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BOR - Papers in Press, published online ahead of print July 5, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062851
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BIOLOGY OF REPRODUCTION 77, 697–706 (2007)
DOI: 10.1095/biolreprod.107.062851
© 2007 by the Society for the Study of Reproduction, Inc.

Autonomous Regulation of Sex-Specific Developmental Programming in Mouse Fetal Germ Cells1

Kazuhiro Iwahashi 3, Hirotaka Yoshioka 3, Eleanor W Low 3, John R McCarrey 4, Ryuzo Yanagimachi 3, and Yukiko Yamazaki 2 3

Institute for Biogenesis Research,3 John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96813 Department of Biology,4 University of Texas at San Antonio, San Antonio, Texas 78249

ABSTRACT

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.

de novo methylation, genomic imprinting, germ cells, meiosis, sex differentiation

INTRODUCTION

In mice, founder primordial germ cells (PGCs) are initially identified as Prdm1-positive cells in the most proximal layer of the epiblast at 6.25 days postcoitum (dpc) [1]. A day later (7.25 dpc), Prdm1-expressing cells are identified within the Ifitm3-positive cell population, and a few of these cells start expressing the definitive PGC marker Dppa3 [1–4]. By 8.5 dpc, PGCs enter the embryo proper, then proliferate and migrate into the genital ridges by 11.5 dpc [5]. In male genital ridges, Sry, the gene that encodes the Y-chromosomal testis-determining factor, is expressed between 10.5 and 12.0 dpc. This gene triggers a complex cascade of events that initiates development of the testes [6]. By 12.5 dpc, morphological sex differentiation has begun in the somatic elements of male and female gonads, whereas germ cells continue to proliferate by mitosis until approximately 13.5 dpc [5, 7]. Male germ cells then enter a mitotic arrest by 15.5–17.5 dpc, and are maintained as type T1 prospermatogonia in the G0/G1 stage of the cell cycle until after birth when, as type T2 prospermatogonia, they resume mitotic activity. On the other hand, female germ cells enter prophase of the first meiotic division at around 13.5–15.5 dpc, and most of them reach the diplotene stage at around birth [5]. Previous evidence suggests that the gonadal environment may be important for initially directing female and male germ cells to enter meiosis or mitotic arrest, respectively [5]. On the other hand, both XX and XY PGCs at 11.5 dpc enter meiosis when they are released from the gonadal environment and cultured in vitro [8, 9]. Very recent reports suggest that retinoic acid produced by somatic cells of the mesonephros specifically directs germ cells to enter meiosis during the fetal period [10, 11]. However, the extent to which ongoing interactions with gonadal somatic cells are required to facilitate maintenance of mitotic or meiotic states in fetal germ cells is not known.

Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of certain genes in a parent-of-origin-specific manner in mammals. Approximately 80 imprinted genes have been identified in mice, and the products of these genes are thought to play important roles in embryogenesis, placental function, fetal growth, and certain maternal-offspring behavioral interactions [12]. The allele-specific expression of these genes is regulated by inherited differences in epigenetic programming. These epigenetic allelic distinctions are propagated during early embryogenesis and are maintained in somatic cells for the lifetime of the individual. However, epigenetic modifications distinguishing imprinted alleles are erased during fetal development of germ cells and subsequently reset in a sex-specific, biallelic manner in preparation for transmission of properly imprinted alleles to the next generation [13–15].

One epigenetic modification that plays a key role in regulating genomic imprinting in eutherian mammals is DNA methylation [16]. In mouse germ cells, the inherited allele-specific methylation imprints are completely erased by 13.5 dpc, and new imprints are reestablished on both alleles according to the sex of the individual. In males, reestablishment of biallelic paternal imprints is completed at around birth. However, it has been shown that the H19 methylation imprint is reestablished asynchronously on the parental alleles during male germ cell development, appearing earlier on the paternal allele than on the maternal allele [17, 18]. Thus, although methylation of the paternal allele is nearly complete by the time of birth, the maternal allele does not become fully methylated until shortly after birth [17, 18]. In females, reestablishment of maternal imprints typically does not begin until after birth, during the oocyte growth phase, but it also shows an asynchronous pattern with acquisition of methylation on maternal alleles earlier than that on the paternal alleles [19, 20].

To study the mechanisms that regulate the development and differentiation of germ cells, many investigators have attempted to use primary culture systems with or without feeder cells, with the latter being especially useful for supporting proliferating PGCs [21–23]. In this regard, it has been suggested that adhesion to surrounding cells is crucial for PGC growth [24]. The effects of soluble growth factors have also been examined using primary culture systems, but these alone could not replicate the beneficial effects of feeder cells [25]. Recently, Farini et al. [9] reported that mouse PGCs isolated at 11.5 dpc survived and proliferated in culture in the absence of feeder cells when the germ cells were cultured on mesh inserts and factors were added to prevent apoptosis.

In the study described here, we have adapted the use of mesh inserts to support the primary culture of fetal germ cells for up to 6 days to examine whether normal development of these cells can occur autonomously in the absence of ongoing interactions with gonadal somatic cells. This method also allowed us to exclude the use of feeder cells that might otherwise mimic the effects of gonadal somatic cells. We investigated the extent to which germ cells isolated from fetal gonads at 13.5 dpc can undergo biallelic resetting of methylation imprints, as well as whether they can either progress into first meiotic prophase (characteristic of female germ cells) or enter mitotic arrest (characteristic of male germ cells) in a proper, sex-specific manner. We found that cultured male germ cells remained mitotic and were able to develop the normal sex-specific patterns of DNA methylation in the differentially methylated domains (DMDs) of the paternally imprinted H19 gene and the maternally imprinted Snrpn gene, whereas cultured female germ cells entered first meiotic prophase but remained hypomethylated at imprinted loci, just as they do during most of the fetal period in vivo. These results indicate that by 13.5 dpc, germ cells have established autonomous sex-specific developmental programming that will subsequently regulate resetting of epigenetic imprints and entry into meiosis or mitotic arrest, even in the absence of ongoing interactions with gonadal somatic cells.

