Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

STUDY QUESTION: Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER: No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 µg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION: This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 µg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA: Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 µg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline.

STUDY QUESTION: How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline? SUMMARY ANSWER: Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells. WHAT IS KNOWN ALREADY: The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L(+/-) males. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study of DNA methylation in Dnmt3L(+/+) versus Dnmt3L(+/-) male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2-20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L(+/+) and Dnmt3L(+/-) males following ≈ 2 months in culture. MATERIALS, SETTING, METHODS: DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning. MAIN RESULTS AND THE ROLE OF CHANCE: At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L(+/-) SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6-28. While the methylation of most loci was stable in culture over time, differences at ≈ 1% of sites were found between Dnmt3L(+/-) and Dnmt3L(+/+) cultures. LIMITATIONS, REASONS FOR CAUTION: Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment. WIDER IMPLICATIONS OF THE FINDINGS: The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a 'window' of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.

Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure.

Persistent organic pollutants (POPs) can induce epigenetic changes in the paternal germline. Here, we report that folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs in a rat model. Pregnant founder dams were exposed to an environmentally relevant POPs mixture (or corn oil) ± FA supplementation and subsequent F1-F4 male descendants were not exposed to POPs and were fed the FA control diet. Sperm miRNA profiles of intergenerational (F1, F2) and transgenerational (F3, F4) lineages were investigated using miRNA deep sequencing. Across the F1-F4 generations, sperm miRNA profiles were less perturbed with POPs+FA compared to sperm from descendants of dams treated with POPs alone. POPs exposure consistently led to alteration of three sperm miRNAs across two generations, and similarly one sperm miRNA due to POPs+FA; which was in common with one POPs intergenerationally altered sperm miRNA. The sperm miRNAs that were affected by POPs alone are known to target genes involved in mammary gland and embryonic organ development in F1, sex differentiation and reproductive system development in F2 and cognition and brain development in F3. When the POPs treatment was combined with FA supplementation, however, these same miRNA-targeted gene pathways were perturbed to a lesser extend and only in F1 sperm. These findings suggest that FA partially mitigates the effect of POPs on paternally derived miRNA in a intergenerational manner.

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.

Characterization of the murine beta-hexosaminidase (HEXB) gene.

The murine HEXB gene, encoding the beta-subunit of the lysosomal hydrolase, beta-hexosaminidase, was isolated from a mouse cosmid library as a single cosmid clone. The entire gene spans 22 kb, considerably less than the 40 kb spanned by its human counterpart. It is highly homologous to the human gene. The 14 intron-exon junctions are entirely conserved, although the intron sequences diverge rapidly. Upstream of the coding region, a 1.3 kb segment was sequenced and shown to function as a promoter when fused with a reporter gene and expressed in monkey COS-7 cells. A short sequence (100 bp), near the start of the coding region, exhibits strong homology to the human HEXB promoter. Analysis of the tissue distribution of the HEXB mRNA in 129/Sv male mice revealed up to 28-fold tissue-specific variations in transcript levels. The kidney and the epididymis had the highest mRNA levels consistent with past surveys of enzyme activity.

Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology.

Genomic methylation patterns originate during gametogenesis and are postulated to be involved in important developmental events, including gene regulation, embryogenesis, and genomic imprinting. In previous work, treatment of male rats with 5-azacytidine, a drug that blocks DNA methylation, resulted in abnormal embryo development when germ cells were exposed throughout spermatogenesis, encompassing mitotic, meiotic, and postmeiotic development, but not if they were only exposed postmeiotically. To explore the mechanisms underlying the effects of 5-azacytidine on sperm function, we determined the effects of the drug on testicular morphology, assessed whether exposure of meiotic spermatocytes resulted in abnormal pregnancy outcome, and examined the role of germ cell genomic demethylation in mediating the effects of 5-azacytidine on spermatogonia and spermatocytes. Male Sprague Dawley rats were treated three times a week with saline or 5-azacytidine (2.5 and 4.0 mg/kg) for 6 weeks (meiotic and postmeiotic germ cell exposure) and 11 weeks (mitotic, meiotic, and postmeiotic exposure). Six weeks of paternal treatment with the highest dose of 5-azacytidine resulted in an increase in preimplantation loss (corpora lutea minus implantation sites) without affecting testicular morphology or altering sperm DNA methylation levels. Eleven weeks of 5-azacytidine treatment at doses that cause preimplantation loss resulted in severe abnormalities of the seminiferous tubules, such as degeneration and loss of germ cells, atrophy of seminiferous tubules, presence of multinuclear giant cells, and sloughing of immature germ cells into the lumen, and a 22-29% decrease in genomic methylation levels in epididymal sperm. On closer evaluation of testicular histology using terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling detection in situ, both 6 and 11 weeks of 5-azacytidine treatment resulted in an increase over the control value in the number of apoptotic germ cells in the seminiferous tubules. Analysis of DNA methylation levels in isolated germ cells of treated males indicated that spermatogonia were more susceptible to the hypomethylating effects of 5-azacytidine than were spermatocytes. These studies provide evidence of an association between demethylation of germ cell DNA and alterations in testicular histology.

Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis.

Cellular junctions in the testis and epididymis play crucial roles for the development and maturation of spermatozoa. In the testis, tight junctions between Sertoli cells form a functional blood testis barrier between 10 and 16 days of age, whereas the tight junctional blood epididymal barrier between adjacent epithelial cells is formed between days 18 and 21. In the present study, occludin, a constituent integral membrane protein of tight junctions, was localized by immunofluorescent confocal microscopy in embryonic (days 13.5-18.5), postnatal (days 5-23) and adult (day 70) mouse testes and epididymides to correlate its expression with the onset of tight junctions and eventual formation of these barriers. At embryonic days 13.5 and 16.5, low diffuse cytoplasmic levels of occludin were observed in cells of the testicular cords. By embryonic day 18.5, the level of occludin was still low but appeared as a filiform-like network streaming toward the center of the cord. At postnatal days 5 and 7 immunostaining became more intense and appeared to outline the periphery of Sertoli cells of seminiferous tubules. Postnatal day 14 marked the appearance of an intense, focal band-like localization of occludin at the base of the tubules, correlating with the appearance of a functional blood-testis barrier. By day 23 and in adults, expression of occludin was noted at the base of the tubule appearing as intense, wavy, discontinuous bands similar in appearance irrespective of the stage of the seminiferous epithelium cycle. In the developing epididymis, intense cytoplasmic immunostaining was present in epithelial cells of many epididymal tubules at embryonic day 13.5. By embryonic day 16.5, intense occludin immunostaining appeared along the lateral plasma membranes of epithelial cells, whereas at embryonic day 18.5, immunostaining was punctate and apically located, suggesting the presence of tight junctions by this age; similar immunostaining was noted at postnatal days 5 and 7. In the adult epididymis, distinct punctate apical staining was observed between adjacent principal cells of all epididymal regions except the proximal initial segment, where occludin was found only in association with narrow cells. These results indicate that in the epididymis, the appearance of occludin at apical sites between adjacent epithelial cells occurs during embryonic development suggesting that tight junctions form earlier than in the testis. While occludin was expressed in a similar pattern between Sertoli cells at all stages of the cycle in the adult testis, its expression in the adult epididymis was cell- and region-specific. Taken together these data suggest that different factors regulate occludin expression in the testis and epididymis.

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis.

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.

Effects of cyclophosphamide on selected cytosolic and mitochondrial enzymes in the epididymis of the rat.

