Differences in DNA Methylation Durin.G Oogenesis and Spermatogenesis and Their Persistence During Early Embryogenesis in the Mouse

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Differences in DNA methylation durin.g oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse

J.P. Sanford, 1,3 H.J. Clark, 2 V.M. Chapman, 1 and J. Rossant a ~Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263 USA; 2Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute and the Department of Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X5, Canada

We have examined the relative methylation levels of several dispersed repeated and low-copy-number gene sequences during gametogenesis and early embryogenesis. Southern blot analyses revealed that L1, intercisternal A particle (IAP), and major urinary protein (MUP) sequences were undermethylated extensively at Mspl sites in DNA from diplotene oocytes. In contrast, the same sequences were highly methylated in DNA from pachytene spermatocytes, round spermatids, and epididymal sperm. These results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavage-stage embryos and inner cell masses (ICM) were methylated at intermediate levels, consistent with transient maintenance of gametic methylation levels during early embryogenesis. Gametic differences in DNA methylation observed here indicate that methylation could provide a mechanism for imprinting maternal and paternal genomes resulting in differential regulation of parental genomes during early development. [Key Words: Mouse; DNA methylation; gametogenesis; embryogenesis; genomic imprinting] Received August 28, 1987; revised version accepted October 6, 1987.

Several lines of evidence suggest that maternal and pa- female genomes can be distinguished later in develop- ternal genomes do not play identical roles in mamma- ment. lian embryonic development. First, analysis of partheno- Genomic imprinting must involve epigenetic, heri- genetic development (Graham 1974)and nuclear trans- table differences between paternal and maternal pronu- plantation experiments has shown that embryos clef. Differential methylation of matemal and patemal containing two maternal or two paternal genomes die DNA could provide a mechanism for imprinting. DNA before or shortly after implantation (McGrath and Solter methylation at cytosine residues can be stably preserved 1984a; Surani et al. 1984), reflecting a requirement for through rounds of replication by maintenance methy- the presence of both maternal and paternal genomes lases {Razin and Riggs 1980)and has been postulated to during embryogenesis. Second, during normal develop- play a role in gene regulation {Cedar 1984}. Three recent ment, a paternal X chromosome is preferentially inacti- studies have provided strong supporting evidence that vated in extraembryonic tissues (Takagi and Sasaki DNA methylation could play a role in genomic im- 1975; West et al. 1977). Third, the hairpin tail mutation printing. These studies showed that the methylation (Thp) is lethal only when maternally inherited (McGrath pattems of exogenous DNA sequences in transgenic and Solter 1984b). Fourth, mice that receive both por- mouse lines sometimes varied according to whether the tions of certain chromosomes from one parent develop gene was inherited from the male or female parent {Reik abnormally (Cattanach and Kirk 1985; Searle and Bee- et al. 1987; Sapienza et al. 1987; Swain et al. 19871. In they 1985). None of these observations can be explained one study, expression of the transgene was also affected by a generalized delay in the onset of paternal gene ex- by the gamete of origin (Swain et al. 1987). Differences pression because gene activation from the zygotic in DNA methylation according to gamete of origin were genome occurs by the two-cell stage (Schultz 1986). detected in both mid-gestation (Reik et al. 1987)and Rather, these studies indicate that imprinting of the adult tissues (Reik et al. 1987; Sapienza et al. 1987; genome occurs during gametogenesis such that male and Swain et al. 1987) but were not directly compared between oocytes and sperm. If DNA methylation were to act as the imprinting 3Present address: Department of Biological Sciences, State University of mechanism in mammalian development, one might ex- New York at Buffalo, Buffalo, New York 14260 USA. pect to find different patterns of DNA methylation in