MATERIALS AND METHODS

Mice

Pou5f1-green fluorescent protein (GFP) transgenic mice (Tg OG2) generated by microinjecting (CBA/CaJ x C57BL/6J) F2 zygotes express germ cell-specific GFP driven by the Pou5f1 gene promoter/enhancer (a generous gift from Dr. J.R. Mann, University of Melbourne, Melbourne, Australia) [26]. The B6(CAST 7) substrain (B6/CAST substrain) of mice has a Mus musculus castaneus (CAST) chromosome 7 on a C57BL/6J (B6) background (a generous gift from Dr. M.S. Bartolomei, University of Pennsylvania, Philadelphia, PA) [27]. Several polymorphisms have been described that distinguish B6 and CAST alleles of imprinted genes on chromosome 7 [27, 28]. Female B6/CAST mice were mated with male Tg OG2 mice to produce (B6/CAST x OG2) F1 hybrid fetuses [29]. GFP-positive germ cells were collected from these F1 fetuses to perform all experiments. For gene expression assay, we also used GFP-positive germ cells derived from (C57BL/6J x OG2) F1 hybrid fetuses. All relevant experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Hawaii.

Media

Gonads and germ cells were collected in Hepes-Dulbecco modified Eagle medium (DMEM; Gibco) with 20% fetal bovine serum (FBS; Hyclone). Germ cells were cultured in high-glucose DMEM (Gibco), supplemented with 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 20% FBS.

Germ Cell Culture

Male and female gonads were obtained from F1 hybrid fetuses at 13.5 dpc. Gonads were dissected in Hepes-DMEM (Gibco) with 20% FBS and incubated in 0.02% EDTA/phosphate solution for 10 min at room temperature. After incubation, gonads were gently pipetted and filtered through a 40-µm cell strainer (BD Falcon) to prepare a single-cell suspension.

For gonadal cell cultures, primary mouse embryonic fibroblasts prepared from 15.5-dpc fetuses were used as feeder cells. Single-cell suspensions of gonadal cells (mixtures of germ cells and somatic cells) were directly seeded onto mitomycin C (Sigma)-treated feeder cells. For germ cell cultures (purified germ cells), GFP-positive germ cells were sorted using the Epics Altra cell sorter (Beckman Coulter). The excitation wavelength was 488 nm (argon laser). After sorting, the purity of GFP-positive cells was >95%. About 4000–7000 germ cells were cultured on collagen-coated mesh inserts (pore size: 0.4 µm; Corning). The mesh inserts were placed into wells of a 24-well cell culture plate (Corning) with or without feeder cells preseeded on the bottom of each well. In some cases, leukemia inhibitory factor (LIF; 1000 U/ml; Chemicon International) was added to the germ cell culture media. Germ cells were cultured at 37°C with 5% CO2 in air for 2, 4, or 6 days. Endogenous GFP-positive germ cells were collected manually from gonadal cell suspensions of fetuses at 13.5, 15.5, 17.5, and 19.5 dpc (0 days postpartum) using a fluorescence microscope equipped with a micropipette. These cells served as controls and so were not cultured, but rather were examined directly. In addition, tail tissue from F1 neonates at 3 days postpartum was used as somatic cell control.

Bisulfite Genomic Sequencing

Allele-specific methylation patterns at imprinted loci were examined using bisulfite genomic sequencing as described [30], with some modifications. One set of genomic DNA prepared from 120–200 GFP-positive germ cells was used for one PCR amplification [29]. For endogenous control and cultured female germ cells, one to two sets of genomic DNA were prepared for each stage. For cultured male germ cells, three to four sets of genomic DNA were prepared for each stage. Bisulfite modification of DNA was performed using a methylation assay kit (Zymo Research). Bisulfite-converted DNA was subjected to PCR amplification of the H19 and Snrpn DMDs. Primer pairs specific for the H19 and the Snrpn genes were used for nested amplification of the H19 and Snrpn DMDs, respectively, as previously described [19, 29]. PCR products were subcloned into the pGEM-T Easy vector (Promega) and sequenced. Subcloning was repeated two to four times for each PCR product, and 6–20 subclones were analyzed in each case. Only those sequences that showed at least 98% conversion of Cs to Ts after the bisulfite treatment were included in the analysis shown. Maternal and paternal alleles were distinguished on the basis of a DNA polymorphism unique to CAST and not present in M. m. musculus, as described [27, 28]. The methylated percentage (%) of all CpG sites combined was calculated for paternal and maternal alleles, respectively.

Quantitative Gene Expression Analysis

After culture for 2–6 days, 50–150 GFP-positive germ cells were collected as one set, and cDNA was synthesized using the Superscript III CellsDirect cDNA synthesis system (Invitrogen). We used two to six sets of germ cells in each stage for gene expression assay. We examined quantitative expression of four imprinted genes (H19, Igf2, Peg3, and Snrpn), two DNA methyltransferases (Dnmt1 and Dnmt3a), and a methyltransferase-like gene (Dnmt3L). The primers were prepared as previously described [27, 31–33]. Quantitative real-time PCR was performed using the iCycler system (Bio-Rad). Results were normalized to the ß-actin gene expression and calibrated according to the result from 13.5 dpc male and female germ cells. Data are presented as means of two to six replicate analyses of each cell type.

Viability of Cultured Germ Cells

To assess the viability of germ cells after 6 days of culture, the cells were incubated with 0.3% trypan blue to detect the dead cells. In addition to exclusion of trypan blue, living cells were recognized on the basis of expression of GFP and/or healthy morphology. Each microscopic field was divided into nine subregions, and 20–50 cells were examined under 400x magnification in each subregion to determine the percentages of viable and nonviable cells. Data are presented as means with their standard errors.

Detection of Meiotic Cells

After 4 days of culture, the male or female germ cells were placed on poly-L-lysine-coated glass slides. After fixation with 4% (w/v) paraformaldehyde, they were washed twice with 3% (w/v) BSA in PBS (-) and were permeabilized with 0.2% (v/v) Triton X-100, 3% BSA in PBS (-) for 15 min each. They were then incubated with a 1:100 dilution of anti-SCP3 monoclonal antibody (Abcam) for 60 min at room temperature. After repeated washing with 3% BSA in PBS (-), the germ cells were incubated with a 1:100 dilution of goat anti-mouse Alexa 568 IgG (Molecular Probes) as a secondary antibody. After washing, immunofluorescence was visualized using an FV 1000 confocal laser scanning microscope (Olympus).

Statistical Analysis

For comparisons of cell viability, data were analyzed by ANOVA with Dunnett's test. Significance was considered at a P value less than 0.05.