The anticancer and immunosuppressive drug cyclophosphamide is extensively used in clinical practice and is known to alter fertility in man. We showed previously that treatment of male rats with low daily doses of cyclophosphamide over a 9-week period caused fetal malformations, a high rate of postimplantation loss and affected epididymal and sperm histology. In the present study, five biochemical measures of epididymal function were used to characterize further the effects of cyclophosphamide on the epididymis. For 1, 3, 6, or 9 weeks, adult Sprague-Dawley rats were gavage-fed daily with saline (control), 5.1 (low dose), or 6.8 (high dose) mg/kg of cyclophosphamide. The specific activities of the two glycolytic enzymes aldolase and lactate dehydrogenase (LDH), the mitochondrial enzyme succinate dehydrogenase, the cytosolic enzyme carnitine acetyltransferase and the lysosomal enzyme acid phosphatase were determined in cytosolic and mitochondrial subcellular fractions from four segments of the epididymis. Cyclophosphamide caused decreases in protein concentrations in all segments of the epididymis only after 6 weeks of treatment with the high dose. The specific activities of aldolase, LDH and succinate dehydrogenase did not differ from control with respect to dose or duration of treatment. In contrast, there were significant effects of cyclophosphamide on carnitine acetyltransferase and acid phosphatase specific activity. After 1 week of treatment, there was a transient dose-related decrease in the specific activity of carnitine acetyltransferase, which was most striking for the corpus epididymidis (76% of control), but which did not differ from control after 3, 6, and 9 weeks. After 6 weeks of treatment with the high dose of cyclophosphamide, carnitine acetyltransferase specific activity in the initial segment and the corpus epididymidis was elevated to 165 and 140%, respectively, as compared with the 1-week high dose values. The specific activity of acid phosphatase did not differ from control after 1 and 9 weeks of treatment. At 3 and 6 weeks, however, there was a dose-related increase in acid phosphatase specific activity for all regions of the epididymis that was most marked in the cauda after the 6-week treatment (140% of control). Therefore, low dose, daily treatment of male rats with cyclophosphamide not only alters specific enzymes in specific segments of the epididymis, but acts in a dose- and time-dependent manner. It is possible that these changes could be mediated by direct, toxic effects of the drug on the epithelium or be secondary to alterations in the spermatozoa as a result of the treatment.

I. Abnormalities in cells of the testis, efferent ducts, and epididymis in juvenile and adult mice with beta-hexosaminidase A and B deficiency.

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two major isoenzymes: Hex A (subunit structure, alphabeta) and Hex B (betabeta). The presence of Hex in the testis and epididymis suggests important roles for the enzyme and its substrates in male fertility and reproductive functions. Disruption of the Hexb gene encoding the beta-subunit of Hex has led to the generation of a mouse model of human Sandhoff disease that survives to adulthood, enabling us to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. At 1 and 3 months of age, the testes, efferent ducts, and epididymides of Hex-deficient (Hexb -/-) and wild-type (Hexb +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy (LM and EM, respectively) as well as with immunocytochemistry employing antibodies to lysosomal proteins. In the testis, the morphological appearance and topographical arrangement of the cell types of the seminiferous epithelium of Hexb -/- mice were similar to those of wild-type animals at both ages. Both Sertoli and germ cells appeared to be unaffected. However, at both ages, myoid cells and macrophages showed an increased number of lysosomes in their cytoplasm as compared with the number seen in controls. The epithelial cells of the efferent ducts also showed an accumulation of lysosomes that increased with age as compared with controls. Principal cells of the entire epididymis revealed an increase in the size and number of lysosomes at 1 month of age as compared with those of controls, and by 3 months, these lysosomes often filled the supranuclear and basal regions of the cells. Narrow cells of the distal initial segment and intermediate zone, normally slender cells showing several lysosomes, became greatly enlarged and entirely filled with lysosomes in Hexb -/- mice. Clear cells of the caput, corpus, and cauda regions also showed a progressive increase in the size and number of lysosomes with age as compared with controls; the clear cells of the mutant mice were often enlarged and at times bulged into the lumen. Some basal cells of each epididymal region in Hexb -/- mice were similar to controls at 1 and 3 months, showing few lysosomes, while others showed an accumulation of lysosomes. Lysosomes of all affected epithelial cells were of varying sizes, but many large ones were present, apparently resulting from lysosomal fusion. Although pale stained, their identification as lysosomes was confirmed by EM immunocytochemistry with anti-cathepsin D and anti-Hex A antibodies. Predominantly in the proximal initial segment, large, pale cellular aggregates were noted in the LM analysis at the base of the epithelium, which by EM analysis were identified as belonging to two different cell types, narrow cells and halo cells. Taken together, these data reveal an increase in the size and number of lysosomes in all epithelial cell types lining the efferent ducts and entire epididymis as well as in myoid cells and macrophages of the testis. In the light of data showing epididymal defects restricted predominantly to the initial segment in Hexa -/- (Hex A-deficient) mice, our data on the Hexb -/- mice demonstrate a major role for Hex that can be fulfilled by either Hex A or Hex B in the epididymis.

II. Characterization and development of the regional- and cellular-specific abnormalities in the epididymis of mice with beta-hexosaminidase A deficiency.