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Sanfotd et al. eggs and sperm and to observe perpetuation of these differences at least through early development. In fact, oocyte sperm liver little is known about the relative methylation levels of H M H M H M X kb endogenous gene sequences during oogenesis and sper- .,,, Q matogenesis. In a previous study, we noted that dispersed repetitive L 1 sequences were undermethylated in DNA from fetal ovaries when compared with DNA from m - 23.1 mature sperm (Sanford et al. 1984). This comparison of somatic cell-contaminated tetraploid fetal oocytes and ~. -9.4 haploid sperm could not demonstrate conclusively, however, that extensive DNA methylation differences .~ -6.6 existed during comparable stages of male and female gametogenesis nor that such differences could persist into -4.4 embryogenesis. In the present study, we have compared om O I the methylation status of purified fetal oocytes with that of tetraploid spermatocytes and haploid spermatids. We have shown that several repeated and low copy se- - 2.3 quences that are heavily methylated at all stages of sper- - 2.0 matogenesis are undermethylated in the oocyte genome. Furthermore, we have shown that DNA methylation patterns in preimplantation embryos and inner cell masses (ICMs) are consistent with perpetuation of differences in gametic DNA methylation into early development.

Results 1 2 3 4 5 6 Comparison of methylation levels of repeated and low copy sequences m fetal oocytes and mature sperm Figure 1. Methylation of L1 sequences in oocyte and sperm DNA. In each lane, 2 ~tg of genomic DNA, digested with HpaII (H, odd-numbered lanes} or MspI (M, even-numbered lanes), Tetraploid dictyate oocytes arrested in prophase I of was loaded. [Lanes 1,2)Oocyte; {lanes 3,4)sperm; {lanes 5,6) meiosis were dissected from 16- to 17-day female fe- liver. tuses. At this stage the number of oocytes recovered from the ovary is maximal because oocytes become This result indicates that these MspI sites are heavily closely associated with contaminating somatic follicle methylated in sperm. cells at later stages (Whittingham and Wood 1983). Oo- To determine whether hypomethylation of oocyte cyte samples used in this study were collected using a DNA was limited to highly repeated sequences, the procedure that gives a high yield of oocytes with min- HpaII digestion patterns of other DNA sequences were imum somatic cell contamination (DeFelici and examined. Intracistemal A particle {lAP)sequences rep- McLaren 1982). One hundred and fifty fetuses yielded 30 resent a retroviral-like class of sequence repeated ap- ~g of oocyte DNA. Cytological analysis of oocyte prepa- proximately 1000 times per haploid mouse genome rations indicated that only 1-5% of the cells were so- (Lueders and Kuff 1980). IAP transcripts are made in the matic (not shown). Using this pure population of oo- oocyte and early embryo but not in later development cytes, we reexamined the methylation at MspI restric- (Piko et al. 1984). We observed significant HpaII digestion sites in L1 sequences in oocyte DNA. The tion of MspI sites in IAP sequences in oocyte DNA and abundance of L1 fragments generated by HpaII digestion determined by densitometry that 75-100% of the major was comparable to that in the MspI digest (Fig. 1, lanes 1 MspI IAP sites were cleaved by HpalI (Fig. 2, lanes 1 and and 2). Densitometric analysis indicated that more than 2). In contrast, IAP MspI sites were not cleaved by HpaII 95% of the L1 MspI sites were digested by HpaII in oo- in sperm and liver DNAs. We also examined methyl- cyte DNA. These results indicate that the MspI sites ation of MspI sites in a tandemly repeated low-copy gene that generate the 3.5- and 5.0-kb fragments are unmeth- family of 35 genes encoding mouse urinary proteins ylated in nearly all of the estimated 30,000 copies of L1 (MUPs), which, unlike the lAP sequences, are not MspI sites present in the oocyte genome. Our previous known to be transcribed during oogenesis (Shaw et al. observation of a sizable proportion of methylated L1 se- 1983). Fragments recognized by the MUP cDNA clone quences in fetal oocytes (Sanford et al. 1984) is most represent both coding and intragenic noncoding regions likely due to extensive somatic cell contamination of in the MUP locus (Clark et al. 1984). We found no signif- the oocytes following the trypsin dissociation procedure icant differences between HpaII and MspI digestion pat- used previously. In contrast to the results obtained using terns for MUP sequences in oocyte DNA (Fig. 3, lanes 1 oocytes, L1 sequences were virtually undigested by and 2), and 90-100% of the MspI sites were recognized HpaII in DNA from sperm and somatic tissue (Fig. 1). by HpaII according to densitometric analysis. These re-

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DNA methylation in gametogenesis sults suggested that nearly all MUP MspI sites examined oocyte sperm liver were unmethylated in oocyte DNA. In contrast, HpaII H M H M H M digestion of sperm DNA revealed negligible digestion of MUP sequences.

Methylation status of repeated and low-copy genes at - 23.1 different stages of spermatogenesis The results described in the previous section demon- -9.4 strated that oocyte DNA was undermethylated consis- tently when compared with DNA from mature sperm. -6~3 Because fetal oocytes have not completed the first meiotic division and sperm have completed both -4.4 meiotic divisions, this comparison did not prove directly that male and female germ cells at the same stage of the meiotic cycle possess different levels of DNA methylation. Thus, we also examined the methylation status of 43:': L1 and MUP sequences in DNA from separated spermatogenic populations from adult testes. Enzymatic dissociation of the testes yielded between 2.5 x 107 and 3.0 x 107 cells per testis. These cells from six testes were separated into fractions enriched in specific cell 1 2 3 4 5 6 types by gravity sedimentation using the Staput apparatus. Fractions 21-28 contained greater than 85% pa- Figure 3. Methylation of MUP sequences in oocyte and sperm chytene spermatocytes. The remaining cells were DNA. In each lane, 10 t~g of genomic DNA, digested with HpaI (H, odd-numbered lanes)or MspI (M, even-numbered lanes), chiefly spermatids. These cells contain one-quarter of was loaded. (Lanes 1,2)Oocyte; (lanes 3,4) sperm; (lanes 5,6) the DNA content of spermatocytes and so would be a liver.

oocyte sperm liver minor contaminant in any DNA preparation from this fraction. A small number of spermatogonia and Sertoli kb H M H M H M X kb cells were also seen. Fractions 37-42 contained almost exclusively (>90%)round spermatids. The remaining cells were chiefly primary spermatocytes. Fractions - 23.1 21-28 and 37-42 were pooled and designated as pachytene spermatocytes and round spermatids, respectively. III I - 9.4 The DNA of these fractions was extracted and examined by Southern blot hybridization. The spermatocyte - 6.6 fraction yielded 0.23 mg of DNA, whereas the spermatid fraction yielded 0.37 mg of DNA. Figure 4 shows that - 4.4 neither L1 sequences nor the MUP sequences of the pa o chytene spermatocytes were digested at MspI sites by 3.0-- HpaII. Identical results were achieved from two separate 2.4-, - 2.3 restriction enzyme digests of the extracted DNA (not shown). Therefore, the DNA of these sequences is 1.7-' Or - 2.0 heavily methylated at this stage of spermatogenesis. This result contrasts with the lack of methylation observed in the same sequences at meiotic prophase of oo- .87- genesis. Figure 4 also shows that neither the L1 nor the MUP - .56 sequences of the round spermatids were digested at MspI .54 - sites by HpaII. Together with the results obtained using epididymal sperm described above, these observations suggest that the level of DNA methylation does not k change substantially during spermatogenesis. 1 2 3 4 5 6

Figure 2. Methylation of LAP sequences in oocyte and sperm Methylation levels of repeated sequences in DNA. In each lane, 2 p.g of genomic DNA, digested with HpaII preimplantation embryos (H, odd-numbered lanes)or MspI (M, even-numbered lanes), was loaded. (Lanes 1,2)Oocyte; (lanes 3,4)sperm; (lanes 5,6) To determine whether maternal and paternal methyl- liver. This blot is a rehybridization of the blot in Fig. 1. ation differences were propagated into early embryo-

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Sanford et al.

AMHMH BMHMH genesis. Tetraploid dictyate stage oocytes from day-17 Kb fetal ovaries showed almost no methylation of MspI sites in L1- and IAP-dispersed repeated sequences and !,° low copy MUP sequences. In contrast, L1 and MUP sequences were almost completely methylated at MspI sites in tetraploid pachytene spermatocytes. Meiosis

- 9.4 - begins in the fetus in the female mouse but not until after birth in the male, so that tetraploid oocytes and - 6.6 - spermatocytes are not derived from the same stage of 4,4 m gonad development. However, there is some indication that the differences in methylation between male and female germ cells may arise prior to the onset of male meiosis, as L1 sequences showed a possible difference in methylation of MspI sites between male and female gonads at 16.5 days of gestation (Monk et al. 1987). The sequences that were highly methylated in pachy- 1234 1 2 34 tene spermatocytes were also highly methylated in spermatids and epididymal sperm, indicating that this state Figure 4. Methylation of L1 or MUP sequences in spermato- persisted throughout spermatogenesis, as observed pre- genesis. In each lane, 2.5 ~g of genomic DNA, digested with viously for several low copy genes (Rahe et al. 1983). It MspI (M, odd-numbered lanes)or HpaII (H, even-numbered was not possible to prove that the undermethylation of lanes), was loaded. (A ) Hybridization with L1 probe. (Lanes 1,2) Pachytene spermatocytes; (lanes 3,4)round spermatids. (B) Hy- the same sequences observed in dictyate oocytes also bridization with MUP probe. (Lanes 1,2) Pachytene spermato- persisted throughout oogenesis, because meiosis is not cytes; (lanes 3,4)round spermatids. completed in the female until after fertilization. A direct comparison of the methylation status of male- and fe- genesis, we isolated DNA from 1000 8-cell embryos, 32- cell embryos, and ICMs and analyzed the methylation Im/eb 8-cell ICM status of minor satellite, L1, and MUP sequences. Figure 5 shows that the MspI sites in Ll-dispersed sequences in 1 2 3 4 5 6 DNA from early embryos were digested by HpaII (lanes Kb 1, 3, and 5) more extensively than in DNA from sperm but less extensively than in oocyte DNA. Densitometric analysis indicated that approximately 50% of each major 23.1 - fragment in the MspI digest was present in the HpaII digest. The intermediate levels of L1 undermethylation in 9.4- the early embryos are consistent with the possibility that maternal and paternal methylation patterns are propagated into early embryogenesis. In addition, minor 6.6 m satellite sequences, which are equally undermethylated in oocytes and sperm (Sanford et al. 1984), were digested by HpaII apparently to the same extent in embryonic 4.4- and germ cell DNAs (Fig. 6, lanes 1, 3, and 5), which supports this hypothesis and indicates that de novo ! methylation does not occur in the preimplantation embryo. Both repetitive sequences were essentially undigested by HpaII in DNA from liver and other somatic tissue DNAs (not shown). The small amount of DNA recovered from the early embryos precluded extensive analysis of the methylation of lower copy sequences. 2.3- / However, MspI sites in MUP gene sequences from eight- cell embryonic DNA were also cleaved by HpaII though, like L1 sequences, they were not digested as completely as in oocyte DNA (data not shown).

Discussion Figure 5. Methylation of L1 sequences in preimplantation embryo DNA. In each lane, 20-100 ng of genomic DNA, di- We have demonstrated that MspI sites in several endoge- gested with HpalI (H, odd-numbered lanes)or MspI (M, even- nous sequences, both repeated and low copy, show con- numbered lanes), was loaded. (Lanes 1,2)32-cell embryo {late siderable differences in the extent of cytosine methyl- morula/early blastocyst); (lanes 3,4) 8-cell embryo; (lanes 5,6) ation at comparable stages of oogenesis and spermato- ICM.

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DNA methylation in gametogenesis

Im/eb 8-cell ICM sperm liver Kb 1 2 34 56 7 8 9 10 Kb

23.1

9.4

6.6

4.4

- 1.7

2.3 Figure 6. Methylation of minor satellite sequences - .36 in preimplantation embryo DNA. In each lane i 20-100 ng of genomic DNA, digested with HpaII - 12 (H, odd-numbered lanes)or MspI (M, even-numbered lanes), was loaded. (Lanes 1,2)32-cell embryo (late morula/early blastocyst); (lanes 3,4) 8-cell embryo; (lanes 5,6)ICM; (lanes 7,8)sperm; (lanes 9,10) liver. male-derived genomes at fertilization would require traembryonic tissues, preferential inactivation of the pa- analysis of DNA from isolated male and female pronu- ternal X chromosome begins in the trophectoderm clei, a task that is technically daunting. However, around the 32-cell stage (Takagi et al. 1978), when wide- Southern blots of DNA from 8- and 32-cell embryos and spread differences in DNA methylation, which could ICMs revealed that approximately 50% of the MspI sites mark the parental origin of the two X chromosomes, ap- in L1 sequences were cleaved by HpaII at all stages ex- parently still exist. In contrast, X chromosome inactiva- amined. Although accurate quantitation of such blots is tion in the primitive ectoderm does not occur until after difficult, these patterns of methylation are consistent 6 days of gestation (Monk and Harper 1979), by which with differential methylation of L1 and possibly MUP time major differences in methylation between maternal sequences in male and female genomes at the time of and paternal X chromosomes may have been erased by fertilization and perpetuation of these differences into de novo methylation, resulting in random inactivation the early embryo. Minor satellite sequences were under- in the embryo proper. methylated to similar extents in oocytes, sperm, and Despite the evidence that by 7.5 days post coitum early embryos, which also suggests that there is essen- (p.c.), de novo methylation has eliminated all the differ- tially no change in gametic methylation levels during ences in DNA methylation between male- and female- early embryogenesis. Thus, we have demonstrated that derived DNAs that we have observed here, there is both major differences in methylation of endogenous se- genetic and molecular evidence that some chromosomal quences occur during male and female gametogenesis, imprinting must persist later in development. Certain and we have provided at least circumstantial evidence phenotypic effects of parental origin of specific chromo- that these differences persist throughout preimplanta- somal regions are manifest much later in development tion development. These findings are consistent with a (Bennett 1975; Cattanach and Kirk 1985), and some role for DNA methylation in genomic imprinting in transgenic sequences show differential DNA methyl- mammalian development. ation according to gamete of origin in DNA from adult By 7.5 days of development, all the sequences exam- tissues (Reik et al. 1987; Sapienza et al. 1987). This sug- ined here are highly methylated in the embryonic por- gests that subtle differences in DNA methylation may tion of the conceptus (Chapman et al. 1984; Rossant et persist after the time of extensive de novo methylation al. 1986 and unpubl.), indicating that the differences be- in the embryonic lineage and affect a limited subset of tween male- and female-derived DNA have been obliter- genes that show differential parental effects later in de- ated by de novo methylase activity. Persistence of wide- velopment. spread methylation differences between paternal and Although widespread differences in DNA methylation maternal DNAs until at least implantation would be between oocytes and sperm of the type reported here sufficient to explain some known effects of genomic im- may affect events in early embryogenesis, it seems pos- printing. For example, nuclear transfer experiments have sible that they arose primarily in response to events in shown that the difference in potential of the male and oogenesis and spermatogenesis, such as X-chromosome female genomes in the egg persists until at least the 16- reactivation in oogenesis (Kratzer and Chapman 1981) cell stage of development (Surani et al. 1986). In ex- and condensation of DNA in the sperm head in sper-