RESULTS

Establishment of Genomic Imprints in Endogenous Germ Cells

As a control, we first examined the status of allele-specific methylation in the differentially methylated domains of the H19 and Snrpn genes in endogenous somatic and germ cells that had not been subjected to culture. Our results confirmed previous reports. Thus, in mouse somatic cells, the paternal allele of H19 was hypermethylated and the maternal allele was hypomethylated, whereas the situation was reversed for the Snrpn gene, where the maternal allele was hypermethylated and the paternal allele was hypomethylated, as previously described (Fig. 1A) [28, 34]. These allele-specific methylation patterns are retained in somatic cells throughout the lifetime of the individual. However, in both male and female germ cells this methylation is completely erased by 13.5 dpc, followed by the establishment of new biallelic methylation patterns in a sex-specific manner [18, 19]. This process is shown in Figure 1. In male germ cells, remethylation of the H19 gene had begun by 15.5 dpc and proceeded in an allele-specific manner, with the paternal alleles regaining methylation slightly prior to the maternal alleles, as previously reported [18]. In contrast to the H19 gene, both alleles of the Snrpn gene remained almost completely unmethylated in male germ cells at all stages examined (Fig. 1B). This represents the erasure and resetting mechanism that establishes male-specific, biallelic imprints in the sperm genome. In female germ cells, on the other hand, the de novo methylation of maternally imprinted genes takes place predominantly after birth [20]. Thus, we found that both the H19 and Snrpn genes remained unmethylated at both alleles throughout fetal development of female germ cells at 13.5–19.5 dpc (Fig. 1C), as previously described [19, 20].


Figure 01
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  		FIG. 1 Allele-specific methylation status of the H19 and Snrpn DMDs in endogenous germ cells. A) Maps indicate the location of the H19 upstream DMD and the Snrpn promoter DMD 1 as gray boxes, with 16 CpG dinucleotides represented as open circles within the analyzed region in each DMD, each of which is designated as a closed diamond. For each gene, an analysis of the allele-specific DNA methylation status in somatic cells is shown. Closed circles represent methylated CpGs, and open circles represent unmethylated CpGs. Each line of circles corresponds to an individually analyzed strand of DNA. Sequences are based on GenBank database information: H19 DMD is GenBank accession no. U19619, and Snrpn DMD 1 is GenBank accession no. AF081460. Paternal (P) and maternal (M) alleles were distinguished by DNA polymorphisms in the DMD sequence distinguishing M. musculus castaneus (B6/CAST) and M. musculus musculus (Tg OG2). Also, tail tissue from (B6/CAST X OG2) F1 neonates at 3 days postpartum was used as a somatic cell control. B, C) Purified endogenous male (B) and female (C) germ cells were recovered from (B6/CAST x OG2) F1 fetuses at 13.5, 15.5, 17.5, and 19.5 dpc. Allele-specific methylation in the H19 DMD and the Snrpn DMD 1 was assessed in 200 germ cells collected from each sex at each stage.

Establishment of Genomic Imprints in Germ Cells Cultured in Association with Gonadal Somatic Cells on Feeder Cells

To determine whether sex-specific imprints can become established in fetal germ cells maintained in culture in association with gonadal somatic cells, 13.5-dpc gonads (consisting of germ cells plus somatic cells) were dissociated into single-cell suspensions and cultured on feeder cells for up to 6 days, and then germ cells were collected for bisulfite genomic sequencing. In male germ cells, methylation was analyzed on three to four sets of cultured cells per time point. After 2 days of male germ cell culture, methylated CpG sites were slightly increased in the H19 DMDs of both parental alleles during this time (4% of all CpGs in both paternal and maternal alleles; Fig. 2A). This progressed to 17%–22% methylated CpGs of both alleles after 4 days of culture, and 77%–78% methylated CpGs of both alleles after 6 days of culture. These results showed that although the timing of de novo methylation of the H19 gene was delayed in cultured germ cells compared with endogenous germ cells (Fig. 1B), it is possible for male germ cells to establish a male-specific imprint in culture in the presence of gonadal somatic cells, albeit in the absence of the normal three-dimensional structure of the developing gonad. In contrast, the normally maternally methylated Snrpn gene retained a hypomethylated state on both alleles in male germ cells throughout the culture period (Fig. 2A). This demonstrates that establishment of imprints in cultured male germ cells follows the normal gene-specific pattern observed in endogenous germ cells (Fig. 1B).


Figure 02
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  		FIG. 2 Allele-specific methylation status of the H19 and Snrpn DMDs in germ cells cultured on feeder cells. A) Analysis of allele-specific DNA methylation in the DMD regions of the H19 and Snrpn genes in male germ cells recovered at 13.5 dpc and cultured with gonadal somatic cells for up to 6 days on feeder cells. DNA methylation was analyzed on three to four sets of germ cells at each stage. The progressive acquisition of a biallelic methylation imprint is seen in the paternally imprinted H19 DMD, whereas the maternally imprinted Snrpn DMD remains unmethylated on both alleles. B) A similar analysis of female germ cells recovered at 13.5 dpc and cultured with gonadal somatic cells for 6 days on feeder cells shows that both the H19 and Snrpn DMDs remained unmethylated in these cells during the culture period.

In female germ cells, methylation was analyzed on two sets of cultured cells per time point. The results show that parental alleles of both the H19 and Snrpn genes remained totally unmethylated throughout the 6 days of culture period (Fig. 2B), mimicking the delayed timing of imprint establishment in endogenous female germ cells (Fig. 1C). This indicates that establishment of imprints in fetal germ cells cultured in association with gonadal somatic cells follows the normal sex-specific timing observed in endogenous germ cells.

Establishment of Genomic Imprints in Germ Cells Cultured in the Absence of Gonadal Somatic Cells or Feeder Cells

To determine whether cultured fetal germ cells can establish gene- and sex-specific genomic imprints autonomously in the absence of somatic support, fetal germ cells isolated from gonads at 13.5 dpc were cultured on mesh inserts without gonadal somatic cells or feeder cells for up to 6 days. Bisulfite genomic sequencing was repeated using three to four sets of germ cells at each stage. After 2 days of culture of isolated male germ cells, both paternal and maternal alleles of the H19 gene remained almost completely unmethylated (Fig. 3A). However, following 4 days of culture, isolated male germ cells had initiated de novo methylation of the H19 gene (10%–24% of CpGs in maternal and paternal alleles). After 6 days of culture, 74%–85% CpGs of maternal and paternal H19 DMDs were biallelically hypermethylated in male germ cells (Fig. 3A). As was the case with the germ cells cultured in association with gonadal somatic cells on feeder cells (Fig. 2A), the timing of de novo methylation of the H19 DMDs in cultures of isolated germ cells tended to be delayed relative to that seen in endogenous fetal germ cells (Fig. 1B). As was also seen when germ cells were cultured in association with gonadal somatic cells, the Snrpn gene remained hypomethylated on both alleles throughout the 6-day culture period of isolated fetal male germ cells (Fig. 3A). Because of the limiting numbers of isolated female germ cells that survived the culture period, no analysis of DNA methylation was performed on these cells. These results indicate that, at least in male germ cells, the ability to establish gene- and sex-specific imprints is maintained in culture and does not require ongoing influences from either gonadal somatic cells or feeder cells, and is, therefore, autonomously programmed in the isolated germ cells.


