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Upregulation of the BRCA1 gene in human germ cells and in preimplantation embryos

2005; Elsevier BV; Volume: 84; Issue: 3 Linguagem: Inglês

10.1016/j.fertnstert.2005.02.037

1556-5653

Sandrine Giscard d’Estaing, Delphine Perrin, Gilbert Lenoir, Jean François Guérin, Robert Dante,

BRCA gene mutations in cancer

The quantification of BRCA1 messenger RNA molecules by a quantitative competitive one-step reverse transcriptase polymerase chain reaction method indicates that BRCA1 is upregulated both in human male and female germ cells and in preimplantation embryos. Because BRCA1 is involved in several pathways that participate in preserving intact chromosome and genome integrity, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility. The quantification of BRCA1 messenger RNA molecules by a quantitative competitive one-step reverse transcriptase polymerase chain reaction method indicates that BRCA1 is upregulated both in human male and female germ cells and in preimplantation embryos. Because BRCA1 is involved in several pathways that participate in preserving intact chromosome and genome integrity, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility. A direct involvement of BRCA1 germ line mutations in breast and ovarian cancers is now well established, and several functions have been associated with BRCA1 proteins, including induction of apoptosis, DNA repair, and transactivation activity (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar).Among these potential functions, it seems that BRCA1 might play an important role in spermatogenesis and embryogenesis (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar).Whether BRCA1 participates in the initiation of X inactivation in the embryo is still unknown. However, it has been suggested that BRCA1 dysfunction increases the risk of failure of the maintenance of X chromosome inactivation (2Ganesan S. Silver D.P. Greenberg R.A. Avni D. Drapkin R. Miron A. et al.BRCA1 supports XIST RNA concentration on the inactive X chromosome.Cell. 2002; 111: 393-405Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Nevertheless, BRCA1 is involved in several fundamental processes, including DNA repair and homologous recombination, suggesting that loss of BRCA1 function might affect early embryogenesis and, therefore, has an indirect effect on Xi structure and silencing (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar).Taken together, these data indicate that BRCA1 might be a key molecule in maintaining genome integrity during gametogenesis and early embryogenesis.Because BRCA1 expression in human oocytes and blastocysts has never been investigated, these observations prompted the question of whether BRCA1 is highly expressed at these early stages and whether maternal transcripts are already present in oocytes.We therefore investigated the level of BRCA1 gene expression in human germ cells and in blastocysts.Granulosa cells were isolated from follicular medium after oocytes pick-up in an IVF program. Cells were stratified on a Ficoll gradient and centrifuged and rinsed in phosphate-buffered saline 1×.Human testicular biopsies were obtained from patients undergoing an IVF program in cases of obstructive azoospermia (Laboratory of Reproduction Biology, Hôpital E. Herriot, Lyon, France). The preparation's purity was microscopically verified. Mainly immature germ cells as primary spermatocytes and spermatids were retained after selection of spermatogenesis cell lines on Percoll gradient (3Gandini L. Lenzi A. Lombardo F. Pacifici R. Dondero F. Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen.Hum Reprod. 1999; 14: 1022-1027Crossref PubMed Scopus (41) Google Scholar).Immature oocytes (metaphase I) from the intracytoplasmic sperm injection program or metaphase II oocytes that failed to fertilize 24 hours after conventional IVF were collected from the IVF laboratory. Follicular cells linked to the zonae pellucidae were removed by pipetting and enzymatic methods (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar).Human blastocysts were collected from the IVF laboratory. Those embryos were discarded (i.e., neither transferred nor frozen) because of poor morphologic quality. They were treated with the same method described above for the oocytes. Both germ cells and embryos were obtained with the couples' consent. The research