S6 phosphorylation is regulated at multiple levels in Xenopus laevis oocytes

It is known that the 40s ribosomal protein S6 undergoes a dramatic increase in its level of phosphorylation during Xenopus oocyte meiotic maturation in response to progesterone stimulation. During prophase arrest, the majority of S6 has 0 moles phosphate per mole protein; this increases to 4-5 moles phosphate per mole protein by the time of germinal vesicle breakdown (GVBD). Our in vitro and in vivo studies indicate that the accumulation of phosphate on S6 is the net result of a 4-5-fold increase in S6 kinase activity and a 30-50% decrease in the rate of dephosphorylation and/or turnover of phosphate groups on S6 in maturing oocytes. In addition, the level of phosphorylation of S6 on 80s monosomes injected into non-hormone-stimulated oocytes was unexpectedly high. This indicates that the S6 kinase/phosphatase ratio in prophase arrested oocytes is higher than anticipated from previous studies. This observation implies that the majority of the oocyte ribosomes may be sequestered from any S6 kinase during meiotic prophase. Furthermore, these observations suggest that a portion of the increased accumulation of phosphate on S6 may be the result of increased accessibility of the ribosomes to S6 kinase during oocyte meiotic maturation.     

Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation.

We previously demonstrated that a protein of M(r) 75,000 (p75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K.E. Pierce, M. C. Siebert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Dev. Biol. 141, 381-392). To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocytes, which were isolated from juvenile mice, were incubated for 4 hr in medium containing [35S]methionine, and radiolabeled proteins were immunoprecipitated using an antiserum that detects p75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (less than 20 microns). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 microns size and then remains constant throughout the remaining period of oocyte growth (40-70 microns). In the fully grown, germinal vesicle (GV)-intact oocyte (70-80 microns), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolabeled protein. Three hours after the transfer of these oocytes to a medium that supports resumption of meiosis and GV breakdown in vitro, oocytes subjected to a 1-hr labeling pulse display a 35% decrease in the relative level of p75 synthesis. By 15 hr of maturation, p75 synthesis was reduced to 14% of that in the fully grown, GV-intact oocyte and this is similar to the level of p75 synthesis in ovulated eggs. The level of p75 synthesis following in vitro translation of total egg RNA is only 38% lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following in vitro translation of oocyte, but not egg, poly(A)+ RNA. These results are consistent with p75 synthesis during oocyte maturation being under translational control.     

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.     

Interaction between rat brain microtubule associated proteins (MAPs) and free ribosomes from Xenopus oocyte: a possible mechanism for the in ovo distribution of MAPs

The binding of microtubule associated proteins (MAPs) to free 80 S ribosomes isolated from Xenopus laevis oocytes inhibits in vitro tubulin assembly (Jessus et al., 1984). The inhibition of tubulin polymerisation was shown to be dependent upon GTP. The dose of GTP needed to induce 50% of the maximal effect was 0.5 mM. Furthermore, the inhibition is enhanced by pretreatment of the ribosomes with ATP-gamma-S, and partially abolished after phosphatase treatment, which strongly suggests that protein phosphorylation regulated the inhibitory effect. When fluorescent purified MAPs are microinjected into Xenopus laevis oocyte, they cap 1 h later the basal nuclear envelope; in contrast, when the fluorescent MAPs-ribosome complex is injected, the fluorescent MAPs remain in the cytoplasm and never reach the region underlying the nuclear envelope.     

Cellular localization of DNA polymerase activities in full-grown oocytes and embryos of Xenopus laevis

In full-grown oocytes of Xenopus laevis more than 80 % of the total DNA polymerase activity is found in the germinal vesicle (nucleus) and only about 8% in the cytoplasm. The intracellular distribution of the multiple DNA polymerase forms has been studied in oocytes and in embryonic cells. The oocyte nucleus contains a major DNA polymerase species, sedimenting at about 7S, and a minor species sedimenting at about 5S. These enzymes are comparable, respectively, with the DNA polymerases α and β described in other biological systems. In the oocyte cytoplasm, besides a small amount of the 7S form, an 8–9S DNA polymerase activity is also detectable. In the nuclei of embryonic cells, in addition to the DNA polymerase forms present in the oocyte nucleus, a new major form which seems specific for the eggs and embryos is detectable by DEAE chromatography.     