Figure 03
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  		FIG. 3 Allele-specific methylation status of the H19 and Snrpn DMDs in isolated male germ cells cultured on mesh inserts without somatic cells or feeder cells. A) Analysis of allele-specific DNA methylation in the DMD regions of the H19 and Snrpn genes in purified male germ cells recovered at 13.5 dpc by fluorescent-activated cell sorting and cultured for up to 6 days without gonadal somatic cells or feeder cells. DNA methylation status was analyzed on three to four sets of germ cells at each stage. The progressive acquisition of a biallelic methylation imprint is seen in the paternally imprinted H19 DMD, whereas the maternally imprinted Snrpn DMD remains unmethylated on both alleles. B) A similar analysis of purified male germ cells recovered at 13.5 dpc and cultured for 6 days in the presence of LIF shows a similar, although slightly retarded acquisition of biallelic methylation imprints in the H19 DMD.

To examine whether the establishment of genomic imprints is influenced by factors regulating pluripotency, isolated male germ cells were cultured in the presence of LIF. Under these conditions, CpGs in the H19 DMD became partially methylated on both alleles after 4 days of culture, and the proportion of methylated CpGs on both alleles continued to increase by 6 days of culture (Fig. 3B). However, after 6 days of culture, the extent of methylation in the H19 DMDs was relatively lower in the germ cells cultured in the presence of LIF compared with those cultured without LIF (58% and 80% of CpGs in both alleles, respectively; Fig. 3). This result suggests that the establishment of the male-specific genomic imprint might be delayed overall in the presence of LIF.

Gene Expression in Cultured Germ Cells

As an indication of the extent of proper epigenetic programming in cultured germ cells, we examined the expression of four imprinted genes (H19, Igf2, Peg3, and Snrpn) in isolated male germ cells cultured on mesh inserts for 6 days without somatic cells and compared this to the expression of these same genes in endogenous fetal male germ cells during the equivalent developmental period (Fig. 4). Examination of endogenous male germ cells at 19.5 dpc revealed a pattern of very low expression of H19, expression of Igf2 at slightly higher than background levels, expression of Peg3 at a medium level, and high-level expression of Snrpn. Isolated male germ cells cultured on mesh inserts for 6 days displayed a similar pattern of expression of these four imprinted genes, although the absolute levels of expression of the Peg3 and Snrpn genes were reduced compared with those observed in endogenous male germ cells.


Figure 04
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  		FIG. 4 Quantitative expression of imprinted genes in male germ cells. Relative expression levels of four different imprinted genes, H19, Igf2, Peg3, and Snrpn, were assessed by quantitative RT-PCR in endogenous male germ cells recovered at 19.5 dpc and in purified male germ cells recovered by fluorescent-activated cell sorting at 13.5 dpc and cultured on mesh inserts without gonadal somatic cells or feeder cells for 6 days. Results were normalized to levels of ß-actin gene expression. Note that although the absolute level of expression of the Peg3 and Snrpn genes was lower in cultured male germ cells than in endogenous male germ cells, the overall expression profile of these four imprinted genes was similar in the endogenous and cultured cells. We used two or three sets of 150 male germ cells in each stage (endogenous or cultures). A sample of cDNA prepared from each set of germ cells was divided into five aliquots for analysis of expression of the four imprinted genes and ß-actin in each experiment. In endogenous germ cells, each column represents the means of two experiments, and two dots show the variation. In cultured germ cells, each column represents the mean of three experiments, with standard errors shown as bars.

We also examined the expression of three genes that regulate DNA methylation status (Dnmt1, Dnmt3a, and Dnmt3L) in both male and female germ cells (Fig. 5). The product of the Dnmt1 gene is the normal "maintenance methyltransferase" that is responsible for converting hemimethylated sites back to fully methylated sites following DNA replication [35, 36]. In this study, endogenous germ cells maintained relatively constant expression levels of Dnmt1 in both males and females at 13.5–19.5 dpc (Fig. 5A). This likely reflects that Dnmt1 is not responsible for the genome-wide de novo methylation events that take place in the male germ line prenatally [33, 37]. In male and female germ cells cultured for 2–6 days, expression levels of Dnmt1 were very similar to those in endogenous germ cells (Fig. 5A). To the contrary, Dnmt3a is a so-called de novo methyltransferase, which is required for the establishment of parental imprints [38, 39]. Furthermore, Dnmt3L is normally required to direct proper de novo DNA methylation in association with the product of the Dnmt3a gene [38–40]. We next examined the expression dynamics of Dnmt3a and Dnmt3L in male and female germ lines. In endogenous cells, Dnmt3a displayed unique developmental profiles that showed marked sex-specific differences. In males, Dnmt3a expression was gradually elevated between 15.5 and 17.5 dpc (Fig. 5B), which correlates exactly with the normal period of de novo methylation in these cells. In females, on the other hand, only low expression levels of Dnmt3a between 13.5 and 17.5 dpc were maintained (Fig. 5B). This same sexually dimorphic pattern was mimicked by cultured germ cells. In the female germ cells, Dnmt3a expression remained constant throughout the culture period for up to 4 days (Fig. 5B). On the contrary, in isolated male germ cells, levels of Dnmt3a expression did increase slightly after 2 days of culture, and it peaked after 4 days of culture, followed by the decrease after 6 days of culture. It must be noted, however, at all stages examined, the expression levels were consistently lower than those detected in endogenous controls. Last, we examined the expression levels of Dnmt3L in male and female germ lines. In endogenous male germ cells, the expression levels of the Dnmt3L gene were extremely low in endogenous germ cells in males at 13.5 dpc. However, the Dnmt3L expression level was increased at 15.5 dpc and peaked at 17.5 dpc (Fig. 5C). On the other hand, expression of Dnmt3L remained completely repressed in endogenous female germ cells between 13.5 and 19.5 dpc. Our results showed that the sex-specific expression patterns of Dnmt3L are very similar to those of Dnmt3a in endogenous germ cells. We also found sex-specific expression patterns of Dnmt3L in cultured germ cells (Fig. 5C). No expression above background was detected in isolated female germ cells cultured for up to 4 days. On the contrary, in isolated male germ cells, levels of Dnmt3L expression did increase slightly after 2 days of culture, and further after 4 days of culture, but then decreased again after 6 days of culture, although at all stages examined the expression levels were consistently lower than those detected in endogenous controls.


