Origin of DNA methylation patterns in the male germ line : roles of the novel DNA methyltransferases in male germ cells

Formation of gametes capable of supporting development is dependent on a number of genetic and epigenetic events. DNA methylation is an epigenetic modification catalyzed by enzymes named DNA methyltransferases (DNMTs). In the mouse, methylation of DNA is associated with the control of gene expression and proper embryo development. Methylation patterns are established in a sex- and sequence-specific manner during male and female germ cell development and further modified during early embryonic development. Even though new DNMTs have recently been identified, little information is known on the origin of the methylation marks during male germ cell development (spermatogenesis). The main goal of the work presented in this thesis was to gain a better understanding of the enzymes involved in creating the epigenetic program of the male genome. The first step in doing so involved comparing the temporal expression profiles of DNA methyltransferases in the developing testis and ovary. The expression profiles obtained indicated that in the male, DNMT3a and DNMT3L could be involved in de novo methylation while DNMT3b and DNMT1 could be responsible for maintaining methylation patterns following DNA replication. Next, characterization of Dnmt3a and Dnmt3b expression in isolated postnatal male germ cells revealed how tightly regulated the expression of these genes is during spermatogenesis: specific transcript variants and protein isoforms of each DNMT are differentially expressed during male germ cell development. Finally, assessing the effect of Dnmt3L inactivation on the male germ line exposed the presence of a mitotic defect in germ cells lacking this protein. DNA methylation analyses revealed that many loci throughout the genome are marked for methylation by DNMT3L, indicating a more global role for this enzyme than that previously reported in genomic methylation patterning in the male germ line. As methylation patterns instituted during spermatogenesis have to be properly established for accurate transmission of epigenetic information to the next generation, the studies presented here contribute to our knowledge of the events leading to the creation of the epigenetic program necessary for the formation of healthy gametes

Coordinate regulation of DNA methyltransferase expression during oogenesis

<p>Abstract</p> <p>Background</p> <p>Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of <it>Dnmt3a </it>and <it>Dnmt3b</it>, as well as a regulator of DNA methylation, <it>Dnmt3L</it>, in isolated female germ cells.</p> <p>Results</p> <p>Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated <it>Snrpn</it>, <it>Peg3 </it>and <it>Igf2r </it>DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the <it>de novo </it>methyltransferase <it>Dnmt3b</it>, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.</p> <p>Conclusion</p> <p>Together these results provide a better understanding of the developmental regulation of <it>Dnmt3a</it>, <it>Dnmt3b </it>and <it>Dnmt3L </it>at the time of <it>de novo </it>methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.</p

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

<p>Abstract</p> <p>Background</p> <p>Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development.</p> <p>Results</p> <p>A developmental study covering the first 12 days following birth was conducted on a <it>Dnmt3L </it>mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in <it>Dnmt3L</it>^-/-males, pointing to a mitotic defect. A temporal expression study showed that expression of <it>Dnmt3L </it>is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in <it>Dnmt3L</it>^+/+germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content.</p> <p>Conclusion</p> <p>Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed.</p

Critical Period of Nonpromoter DNA Methylation Acquisition during Prenatal Male Germ Cell Development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility

Isolation and short-term culture of mouse spermatocytes for analysis of meiosis.

Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmacological induction of the prophase-to-division phase transition

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

Background: Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development. Results: A developmental study covering the first 12 days following birth was conducted on a Dnmt3L mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in Dnmt3L-/- males, pointing to a mitotic defect. A temporal expression study showed that expression of Dnmt3L is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in Dnmt3L+/+ germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content. Conclusion: Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed

Spata22, a novel vertebrate-specific gene, is required for meiotic progress in mouse germ cells.

The N-ethyl-N-nitrosourea-induced repro42 mutation, identified by a forward genetics strategy, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression