- Email: [email protected]
Female meiosis and beyond: more questions than answers?
Reproductive BioMedicine Online (2012) 24, 589– 590
www.sciencedirect.com www.rbmonline.com
EDITORIAL
Female meiosis and beyond: more questions than answers? Three years ago, there was some excitement about a publication reporting that female germline stem cells are present in the adult mouse ovary, can be isolated and propagated in vitro, and can generate oocytes, embryos and healthy live pups after transplantation into recipient, infertile females (Zou et al., 2009). More recently, it was reported that haploid cells could consistently be derived from human induced pluripotent stem cells of non-ovarian male- or female-derived tissues (Eguizabal et al., 2011). Therefore, it does not come as such a surprise to read a paper from Professor Johnathan Tilly’s group in Harvard reporting that the induction of meiosis in stem cells derived from human adult ovary has been achieved (White et al., 2012). Embryonic stem cells have also been obtained from cultured human ovarian surface epithelium (Parte et al., 2011), but meticulous analysis of the survival of stem cells/oogonia in the human ovary suggests that oogonia die within the first two years after birth, bringing into question a physiological role for germline-like or oogonial stem cells in neo-oogenesis in humans (Johnson et al., 2004; Byskov et al., 2011). However, the presence of the rare cells, termed ‘oogonial stem cells (OSC)’ by the Tilly group, might be difficult to identify in fixed ovaries. The Tilly group was able to enrich and study gene expression in GFP-expressing human oogonial stem cells after modifying the isolation and cultivation procedure originally described by Zou et al. (2009). Others, however, have failed so far to repeat this approach. They also report that these stem cells formed GFP-labelled oocyte-like cells within follicle-like structures after their transplantation into mice (White et al., 2012). I must confess that I remain sceptical about the significance of the generation of haploid germ-cell-like cells from mouse or human adult ovarian tissue for fertility preservation in humans (White et al., 2012; Zou et al., 2009). I am sceptical not only because of the controversies about the possible physiological role of OSC, but also because of the well-known species-specific differences in germ cell formation in the human female compared with the rodent (e.g. relative duration of oogenesis, period and extent of growth, and genomic imprinting to achieve full developmental com-
petence). In addition, there are difficulties in maintaining, and certainly in entirely reconstituting, folliculogenesis, oocyte growth and in-vivo and in-vitro maturation in the human. Regrettably, many questions remain about the efficiency of the new protocols and the perspectives for neo-oogenesis in humans, and so it is premature to raise hopes in the public, and particularly in desperate sub- or infertile women, that regaining or preserving fertility will be possible using such approaches. Thus, haploid status as reported by White et al. (2012) is characteristic of germ cells in males but not in females, in which the oocytes undergo a reductional separation at first meiosis prior to ovulation, thus generating a first polar body with a complete set of dyads (chromosomes consisting of two sister chromatids). Healthy, normal, and competent oocytes arrest at metaphase II such that second meiosis is completed only after fertilization by sperm (or after spontaneous or artificial parthenogenetic activation), and only then is a haploid second polar body formed. The oocyte is thus never ‘haploid’ in the true sense of the word, as it contains a diploid set of maternal chromosomes prior to fertilization and a haploid set of maternal chromosomes plus the haploid genome from the sperm (in the female and male pronuclei respectively) in the one-cell zygote. The haploid second polar body should not dislodge from the oocyte as it is contained within the thick zona pellucida. Moreover, Hu ¨bner et al. (2003) reported many years ago that it is possible to generate oocyte-like cells and blastocysts from mouse embryonic stem cells in vitro. These oocytes failed to arrest at metaphase II and underwent spontaneous parthenogenetic activation (Hu ¨bner et al., 2003). Interestingly, it was more efficient to induce meiosis and oocyte-like cells from cultures of male embryonic stem cells. Importantly, spontaneous activation, or parthenogenesis, can be the basis of pathologies underlying recurrent inability to maintain a normal pregnancy following assisted reproduction treatment (Combelles et al., 2011). It remains open to question whether ovarian tissues used in the recently published report by White et al. (2012), that may
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.04.007