Figure 05
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  		FIG. 5 Quantitative expression of two DNA methyltransferases and a DNA methyltransferase-like gene in male and female germ cells. Relative expression levels of two different DNA methyltransferase genes, Dnmt1 (A) and Dnmt3a (B), and a DNA methyltransferase-like gene, Dnmt3L (C), in endogenous and cultured germ cells are shown. In each case, expression of each gene was measured in purified endogenous male germ cells (recovered at 13.5, 15.5, 17.5, and 19.5 dpc) and female germ cells (recovered at 13.5, 15.5, and 17.5 dpc). These expression profiles were compared with those detected in isolated germ cells cultured on mesh inserts without gonadal somatic cells or feeder cells for 0 (13.5), 2 (+ 2d), 4 (+ 4d), or 6 (+ 6d) days in males and for 0, 2, or 4 days in females. Although the developmental expression profiles of these three genes differ, the profiles for each gene are similar in endogenous and cultured germ cells of the same sex. We used three to six sets of 50 germ cells in each stage (endogenous or cultured). A cDNA sample prepared from each set of germ cells was divided into two aliquots for analysis of expression of one DNA methyltransferase gene and ß-actin in each experiment. Each column represents the mean of three to six replicate experiments, with standard errors shown as bars.

Viability of Cultured Germ Cells

We compared the viability of male and female germ cells cultured for 6 days on mesh inserts under various conditions (Fig. 6). About 32% of male germ cells survived for 6 days on mesh inserts in the absence of feeder cells, which approximated the survival rate of germ cells cultured directly on feeder cells without mesh insert (36%; Fig. 6A). When male germ cells were cultured on mesh inserts above feeder cells, the viability increased to 43%. On the other hand, in the presence of LIF, the viability of isolated male germ cells was markedly decreased with or without feeder cells. When isolated female germ cells were cultured on mesh inserts without feeder cells, 3% of the cells remained viable after 6 days, representing a marked decrease from the 14% survival rate when these cells were cultured directly on feeder cells (Fig. 6B). When these cells were cultured on mesh inserts above feeder cells, their viability doubled to 7%. On the other hand, the viability of female germ cells was not improved in the presence of LIF (3% survival). These results suggest that male germ cells are significantly more resistant to the culture condition we tested than female germ cells.


Figure 06
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  		FIG. 6 Viability of cultured germ cells. The viability of isolated male and female germ cells cultured for 6 days in different conditions is shown. Isolated germ cells (3000–7000) were recovered by fluorescent-activated cell sorting and cultured for 6 days for each analysis. The viability of isolated male (A) and female (B) germ cells is shown for each of five different culture conditions, including (left to right), isolated germ cells cultured on mesh inserts without feeder cells, isolated germ cells cultured on mesh inserts above feeder cells, isolated germ cells cultured on mesh inserts without feeder cells but with the addition of LIF to the media, isolated germ cells cultured on mesh inserts above feeder cells with the addition of LIF to the media, and isolated germ cells cultured directly on feeder cells. Each column represents the mean of three experiments, with standard errors shown as bars. Asterisks on error bars indicate a significant difference (*P < 0.05, **P < 0.01) compared with the data in germ cells cultured on mesh inserts without feeder cells. The viability of male germ cells cultured on mesh inserts without feeder cells but with the addition of LIF was significantly (P < 0.01) lower than that of male germ cells cultured on mesh inserts without feeder cells. The viability of female germ cells cultured directly on feeder cells is significantly (P < 0.05) higher than that of female germ cells cultured on mesh inserts without feeder cells.

Detection of Meiotic Germ Cells

To determine whether fetal germ cells entered meiosis during the culture period, we stained the cultured cells with anti-SCP3 antibody, which recognizes a synaptonemal complex protein that appears only in meiotic cells [41–43]. Sorted, GFP-positive germ cells were cultured on mesh inserts for up to 4 days in the absence of gonadal somatic cells or feeder cells. As a positive control, endogenous female germ cells were recovered at 13.5, 15.5, and 17.5 dpc and were directly stained for SCP3 without being subjected to culture. This revealed staining patterns characteristic of preleptotene/leptotene, zygotene, and pachytene stages of first meiotic prophase (Fig. 7, A–C). Approximately 35% of endogenous female germ cells at 13.5 dpc had already entered meiosis at preleptotene/leptotene. After 2 days of culture, more than 80% of female germ cells showed progression to the zygotene-pachytene stages (Fig. 7E). About 70%–80% of female germ cells progressed to the pachytene stage after 4 days of culture (Fig. 7F). Culture in the presence of LIF produced no remarkable difference in the proportion of isolated female germ cells that entered meiosis (Fig. 7G). The kinetics of entry into meiosis of endogenous and cultured germ cells is summarized in Figure 7H. This shows that progression of isolated fetal female germ cells into meiotic prophase occurs at a similar rate during the first 2 days in culture as it does in endogenous female germ cells, and that this continues, albeit at a slightly retarded rate compared with endogenous female germ cells, during the next 2 days in culture. On the contrary, isolated male germ cells showed no staining for SCP3 after culture for 4 days on mesh inserts (Fig. 7D), indicating no entry into meiotic prophase. Importantly, only isolated female germ cells entered meiosis in culture. This reflects the normal sex-specific discrepancy in the development of endogenous germ cells, with female germ cells entering meiosis during the fetal period, whereas male germ cells do not enter meiosis until the time of puberty, well after birth.