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Sanford et al. matogenesis (Wagner and Minhas 1982). Major differ- small fragments. The tubules were washed several times in ences in methylation between oocytes and sperm could RPMI-H, resuspended in RPMI-H containing trypsin {Sigma, then be perpetuated by maintenance methylation during final concentration 250 ~g/ml)and DNase (Sigma, final concen- early development, allowing them to acquire the sec- tration 5 ~g/ml), and shaken in the water bath for 20 mm. ondary function of marking the parental origin of ho- Clumps of cells that remained were dispersed by pipetting. Bo- mologous chromosomes. vine serum albumin (BSA, Sigma, final concentration 0.5% wt/ vol) was added to the cell suspension, which was then centrifuged Thus, there is increasing evidence that DNA methyl- at 1500g for 7 rain. The cell pellet was resuspended in RPMI-H ation may be involved in genomic imprinting in containing BSA and DNase, centrifuged, resuspended in 10 ml of mammals. Widespread differences occur in methylation RPMI-H containing BSA, and filtered through nylon mesh (Sar- of specific sequences during oogenesis and sperrnatogen- gent-Welch, pore size 75 ~m). Cell concentration was determined esis, and these differences probably persist through early using a hemocytometer. cleavage. It is also possible, although not yet proven for The cells in suspension were separated by gravity sedimenta- endogenous sequences, that some differences persist tion using the Staput sedimentation apparatus (Miller and much later in development. All of these properties, Phillips 1969). The cell suspension was loaded into a Staput combined with the known effects of DNA methylation SP-120 chamber, which was then loaded with 550 ml of 2-4% on gene expression, are consistent with the predicted (wt/vol) BSA gradient in RPMI-H. After 2.5 hr, 10-ml fractions were collected from the chamber at 15 ml/min. properties of any imprinting mechanism. However, all To determine the cell types present in each fraction, an ali- the evidence is circumstantial, and proof of the impor- quot was pipetted onto a microscope slide, allowed to air-dry tance of DNA methylation in genomic imprinting for a few minutes, and then fixed in three parts ethanol/one awaits the isolation and characterization of specific gene part acetic acid. The slides were stained using Giemsa (BDH) in sequences that are differentially expressed from the PBS. The cells were identified according to the criteria of Meis- male and female genomes. trich et al. (1973). The remainder of each fraction was centrifuged, and the pellet was resuspended in 1 ml of RPMI-H, transferred to an Materials and methods Eppendorf tube, and centrifuged again. The supernatant was re- Mice moved, and the pellet was frozen at -70°C until the DNA was extracted. Random bred mice of Ha/ICR (West Seneca Laboratory of Ros- well Park Memorial Institute, Buffalo, NY) or CD-1 (Charles River, St. Constant, Quebec)strains were used throughout this Morula and blastocyst collection study. Mice were 6-16 weeks of age. Females were superovulated by intraperitoneal injection of 5 IU of pregnant mare's serum gonadotropin (PMS, Sigma), 48 hr be- Oocyte and sperm collection fore administering 5 IU human chorionic gonadotropin (hCG, Sigma). Treated females were mated and 8- and 32-cell embryos Ovaries were dissected from 16- to 17-day female fetuses (day were collected at 72 and 84 hr post-hCG, respectively. Blasto- 1 = day of vaginal plug detection) and incubated in 0.02% cysts were obtained at 96 hr post-hCG. Preimplantation em- EDTA/phosphate-buffered saline (PBS) for 30 rain at room tem- bryos were flushed from the oviducts or uteri with PB-1 me- perature to chelate Ca 2+ and perturb cell junctions (DeFelici dium (Whittingham and Wales 1969)plus 10% fetal calf serum, and McLaren 1982). The enclosed oocytes were released by pelleted, resuspended in PB, and frozen. teasing the ovary with fine needles. Oocytes were collected, pelleted, resuspended in Pronase buffer [PB: 50 mM Tris, 150 mM NaC1, 100 mM EDTA (pH 10)], and frozen at -20°C. Oo- ICM collection cytes collected in this manner were 95-99% pure based on cy- Immunosurgery (Solter and Knowles 1975)was used to isolate tological analysis. ICMs. Blastocysts were exposed to acidic Tyrode's solution {pH Caudae epididymidum were dissected from adult males and 2.5) for a few seconds at room temperature to remove the zona minced in Brinster's medium (GIBCO) for 10 min at room tem- pellucida and incubated in rabbit anti-mouse serum diluted perature. Free-swimming sperm were pelleted and resuspended 1:8 in PB-1 for 45 min at 37°C. Following several rinses in in PB containing 2 mM dithiothreitol (DTT) to facilitate break- PB-1, outer trophectoderm cells were lysed by incubation in down of the condensed sperm DNA. rabbit complement (Low-tox, Cedarlane Laboratories, Homby, Ontario}, diluted 1:10 in PB-1, for 35 min at 37°C. The re- Separation of spermatogenic populations maining ICMs were rinsed in PB-1, pelleted, resuspended m PB, and frozen. Purified populations of primary spermatocytes and round spermatids were prepared using techniques described previously (Romrell et al. 1976; Gold et al. 1983). Sexually mature male DNA extractions and Southern blotting CD-1 mice were sacrificed, and their testes were removed and DNA extractions and Southem blotting were performed as de- stripped of the tunica albuginea, using forceps. The testes were scribed previously {Chapman et al. 1984). Phage k DNA was placed in medium designated RPMI-H, which is similar to me- added to restriction digests and stained with ethidium bromide dium RPMI 1640, except that amino acids and vitamins have to assure complete digestion. Some blots were also rehybridized been omitted and NaHCOa has been replaced by 24 mM HEPES. with a mitochondrial DNA probe, p501-1, which showed iden- Collagenase IA (Sigma)was added (final concentration 1 tical digestion pattems with MspI and HpaII because mito- mg/ml), and the testes were shaken at 120 cycles/min in a 34°C chondrial DNA is unmethylated {Hecht et al. 1984). DNA water bath for about 15 min until the seminiferous tubules had probes were labeled with [c~-a2P]dATP or dCTP (400 or 3000 Ci/ become separated from each other. Prolonged shaking was mmole, Amersham), using the oligolabeling method (Feinberg avoided because it caused the individual tubules to break into and Vogelstein 1983}. The resulting probes had specific activi-