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two isoenzymes: Hex A (subunit structure alphabeta) and Hex B (betabeta). Its presence in the testis and epididymis suggests important roles for Hex and its substrates in male fertility and reproductive functions. Disruption of the Hexa gene encoding the alpha-subunit of Hex has led to the generation of a mildly affected mouse model of human Tay-Sachs disease, allowing us the opportunity to analyze the effects of isolated Hex A deficiency on epithelial cellular morphology of the male reproductive tract. At 5 weeks and at 3, 5, and 12 months, the testes, efferent ducts and epididymides of Hex A-deficient (Hexa -/-) and wild-type (Hexa +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy as well as with immunocytochemistry employing antibodies to lysosomal enzymes. In the testis, the seminiferous epithelium of Hexa -/- mice appeared comparable to that of wild-type mice in appearance and topographical arrangement of its cell types at all ages examined. Also, no differences were noted for the efferent ducts. In contrast, there were striking abnormalities in the epididymides of the mutant mice; however, the abnormalities were mainly restricted to the initial segment and intermediate zone. Principal cells of these regions at 5 weeks showed a dramatic increase in the number of lysosomes as compared with those from wild-type animals, and this progressed with increasing age. Furthermore, unlike the few small lysosomes present in wild-type mice, those of Hexa -/- mice were at times enlarged and often filled the supranuclear and basal regions of these cells. In the light microscope, large, dense cellular aggregates were noted at the base of the epithelium in the proximal initial segment that corresponded in the electron microscope to two different cell types, both of which increased in size with age. One aggregate was considered to belong to narrow cells on the basis of the presence of numerous cup-shaped vesicles characteristic of these cells; they appeared to be dislocated from the upper half of the epithelium. In the distal initial segment and intermediate zone, narrow cells were readily identified, but rather than being slender as in the control animals, they were greatly enlarged and filled with pale lysosomes in mutant mice. The second type of cellular aggregate noted in the proximal initial segment corresponded to halo cells. They contained numerous small and large lysosomes and small, Golgi-related, dense, core granules characteristic of halo cells. On the basis of the large size of these cells, they appeared to be actively internalizing substances from the intercellular space. In contrast, principal and clear cells of the caput, corpus, and cauda regions did not appear to show a significant increase in number or size of lysosomes as compared with those of wild-type animals. All structures identified as lysosomes in the various cell types were immunoreactive for cathepsin D. The present data thus reveal that isolated Hex A deficiency results in region- and cell-specific abnormalities in the epididymis but in no apparent abnormalities in the testis or efferent ducts. Specific roles for Hex A that cannot be compensated for by other isozymes of Hex appear to exist within lysosomes of epithelial cells predominantly of the initial segment and intermediate zone. Taken together, the results also suggest that the inability to degrade endocytosed substrates normally acted upon by Hex A in lysosomes of principal and narrow cells leads to their accumulation, eventual fusion, and increased size.

Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells.

The development of germ cells is a highly ordered process that begins during fetal growth and is completed in the adult. Epigenetic modifications that occur in germ cells are important for germ cell function and for post-fertilization embryonic development. We have previously shown that male germ cells in the adult mouse have a highly distinct epigenetic state, as revealed by a unique genome-wide pattern of DNA methylation. Although it is known that these patterns begin to be established during fetal life, it is not known to what extent DNA methylation is modified during spermatogenesis. We have used restriction landmark genomic scanning (RLGS) and other techniques to examine DNA methylation at multiple sites across the genome during postnatal germ cell development in the mouse. Although a significant proportion of the distinct germ cell pattern is acquired prior to the type A spermatogonial stage, we find that both de novo methylation and demethylation occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. Alterations include predominantly non-CpG island sequences from both unique loci and repetitive elements. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage. These studies better define the developmental timing of genome-wide DNA methylation pattern acquisition during male germ cell development.

A unique configuration of genome-wide DNA methylation patterns in the testis.

