Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases that are widely distributed in eukaryotic cells. Studies in the last decade revealed that MAPK cascade plays pivotal roles in regulating the meiotic cell cycle progression of oocytes. In mammalian species, activation of MAPK in cumulus cells is necessary for gonadotropin-induced meiotic resumption of oocytes, while MAPK activation is not required for spontaneous meiotic resumption. After germinal vesicle breakdown (GVBD), MAPK is involved in the regulation of microtubule organization and meiotic spindle assembly. The activation of this kinase is essential for the maintenance of metaphase II arrest, while its inactivation is a prerequisite for pronuclear formation after fertilization or parthenogenetic activation. MAPK cascade interacts extensively with other protein kinases such as maturation-promoting factor, protein kinase A, protein kinase C, and calmodulin-dependent protein kinase II, as well as with protein phosphatases in oocyte meiotic cell cycle regulation. The cross talk between MAPK cascade and other protein kinases is discussed. The review also addresses unsolved problems and discusses future directions.     

Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro

Sequential protein synthesis and protein phosphorylation patterns were generated by radiolabelling bovine cumulus-oocyte complexes after various periods of culture with [35S]methionine and [32P]orthophosphate respectively. The radiolabelled oocytes were assessed for their nuclear status and used individually for gel electrophoresis. Marked changes in the protein synthesis patterns were observed exclusively after germinal vesicle breakdown (GVBD), whereas oocytes which remained in the germinal vesicle stage showed a consistent protein synthesis pattern. The changes were observed after 8 and 16 h or culture, shortly after GVBD and before first polar body extrusion. From 3 h of culture, dominant phosphoprotein bands with apparent molecular weights of 24,000 and two between 50,000 and 60,000 were observed. The latter bands displayed slight molecular weight changes, which were not closely time related. After GVBD, the phosphoprotein band with Mr 19,000 was no longer observed. This study demonstrates that specific changes in protein synthesis and protein phosphorylation are programmed during bovine oocyte maturation.     

Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2'-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.     

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

Protein phosphorylation patterns during in vitro maturation of the goat oocyte

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [³²P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley-Liss, Inc.     

Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.     

Analysis of site-specific phosphorylation of the retinoblastoma protein during cell cycle progression

Differential phosphorylation of the retinoblastoma protein plays a pivotal role in cell cycle regulation. The retinoblastoma protein is specifically phosphorylated during the cell cycle by cyclin-dependent kinase complexes which intersect with many cellular signaling networks. Since the loss of the retinoblastoma signaling pathways occurs in a wide variety of human tumors, understanding the significance of site-specific phosphorylation can clarify the role of selected cyclin-dependent kinase complexes during cell cycle progression. Here we describe the phosphospecificity and cellular characterization of a panel of polyclonal antibodies that recognize unique phosphorylation sites within the retinoblastoma protein. These reagents were used to validate authentic cellular retinoblastoma phosphorylation sites at amino acids 780, 795, and 807/811 correlating with the G1-S transition.     

Regulation of a mitogen-activated protein kinase kinase kinase,MLTK by PKN

PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p38gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.     

Effects of wortmannin on the kinetics of GVBD and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro

The present study was conducted with the objective of examining the effect of wortmanin, a specific PI 3-kinase inhibitor, on the kinetic of GVBD, and on the activities of the maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase during bovine oocyte maturation. The time sequence for GVBD was not different between oocytes cultured with or without wortmannin. Most of the cultured oocytes were at the filamentous bivalents stage after 4 h of culture. Six hours after the start of culture, most of the oocytes possessed germinal vesicles with condensed bivalent, and by 10 h of culture nearly all of the cultured oocytes underwent GVBD. A gradual increase in MPF activity until 12 h of culture was observed in the presence and absence of wortmannin. A sharp decrease in MPF activity in oocytes cultured without wortmannin treatment was recorded at 14 h of culture. Thereafter, MPF regained activity, reaching a maximum level at 20 to 24 h of culture. For oocytes cultured with wortmannin, no decline in the activity of MPF was observed during the interval from 12 to 24 h of culture. For these oocytes the MPF activity remained nearly stable during this transition until the end of incubation. The presence of wortmannin in the maturation medium did not alter MAP kinase activity. Taken together, these observations indicate that inhibition of PI 3-kinase does not modulate the time sequence of GVBD or the pattern of MAP kinase activity in bovine oocytes. However, PI 3-kinase might be one of the molecules that regulate the sharp reduction in the activity of MPF during the MI/MII transition.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Effect of IGF-I on pig oocyte maturation,fertilization, and early embryonic development in vitro,and on granulosa and cumulus cell biosynthetic activity

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on ³H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. ³H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in ³H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.     

Regulatory roles of ubiquitin-proteasome pathway in pig oocyte meiotic maturation and fertilization

The ubiquitin-proteasome pathway is involved in the degradation of proteins related to cell cycle progression including cyclins. The present study, using two specific proteasome inhibitors, for the first time investigated the roles of ubiquitin-proteasome in cell cycle progression during pig oocyte meiotic maturation and after fertilization. In contrast to its effect in rodent oocytes, proteasome inhibition strongly prevented germinal vesicle breakdown (GVBD). After GVBD, proteasome inhibition disrupted meiotic apparatus organization, cell cycle progression, and first polar body (PB1) extrusion. Sperm penetration into the oocytes was completely inhibited when proteasome inhibitors were added at the beginning of insemination. However, sperm chromatin decondensation and metaphase-interphase transition were not affected when inhibitors were added once sperm penetrated. The results suggest that ubiquin-proteasome complex is one of the critical regulators of meiotic cell cycle, but proteasome inhibitors do not affect major fertilization events when added after sperm penetration into the oocytes in the pig.     

Dual stimulation of Ras/mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.     

Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization

At least five activated protein kinases were detectable in soluble extracts from maturing as compared to immature sea star oocytes. These kinases could be distinguished on the basis of the time courses of their activation following exposure of the oocytes to 1-methyladenine, their substrate specificities, and their chromatographic properties on DEAE-Sephacel and Sephacryl S-200. A histone H1 kinase (HH1K) (Mr 110,000) underwent maximal activation near the time of 1-methyladenine-induced germinal vesicle breakdown (GVBD). When myelin basic protein (MBP) was used as a substrate, HH1K and two additional kinases (MBPK-I and MBPK-II) were detectable. MBPK-II (Mr 110,000) was fully activated at the time of GVBD, whereas peak activation of MBPK-I (Mr 45,000) occurred after this event. Two "ribosomal protein S6 kinases" (S6K-I and S6K-II) could be detected with a synthetic peptide (RRLSSLRA), which was patterned after a major phosphorylation site in S6. The two S6 kinases (Mr 110,000 for both) underwent activation post-GVBD. HH1K and S6K-I coeluted from DEAE-Sephacel at a conductivity of 5.5-6.0 mmho, whereas MBPK-I, MBPK-II, and S6K-II coeluted from this resin in a second peak at a conductivity = 10-11 mmho. The HH1K and MBPK-II activities both declined prior to the emission of the first polar body (i.e., meiotic cell division), but the MBPK-I, S6K-I, and S6K-II activities remained elevated during this time. The activities of these kinases were also examined during the early cell divisions in sea urchin embryos. Within 5 min after fertilization, the high level of MBPK-I activity in sea urchin eggs rapidly declined. However, along with the HH1K and MBPK-II activities, the MBPK-I activity was transiently increased prior to each cell division. No appreciable postfertilization changes in the S6K-I and S6K-II activities were apparent during the first three cycles of cell division.