program was approved by the Commission Nationale de Médecine et Biologie de la Reproduction (Ministère de l'Emploi et de la Solidarité).Ribonucleic acid was extracted from granulosa cells and sperm cells with the RNeasy kit (Qiagen, Courtaboeuf, France) and quantitated by optical density and by densitometry. BRCA1 transcripts in granulosa cells and in immature male germs cells were determined by a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay, as previously described (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar); that is, equal amounts (4 ng) of total RNA samples were co-amplified with increasing amounts of competitor RNAs in a final volume of 100 μL.Owing to the low quantity of oocytes and embryos, RT-PCR assays were performed directly from the samples, after lysis for 5 minutes at 80°C in buffer containing 5 mmol/L dithiothreitol (Roche Molecular Biochemicals, Meylan, France), 0.8 % NP40, and 1 U RNAsin (Roche Molecular Biochemicals). Oocytes RNA (six oocytes per point) was co-amplified with increasing amounts of RNA competitor (5 × 103, 10 × 103, and 50 × 103 copies of competitor) and blastocyst RNA (two blastocysts per point) with increasing amounts of RNA competitor (103, 5 × 103, and 10 × 103 copies of competitor). A representative assay is shown in Figure 1A.Because, in this assay, the amount of used RNA in the RT-PCR assay cannot be quantified, we evaluated the amount of RNA extracted by this lysis method. Pool oocytes (four to six per tube) and blastocysts (two to three per tube) were lysed in the buffer described above, in a final volume of 11.5 μL, and then, after deoxyribonuclease digestion, mixed with an equal volume of a 200-fold diluted solution of RiboGreen (Molecular Probes, Interchim, Montluçon, France), an ultrasensitive fluorescent nucleic acid stain, and quantified by fluorometry with the Fluor fluorometer (Biorad, Ivry, France) and Quantity One software (Biorad, Ivry, France) in comparison with serial dilutions of a standard ribosomal RNA (Roche Molecular Biochemicals).Using this method, we found 0.51 ± 0.02 ng of total RNA per oocyte and 1.8 ± 0.5 ng of total released RNA per blastocyst (number of independent determinations: 3).Each RT-PCR determination was deduced from the values obtained from three points, and at least two other determinations were done with a wider range of competitor dilution. Data obtained from hundreds of samples analyzed to date indicate that the variations for each category of cells were less than 15%.The amount of BRCA1 messenger RNA (mRNA) in immature sperm cells (11 × 106 copies per microgram of total RNA; Fig. 1B) was found to be 20–50 times higher than the amount in granulosa cells (0.17 × 106 copies per microgram of RNA; Fig. 1B). This difference of expression is also comparable to the one observed in the case of other different somatic cells, such as fetal, adult mammary gland, or epithelial cells in previously described studies (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar, 6Magdinier F. Dalla Venezia N. Lenoir G.M. Frappart L. Dante R. BRCA1 expression during prenatal development of the human mammary gland.Oncogene. 1999; 18: 4039-4043Crossref PubMed Scopus (22) Google Scholar).Because the number of germ cells and blastocysts available for these analyses was very limited, we have scaled down the method to very small amounts of cells. The RT-PCR assays were performed directly from lysed oocytes or blastocysts.The total amount of BRCA1 transcripts was calculated to be 4 × 106 copies per microgram of RNA in oocytes and 0.7 × 106 copies per microgram of RNA in blastocysts (Fig. 1B). Although in blastocysts the BRCA1 RNA copy number is lower than in sperm germ cells or oocytes, the expression of BRCA1 remains very high in the blastocysts, because all the human embryonic or adult somatic tissues analyzed to date exhibit a lower level of BRCA1 expression (0.5 × 106 to 0.1 × 106 copies per microgram of RNA) (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar, 6Magdinier F. Dalla Venezia N. Lenoir G.M. Frappart L. Dante R. BRCA1 expression during prenatal development of the human mammary gland.Oncogene. 