Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes

In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.     

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.     

Changes in protein phosphorylation during the maturation of mammalian oocytes in vitro

Cumulus-enclosed sheep oocytes were cultured in gonadotrophin-containing medium for up to 9 hr and were then incubated for 3 hr in the presence of [32P]phosphate. The incorporation of 32P into TCA-insoluble material was measured, and oocyte proteins were separated by one- and two-dimensional gel electrophoresis. Incorporation of [32P]phosphate into protein increased after 3 hr culture and again after 9 hr, the time of germinal vesicle breakdown (GVBD). Qualitative and quantitative changes in the phosphorylation of proteins occurred over the 12-hr period studied. One of the most prominent changes was the appearance of a band of Mr 33,000, which was absent at 0-3 hr but appeared with increasing intensity with longer periods of culture. Two-dimensional electrophoresis revealed that the bulk of material in this band was a neutral polypeptide. No significant incorporation of [32P]phosphate was found in ribosomal extracts of oocytes.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.     

Stabilization of the maturation promoting factor (MPF) from Xenopus laevis oocytes. Protection against calcium ions

Maturation promoting factor (MPF) activity was recovered from progesterone-matured Xenopus oocytes cytosol, fractionated by polyethylene glycol-20,000 or ethanolic precipitation. An improved stabilization of the biological activity was obtained by adding adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) (50 microM) to the preparation buffers. Neither Ca2+ ions nor calmodulin inhibit the partially purified MPF.     

Amino acids can induce nondisjunction during maturation of mammalian oocytes in vitro

Certain chromosomal disorders in mammalian embryos are traceable to meiotic errors during oocyte maturation. This report evaluates the influence of amino acids on meiotic maturation in vitro of oocytes from pigs, hamsters, and rats. The results indicate that maturing porcine oocytes respond not only to 1-glutamine (1-gln) but also to 1-isoleucine (1-ileu) in complex or chemically defined media by exhibiting significantly (P less than .05) increased incidences of nondisjunction when compared with oocytes in control medium. Nondisjunction was highly correlated (r = 0.981) with dose of 1-gln in porcine oocytes (incidences of maturing oocytes exhibiting nondisjunction were 19.3%, 39.7%, 41.5%, 66.2%, and 88.5% at 1-gln concentrations of 0 mM, 0.5 mM, 1.0 mM, 3.0 mM and 10.0 mM, respectively). Hamster oocytes also exhibited significantly (P less than .05) increased nondisjunction when cultured in medium containing 1-gln (52% of maturing cumulus-enclosed oocytes exhibited nondisjunction in medium with 3.0 mM 1-gln vs. 6.3% in control medium). Denuded hamster oocytes also responded to 1-gln (35% exhibited nondisjunction in 4.0 mM 1-gln, 8% in control medium). In contrast the incidence of nondisjunction in rat oocytes was not increased significantly by 1-gln over a concentration range of 0-12.0 mM. This study demonstrates that maturing oocytes respond to certain environmental conditions by undergoing chromosomally abnormal maturation. Specifically, amino acids can induce nondisjunction when present in elevated concentrations during oocyte maturation. The amino acid influence not only was dose dependent for porcine oocytes but also was species specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte-cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199-based medium in 5% CO₂ in humidified air at 38.5°C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48-hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes. Mol. Reprod. Dev. 47:191–199, 1997. © 1997 Wiley-Liss, Inc.     

Nuclear maturation in pig and rabbit oocytes after interspecific fusion

Porcine ovarian oocytes were fused with either homologous (porcine) or heterologous (rabbit) oocytes, both at different stages of maturation. The maturation-promoting factor (MPF) present in maturing porcine oocytes or ovulated rabbit oocytes induced rapid chromosome condensation of the oocytes with intact germinal vesicles (GVs). In the case of activation of ovulated rabbit oocyte, germinal vesicle breakdown (GVBD) of porcine oocytes was incomplete or did not occur. In the giant cells consisting of two immature porcine oocytes, meiotic maturation proceeded in the same manner as in unfused oocytes. However, in cells derived from fusion of immature porcine and rabbit oocytes, two metaphase groups of chromosomes were observed 6 h after fusion. It may be concluded that GVBD is governed after fusion by the cytoplasm originating from the oocytes of more advanced stages of maturation or from those which mature faster.     

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.     

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.     

Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles

The limited knowledge on the regulation of oocyte formation, the different steps of folliculogenesis and the required conditions for oocytes to undergo proper growth, differentiation and maturation are major causes of the failure in obtaining viable offspring from in vitro cultured early oocytes from domestic animals and humans. This review highlights the factors that at present are known to be involved in the formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.     

Activity of the maturation-promoting factor and the extent of protein phosphorylation oscillate simultaneously during meiotic maturation of starfish oocytes

Maturation-promoting factor (MPF) activity and the protein phosphorylation pattern were monitored throughout the time course of meiotic maturation following hormonal stimulation of prophase-arrested starfish oocytes. MFP activity disappeared or decreased dramatically during the first and second meiotic cleavages. MPF activity came back to a very high level after the first but not the second meiotic cleavage. The state of protein phosphorylation was monitored using both tracer experiments and direct measurements of the absolute amount of phosphate in phosphoproteins. High and low levels of MPF activities were, respectively, associated with high and low levels of protein phosphorylation. It is suggested that the turn over of phosphate already bound to proteins in prophase-blocked oocytes does not change following hormone addition.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.