Observations on the Movements of the Living Primordial Germ Cells in the Mouse

Blandau, Richard J.; White, Beverly J.; Rumery, Ruth E.

doi:10.1016/s0015-0282(16)34981-0

Cited by 97 publications

(27 citation statements)

References 4 publications

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“…From each litter, a few ovaries were set aside for cytological examination and the remaining ovaries were pooled on a siliconized microscope slide. Under gentle pressure between two siliconized microscope slides, germ cells are preferentially released (28). The individual cells were sucked up in a drawn Pasteur pipette and transferred to a 0.4-ml Microfuge tube (Brinkman).…”

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confidence: 99%

X chromosome reactivation in oocytes of Mus caroli.

Kratzer¹,

Chapman²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Mature mammalian oocytes have both of their X chromosomes active, while somatic cells from the same individual have one of their X chromosomes in an inactive state. We asked whether the X chromosomes of the germ cells never undergo inactivation in their ontogeny or whether inactivation of an X chromosome does occur but is followed by a subsequent reactivation event. Our approach has used an electrophoretic polymorphism for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in the mouse species Mus caroli. G6PD is dimeric, and a heterodimer is produced'in cells from heterozygous females ifand only ifboth X chromosomes are active. Ovaries from heterozygous fetuses at different gestational ages were dissected and either studied cytologically or pressed between microscope slides to obtain germ cell-rich and germ cell-poor preparations. No heterodimer band was detected on the 10th day of development in germ cellrich preparations. On subsequent days, an increasingly intense heterodimer band was detected, which, by the 13th day, was approximately twice as intense as the corresponding homodimer bands. Consideration of (i) the G6PD activity per germ cell and per somatic cell and (ii) the percentage of germ cells in the germ cell-rich preparations indicated that a heterodimer band should have been visible on the 10th day had both X chromosomes been active. Cytological examinations showed that the earliest germ cells enter meiotic prophase on the eleventh day. These results demonstrate that oogonia have a single active X chromosome and that the inactive X chromosome is reactivated at or, more likely, shortly before entry into meiotic prophase.The Lyon hypothesis (1), which states that only one X chromosome is active in the somatic cells offemale mammals, is well supported by experimental data (2). Both X chromosomes are apparently active in the preimplantation embryo (3-6), and random inactivation of one X chromosome occurs in the fetal tissues soon after implantation (7,8). The inactive state of the X chromosome is then stably inherited in somatic cells (9-12). The state ofX-chromosome activity in the germ line is not yet clear. Based on gene product studies for several X-linked genes (13-16), mature oocytes have two active X chromosomes, but this could result either from failure of inactivation in the germ line or from reactivation ofa previously inactive X chromosome.The question of germ line X chromosome activity has been investigated previously. Cytologically, Ohno (17) reported the presence ofa chromatin body in migrating primordial germ cells of the mouse but not in oogonia in the ovaries of the rabbit and human (18,19). Gartler et al. (20) has recently observed a heterochromatic chromosome in oogonial mitoses in the mouse. Biochemically, Gartler et al. (21) presented evidence that both X chromosomes are active in germ cells from human ovaries at 13 weeks, but not at 12 weeks, of gestation. Using the same approach, however, Migeon and Jelalian (22) reported that both X chromosomes were active at...

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mentioning

confidence: 99%

X chromosome reactivation in oocytes of Mus caroli.

Kratzer¹,

Chapman²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…The ovaries were collected in phosphate-buffered saline (PBS) solution and processed within 24 h of termination. Oocytes were isolated by a squash technique developed by Blandau et al (1963). The cells were fixed and slides were made according to previously published methods (Cheng and Gartler 1994).…”

Section: Methodsmentioning

confidence: 99%

Analysis of a paracentric inversion in human oocytes: nonhomologous pairing in pachytene

1999

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A cytological analysis of the pairing configurations in meiosis in a 19-week human fetus with a de novo paracentric inversion of chromosome 7 (q11.23)(q21.1) is reported, using fluorescent in situ hybridization with a chromosome 7 DNA library, a DNA probe for the centromeric region of chromosome 7, and a probe for the William Syndrome Critical Region (WSCR) at 7q11.23. Of 1079 pachytene cells, 58% exhibited complete heterosynapsis of the inverted region while only 10.3% of cells exhibited the expected loop formation. Meiotic progression was observed to be normal.

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“…The cells released from the ovary, which were preferentially (but not exclusively) germ cells, were picked up with a finely drawn Pasteur pipette (25).…”

Section: Methodsmentioning

confidence: 99%

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Singer-Sam

Goldstein

Dai

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGKI). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.Female primordial germ cells of mammals resemble somatic cells in having both an active and an inactive X chromosome. Evidence for inactivity of one X chromosome of oogonia has been found both cytologically and by studies on glucose-6-phosphate dehydrogenase heterodimer formation (1, 2). After migration of the oogonia to the genital ridge at, or shortly before, the time of entry into meiosis, reactivation of the previously inactive X chromosome occurs (1-6). DNA methylation has been correlated with X chromosome inactivation in many studies (reviewed in refs. 7-9) and it is of interest to determine whether a loss of methylation is concomitant with X chromosome reactivation during oogenesis. Data on DNA methylation of oogenic cells are sparse. Several years ago evidence was obtained for undermethylation of some repeated sequences in female germ-line DNA (10, 11), and more recently, Driscoll and Migeon (12) obtained evidence for lack of methylation of several X-linked and autosomal genes in oogenic and spermatogenic cell fractions.As the latter study was done on tissue fractions rather than preparations free of somatic cells, we thought it was important to measure DNA methylation of highly purified germ cells. As such cells are isc. table only in small quantities, we used a polymerase chain raction (PCR)-based assay previously used to measure DNA methylation at a CCGG site in individual mouse embryos (13).The site we chose to assay was Hpa II site H8 in the 5' untranslated coding region of the human X-linked PGKI gene, 20 base pairs (bp) downstream of the major transcription start site (14). The 5' region of the PGKI gene is now the most highly characterized X-linked promoter region, and the relationship between methylation, transcription, and transcription factors has been studied both in normal human lymphocytes and in cultured cell lines (15)(16)(17)(18)(19). Recently, ligation-mediated genomic sequencing has been used to establish that the promoter region of the PGKI gene on the inactive X chromosome is methylated at 60 of 61 CpG sites, whereas the active promoter has no methylated sites (19). Prior to these studies, methylation-sensitive restriction enzymes had been used to investigate methylation at the 8 Hpa II sites in the PGKI 5' region in lymphocytes (15) and in cell lines before and after...

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Observations on the Movements of the Living Primordial Germ Cells in the Mouse

Cited by 97 publications

References 4 publications

X chromosome reactivation in oocytes of Mus caroli.

X chromosome reactivation in oocytes of Mus caroli.

Analysis of a paracentric inversion in human oocytes: nonhomologous pairing in pachytene

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

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