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DNA methylation in gametogenesis ties of 0.2 x 109 to 2.0 x 109 cpm/~g. For rehybridizations, multi-gene family; sequence analysis of cDNA clones and filters were washed in 0.01 x SSC, 0.1 x Denhardt's at 70°C for differential regulation in the liver. Nucleic Acids Res. 4 hr. 12: 6073-6090. Lueders, K.K. and E.L. Kuff. 1980. Intracisternal A-particle Plasmid probes genes: Identification in the genome of Mus musculus and comparison of multiple isolates from a mouse gene library. Plasmids containing repeat.ed sequence inserts were isolated Proc. Natl. Acad. Sci. 77: 3571-3575. from a repetitive DNA library constructed from Mus musculus McGrath, J. and D. Solter. 1984a. Completion of mouse em- genomic DNA (Pietras et al. 1983). pMR150 and pMR134 bryogenesis requires both the maternal and paternal (plasmid mouse repetitive)represent minor satellite and dis- genomes. Cell 37:179-183. persed repeated L1 sequences (Voliva et al. 1983), respectively. 1984b. Maternal T hp lethality in the mouse is a nuclear, The MUP plasmid, p499, was kindly provided by Dr. W. Held not cytoplasmic, defect. Nature 308: 550-551. (Kuhn et al. 1984). The IAP plasmid, pMIA1, is from Dr. Meistrich, M.L., W.R. Bruce, and Y. Clermont. 1973. Cellular Lueders (Leuders and Kuff 1980). The MUP cDNA insert was composition of fractions of mouse testis ceils following ve- isolated as described (Feinberg and Vogelstein 1983). locity sedimentation separation. Exp. Cell Res. 79: 213- 227. Miller, R.G. and R.A. Phillips. 1969. Separation of cells by ve- Acknowledgments locity sedimentation. I. Cell Physiol. 73: 191-197. We thank Rudi Balling, Carmen Sapienza, Linda Mullins, Monk, M. and M.I. Harper. 1979. Sequential X-chromosome in- Dennis Stephenson, and Steve Grant for useful discussion. This activation coupled with cellular differentiation in early work was supported by grants from the National Institutes of mouse embryos. Nature 281:311-313. Health to V.M.C. and the Medical Research Council and Nat- Monk, M., M. Boubelik, and S. Lehnert. 1987. Temporal and ural Sciences and Engineering Research Council of Canada to regional changes in DNA methylation in the embryonic, ex- J.R.J.R. is a Research Associate of the National Cancer Insti- traembryonic and germ cell lineages during mouse embryo tute of Canada, and H.C. is supported by a fellowship from the development. Development 99: 371-382. Medical Research Council of Canada. Pietras, D.F., K.L. Bennett, L.D. Siracusa, M. Woodworth-Gutai, K.W. Gross, C. Kane-Haas, and N.D. Hastie. 1983. Con- struction of a small Mus musculus repetitive DNA library: References Identification of a new satellite sequence in Mus musculus. Nucleic Acids Res. 11: 6966-6983. Bennett, D. 1975. The T-locus of the mouse. Cell 6: 441-454. Piko, L., M.D. Hammons, and K.D. Taylor. 1984. Amounts, Cattanach, B.M. and M. Kirk. 1985. 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Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse.

J P Sanford, H J Clark, V M Chapman, et al.

Genes Dev. 1987, 1: Access the most recent version at doi:10.1101/gad.1.10.1039

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