In the mammalian lifecycle, the two periods of genome-wide epigenetic reprogramming are in the early embryo, when somatic patterns are set, and during germ cell development. Although some differences between the reprogrammed states of somatic and germ cells have been reported, overall patterns of genomic methylation are considered to be similar. Using restriction landmark genomic scanning to examine approximately 2,600 loci distributed randomly throughout the genome, we find that the methylation status of testicular DNA is highly distinct, displaying eightfold the number of hypomethylated loci relative to somatic tissues. Identification and analysis of >300 loci show that these regions are generally located within nonrepetitive sequences that are away from CpG islands and 5' regions of genes. We show that a contributing factor for these differences is that the methylation state of non-CpG-island DNA is correlated with the regional level of GC content within chromosomes and that this relationship is inverted between the testis and somatic tissues. We also show that in Dnmt3L-deficient mice, which exhibit infertility associated with abnormal chromosomal structures in germ cells, this unique testicular DNA methylation pattern is not established. These special properties of testicular DNA point to a broad, distinct epigenetic state that may be involved in maintaining a unique chromosomal structure in male germ cells.

Sex-specific promoters regulate Dnmt3L expression in mouse germ cells.

BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT-PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/- females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.

Origin and roles of genomic methylation patterns in male germ cells.

The epigenetic modification of DNA by methylation at cytosine residues is initiated in the germ line and is required for normal embryonic development in mammals. Marked differences in genomic methylation between male and female gametes arise during gametogenesis and have been implicated in genomic imprinting. While DNA methylation patterns for different types of gene sequences are known to change during spermatogenesis, the precise role(s) of DNA methylation in the normal development of male germ cells is poorly understood. The expression of the one known active form of DNA methyltransferase is highly regulated during spermatogenesis and may provide insight into mechanisms underlying the establishment of methylation patterns in germ cells.

Developmental expression of DNA methyltransferase messenger ribonucleic acid, protein, and enzyme activity in the mouse testis.

Sex- and sequence-specific patterns of methylation of mammalian DNA are established during gametogenesis and are believed to be important for genomic imprinting and developmental gene regulation. DNA methylation in mammalian cells is performed predominantly by the enzyme DNA (cytosine-5)-methyltransferase (DNA MTase). For a better understanding of how DNA methylation events are regulated during spermatogenesis, a developmental study comparing the expression of DNA MTase mRNA, protein, and enzyme activity was performed. Northern and Western blotting and enzyme activity assays were carried out on testes and purified populations of cells from the testes of mice aged 6-70 days. The 5.2-kb DNA MTase transcript was most abundant in testes of mice aged 6-10 days (2-3-fold the 70-day values); it had decreased in abundance by 40% by Day 20 and reached steady adult levels by 63 days. On Western blot analysis, developmental changes in the relative abundance of DNA MTase protein paralleled the changes seen in mRNA concentrations. DNA MTase enzyme activity in the testis was highest at 6 days of age (10-fold the 70-day values); it had decreased by more than 65% by 20 days and reached steady adult levels at 35 days. Analysis of purified germ cells from the adult testis revealed high levels of expression of both DNA MTase mRNA and protein in haploid round spermatids. In conclusion, DNA MTase is clearly developmentally regulated during spermatogenesis at the level of mRNA, protein, and enzyme activity. These results argue for an important role for this DNA-methylating enzyme during spermatogenesis.

Developmental exposure of male germ cells to 5-azacytidine results in abnormal preimplantation development in rats.

Methylation of cytosine residues in mammalian DNA is established during gametogenesis and embryogenesis; it plays an important role in gene regulation and normal embryonic development and has also been implicated in genomic imprinting. In the present study, we evaluated whether paternal administration of 5-azacytidine, a drug that is incorporated into DNA and blocks DNA methylation, could alter male germ cell development and function. A drug that does not block methylation, 6-azacytidine, served as a control. Adult male Sprague-Dawley rats (n = 4-8 per group) were treated i.p., three times per week for 4 and 11 wk, with saline or 2.5 (low dosage) or 5.0 (high dosage) mg/kg of 5-azacytidine and 6-azacytidine. After each of the treatment periods, males were mated to determine effects on fertility and embryo development. Although neither 6-azacytidine nor 4 wk of 5-azacytidine treatment affected male reproductive organ weights or sperm counts, 11 wk of 5-azacytidine resulted in dose-dependent reductions in testis and epididymal weights and sperm counts. Both dosages of 5-azacytidine resulted in significant increases in preimplantation loss, and the high dosage of 5-azacytidine caused a decrease in fertility. Examination of embryos on Day 2 of gestation revealed a striking dose-dependent increase in the average number of abnormal embryos per litter sired by the males treated with 5-azacytidine (saline, 0.33 +/- 0.24; low dosage, 2.64 +/- 0.92; high dosage, 10.09 +/- 0.95). In summary, paternal administration of 5-azacytidine interfered with normal male germ cell development and resulted in alterations in fertilization and early embryo development. We suggest that 5-azacytidine-induced alterations in germ cell DNA methylation patterns may be one of the underlying mechanisms, since similar dosages of the analogue 6-azacytidine had no effect on male reproduction and progeny outcome.