Figure 07
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  		FIG. 7 Immunohistochemistry to detect SCP3 in meiotic germ cells. A) Endogenous female germ cell at 13.5 dpc in the preleptotene/leptotene stage of first meiotic prophase. B) Endogenous female germ cell at 15.5 dpc in the zygotene stage of first meiotic prophase. C) Endogenous female germ cell at 17.5 dpc in the pachytene stage of first meiotic prophase. D) Male germ cell recovered at 13.5 dpc and cultured for 4 days showing no SCP3 staining indicative of a mitotic cell. E) Female germ cells isolated at 13.5 dpc and cultured for 2 days reached the zygotene stage of first meiotic prophase. F) Female germ cells isolated at 13.5 dpc and cultured for 4 days reached the pachytene stage of first meiotic prophase. G) Female germ cells isolated at 13.5 dpc and cultured for 4 days in the presence of LIF also reached the zygotene stage (white arrow) and pachytene stage (white arrowheads) of first meiotic prophase. H) Proportions of cultured female and male germ cells entering meiosis. The percentages of endogenous female cells at different stages of first meiotic prophase at 13.5, 15.5, and 17.5 dpc are compared with those of isolated female germ cells recovered at 13.5 dpc and cultured on mesh inserts without gonadal somatic cells or feeder cells for 2 (+ 2d) or 4 (+ 4d) days without the addition of LIF or with the addition of LIF (+ LIF) to the culture media. On the contrary, isolated male germ cells were recovered at 13.5 dpc and cultured for 4 days without somatic cells, feeder cells, or LIF, none of which stained for SCP3. Note that isolated female germ cells cultured for 2 days with or without LIF show a distribution of cells throughout first meiotic prophase that is similar to that seen among endogenous female germ cells at 15.5 dpc, whereas isolated female germ cells cultured with or without LIF for 4 days show slightly retarded progress through first meiotic prophase compared to endogenous female germ cells at 17.5 dpc. Data represent the mean percentage of three scores performed in one experiment on a total of 70–210 cells for each stage. The second set of experiments also showed a similar pattern. Bar = 20 µm.

DISCUSSION

Sexually dimorphic characteristics in fetal germ cells are the establishment of sex-specific imprinting at certain loci, as well as entry into meiosis by fetal female germ cells as opposed to mitotic arrest by fetal male germ cells. Relatively little is known about how either of these processes is regulated. Specifically, it remains unknown to what extent these processes are autonomously regulated within the germ cells themselves or are induced by the surrounding, sexually dimorphic gonadal environments in each sex. To examine this question, we adapted a novel culture method with a mesh insert instead of feeder cells. Previous reports showed that feeder cells could be used to sustain fetal germ cells in culture [24], and subsequent reports showed that apoptosis of cultured germ cells could be suppressed by either direct contact with feeder cells or the inclusion of certain soluble factors in the culture media in the absence of feeder cells [44]. Recently, Farini et al. [9] demonstrated that adhesion to an acellular substrate, such as a mesh insert, could be used in lieu of feeder cells to sustain germ cells in culture. Using this system, we could directly test the ability of germ cells to undergo sex-specific differentiation in the absence of somatic influences.

We found that 32% of 13.5-dpc male germ cells could survive for 6 days when cultured in the absence of somatic support. In contrast, 3% of 13.5-dpc female germ cells survived 6 days of culture in the same conditions (Fig. 6). It is known that both male and female germ cells are normally subject to apoptosis during the fetal period and that this effect is typically two to three times more pronounced in females than in males, especially from 15.5 dpc and later [25, 45–47]. Thus, the differential viability we observed between male and female germ cells in culture reflects a similar, naturally occurring discrepancy that occurs during the equivalent period of development of endogenous male and female germ cells. The mechanism(s) responsible for this difference in apoptotic activity in male and female germ cells remain unknown. The meiotic process may critically interact with the mechanisms of apoptotic selection in germ cells. Also, it remains a possibility that some germ cells that fail to develop proper patterns of imprinting are selectively lost. However, it is noteworthy that this effect correlates directly with another primary distinction between males and females—the entry of female germ cells into meiosis during the fetal period while male germ cells enter mitotic arrest during this same period.

LIF is known to promote proliferation and survival of early-stage PGCs and to maintain these cells in an undifferentiated state in vitro [25, 48, 49]. Because PGCs express a LIF receptor, it has been suggested that PGCs are a direct target of LIF action in vitro [49]. However, when added to the culture media for 13.5-dpc germ cells in our study, LIF led to a significant decrease in viability of male germ cells cultured for 6 days. Similarly, LIF was previously shown to sustain 11.5-dpc PGCs in an undifferentiated state and to inhibit entry of these cells into meiosis in culture [8, 9]. However, we observed no beneficial effects of LIF on the survival of 13.5-dpc fetal germ cells (Fig. 6), nor did we see any inhibition of these cells from entering meiosis following 4 days in culture (Fig. 7H). These results show that the effects of LIF on PGC growth change around the time that PGCs cease mitotic division and undergo differentiation.

A significant distinction between males and females in our study was the sex-specific entry into meiosis of cultured female germ cells but not of cultured male germ cells. This observation extends earlier findings reported by De Felici and McLaren [21] demonstrating that this sexually dimorphic trait is autonomously programmed in germ cells by 13.5 dpc, and by Adams and McLaren [50] showing that male germ cells in an intact developing testis are committed to spermatogenesis from 12.5 dpc and later. This is also consistent with the suggestion that germ cells in the male genital ridge become blocked from entry into meiosis at about 12.5 dpc, coincident with the differentiation of Sertoli cells and the initial formation of testicular cords [5]. However, when germ cells of either sex are removed from the genital ridges before 12.5 dpc and cultured in vitro, they enter meiosis regardless of their sex chromosomal constitution (XX or XY) [8, 9].

Recent reports have elucidated key molecular regulators of entry of fetal germ cells into meiosis, including the sex-specific expression of the Stra8 gene induced by retinoic acid in the female gonads specifically between 11.5 and 13.5 dpc [11] and the coincident repression of retinoic acid induction of Stra8 expression as a result of sex-specific expression of the retinoid-degrading enzyme Cyp26b1 in the male gonads [10, 11]. Our results indicate that these (and possibly other) molecular events that occur in the developing gonads by 13.5 dpc are sufficient to irreversibly program fetal germ cells to subsequently enter meiosis in females or mitotic arrest in males. Our studies showed that the normal sexually dimorphic pattern of biallelic acquisition of methylation imprints is accurately recapitulated in cultured germ cells, either in isolation or in association with gonadal somatic cells and feeder cells (Figs. 1–3). This demonstrates that neither the normal three-dimensional structure of the fetal gonads nor an ongoing association with gonadal somatic cells or even with generic supporting (feeder) cells is required to facilitate this process. Thus, if this process is normally dependent on instructive interactions between germ cells and somatic cells, those interactions must take place prior to 13.5 dpc. If this is the case, the regulation of sex-specific genomic imprinting would be directly analogous to that of sex-specific entry into meiosis or mitotic arrest in these cells. Indeed, these mechanisms could be related. Alternatively, the process of genomic imprinting may be regulated by a completely autonomous mechanism operating within the gonadal germ cells themselves.