590 have been derived from women treated with steroids before tissue donation, should be considered ‘normal’ (LovellBadge, 2012), but might promote ‘male’ rather than ‘female’ meiosis, or undergo spontaneous parthenogenetic activation. Thus, the recent report does not provide convincing evidence for a gender-specific oogenetic pattern of recombination, not even for the characteristic presence of meiotic cells with thread-like assemblies of proteins of the synaptonemal complex for efficient pairing and recombination, and completion of normal meiotic progression to metaphase II, including establishment of an efficient meiotic arrest. Last but not least, there is no information on the epigenetic status of the oocyte-like cells, or of embryos derived from germline-like stem cells from adult ovary in the mouse. Epigenetic ‘mutations’ can induce implantation failures, congenital abnormalities, foetal death and, notably, epigenetic diseases like Angelman syndrome, Beckwith Wiedeman syndrome, Silver-Russel syndrome or predisposition to cardiovascular disease and childhood cancers (recently discussed by Tomizawa and Sasaki, 2012; van Montfoort et al., 2012). Erasure of genomic imprints is a prerequisite to gender-specific genomic imprinting during gametogenetis (Hackett et al., 2012; van Montfoort et al., 2012). Unlike in spermatogenesis, genomic imprinting occurs during the oocyte growth phase in the female (Lucifero et al., 2004), and there is evidence that even those immature human oocytes that undergo growth in vivo in stimulated cycles, but do not complete maturation to metaphase II, harbour imprint defects after in-vitro maturation and are susceptible to epigenetic and chromosomal instability (Borghol et al., 2006; Magli et al., 2006). Therefore, any claims that viable, healthy oocytes can be obtained from stem cells from adult mouse, and particularly from human ovarian tissue, must provide convincing evidence that recombination occurs and that imprint patterns before and after oocyte growth are normal. Furthermore, oocytes derived from the manipulations should be capable of remodelling the male chromatin and of protecting the genomic imprints from male and female germ cells from an untimely erasure after fertilization. The true benefit of the new approach to isolation and propagation of post-natal ovarian cells with a potential to form a functional follicular unit that can develop towards a mature oocyte lies in the opportunities to study these processes and their regulation in vitro. However, before there is proof that imprinting and bi-directional signalling within newly formed follicles are normal, and that the approach will yield high-quality oocytes with reasonable efficiency, it is prudent to be sceptical about the clinical relevance of the findings. This is not to say that one should not retain an open and enthusiastic mind towards challenges to dogmas, innovations in techniques that are promising in the treatment of sub-fertile patients, and particularly, novel developments in stem cell biology that provide models and information on the molecular bases of germ cell development, fertility and health of offspring. Just do not overhype prematurely!
Editorial
References Borghol, N., Lornage, J., Blache `re, T., Sophie, Garret.A., Lefe `vre, A., 2006. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 87, 417–426. Byskov, A.G., Høyer, P.E., Yding, Andersen.C., Kristensen, S.G., Jespersen, A., Møllga ˚rd, K., 2011. No evidence for the presence of oogonia in the human ovary after their final clearance during the first two years of life. Hum. Reprod. 26, 2129–2139. Combelles, C.M., Kearns, W.G., Fox, J.H., Racowsky, C., 2011. Cellular and genetic analysis of oocytes and embryos in a human case of spontaneous oocyte activation. Hum. Reprod. 26, 545– 552. Hackett, J.A., Zylicz, J.J., Surani, M.A., 2012. Parallel mechanisms of epigenetic reprogramming in the germline. Trends Genet. (Epub ahead of print). Hu ¨bner, K., Fuhrmann, G., Christenson, L.K., Kehler, J., Reinbold, R., De La Fuente, R., Wood, J., Strauss 3rd, J.F., Boiani, M., Scho ¨ler, H.R., 2003. Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256. Johnson, J., Canning, J., Kaneko, T., Pru, J.K., Tilly, J.L., 2004. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145–150. Lovell-Badge, R., 2012. Hype, hope and heresy – or why it is bad to eggsaggerate. BioNews 648. Lucifero, D., Mann, M.R.W., Bartolomei, M.S., Trasler, J.M., 2004. Gene-specific timing and epigenetic memory in oocyte imprinting. Hum. Mol. Genet. 13, 839–849. Magli, M.C., Ferraretti, A.P., Crippa, A., Lappi, M., Feliciani, E., Gianaroli, L., 2006. First meiosis errors in immature oocytes generated by stimulated cycles. Fertil. Steril. 86, 629–635. Parte, S., Bhartiya, D., Telang, J., Daithankar, V., Salvi, V., Zaveri, K., Hinduja, I., 2011. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev. 20, 1451–1464. Tomizawa, S., Sasaki, H., 2012. Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell. J. Hum. Genet. 57, 84–91. van Montfoort, A.P., Hanssen, L.L., de Sutter, P., Viville, S., Geraedts, J.P., de Boer, P., 2012. Assisted reproduction treatment and epigenetic inheritance. Hum. Reprod. Update 18, 171–197. White, Y.A., Woods, D.C., Takai, Y., Ishihara, O., Seki, H., Tilly, J.L., 2012. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat. Med. 18, 413–421. Zou, K., Yuan, Z., Yang, Z., Luo, H., Sun, K., Zhou, L., Xiang, J., Shi, L., Yu, Q., Zhang, Y., Hou, R., Wu, J., 2009. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat. Cell Biol. 11, 631–636.
