Component Of Cytostatic Factor Research Articles

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.

Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest.

The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.

Mos and the cell cycle: the molecular basis of the transformed phenotype

The product of the mos proto-oncogene is a serine/threonine kinase that is expressed at high levels in germ cells. Mos is a regulator of meiotic maturation, and is required for the initiation and progression of oocyte meiotic maturation that leads to the production of unfertilized eggs. Mos is also a component of cytostatic factor, an activity that is believed to arrest oocyte maturation at meiotic metaphase II. There is evidence showing that the Mos protein is associated with tubulin in unfertilized eggs and transformed cells, raising the possibility that it is involved in the microtubular reorganization that occurs during M-phase. Inappropriate expression of its M-phase activity during interphase of the cell cycle may be responsible for its transforming activity.

Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor.

Expression of the mosxe protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mosxe functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mosxe or kinase-inactive mosxe(R90), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mosxe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mosxe, this indicates that the major phosphopeptides of mosxe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mosxe protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mosxe were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine----alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mosxe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine----alanine mosxe mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mosxe mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF.

C- mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes

Recently, it has been shown that the product of the c- mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c- mos product in metaphase II mouse oocytes and soon after activation. The amount of c- mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c- mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c- mos is detectable during the first zygotic interphase. Our data show that the degradation of c- mos is not involved in the release from the metaphase arrest.

Progression from meiosis I to meiosis II in Xenopus oocytes requires de novo translation of the mosxe protooncogene.

The meiotic maturation of Xenopus oocytes exhibits an early requirement for expression of the mosxe protooncogene. The mosxe protein has also been shown to be a component of cytostatic factor (CSF), which is responsible for arrest at metaphase of meiosis II. In this study, we have assayed the appearance of CSF activity in oocytes induced to mature either by progesterone treatment or by overexpression of mosxe. Progesterone-stimulated oocytes did not exhibit CSF activity until 30-60 min after germinal vesicle breakdown (GVBD). Both the appearance of CSF activity and the progression from meiosis I to meiosis II were inhibited by microinjection of mosxe antisense oligonucleotides just prior to GVBD. These results demonstrate a translational requirement for mosxe, which is temporally distinct from the requirement for mosxe expression at the onset of meiotic maturation. In contrast to progesterone-treated oocytes, oocytes that were induced to mature by overexpression of mosxe exhibited CSF activity at least 3 hr prior to GVBD. Despite the early appearance of CSF, these oocytes were not arrested at meiosis I. These results indicate that, although CSF activity is capable of stabilizing maturation-promoting factor (MPF) at meiosis II and in cleaving embryos, it is incapable of stabilizing MPF prior to or at meiosis I. These studies show that the complex regulation of the cell cycle during meiosis differs significantly from the regulation of the cell cycle during mitosis.

Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos.

Previous work has demonstrated that the Xenopus protooncogene mosxe can induce the maturation of prophase-arrested Xenopus oocytes. Recently, we showed that mosxe can transform murine NIH3T3 fibroblasts, although it exhibited only 1-2% of the transforming activity of the v-mos oncogene. In this study we have investigated the ability of the v-mos protein to substitute for the mosxe protein in stimulating Xenopus oocytes to complete meiosis. Microinjection of in vitro synthesized RNAs encoding either the mosxe or v-mos proteins stimulates resting oocytes to undergo germinal vesicle breakdown. Microinjection of an antisense oligonucleotide spanning the initiation codon of the mosxe gene blocked progesterone-induced oocyte maturation. When oocytes were microinjected first with the mosxe antisense oligonucleotide, and subsequently with in vitro synthesized v-mos RNA, meiotic maturation was rescued as evidenced by germinal vesicle breakdown. The v-mos protein exhibited in vitro kinase activity when recovered by immunoprecipitation from either microinjected Xenopus oocytes or transfected monkey COS-1 cells; however, in parallel experiments, we were unable to detect in vitro kinase activity associated with the mosxe protein. Microinjection of in vitro synthesized v-mos RNA into cleaving Xenopus embryos resulted in mitotic arrest, demonstrating that the v-mos protein can function like the mosxe protein as a component of cytostatic factor. These results exemplify the apparently conflicting effects of the v-mos protein, namely, its ability to induce maturation of oocytes, its ability to arrest mitotic cleavage of Xenopus embryo, and its ability to transform mammalian fibroblasts.