Dimethyl sulfoxide inhibits spontaneous oocyte fragmentation and delays inactivation of maturation promoting factor (MPF) during the prolonged culture of ovulated murine oocytes in vitro

In this study, the effects of dimethyl sulfoxide (DMSO) on the spontaneous aging of ovulated murine oocyte were evaluated in vitro. When ovulated oocytes were cultured continuously in vitro without fertilization stimulation, they underwent several phenotypic changes, including non-activation, activation, fragmentation, and lysis. To investigate the effects of DMSO on these changes, I cultured ovulated oocytes with various concentrations of DMSO and evaluated the phenotypic changes for up to 3 days. After 3 days of culture, the frequency of oocyte fragmentation was significantly lower in oocytes treated with 2 and 4% DMSO (7 and 5%, respectively) than in control oocytes (80%). All control oocytes were activated or fragmented after 3 days of culture in vitro. However, more than 80% of the oocytes cultured with 4% DMSO for 3 days contained spindles and condensed chromosomes, although they displayed abnormal spindle structures. Next Cdk1 activity in DMSO-treated oocytes was examined. The results showed that DMSO treatment prevented the reduction in Cdk1 activity during prolonged culture. Moreover, DMSO inhibited the degradation of cyclin B. These results suggest that DMSO inhibits spontaneous oocyte fragmentation and maintains Cdk1 activity in ovulated murine oocytes during prolonged culture in vitro, possibly by inhibiting cyclin B degradation.     

Control of oocyte maturation in cows--biological factors

Since bovine in vitro fertilization became possible in the early 80s, a lot of effort has been done to clarify the mechanisms of what seems more and more one of the crucial steps in this procedure, being oocyte maturation. Undoubtedly, many biological factors act together to prepare the immature oocyte for a successful development to a competent embryo after fertilization. Defects in oocyte maturation can possibly be caused by an inadequate nuclear or cytoplasmic maturation or even by a failure of both. There is a general agreement upon the fact that the origin of the oocyte can play an important role. Oocytes derived from very small follicles show a lower rate of maturation and lower blastocyst development with currently used maturation protocols. Parthenogenetic activation of small size follicle derived oocytes suggests that their poor development was not caused by fertilization problems but more likely by intrinsic oocyte factors. Similar developmental rates achieved through nuclear transfer and parthenogenetic activation suggests that the nucleus of the incompetent oocyte may not be the sole reason for a poor development. Another important factor appears to be the donor animal age. The younger the donor animal, the more impaired is its oocyte's developmental competence in most of the embryo IVP systems. Treatment with exogeneous gonadotropins can be beneficial in young donors on the oocyte cleavage rates but does not always increase the final blastocyst outcome. This review briefly documents some of the biological factors and their possible effects on the developmental capacities of the bovine oocyte in vitro.     

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo

Protein phosphatases 1 and 2B from rabbit skeletal muscle were found to catalyze the dephosphorylation of ribosomal protein S6 in vitro. Phosphorylation of protein phosphatase-1 by the transforming protein of Rous sarcoma virus, pp60v-src, abolished S6 dephosphorylation by the purified enzyme. Analysis of the dephosphorylation of phosphorylase a and phosphorylase kinase in Xenopus oocyte extracts and after microinjection indicated the presence of oocyte enzymes similar to protein phosphatases-1 and -2B. Studies with 32P-labeled 40 S ribosomal subunits suggested that these enzymes were functioning as S6 phosphatases in oocytes. These findings support the hypothesis that regulation of protein phosphatase activity may be involved in the increase in S6 phosphorylation observed after mitogenic stimulation.