Ursula Eichenlaub-Ritter Section Editor E-mail address: [email protected]
www.sciencedirect.com www.rbmonline.com
EDITORIAL
Female meiosis and beyond: more questions than answers? Three years ago, there was some excitement about a publication reporting that female germline stem cells are present in the adult mouse ovary, can be isolated and propagated in vitro, and can generate oocytes, embryos and healthy live pups after transplantation into recipient, infertile females (Zou et al., 2009). More recently, it was reported that haploid cells could consistently be derived from human induced pluripotent stem cells of non-ovarian male- or female-derived tissues (Eguizabal et al., 2011). Therefore, it does not come as such a surprise to read a paper from Professor Johnathan Tilly’s group in Harvard reporting that the induction of meiosis in stem cells derived from human adult ovary has been achieved (White et al., 2012). Embryonic stem cells have also been obtained from cultured human ovarian surface epithelium (Parte et al., 2011), but meticulous analysis of the survival of stem cells/oogonia in the human ovary suggests that oogonia die within the first two years after birth, bringing into question a physiological role for germline-like or oogonial stem cells in neo-oogenesis in humans (Johnson et al., 2004; Byskov et al., 2011). However, the presence of the rare cells, termed ‘oogonial stem cells (OSC)’ by the Tilly group, might be difficult to identify in fixed ovaries. The Tilly group was able to enrich and study gene expression in GFP-expressing human oogonial stem cells after modifying the isolation and cultivation procedure originally described by Zou et al. (2009). Others, however, have failed so far to repeat this approach. They also report that these stem cells formed GFP-labelled oocyte-like cells within follicle-like structures after their transplantation into mice (White et al., 2012). I must confess that I remain sceptical about the significance of the generation of haploid germ-cell-like cells from mouse or human adult ovarian tissue for fertility preservation in humans (White et al., 2012; Zou et al., 2009). I am sceptical not only because of the controversies about the possible physiological role of OSC, but also because of the well-known species-specific differences in germ cell formation in the human female compared with the rodent (e.g. relative duration of oogenesis, period and extent of growth, and genomic imprinting to achieve full developmental com-
petence). In addition, there are difficulties in maintaining, and certainly in entirely reconstituting, folliculogenesis, oocyte growth and in-vivo and in-vitro maturation in the human. Regrettably, many questions remain about the efficiency of the new protocols and the perspectives for neo-oogenesis in humans, and so it is premature to raise hopes in the public, and particularly in desperate sub- or infertile women, that regaining or preserving fertility will be possible using such approaches. Thus, haploid status as reported by White et al. (2012) is characteristic of germ cells in males but not in females, in which the oocytes undergo a reductional separation at first meiosis prior to ovulation, thus generating a first polar body with a complete set of dyads (chromosomes consisting of two sister chromatids). Healthy, normal, and competent oocytes arrest at metaphase II such that second meiosis is completed only after fertilization by sperm (or after spontaneous or artificial parthenogenetic activation), and only then is a haploid second polar body formed. The oocyte is thus never ‘haploid’ in the true sense of the word, as it contains a diploid set of maternal chromosomes prior to fertilization and a haploid set of maternal chromosomes plus the haploid genome from the sperm (in the female and male pronuclei respectively) in the one-cell zygote. The haploid second polar body should not dislodge from the oocyte as it is contained within the thick zona pellucida. Moreover, Hu ¨bner et al. (2003) reported many years ago that it is possible to generate oocyte-like cells and blastocysts from mouse embryonic stem cells in vitro. These oocytes failed to arrest at metaphase II and underwent spontaneous parthenogenetic activation (Hu ¨bner et al., 2003). Interestingly, it was more efficient to induce meiosis and oocyte-like cells from cultures of male embryonic stem cells. Importantly, spontaneous activation, or parthenogenesis, can be the basis of pathologies underlying recurrent inability to maintain a normal pregnancy following assisted reproduction treatment (Combelles et al., 2011). It remains open to question whether ovarian tissues used in the recently published report by White et al. (2012), that may
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.04.007
