified, containing 29 CpGs (GenBank Accession n° Y10620: nt 609–898). The PCR reaction contained 3 μl of agarose-embedded modified DNA, 1x buffer with 1.5 mM MgCl

2, 0.12 mM of each dNTP (Invitrogen,

Carlsbad, CA), 0.5 μM of each primer (Thermo Electron) and 1.5 U HotStarTaq enzyme (5 U/μl, Qiagen, Hilden, Germany). The PCR conditions were: initial strand denaturation (15 min, 95°C), followed by 45 amplification cycles (denaturation, 1 min, 94°C; primer annealing, 1 min, 60°C; strand elongation,

The present results also showed that DNMT1, DNMT3A and DNMT3B proteins are present in all spermatogenic cell stages. Interestingly, we observed that all three enzymes co-localize in the nucleus of pachytene and secondary spermatocytes and in elongated spermatids, although with a fainter staining in the lat-ter. This suggests that re-methylation events might occur during meiotic recombination and before the second meiotic division, as well as in the late spermatid stage associated with histone to protamine exchange, possibly preventing methylation errors to be transmitted by the male gamete. Moreover, cytoplasmic localiza-tion of DNMT proteins seems to correlate with mRNA peaks of expression, especially in primary spermatocytes and round sper-matids. Additionally, in purified ejaculated sperm, DNMT1 was confined to the equatorial region, DNMT3A to the midpiece and DNMT3B to the anterior half of the sperm head. These results show that, in humans, the three enzymes are present in all germ cell stages, with stage specific nuclear-cytoplasm shuttling and specific compartment trapping in mature sperm. Previous analy-sis at the protein level, in mouse germ cells, revealed that Dnmt3a and Dnmt3b are present from spermatogonia to round spermatids and are absent in elongated spermatids,41 whereas Dnmt1 is pres-ent in the nucleus of spermatogonia and leptotene/zygotene sper-matocytes, and in round spermatids, being absent in pachytene spermatocytes and almost undetected in elongated spermatids.44

In conclusion, our data shows that imprinting marks are already established in human spermatogonia and kept in subse-quent stages, with the exception of a few CpGs that present an altered methylation status (which were also previously observed in sperm from normozoospermic control individuals).35 Since the position of these CpGs seems to vary between cell stages, we propose that site-specific re-methylation and demethylation events might occur throughout spermatogenesis. Supporting this hypothesis, maintenance and de novo methyltransferases were detected in all spermatogenic cell stages, both at the mRNA and protein level, suggesting that maintenance of paternal imprints during human spermatogenesis is a dynamic process.

Table 1. Relative expression of DNMT1 against DNMT3A and DNMT3B

DNMT1/DNMT3A DNMT1/DNMT3B

SGA 3.68 22.99

ST1 4.62 2.82

ST2 11.66 nd

Sa 2.18 1.64

Sd nd nd

Sz 4.96 nd

T1 11.19 51.77

T2 7.31 11.57

SGA, spermatogonia A; ST1, primary spermatocytes; ST2, secondary spermatocytes; Sa, round spermatids; Sd, elongated spermatids; Sz, ejaculated spermatozoa; T1, whole testis; T2, Human testis total RNA (Clontech). nd, not detected.

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of amplified products was confirmed by melting curve analysis and gel electrophoresis.

Indirect immunofluorescence detection of DNMT1, DNMT3A and DNMT3B proteins. Germ cells from a testicular biopsy and ejaculated spermatozoa were fixed in 4% paraformal-dehyde in PBS, for 60 min at room temperature or overnight at 4°C, and stored at -20°C until use. Samples were washed twice with cold PBS and incubated for 10 min with 0.25% Triton X-100 in PBS for permeabilization. After three washes with PBS, sam-ples were incubated with 1% BSA in PBS for 30 min to block unspecific binding of the antibodies. Cells were then incubated with primary monoclonal mouse antibodies (one antibody per slide) against DNMT1 (1 μg/ml), DNMT3A (1 μg/ml) and DNMT3B (5 μg/ml) (Imgenex, San Diego, CA), in 1% BSA in PBST (0.5% Tween20 in PBS), overnight at 4°C. After washing 3 times with PBS, cells were incubated with the secondary antibody, Alexa Fluor 568 goat anti-mouse IgG1 γ1 (4 μg/ml) (Molecular Probes, Carlsbad, CA), in 1% BSA in PBST, for 1 hour at room temperature, in the dark. After three washes with PBS, slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and stored at 4°C until observed in a fluorescence microscope (Zeiss Axio Imager.Z1).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was partially supported by the Portuguese Foundation for Science and Technology (FCT) with a Ph.D., fellowship (SFRH/BD/19967/2004 to C.J.M.) and research grants (POCI/SAU-MMO/60555/04, 60709/04, 59997/04; UMIB).

We would like to acknowledge the valuable help of Joern Walter (University of Saarlandes, Saarbruecken, Germany) for training in bisulphite treatment with agarose beads, Sengul Tozlu and Sophie Vacher (Institut Curie—Hôpital René Huguenin) for

1 min, 72°C) and a final extension (20 min, 72°C). In some cases it was necessary to perform a hemi-nested PCR since no amplification was detected in the first-round PCR.32 The same conditions were used for the second-round PCR but the number of cycles was reduced to 30. PCR products were puri-fied with GFX PCR-DNA and Gel Band Purification Kit (GE Healthcare) and cloned with TOPO TA cloning kit (Invitrogen). Clones were sequenced in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA); to assure the efficiency of the bisulphite treatment, only sequences with more than 95% of non-CpG cytosines converted were validated.

Quantitative/real-time RT-PCR. On isolated spermatogenic cells (SGA = 2,650, ST1 = 3,700, ST2 = 3,600, Sa = 3,600 and Sd = 3,600), RNA extraction and Reverse Transcription (RT) reac-tion was performed using Superscript III Platinum CellsDirect Two-Step qRT-PCR Kit with SYBR Green (Invitrogen), accord-ing to manufacturers’ instructions. RNA from spermatozoa was isolated using TriPure reagent (Roche) and from testicular biopsies using RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was carried out with Superscript First-Strand Synthesis System for RT-PCR, using random hexamers as the priming method (Invitrogen, Carlsbad, CA). Human testis total RNA from Clontech (Palo Alto, CA) was used as a positive control. Real time PCR reactions were carried out as described in reference 48, for RPLP0, ACTB and UBB (reference genes) and for DNA Methyltransferases, DNMT1, DNMT3A, DNMT3B and DNMT3L. All primers were designed to amplify only cDNA (at least one of the primers is exon-spanning) and are complementary to regions that are present in all the major protein coding alternative transcripts of DNMTs (Table 2). Real time PCR was performed in an ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and relative expression was calculated using qBase software,49 using the effi-ciency of amplification for each gene (determined by a standard curve) and normalizing against three reference genes. Specificity

Table 2. Primers for real-time RT-PCR for detection of human DNMTs expression

Gene Primer sequence (5'-3') Sequence amplifieda Product size

RPLP0F-GGC GAC CTG GAA GTC CAA CT

NM_001002: nt 195–343 149 bpR-CCA TCA GCA CCA CAG CCT TC

ACTBF-AGA GCC TCG CCT TTG CCG AT

NM_001101: nt 21–206 187 bpR-CCA TCA CGC CCT GGT GCC T

UBBF-TCC GCT AAC AGG TCA AAA TGC A

NM_018955: nt 122–249 129 bpR-GGG AAT GCC TTC CTT ATC CTG

DNMT1F-TGG ACG ACC CTG ACC TCA AAT

NM_001379, nt 1,316–1,483 168 bpR-GCT TAC AGT ACA CAC TGA AGC A

DNMT3AF-TAT TGA TGA GCG CAC AAG AGA GCb

AF331856, nt 1,627–1,737 111 bpR-GGG TGT TCC AGG GTA ACA TTG AGb

DNMT3BF-GGC AAG TTC TCC GAG GTC TCT Gb

AF331857, nt 1,120–1,232 113 bpR-TGG TAC ATG GCT TTT CGA TAG GAb

DNMT3LF-GGC CCT TCT TCT GGA TGT TCG T

AF194032, nt 1,317–1,410 94 bpR-ATG GTG ACT GGC TCC ATC TCC A

F, forward; R, reverse; aGenBank accession numbers; bGirault et al. 2003.

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Note

Supplemental materials can be found at: www.landesbioscience.com/journals/epigenetics/article/17993

help with real-time PCR, Luis Ferraz (MD, Urologist, Director, Department of Urology, Hospital Centre of Vila Nova de Gaia, Portugal) for the andrology work and Joaquina Silva (MD), Mariana Cunha, Paulo Viana and Ana Gonçalves (MSc) for the IVF work.

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