Interestingly, the acquisition of de novo DNA methylation in the cultured germ cells was delayed about 2 days relative to that observed in endogenous germ cells in each sex. This could be due to a general disruption of cellular processes that results from the dissociation and plating procedures, from which the cultured cells must recover prior to proceeding with normal differentiative events, and/or this may be due to diminished levels of expression of Dnmt3a and Dnmt3L in cultured male germ cells (Fig. 5).

A final variable tested in relation to the establishment of methylation imprints in isolated male germ cells was the presence or absence of LIF, which normally sustains a state of pluripotency in cultured cells [49]. We observed that de novo methylation of the H19 DMD occurs at lower levels in the presence of LIF. To the extent that acquisition of methylation at the H19 locus represents male-specific differentiation during germ cell development, this process may be repressed or retarded by the effects of LIF that would otherwise promote a more undifferentiated state in these cells.

Perhaps the most significant observation regarding the normalcy of acquisition of methylation imprints in cultured germ cells is that it occurred in a sex-specific manner that directly reflected the normal sexually dimorphic acquisition of gene-specific methylation imprints in developing germ cells. The differential methylation status of imprinted genes in endogenous and cultured fetal male germ cells suggests that these genes might also be differentially expressed, with the expectation being that the more methylated genes would be repressed and the less methylated genes expressed. Our analysis of endogenous male germ cells recovered at 19.5 dpc demonstrates that this indeed is normally the case. The similar, albeit somewhat reduced profile of expression of these genes in isolated male germ cells maintained in culture for 6 days indicates that this effect is accurately recapitulated in culture (Fig. 4). Thus, both the normal acquisition of sex- and gene-specific methylation imprints and the concomitant gene-specific expression patterns of imprinted genes are autonomously regulated in fetal germ cells, and both effects can now be accurately recreated in culture.

The sex-specific acquisition of genomic imprints in cultured fetal germ cells implies that regulators of epigenetic programming may themselves be differentially expressed in these cells, just as they have been shown to be in endogenous fetal germ cells. We confirmed that this is indeed the case for the Dnmt3a and Dnmt3L genes, which have been shown to be essential for the establishment of germline-specific methylation imprints associated with imprinted genes [38, 39, 51]. Our analysis of the expression of the Dnmt3a and Dnmt3L genes in cultured fetal germ cells revealed dramatic sex-specific differences in the expression levels and timing that were typical features in endogenous controls (Fig. 5, B and C). Because Dnmt3a and Dnmt3L expression levels in male germ cells cultured for 4 days were about 65% and 20% of those in endogenous controls at 17.5 dpc, respectively, this shortage might affect the lower DNA methylation status of the imprinted loci in cultured male germ cells (Figs. 2 and 3). As a conclusion, differential expression of Dnmt3 gene family involved in epigenetic programming is also autonomously regulated in fetal germ cells at 13.5 dpc and later.

In summary, we have demonstrated that it is possible to maintain isolated mouse fetal germ cells in culture for a period that mimics the later portion of normal fetal development. As a result, we conclude that sex-specific differentiation of germ cells is autonomously programmed in these cells by 13.5 dpc, such that no additional instructive interactions with gonadal environment are required to drive these differentiative events.

ACKNOWLEDGMENTS

We thank Drs. Marisa S. Bartolomei and Jeff R. Mann for generously providing the B6/CAST and OG2 transgenic mice. We also acknowledge Dr. Richard Allsopp for technical assistance with the cell sorting, and Drs. Yusuke Marikawa, Mary Ann Handel, and W. Steven Ward for their advice during the preparation of this manuscript. We are grateful to Drs. John Grove, Peter Holck, and Hitoshi Ishimoto for their advice on statistical analysis.

FOOTNOTES

1Supported by National Institutes of Health grant HD042772 to Y.Y., R.Y., and J.R.M.; National Institutes of Health grant P20RR16467 to Y.Y.; and the Victoria S. and Bradley L. Geist Foundation grant HCF 20050392 to Y.Y. Back

Correspondence: 2FAX: 808 692 1962; e-mail: yyamazak{at}hawaii.edu

Received: 15 May 2007.

First decision: 5 June 2007.

Accepted: 3 July 2007.

