The process of vertebrate egg development consists of oocyte growth coupled with specific pauses at various stages of maturation. The final pause is known as the metaphase II arrest, from which eggs are released only after being fertilised by sperm. The cellular activity that ensures this arrest has been known for 30 years by the alias 'cytostatic factor' (CSF), ever since Masui and Markert (1) showed that egg development is arrested by CSF before fertilisation. Subsequently it has been established that a signalling pathway involving the Mos protein is a key component of CSF activity. However, inactivation of CSF and the Mos pathway occurs after fertilisation triggers the resumption of development, suggesting that as-yet-uncharacterised elements of CSF are required to maintain the developmental arrest. Reimann and Jackson (2) define a new, Mos-independent component of CSF, which regulates the degradation of proteins required for developmental arrest in eggs. Their work provides new insight into the regulation of CSF, and raises questions about the contribution of the Mos pathway to CSF activity.
There are two processes by which cells normally divide: mitosis and meiosis. During mitosis, cells replicate their chromosomes (producing a '4n' DNA content) and segregate them equally to each of two daughter cells (which are therefore '2n'). By contrast, during meiosis of female germ cells (oocytes), the replicated (4n) chromosomes undergo two successive 'reductive' divisions – separating first a set of chromosomes (2n) in the first polar body and second a set of replicated chromosomes after fertilisation – to generate a haploid set (1n) in the egg. By contrast, male meiosis (spermatogenesis) proceeds through the first and second meiotic divisions to produce four (1n) germ cells (sperm). Progression through female meiosis (oogenesis) to produce one unfertilised egg occurs in a stepwise fashion in response to defined extracellular cues. In vertebrates, female germ cells pause just before the first meiotic division and undergo an extended period of growth, which is necessary to accumulate resources for early embryonic stages of development. Having accomplished this, oocytes become competent to respond to hormonal signals and resume meiosis.
Resumption of meiosis is accompanied by the activation of maturation-promoting factor – a complex consisting of a regulatory protein, cyclin B2, and a catalytic protein, Cdc2. The activity of MPF is controlled by the association of these proteins with each other, and by two competing enzymes, the Myt1 kinase and Cdc25, that either add or remove phosphate groups from Cdc2. Active MPF is required for dissolution of the envelope surrounding the nucleus and formation of the 'spindle' apparatus that will segregate homologous chromosomes. Chromosome segregation is then triggered by the anaphase-promoting complex (APC), which mediates the degradation of cyclin B2 and, hence, inactivation of MPF.
Entry into the second meiotic division occurs as MPF is reactivated, when newly synthesised cyclin B2 associates with Cdc2. But now the degradation of cyclin B2 is blocked so eggs arrest division before they separate their replicated chromosomes, at a stage called metaphase II. This arrest may be a safeguard against parthenogenesis – embryonic development without fertilisation – as its release is triggered by fertilisation.
Our first insight into how oocytes arrest in metaphase II came from Masui and Markert (1), who showed that cytoplasm from a metaphase-II-arrested oocyte could induce a similar arrest when injected into a mitotically dividing embryonic cell (blastomere). They concluded that this arrest could be attributed to a specific activity, dubbed CSF, in the cytoplasm of the arrested oocyte. They found that CSF arose in the cytoplasm following the appearance of MPF, roughly when the first meiotic division began; and that CSF levels increased between the first and second meiotic divisions and declined after fertilisation, shortly after the inactivation of MPF1.
But attempts to biochemically purify CSF were frustrated by its instability, and the study of the molecular properties of CSF languished for almost 20 years, until Sagata et al. (3) showed that the Mos protein has many characteristics in common with CSF. Mos was synthesised prior to entry into the first meiotic division and degraded after fertilization; injection of Mos into a mitotic blastomere caused metaphase-II-like arrest; and extracts of metaphase-II-arrested eggs that had been depleted of Mos lacked CSF activity (3). Thus, Mos is a component of CSF.
Mos is a protein kinase that activates the mitogen-activated protein kinase (MAPK) pathway. Like Mos, each component of this pathway, including MAPK and p90rsk, behaves like CSF when injected into blastomeres (4-6). Most recently (7), p90rsk has been shown to phosphorylate and activate the APC inhibitor Bub1. Thus, synthesis of Mos and activation of the MAPK pathway might culminate in activation of Bub1 and inhibition of APC activity, which consequently cannot trigger cyclin B2 degradation and release from metaphase II arrest.
However, although the Mos pathway is a necessary component of CSF, by itself it is insufficient to maintain metaphase II arrest; after fertilisation, cyclin B2 is degraded before inactivation of the Mos pathway. Indeed, it does not even appear to be necessary to maintain metaphase II arrest, as Reimann and Jackson (2) now show that blocking this pathway with a MAPK inhibitor does not cause cyclin B2 degradation in CSF-containing extracts. These authors instead propose that the Emi1 protein, identified previously as an APC inhibitor, is needed to maintain metaphase II arrest.
Reimann and Jackson (2) found that adding extra Emi1 to the CSF-containing extracts prevented the destruction of cyclin B2 that is triggered by the addition of calcium — a process that mimics fertilisation. By contrast, depletion of Emi1 led to cyclin B2 degradation. Furthermore, as shown previously (8), adding Emi1 to mitotic blastomeres arrests their division. Their results suggest that Emi1 arrests the second meiotic division by preventing the destruction of cyclin B2, in a MAPK-independent way, and that fertilisation would lead to the inhibition of Emi1 and activation of the APC, releasing the egg from metaphase II arrest.
