Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

The cell cycle can occur in starfish oocytes and embryos without the production of transferable MPF (maturation-promoting factor)

All cells undergoing the transition from interphase to metaphase have been postulated to contain a "maturation-promoting factor" (MPF) capable of causing meiotic maturation when injected into immature oocytes. We have shown in an accompanying paper (A. Picard, M. C. Harricane, J. C. Labbe, and M. Doreé, 1988, Dev. Biol. 128, 121-128) that the basic oscillator driving the cell cycle still operates in maturing starfish oocytes and fertilized eggs in the absence of germinal vesicle (GV) material. Under such conditions of enucleation, we now show, however, that MPF activity cannot be detected after hormonal stimulation of prophase-arrested oocytes in Astropecten or after the normal time of second meiotic cleavage in Marthasterias. In contrast, cell cycles occur with the production of transferable MPF activity in embryos from which both pronuclei have been removed after fertilization. Reinjection of the entire contents of a GV after the normal time of second meiotic cleavage restores the ability of cytoplasm to induce meiotic maturation in immature recipient oocytes after transfer. Transduction of the hormonal stimulus at the level of the plasma membrane, stimulation of the phosphorylation of cytoplasmic proteins, and activation of a cycling Ca2+- and cyclic nucleotide-independent histone kinase still occur in the absence of GV material. Since previous studies have demonstrated that the presence of GV material in the recipient oocytes is absolutely required in starfish for the amplification of microinjected MPF (Kishimoto et al., 1981; Picard and Doree, 1984), we propose that some unidentified component of the GV is required, at least after the normal time of second meiotic cleavage in donor oocytes and at any time in recipient oocytes, for the successful transfer of MPF activity in starfish.     

Lithium inhibits amplification or action of the maturation-promoting factor (MPF) in meiotic maturation of starfish oocytes

Microinjection of LiCl reversibly inhibits hormone-induced meiotic maturation of starfish oocytes. Microinjection of NaCl (even in ouabain-treated oocytes) or KCl, or external application of LiCl have no such effect. Blockade of meiotic maturation by Li+ occurs even when microinjection is performed after the hormone dependent period has ended, that is the period during which the hormone must be present in the medium in order that meiosis can take place. Li+ microinjection prevents oocytes from meiosis reinitiation following transfer of cytoplasm taken from maturing oocytes, which contain a maturation-promoting factor (MPF). Cytoplasm taken from Li+-injected and hormone-treated oocytes does not trigger meiosis reinitiation when transferred in control immature oocytes. Intracellular pH does not change following LiCl microinjection. Simultaneous microinjection of either K+, Na+, or EGTA does not prevent Li+-dependent inhibition in oocytes.     

Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer

In general, oocytes arrested at metaphase of the second meiotic division (MII) are used as recipient cytoplasts for nuclear transfer (NT) procedures. MII oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which cause nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and have been implicated in nuclear reprogramming. However, the occurrence of NEBD and the extent of PCC are variable between individual oocytes and species and are dependent on donor cell type and cell cycle stage. Enucleation, which removes oocyte cytoplasm, may reduce MPF and MAPK activities and reduce reprogramming; conversely, increasing kinase activities may increase reprogramming. We compared the effects of enucleation of ovine oocytes at anaphase/telophase of the first meiotic division (AI-TI) and at MII. MPF and MAPK activities were maximal at MII; blind enucleation at AI-TI was more efficient than at MII and removed a smaller volume of cytoplasm. Neither protocol significantly affected the activity of either kinase and the fate of the donor nucleus; however, enucleation per se significantly reduced the occurrence of NEBD in NT embryos. Treatment with 10 mM caffeine significantly increased the activities of both kinases and the occurrence of NEBD but did not affect the frequency of development to the blastocyst stage; however, a significant increase in total cell numbers was observed. The results show that caffeine can increase MPF and MAPK activities in ovine oocytes and that this may contribute to an increased reprogramming in NT embryos.     

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.     

The role of RhoA in the germinal vesicle breakdown of mouse oocytes

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.     