Reproductive epigenetics.

Epigenetics refers to covalent modifications of DNA and core histones that regulate gene activity without altering DNA sequence. To date, the best-characterized DNA modification associated with the modulation of gene activity is methylation of cytosine residues within CpG dinucleotides. Human disorders associated with epigenetic abnormalities include rare imprinting diseases, molar pregnancies, and childhood cancers. Germ cell development and early embryo development are critical times when epigenetic patterns are initiated or maintained. This review focuses on the epigenetic modification DNA methylation and discusses recent progress that has been made in understanding when and how epigenetic patterns are differentially established in the male and female germlines, the mouse, and human disorders associated with abnormalities in epigenetic programming in germ cells and early embryos, as well as genetic and other modulators (e.g. nutrition and drugs) of reproductive epigenetic events.

A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems.

We have found previously that daily treatment of male rats for 11 wk with low doses of the anticancer drug cyclophosphamide had no apparent effect on male reproductive organ weights, epididymal sperm counts, or serum hormones at the end of the treatment period; yet, upon breeding to untreated females, these males produced a high rate of post-implantation loss and fetal anomalies. The present study was designed to investigate the time course and dose response of the effects of chronic cyclophosphamide treatment on the male reproductive and hematologic systems. Male Sprague-Dawley rats were gavage-fed for 1, 3, 6 and 9 wk with saline (control), or 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide. After each of the treatment periods, males were mated to determine the effect on pregnancy outcome, then killed, and the effects on the male reproductive and hematologic systems were assessed. After 6 wk of treatment, a sharp increase in mortality was found between the 5.1 and 6.8 mg/kg/day doses of cyclophosphamide. The high dose of cyclophosphamide induced higher levels of pre- and post-implantation loss but fewer fetal anomalies than did the low dose. The low dose of cyclophosphamide did not affect reproductive organ weights; in contrast, the high dose caused decreases in epididymal, ventral prostate, and seminal vesicle weights after 3, 6, and 9 wk. Testicular and epididymal sperm counts were decreased in a dose-dependent manner after 3 wk; in addition, the high dose led to a decrease in epididymal sperm counts after 6 wk of treatment. Another rapidly proliferative tissue, the bone marrow, was dramatically affected by both doses of cyclophosphamide at all time points, with leukocyte counts decreasing to 40% of control by 1 wk. After 9 wk of treatment, effects on the male reproductive system were less marked, compared to earlier time points, whereas those on the hematologic system and pregnancy outcome persisted. Thus chronic low-dose treatment of male rats with cyclophosphamide not only had early and striking effects on the bone marrow and the pregnancy outcome but also affected the male reproductive system in a clear time- and dose-dependent manner.

Regulated synthesis and localization of DNA methyltransferase during spermatogenesis.

Differences in the methylation patterns of male and female gamete DNA are likely to be involved in genomic imprinting. However, little is known of the mechanisms that regulate de novo methylation and demethylation during gametogenesis. We report here that the well-characterized M(r) 190,000 form of DNA methyltransferase (the only known form) is present in isolated mitotic, meiotic, and postmeiotic male germ cells, with the exception of meiotic pachytene spermatocytes, where the protein is undetectable by immunoblot analysis and a novel 6.2-kb DNA methyltransferase transcript is present. Whereas replication and methylation are coupled in somatic cells, the presence of DNA methyltransferase in postreplicative male germ cells is consistent with previously observed de novo methylation events in these cells. Immunofluorescence experiments revealed that DNA methyltransferase is localized to the nuclei of male germ cells, with a subset of spermatogonia and postreplicative leptotene/zygotene spermatocytes displaying prominent nuclear foci that are strongly enriched in DNA methyltransferase. The data suggest that down-regulation of DNA methyltransferase expression during the pachytene stage of meiosis utilizes an mechanism that is associated with the production of a larger mRNA, and that de novo methylation in leptotene/zygotene spermatocytes may take place in spatially restricted nuclear domains that are enriched in DNA methyltransferase.