Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages

DNA methylation is necessary for the silencing of endogenous retrotransposons and the maintenance of monoallelic gene expression at imprinted loci and on the X chromosome. Dynamic changes in DNA methylation occur during the initial stages of primordial germ cell development; however, all consequences of this epigenetic reprogramming are not understood. DNA demethylation in postmigratory primordial germ cells coincides with erasure of genomic imprints and reactivation of the inactive X chromosome, as well as ongoing germ cell differentiation events. To investigate a possible role for DNA methylation changes in germ cell differentiation, we have studied several marker genes that initiate expression at this time. Here, we show that the postmigratory germ cell-specific genes Mvh, Dazl and Scp3 are demethylated in germ cells, but not in somatic cells. Premature loss of genomic methylation in Dnmt1 mutant embryos leads to early expression of these genes as well as GCNA1, a widely used germ cell marker. In addition, GCNA1 is ectopically expressed by somatic cells in Dnmt1 mutants. These results provide in vivo evidence that postmigratory germ cell-specific genes are silenced by DNA methylation in both premigratory germ cells and somatic cells. This is the first example of ectopic gene activation in Dnmt1 mutant mice and suggests that dynamic changes in DNA methylation regulate tissue-specific gene expression of a set of primordial germ cell-specific genes.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis

DNA methylation is essential for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, contribute to the creation of DNA methylation patterns in embryos. We demonstrated that the Dnmt3a and Dnmt3b proteins are expressed at different stages of embryogenesis. Dnmt3b is specifically expressed in totipotent embryonic cells, such as inner cell mass, epiblast and embryonic ectoderm cells, whilst Dnmt3a is significantly and ubiquitously expressed after E10.5. The difference in the expression stages of the Dnmt3a and Dnmt3b proteins may contribute to their distinct functions during the embryogenesis.     

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.     

Asynchronous expression of the homeodomain protein CUX1 in Sertoli cells and spermatids during spermatogenesis in mice

The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.     

A Distinct Expression Pattern of Cyclin K in Mammalian Testes Suggests a Functional Role in Spermatogenesis

Germ cell and embryonic stem cells are inextricably linked in many aspects. Remarkably both can generate all somatic cell types in organisms. Yet the molecular regulation accounting for these similarities is not fully understood. Cyclin K was previously thought to associate with CDK9 to regulate gene expression. However, we and others have recently shown that its cognate interacting partners are CDK12 and CDK13 in mammalian cells. We further demonstrated that cyclin K is essential for embryonic stem cell maintenance. In this study, we examined the expression of cyclin K in various murine and human tissues. We found that cyclin K is highly expressed in mammalian testes in a developmentally regulated manner. During neonatal spermatogenesis, cyclin K is highly expressed in gonocytes and spermatogonial stem cells. In adult testes, cyclin K can be detected in spermatogonial stem cells but is absent in differentiating spermatogonia, spermatids and spermatozoa. Interestingly, the strongest expression of cyclin K is detected in primary spermatocytes. In addition, we found that cyclin K is highly expressed in human testicular cancers. Knockdown of cyclin K in a testicular cancer cell line markedly reduces cell proliferation. Collectively, we suggest that cyclin K may be a novel molecular link between germ cell development, cancer development and embryonic stem cell maintenance.