1999; 18: 4039-4043Crossref PubMed Scopus (22) Google Scholar) (unpublished data).We chose a quantitative RT-PCR method because such assays measure the absolute amount of mRNA in an RNA sample; furthermore, this quantification is not related to the amount of a control mRNA, which might vary between samples or cellular types. Thus, data obtained from different cell lines or samples can be directly compared.In mice, combinations of semiquantitative RT-PCR and Northern blotting have clearly indicated that BRCA1 is highly expressed in meiotic male germ cells and postmeiotic spermatids (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar, 7Blackshear P.E. Goldsworthy S.M. Foley J.F. McAllister K.A. Bennett L.M. Collins N.K. et al.Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.Oncogene. 1998; 16: 61-68Crossref PubMed Scopus (78) Google Scholar). This high level of expression has been also observed in mouse female germ cells with an immunohistochemical approach (7Blackshear P.E. Goldsworthy S.M. Foley J.F. McAllister K.A. Bennett L.M. Collins N.K. et al.Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.Oncogene. 1998; 16: 61-68Crossref PubMed Scopus (78) Google Scholar).In human oocytes and blastocysts, this study found increased levels of transcripts, indicating that the number of BRCA1 mRNA molecules was high when compared with the level of transcripts in somatic cells. Although variations from embryo to embryo have been observed for several genes (8Monk M. Holding C. Goto T. Isolation of novel developmental genes from human germ cell, oocyte and embryo cDNA by differential display.Reprod Fertil Dev. 2001; 13: 51-57Crossref PubMed Scopus (13) Google Scholar), quantification of the BRCA1 transcripts from pooled human oocytes or embryos gave reproducible results, suggesting that these variations were compensated by the average values obtained with this method.Taken together, the data obtained indicate that BRCA1 are highly expressed in germ cells and blastocysts.In human somatic tissues, BRCA1 expression is highly regulated, only minor variations are observed in a given tissue (approximately ±25% in a panel of normal breast tissues), and alterations of this level of expression are associated with major phenotype changes because this gene is down-regulated in breast cancers (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar).Therefore, the data obtained suggest that upregulation of BRCA1 is a necessary event for human gametogenesis and early embryogenesis. In addition, the high level of BRCA1 transcripts in oocytes suggests a high level of the maternal allele at zygote formation. The presence of a high level of maternal transcripts at these stages might have some consequences in BRCA1 mutation carriers because loss of BRCA1 function is associated with alterations of several pathways that participate in preserving intact chromosome and genome integrity.In accordance with these hypotheses, an excess of female offspring has been found in mutated BRCA1 carrier families among the offspring of female carriers (9Feunteun J. Chompret A. Helbling-Leclerc A. Stoppa-Lyonnet D. Belotti M. Nogues C. et al.Sex ratio among the offspring of BRCA mutation carriers.JAMA. 2004; 292: 687-688Crossref PubMed Scopus (8) Google Scholar). Although it has been suggested that women with breast cancer having female offspring might have a greater motivation to undergo genetic testing, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility. In addition, an elevated incidence of nonrandom X chromosome inactivation has been reported in women with ovarian cancers in BRCA1 mutation carrier families (10Buller R.E. Sood A.K. Lallas T. Buekers T. Skilling J.S. Association between nonrandom X-chromosome inactivation and BRCA1 mutation in germline DNA of patients with ovarian cancer.J Natl Cancer Inst. 1999; 91: 339-346Crossref PubMed Scopus (92) Google Scholar).Whether BRCA1 participates in such pathways in the human embryo is yet unknown. These findings are also consistent with (although they do not prove) the hypothesis that BRCA1 dysfunction might have some consequences in human fertility and embryogenesis.The molecular mechanism involved in the upregulation of BRCA1 in germ cells and in preimplantation embryos remains to be determined, because it has been recently shown with a complementary DNA micro-array approach that upregulation and downregulation occur in early human embryogenesis (11Dobson A.T. Raja R. Abeyta M.J. Taylor T. Shen S. Haqq C. et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet. 2004; 13: 1461-1470Crossref PubMed Scopus (199) Google Scholar). It should be noted, however, that the 5′ end of BRCA1 is embedded in a large CpG island, which is regionally methylated in somatic cells and unmethylated in germ cells and preimplantation embryos (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar), and it has been shown that, in somatic cells, the methylation of this region is associated with a reduced expression of BRCA1 (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar). A direct involvement of BRCA1 germ line mutations in breast and ovarian cancers is now well established, and several functions have been associated with BRCA1 proteins, including induction of apoptosis, DNA repair, and transactivation activity (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar). Among these potential functions, it seems that BRCA1 might play an important role in spermatogenesis and embryogenesis (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar). Whether BRCA1 participates in the initiation of X inactivation in the embryo is still unknown. However, it has been suggested that BRCA1 dysfunction increases the risk of failure of the maintenance of X chromosome inactivation (2Ganesan S. Silver D.P. Greenberg R.A. Avni D. Drapkin R. Miron A. et al.BRCA1 supports XIST RNA concentration on the inactive X chromosome.Cell. 2002; 111: 393-405Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Nevertheless, BRCA1 is involved in several fundamental processes, including DNA repair and homologous recombination, suggesting that loss of BRCA1 function might affect early embryogenesis and, therefore, has an indirect effect on Xi structure and silencing (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar). Taken together, these data indicate that BRCA1 might be a key molecule in maintaining genome integrity during gametogenesis and early embryogenesis. Because BRCA1 expression in human oocytes and blastocysts has never been investigated, these observations prompted the question of whether BRCA1 is highly expressed at these early stages and whether maternal transcripts are already present in oocytes. We therefore investigated the level of BRCA1 gene expression in human germ cells and in blastocysts. Granulosa cells were isolated from follicular medium after oocytes pick-up in an IVF program. Cells were stratified on a Ficoll gradient and centrifuged and rinsed in phosphate-buffered saline 1×. Human testicular biopsies were obtained from patients undergoing an IVF program in cases of obstructive azoospermia (Laboratory of Reproduction Biology, Hôpital E. Herriot, Lyon, France). The preparation's purity was microscopically verified. Mainly immature germ cells as primary spermatocytes and spermatids were retained after selection of spermatogenesis cell lines on Percoll gradient (3Gandini L. Lenzi A. Lombardo F. Pacifici R. Dondero F. Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen.Hum Reprod. 1999; 14: 1022-1027Crossref PubMed Scopus (41) Google Scholar). Immature oocytes (metaphase I) from the intracytoplasmic sperm injection program or metaphase II oocytes that failed to fertilize 24 hours after conventional IVF were collected from the IVF laboratory. Follicular cells linked to the zonae pellucidae were removed by pipetting and enzymatic methods (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar). Human blastocysts were collected from the IVF laboratory. Those embryos were discarded (i.e., neither transferred nor frozen) because of poor morphologic quality. They were treated with the same method described above for the oocytes. Both germ cells and embryos were obtained with the couples' consent. The research program was approved by the Commission Nationale de Médecine et Biologie de la Reproduction (Ministère de l'Emploi et de la Solidarité). Ribonucleic acid was extracted from granulosa cells and sperm cells with the RNeasy kit (Qiagen, Courtaboeuf, France) and quantitated by optical density and by densitometry. BRCA1 transcripts in granulosa cells and in immature male germs cells were determined by a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay, as previously described (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar); that is, equal amounts (4 ng) of total RNA samples were co-amplified with increasing amounts of competitor RNAs in a final volume of 100 μL. Owing to the low quantity of oocytes and embryos, RT-PCR assays were performed directly from the samples, after lysis for 5 minutes at 80°C in