REFERENCES

  1. Ohinata Y, Payer B, O'Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, Saitou M, Surani MA. Blimp1 is a critical determinant of the germ cell lineage in mice Nature 2005 436207–213[CrossRef][Medline]
  2. Saitou M, Barton SC, Surani MA. A molecular programme for the specification of germ cell fate in mice Nature 2002 418293–300[CrossRef][Medline]
  3. Sato M, Kimura T, Kurokawa K, Fujita Y, Abe K, Masuhara M, Yasunaga T, Ryo A, Yamamoto M, Nakano T. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells Mech Dev 2002 11391–94[CrossRef][Medline]
  4. Tanaka SS and Matsui Y. Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells Mech Dev 2002 119(suppl 1)S261–S267[CrossRef][Medline]
  5. McLaren A. Primordial germ cells in the mouse Dev Biol 2003 2621–15[CrossRef][Medline]
  6. Koopman P, Munsterberg A, Capel B, Vivian N, Lovell-Badge R. Expression of a candidate sex-determining gene during mouse testis differentiation Nature 1990 348450–452[CrossRef][Medline]
  7. Lawson KA and Hage WJ. Clonal analysis of the origin of primordial germ cells in the mouse Ciba Found Symp 1994 18284–91 discussion
  8. Chuma S and Nakatsuji N. Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling Dev Biol 2001 229468–479[CrossRef][Medline]
  9. Farini D, Scaldaferri ML, Iona S, La Sala G, De Felici M. Growth factors sustain primordial germ cell survival, proliferation and entering into meiosis in the absence of somatic cells Dev Biol 2005 28549–56[CrossRef][Medline]
  10. Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P. Retinoid signaling determines germ cell fate in mice Science 2006 312596–600[Abstract/Free Full Text]
  11. Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. Retinoic acid regulates sex-specific timing of meiotic initiation in mice Proc Natl Acad Sci U S A 2006 1032474–2479[Abstract/Free Full Text]
  12. Swales AK and Spears N. Genomic imprinting and reproduction Reproduction 2005 130389–399[Abstract/Free Full Text]
  13. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development Science 2001 2931089–1093[Abstract/Free Full Text]
  14. Rideout WM III, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome Science 2001 2931093–1098[Abstract/Free Full Text]
  15. Surani MA. Reprogramming of genome function through epigenetic inheritance Nature 2001 414122–128[CrossRef][Medline]
  16. Ferguson-Smith AC and Surani MA. Imprinting and the epigenetic asymmetry between parental genomes Science 2001 2931086–1089[Abstract/Free Full Text]
  17. Davis TL, Trasler JM, Moss SB, Yang GJ, Bartolomei MS. Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis Genomics 1999 5818–28[CrossRef][Medline]
  18. Davis TL, Yang GJ, McCarrey JR, Bartolomei MS. The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development Hum Mol Genet 2000 92885–2894[Abstract/Free Full Text]
  19. Lucifero D, Mertineit C, Clarke HJ, Bestor TH, Trasler JM. Methylation dynamics of imprinted genes in mouse germ cells Genomics 2002 79530–538[CrossRef][Medline]
  20. Lucifero D, Mann MR, Bartolomei MS, Trasler JM. Gene-specific timing and epigenetic memory in oocyte imprinting Hum Mol Genet 2004 13839–849[Abstract/Free Full Text]
  21. De Felici M and McLaren A. In vitro culture of mouse primordial germ cells Exp Cell Res 1983 144417–427[CrossRef][Medline]
  22. De Felici M and Dolci S. Leukemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM4 feeder layers Dev Biol 1991 147281–284[CrossRef][Medline]
  23. Pesce M, Di Carlo A, De Felici M. The c-kit receptor is involved in the adhesion of mouse primordial germ cells to somatic cells in culture Mech Dev 1997 6837–44[CrossRef][Medline]
  24. De Felici M. Regulation of primordial germ cell development in the mouse Int J Dev Biol 2000 44575–580[Medline]
  25. Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M. Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis) Development 1993 1181089–1094[Abstract]
  26. Szabó PE, Hübner K, Schöler H, Mann JR. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells Mech Dev 2002 115157–160[CrossRef][Medline]
  27. Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei MS. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos Biol Reprod 2003 69902–914[Abstract/Free Full Text]
  28. Tremblay KD, Duran KL, Bartolomei MS. A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development Mol Cell Biol 1997 174322–4329[Abstract]
  29. Yamazaki Y, Low EW, Marikawa Y, Iwahashi K, Bartolomei MS, McCarrey JR, Yanagimachi R. Adult mice cloned from migrating primordial germ cells Proc Natl Acad Sci U S A 2005 10211361–11366[Abstract/Free Full Text]
  30. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines Nucleic Acids Res 1994 222990–2997[Abstract/Free Full Text]
  31. Lee J, Inoue K, Ono R, Ogonuki N, Kohda T, Kaneko-Ishino T, Ogura A, Ishino F. Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells Development 2002 1291807–1817[Medline]
  32. Ogawa H, Ono Y, Shimozawa N, Sotomaru Y, Katsuzawa Y, Hiura H, Ito M, Kono T. Disruption of imprinting in cloned mouse fetuses from embryonic stem cells Reproduction 2003 126549–557[Abstract]
  33. La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, Trasler JM. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells Dev Biol 2004 268403–415[CrossRef][Medline]
  34. Shemer R, Birger Y, Riggs AD, Razin A. Structure of the imprinted mouse Snrpn gene and establishment of its parental-specific methylation pattern Proc Natl Acad Sci U S A 1997 9410267–10272[Abstract/Free Full Text]
  35. Bestor T, Laudano A, Mattaliano R, Ingram V. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases J Mol Biol 1988 203971–983[CrossRef][Medline]
  36. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality Cell 1992 69915–926[CrossRef][Medline]
  37. Sakai Y, Suetake I, Itoh K, Mizugaki M, Tajima S, Yamashina S. Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo Cell Struct Funct 2001 26685–691[CrossRef][Medline]
  38. Hata K, Okano M, Lei H, Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice Development 2002 1291983–1993[Medline]
  39. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting Nature 2004 429900–903[CrossRef][Medline]
  40. Chedin F, Lieber MR, Hsieh CL. The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a Proc Natl Acad Sci U S A 2002 9916916–16921[Abstract/Free Full Text]
  41. Dobson MJ, Pearlman RE, Karaiskakis A, Spyropoulos B, Moens PB. Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction J Cell Sci 1994 1072749–2760[Abstract]
  42. Lammers JH, Offenberg HH, van Aalderen M, Vink AC, Dietrich AJ, Heyting C. The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes Mol Cell Biol 1994 141137–1146[Abstract/Free Full Text]
  43. Zickler D and Kleckner N. Meiotic chromosomes: integrating structure and function Annu Rev Genet 1999 33603–754[CrossRef][Medline]
  44. De Felici M, Scaldaferri ML, Lobascio M, Iona S, Nazzicone V, Klinger FG, Farini D. Experimental approaches to the study of primordial germ cell lineage and proliferation Hum Reprod Update 2004 10197–206[Abstract/Free Full Text]
  45. Coucouvanis EC, Sherwood SW, Carswell-Crumpton C, Spack EG, Jones PP. Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis Exp Cell Res 1993 209238–247[CrossRef][Medline]
  46. Wang RA, Nakane PK, Koji T. Autonomous cell death of mouse male germ cells during fetal and postnatal period Biol Reprod 1998 581250–1256[Abstract/Free Full Text]
  47. Kasai S, Chuma S, Motoyama N, Nakatsuji N. Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes Dev Biol 2003 264202–216[CrossRef][Medline]
  48. Matsui Y, Toksoz D, Nishikawa S, Nishikawa S, Williams D, Zsebo K, Hogan BL. Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture Nature 1991 353750–752[CrossRef][Medline]
  49. Cheng L, Gearing DP, White LS, Compton DL, Schooley K, Donovan PJ. Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth Development 1994 1203145–3153[Abstract]
  50. Adams IR and McLaren A. Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis Development 2002 1291155–1164[Abstract/Free Full Text]
  51. Bourc'his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the establishment of maternal genomic imprints Science 2001 2942536–2539[Abstract/Free Full Text]




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