The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a widely distributed protein kinase that regulates numerous physiological functions. Inhibitors of CaMKII are useful tools for investigating the CaMKII functions. Here we identified a novel CaMKII inhibitor protein (CaM-KIIN) from the human dendritic cell cDNA library by large-scale random sequencing. Human CaM-KIIN contains 79 amino acids, which shares 98% identity and 98% positives with rat CaMKII inhibitor protein beta and 65% identity and 78% positives with rat CaMKII inhibitor alpha. Human CaM-KIIN mRNA expression was detectable in various tissues and cell lines by Northern blot and RT-PCR. To investigate its biological functions, full-length human CaM-KIIN was overexpressed in colon adenocarcinoma LoVo cells. When expressed in LoVo cells, it could inhibit cell proliferation, block cell growth, and decrease the viable cell number. These results characterize a potential cellular inhibitor protein of CaMKII that plays an important role in the regulation of cell growth.     

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.     

Premeiotic aster as a device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes

The germinal vesicle (GV) of starfish oocytes stays just beneath the oocyte cortex at the presumptive animal pole during the long period of oogenesis. We subjected oocytes to a centrifugal force field to detach the GV from the cortex. The association between the cortex and the GV persisted and withstood a small amount of centrifugal acceleration at 200 g. The GV was eventually separated from the cortex at 700 g. The amount of acceleration sufficient for the GV separation was lowered when the oocytes were pretreated with Nocodazole and was increased by Taxol pretreatment. Observation of microtubular structures with an anti-alpha-tubulin antibody revealed the presence of a complex of spots and radiating arrays as was described by J. J. Otto and T. E. Schroeder (1984, Dev. Biol. 101, 274-281) and called the premeiotic aster. Nocodazole shortened the astral arrays, and Taxol enhanced them. These observations indicate that the premeiotic aster works as a device to hold the GV in an eccentric position just beneath the oocyte cortex.     

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.     

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.     

Maturation of the reconstructed oocytes by germinal vesicle transfer in rabbits and mice

The present study was designed to evaluate the feasibility of germinal vesicle (GV) transfer in rabbits and mice. The GV oocytes were collected from ovaries and cultured in 20 microg/mL 3-isobutyl-1-methylxanthin (IBMX) in TCM199 medium, which caused oocytes to shrink, enlarging the perivitelline space to facilitate the GV removal and transfer. Pairs of GV-cytoplast complexes were fused with electric pulses, and the fused, reconstructed oocytes were cultured in TCM199 for 24 h. Results are as follows: 1) The exposure time of rabbit GV oocytes to IBMX medium affected the success of GV removal. For oocytes cultured for 2 and 3 h in IBMX medium, removed rates were 56% and 44, respectively, significantly higher (P < 0.05) than removal rates of GV oocytes cultured for 1 and 4 h (27% and 27%, respectively); 2) There was no significant difference (P > 0.1) in fusion and maturation rates of rabbit reconstructed oocytes collected at 72 and 84 h after initiation of FSH injection to donors; 3) eCG in the maturation media improved development of rabbit-to-rabbit GV transferred oocytes but had no positive effect on mouse-to-rabbit GV transferred oocytes; 4) When mouse GV-karyoplasts were injected into enucleated rabbit oocytes, fusion rates of GV-karyoplasts measuring 40- to 50-microm and 80- to 90-microm in diameters obtained were 84% and 93%, respectively. The rates were significantly higher (P < 0.05) than fusion rates after transferring GV-karyoplasts measuring 30- to 35-microm in diameter (63%). The maturation rate (89%) of reconstructed oocytes composed of 80- to 90-microm mouse GV-karyoplasts and rabbit GV-enucleated cytoplasts was higher than that seen for oocytes composed of 40- to 50-microm (77%, P<0.05) or 30- to 35-microm (59%, P<0.01) mouse karyoplasts. Thirty-five of the 63 (56%) mature mouse-to-rabbit reconstructed oocytes had the normal complement of 20 chromosomes.     

Activation at the germinal vesicle stage of starfish oocytes produces parthenogenetic development through the failure of polar body extrusion

Starfish oocytes can be fertilized after germinal vesicle breakdown (GVBD) and artificial parthenogenesis can be induced by activating the oocytes after GVBD (post-GVBD activation). In the present study, parthenogenotes were obtained by the activation of immature oocytes with caffeine before treatment with 1-methyladenine (1-MeAde) to induce oocyte maturation. Most of the caffeine-treated eggs developed as tetraploids, as parthenogenotes produced by the post-GVBD activation. The parthenogenotes were derived only from eggs that failed to extrude polar bodies, mostly from eggs failing to extrude a second polar body. Eggs derived from immature oocytes activated by A23187, treated with 1-MeAde and post-treated with cytochalasin B failed to extrude polar bodies, and eventually developed into parthenogenetic embryos. These results indicate that the present parthenogenesis mechanism shares the same characteristics as that achieved by post-GVBD activation in the suppression of polar body formation as a key means for successful starfish parthenogenesis.     

Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase

G2-phase-arrested immature starfish oocytes contain inactive cdc2 kinase and cdc25 phosphatase, and an inactivator for cdc2 kinase. In this system, we have studied how the regulatory balance is apped toward the initial activation of cdc2 kinase. During the hormone-dependent period (Guerrier, P., and M. Doree, 1975. Dev. Biol. 47:341-348), p34cdc2 and cdc25 protein are already converted, though not fully, to active forms, whereas the inactivators for cdc2 kinase and cdc25 phosphatase are able to exhibit their activities if the hormone were removed. We produced "triggered oocytes," in which due to a neutralizing anticdc25 antibody, the activation of cdc2 kinase is prevented out cdc25 protein is phosphorylated slightly after the maturation-inducing hormonal stimulus. In contrast to control immature oocytes, in triggered oocytes the injected cdc2 kinase is not inactivated, and accordingly the level of cdc2 kinase activity required for meiosis reinitiation is much less. These results imply the presence of a cdc2 kinase activity-independent process(es) that suppresses the inactivator for cdc2 kinase and initially phosphorylates cdc25 protein, although this process is reversible during the initial activation of cdc2 kinase. At the most initial triggering of M-phase, the cdc2 kinase activity-independent process might trip the switch leading to the initial activation of cdc2 kinase. Thereafter, in parallel, the cdc2 kinase-dependent feedback loops described by others may cause further increase in cdc2 kinase activity. We propose that a putative suppressor, which downregulates the inactivator for cdc2 kinase independently of nuclear components, might be a previously unrecognized component of maturation-promoting factor.     

Concanavalin A induces germinal vesicle breakdown in Barnea candida (Mollusca,Pelecypoda) oocytes

Treatment of Barnea candida oocytes with 5 μg/ml Con A or above elicits germinal vesicle breakdown (GVBD), the timing for this event being dose dependent. At 100 μg/ml, GVBD occurs within 20 to 30 min, a lag time corresponding to that observed after fertilization. Con A-induced GVBD requires the presence of 2 mM external calcium during all the treatment period while at 10 mM external Ca²⁺, the calcium-dependent period is slightly reduced. It is sensitive to low pH Na-acetate sea water, 50 μM trifluoperazine, 20μM D-600 or 2,4-dinitrophenol, as well as to 10 μg/ml cytochalasin B. A straightforward interpretation of these data would be that Con A-induced maturation is sustained by an energy-requiring effector mechanism involving intracellular contractile proteins.     

The germinal vesicle nucleus of Xenopus laevis oocytes as a selective storage receptacle for proteins

The amorphous nucleoplasm of the germinal vesicle nucleus of Xenopus laevis oocytes has been selectively extracted under conditions which leave the nuclear formed elements morphologically intact. The nucleoplasm contains about 97% of the total nuclear proteins and on SDS- polyacrylamide gels some 68 polypeptides can be distinguished. On the basis of solubility differences, the nucleoplasmic proteins can be classified into two categories. The first consists of soluble or easily solubilized proteins which comprise about 34 polypeptides making up 87% of the nucleoplasm. A few of these proteins show electrophoretic mobilities similar to those of soluble proteins of the cytoplasm, but most are unique to the nucleus. The residual 13% of the nucleoplasmic proteins are tightly bound to a nucleoplasmic gel and can be extracted only by solubilizing the gel. The solubility characteristics of the proteinaceous gel suggest a complex held together by salt, nonpolar, hydrogen, and possibly disulfide bonding. Some 34 polypeptides can be distinguished in this gel fraction, including prominent and highly enriched polypeptides of about 115,000 and 46,000 daltons. The relatively soluble fraction of the nucleoplasm does not contain informofers and contains little or no nucleic acid. Evidence is presented that if histones are present in the germinal vesicle, they can comprise no more than about 8% of the total protein. The possibility is discussed that the unique polypeptides of the nucleoplasm may be sequestered there by selective adsorption to or in the nuclear gel.