buffer containing 5 mmol/L dithiothreitol (Roche Molecular Biochemicals, Meylan, France), 0.8 % NP40, and 1 U RNAsin (Roche Molecular Biochemicals). Oocytes RNA (six oocytes per point) was co-amplified with increasing amounts of RNA competitor (5 × 103, 10 × 103, and 50 × 103 copies of competitor) and blastocyst RNA (two blastocysts per point) with increasing amounts of RNA competitor (103, 5 × 103, and 10 × 103 copies of competitor). A representative assay is shown in Figure 1A. Because, in this assay, the amount of used RNA in the RT-PCR assay cannot be quantified, we evaluated the amount of RNA extracted by this lysis method. Pool oocytes (four to six per tube) and blastocysts (two to three per tube) were lysed in the buffer described above, in a final volume of 11.5 μL, and then, after deoxyribonuclease digestion, mixed with an equal volume of a 200-fold diluted solution of RiboGreen (Molecular Probes, Interchim, Montluçon, France), an ultrasensitive fluorescent nucleic acid stain, and quantified by fluorometry with the Fluor fluorometer (Biorad, Ivry, France) and Quantity One software (Biorad, Ivry, France) in comparison with serial dilutions of a standard ribosomal RNA (Roche Molecular Biochemicals). Using this method, we found 0.51 ± 0.02 ng of total RNA per oocyte and 1.8 ± 0.5 ng of total released RNA per blastocyst (number of independent determinations: 3). Each RT-PCR determination was deduced from the values obtained from three points, and at least two other determinations were done with a wider range of competitor dilution. Data obtained from hundreds of samples analyzed to date indicate that the variations for each category of cells were less than 15%. The amount of BRCA1 messenger RNA (mRNA) in immature sperm cells (11 × 106 copies per microgram of total RNA; Fig. 1B) was found to be 20–50 times higher than the amount in granulosa cells (0.17 × 106 copies per microgram of RNA; Fig. 1B). This difference of expression is also comparable to the one observed in the case of other different somatic cells, such as fetal, adult mammary gland, or epithelial cells in previously described studies (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar, 6Magdinier F. Dalla Venezia N. Lenoir G.M. Frappart L. Dante R. BRCA1 expression during prenatal development of the human mammary gland.Oncogene. 1999; 18: 4039-4043Crossref PubMed Scopus (22) Google Scholar). Because the number of germ cells and blastocysts available for these analyses was very limited, we have scaled down the method to very small amounts of cells. The RT-PCR assays were performed directly from lysed oocytes or blastocysts. The total amount of BRCA1 transcripts was calculated to be 4 × 106 copies per microgram of RNA in oocytes and 0.7 × 106 copies per microgram of RNA in blastocysts (Fig. 1B). Although in blastocysts the BRCA1 RNA copy number is lower than in sperm germ cells or oocytes, the expression of BRCA1 remains very high in the blastocysts, because all the human embryonic or adult somatic tissues analyzed to date exhibit a lower level of BRCA1 expression (0.5 × 106 to 0.1 × 106 copies per microgram of RNA) (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar, 6Magdinier F. Dalla Venezia N. Lenoir G.M. Frappart L. Dante R. BRCA1 expression during prenatal development of the human mammary gland.Oncogene. 1999; 18: 4039-4043Crossref PubMed Scopus (22) Google Scholar) (unpublished data). We chose a quantitative RT-PCR method because such assays measure the absolute amount of mRNA in an RNA sample; furthermore, this quantification is not related to the amount of a control mRNA, which might vary between samples or cellular types. Thus, data obtained from different cell lines or samples can be directly compared. In mice, combinations of semiquantitative RT-PCR and Northern blotting have clearly indicated that BRCA1 is highly expressed in meiotic male germ cells and postmeiotic spermatids (1Scully R. Livingston D.M. In search of the tumour-suppressor functions of BRCA1 and BRCA2.Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar, 7Blackshear P.E. Goldsworthy S.M. Foley J.F. McAllister K.A. Bennett L.M. Collins N.K. et al.Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.Oncogene. 1998; 16: 61-68Crossref PubMed Scopus (78) Google Scholar). This high level of expression has been also observed in mouse female germ cells with an immunohistochemical approach (7Blackshear P.E. Goldsworthy S.M. Foley J.F. McAllister K.A. Bennett L.M. Collins N.K. et al.Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.Oncogene. 1998; 16: 61-68Crossref PubMed Scopus (78) Google Scholar). In human oocytes and blastocysts, this study found increased levels of transcripts, indicating that the number of BRCA1 mRNA molecules was high when compared with the level of transcripts in somatic cells. Although variations from embryo to embryo have been observed for several genes (8Monk M. Holding C. Goto T. Isolation of novel developmental genes from human germ cell, oocyte and embryo cDNA by differential display.Reprod Fertil Dev. 2001; 13: 51-57Crossref PubMed Scopus (13) Google Scholar), quantification of the BRCA1 transcripts from pooled human oocytes or embryos gave reproducible results, suggesting that these variations were compensated by the average values obtained with this method. Taken together, the data obtained indicate that BRCA1 are highly expressed in germ cells and blastocysts. In human somatic tissues, BRCA1 expression is highly regulated, only minor variations are observed in a given tissue (approximately ±25% in a panel of normal breast tissues), and alterations of this level of expression are associated with major phenotype changes because this gene is down-regulated in breast cancers (5Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Down-regulation of BRCA1 in human sporadic breast cancer analysis of DNA methylation patterns of the putative promoter region.Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar). Therefore, the data obtained suggest that upregulation of BRCA1 is a necessary event for human gametogenesis and early embryogenesis. In addition, the high level of BRCA1 transcripts in oocytes suggests a high level of the maternal allele at zygote formation. The presence of a high level of maternal transcripts at these stages might have some consequences in BRCA1 mutation carriers because loss of BRCA1 function is associated with alterations of several pathways that participate in preserving intact chromosome and genome integrity. In accordance with these hypotheses, an excess of female offspring has been found in mutated BRCA1 carrier families among the offspring of female carriers (9Feunteun J. Chompret A. Helbling-Leclerc A. Stoppa-Lyonnet D. Belotti M. Nogues C. et al.Sex ratio among the offspring of BRCA mutation carriers.JAMA. 2004; 292: 687-688Crossref PubMed Scopus (8) Google Scholar). Although it has been suggested that women with breast cancer having female offspring might have a greater motivation to undergo genetic testing, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility. In addition, an elevated incidence of nonrandom X chromosome inactivation has been reported in women with ovarian cancers in BRCA1 mutation carrier families (10Buller R.E. Sood A.K. Lallas T. Buekers T. Skilling J.S. Association between nonrandom X-chromosome inactivation and BRCA1 mutation in germline DNA of patients with ovarian cancer.J Natl Cancer Inst. 1999; 91: 339-346Crossref PubMed Scopus (92) Google Scholar). Whether BRCA1 participates in such pathways in the human embryo is yet unknown. These findings are also consistent with (although they do not prove) the hypothesis that BRCA1 dysfunction might have some consequences in human fertility and embryogenesis. The molecular mechanism involved in the upregulation of BRCA1 in germ cells and in preimplantation embryos remains to be determined, because it has been recently shown with a complementary DNA micro-array approach that upregulation and downregulation occur in early human embryogenesis (11Dobson A.T. Raja R. Abeyta M.J. Taylor T. Shen S. Haqq C. et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet. 2004; 13: 1461-1470Crossref PubMed Scopus (199) Google Scholar). It should be noted, however, that the 5′ end of BRCA1 is embedded in a large CpG island, which is regionally methylated in somatic cells and unmethylated in germ cells and preimplantation embryos (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar), and it has been shown that, in somatic cells, the methylation of this region is associated with a reduced expression of BRCA1 (4Magdinier F. Billard L.M. Wittmann G. Frappart L. Benchaib M. Lenoir G.M. et al.Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.FASEB J. 2000; 14: 1585-1594Crossref PubMed Google Scholar).