What, then, is the role of the Mos pathway in CSF activity? The observation that mouse oocytes lacking the mos gene fail to arrest at metaphase II and spontaneously complete the second meiotic division (9, 10) indicates that Mos is both necessary and sufficient for entry into metaphase II arrest, especially given that naturally occurring (endogenous) Emi1 fails to cause this arrest in the absence of Mos. Thus it would seem that endogenous Emi1 is not sufficient to activate the CSF activity and that the activity of, or responsiveness to, endogenous Emi1 is regulated in a Mos-dependent fashion.
Given the involvement of Mos in the formation of the meiotic spindle (11-14), we propose that Mos-mediated modification of this spindle, perhaps at the kinetochores (where chromosomes attach), is required for Emi1 activity. If so, then blastomeres injected with Mos would arrest their division because Mos-induced changes in spindle structure increase its sensitivity to the effects of endogenous Emi1. By contrast, exogenous Emi1 might induce blastomere arrest because it compensates for the usual lack of sensitivity of the mitotic spindle to endogenous Emi1. This remains to be seen. But for now, it is satisfying to see that another piece in a 30-year-old puzzle is in place.
References
1. Masui, Y. & Markert, C. L. J. Exp. Zool. 177, 129-145 (1971).
2. Reimann, J. D. R. & Jackson, P. K. Nature 416, 850-854 (2002).
3. Sagata, N., Watanabe, N., Vande Woude, G. F. & Ikawa, Y. Nature 342, 512-518 (1989). 4. Haccard, O. et al. Science 262, 1262-1265 (1993).
5. Gross, S. D., Schwab, M. S., Lewellyn, A. L. & Maller, J. L. Science 286, 1365-1367 (1999).
6. Bhatt, R. R. & Ferrell, J. E. Jr Science 286, 1362-1365 (1999).
7. Schwab, M. S. et al. Curr. Biol. 11, 141-150 (2001).
8. Reimann, J. D. et al. Cell 105, 645-655 (2001).
9. Colledge, W. H., Carlton, M. B., Udy, G. B. & Evans, M. J. Nature 370, 65-68 (1994).
10. Hashimoto, N. et al. Nature 370, 68-71 (1994).
11. Choi, T. et al. Proc. Natl Acad. Sci. USA 93, 7032-7035 (1996).
12. Verlhac, M.-H et al. Development 122, 815-822 (1996).
13. Gross, S. D. et al. Curr. Biol. 10, 430-438 (2000).
14. Bodart, J. F. et al. Dev. Biol. (in the press).
Figures and links to PubMed and ISI abstracts can be found on the full-text page in the 25 April issue of Nature.
Nature (News and Views)
25 April 2002
Dr. Bill Silvia, Associate Professor of Reproductive Physiology in the Department of Animal Sciences at the University of Kentucky, has kindly provided the following commentary in response to the above news item.
In the April 25, 2002 issue of Nature (News and Views), N.S. Duesbery and G.F. Vande Woude discuss a report that appears in the same issue by J.D.R. Reinmann and P.K. Jackson on the regulation of meiosis II in oocytes. The later researchers identified a new regulatory factor (Emi 1) that appears to play a role in the arrest and resumption of meiosis II in Xenopus oocytes. Duesbery and Vande Woude do a fine job of reviewing some of the history and describing the current state of knowledge in this complex and active area of research.
What is meiosis? Meiosis or 'reduction division' is the unique process by which germ cells give rise to gametes. It consists of two divisions, meiosis I and II. In mammalian females, the germ cells initiate the first meiotic division while the developing female fetus is still in utero. However; this division is arrested in prophase of meiosis I. In situ, a properly matured oocyte will resume meiosis (overcome arrest) when the preovulatory follicle is exposed to an ovulatory surge of luteinising hormone. Meiosis will then progress to metaphase of meiosis II. At this point, a second period of meiotic arrest ensues. The resumption of meiosis II is induced by contact with sperm cells and is thus completed as part of fertilisation.
Do experiments conducted with Xenopus (frog) oocytes apply to mammals? For the most part, yes. The mechanisms regulating cell division appear to be highly conserved.
Are there immediate veterinary applications of this research? It is hard for me to envision immediate applications to veterinary or human medicine. Is failure of meiosis II a common problem? Probably not. In in vitro fertilisation settings, only 15-20% of oocytes fail to complete meiosis II. Most of these probably fail to be fertilised (in other words, it is probably rare for an oocyte to be fertilised and meiosis II not proceed normally). Does blocking meiosis II have any application? Theoretically, yes. It could have a contraceptive application. Blocking meiosis II would lead to the formation of triploid embryos that cannot survive through gestation. The drawback is that they can survive for a while, generally long enough to form trophoblast tissue and disrupt normal cyclicity. Delivery of an inhibitor of meiosis II would also be problematic. There are better approaches to contraception already available. Another theoretical application is in the creation of parthenotes (individuals derived from a single parent). Parthenotes have been formed by activating oocytes then blocking meiosis II. However, these individuals are never carried to term because of deficiencies in either the embryo or the placenta. Paternal components of the genome (imprinted) appear to be required for a pregnancy to progress normally.
So then, why should we study the regulation of meiosis II? I believe that it is an extremely important area of basic research. The mechanisms that regulate the arrest and resumption of meiosis in oocytes are complex but may hold the key to controlling undesirable cell division in other cell types. More importantly, meiosis is the essence of sexual reproduction. It is during meiosis that recombination and random assortment of chromosomes occur, ensuring that every gamete contains a unique complement of genetic material. We owe our individuality to the success of meiosis. Studying meiosis is justifiable for that reason alone."