590 have been derived from women treated with steroids before tissue donation, should be considered ‘normal’ (LovellBadge, 2012), but might promote ‘male’ rather than ‘female’ meiosis, or undergo spontaneous parthenogenetic activation. Thus, the recent report does not provide convincing evidence for a gender-specific oogenetic pattern of recombination, not even for the characteristic presence of meiotic cells with thread-like assemblies of proteins of the synaptonemal complex for efficient pairing and recombination, and completion of normal meiotic progression to metaphase II, including establishment of an efficient meiotic arrest. Last but not least, there is no information on the epigenetic status of the oocyte-like cells, or of embryos derived from germline-like stem cells from adult ovary in the mouse. Epigenetic ‘mutations’ can induce implantation failures, congenital abnormalities, foetal death and, notably, epigenetic diseases like Angelman syndrome, Beckwith Wiedeman syndrome, Silver-Russel syndrome or predisposition to cardiovascular disease and childhood cancers (recently discussed by Tomizawa and Sasaki, 2012; van Montfoort et al., 2012). Erasure of genomic imprints is a prerequisite to gender-specific genomic imprinting during gametogenetis (Hackett et al., 2012; van Montfoort et al., 2012). Unlike in spermatogenesis, genomic imprinting occurs during the oocyte growth phase in the female (Lucifero et al., 2004), and there is evidence that even those immature human oocytes that undergo growth in vivo in stimulated cycles, but do not complete maturation to metaphase II, harbour imprint defects after in-vitro maturation and are susceptible to epigenetic and chromosomal instability (Borghol et al., 2006; Magli et al., 2006). Therefore, any claims that viable, healthy oocytes can be obtained from stem cells from adult mouse, and particularly from human ovarian tissue, must provide convincing evidence that recombination occurs and that imprint patterns before and after oocyte growth are normal. Furthermore, oocytes derived from the manipulations should be capable of remodelling the male chromatin and of protecting the genomic imprints from male and female germ cells from an untimely erasure after fertilization. The true benefit of the new approach to isolation and propagation of post-natal ovarian cells with a potential to form a functional follicular unit that can develop towards a mature oocyte lies in the opportunities to study these processes and their regulation in vitro. However, before there is proof that imprinting and bi-directional signalling within newly formed follicles are normal, and that the approach will yield high-quality oocytes with reasonable efficiency, it is prudent to be sceptical about the clinical relevance of the findings. This is not to say that one should not retain an open and enthusiastic mind towards challenges to dogmas, innovations in techniques that are promising in the treatment of sub-fertile patients, and particularly, novel developments in stem cell biology that provide models and information on the molecular bases of germ cell development, fertility and health of offspring. Just do not overhype prematurely!
Editorial
References Borghol, N., Lornage, J., Blache `re, T., Sophie, Garret.A., Lefe `vre, A., 2006. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 87, 417–426. Byskov, A.G., Høyer, P.E., Yding, Andersen.C., Kristensen, S.G., Jespersen, A., Møllga ˚rd, K., 2011. No evidence for the presence of oogonia in the human ovary after their final clearance during the first two years of life. Hum. Reprod. 26, 2129–2139. Combelles, C.M., Kearns, W.G., Fox, J.H., Racowsky, C., 2011. Cellular and genetic analysis of oocytes and embryos in a human case of spontaneous oocyte activation. Hum. Reprod. 26, 545– 552. Hackett, J.A., Zylicz, J.J., Surani, M.A., 2012. Parallel mechanisms of epigenetic reprogramming in the germline. Trends Genet. (Epub ahead of print). Hu ¨bner, K., Fuhrmann, G., Christenson, L.K., Kehler, J., Reinbold, R., De La Fuente, R., Wood, J., Strauss 3rd, J.F., Boiani, M., Scho ¨ler, H.R., 2003. Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256. Johnson, J., Canning, J., Kaneko, T., Pru, J.K., Tilly, J.L., 2004. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145–150. Lovell-Badge, R., 2012. Hype, hope and heresy – or why it is bad to eggsaggerate. BioNews 648. Lucifero, D., Mann, M.R.W., Bartolomei, M.S., Trasler, J.M., 2004. Gene-specific timing and epigenetic memory in oocyte imprinting. Hum. Mol. Genet. 13, 839–849. Magli, M.C., Ferraretti, A.P., Crippa, A., Lappi, M., Feliciani, E., Gianaroli, L., 2006. First meiosis errors in immature oocytes generated by stimulated cycles. Fertil. Steril. 86, 629–635. Parte, S., Bhartiya, D., Telang, J., Daithankar, V., Salvi, V., Zaveri, K., Hinduja, I., 2011. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev. 20, 1451–1464. Tomizawa, S., Sasaki, H., 2012. Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell. J. Hum. Genet. 57, 84–91. van Montfoort, A.P., Hanssen, L.L., de Sutter, P., Viville, S., Geraedts, J.P., de Boer, P., 2012. Assisted reproduction treatment and epigenetic inheritance. Hum. Reprod. Update 18, 171–197. White, Y.A., Woods, D.C., Takai, Y., Ishihara, O., Seki, H., Tilly, J.L., 2012. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat. Med. 18, 413–421. Zou, K., Yuan, Z., Yang, Z., Luo, H., Sun, K., Zhou, L., Xiang, J., Shi, L., Yu, Q., Zhang, Y., Hou, R., Wu, J., 2009. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat. Cell Biol. 11, 631–636.
Ursula Eichenlaub-Ritter Section Editor E-mail address: [email protected]