Germinal vesicle contents are required for the cytoplasmic cycle during meiotic division of starfish oocytes

Enucleated oocytes of starfish still show cyclic changes in cortical tension with a temporal pattern similar to that exhibited by intact oocytes during meiotic division, provided that the enucleation is performed a certain time after the breakdown of the germinal vesicle (K. Yamamoto and M. Yoneda, Dev. Biol. 96, 166-172, 1983). If an oocyte is bisected immediately after germinal vesicle breakdown, the resulting nonnucleate fragment shows some change in tension, but the pattern of change is much less regular than that seen in intact oocytes, suggesting that the dispersion of germinal vesicle (GV) contents into cytoplasm is required for the establishment of the cytoplasmic cycle. In order to demonstrate the role of GV contents directly, nonnucleate fragments derived from immature oocytes were injected with GV contents taken from other immature oocytes. On treatment with 1-methyladenine (1-MA) these fragments showed two rounds of increase in tension as is characteristic of intact maturing oocytes. The first rise in tension was always observed 50-70 min after the treatment with 1-MA, similar to the time of first polar body formation in intact oocytes, regardless of the time of injection of GV contents. Even when GV contents were injected into nonnucleate fragments which had been already treated with 1-MA, these fragments showed two rounds of change in tension. The timing of the first rise in tension was found to be 38 +/- 7 min after injection, irrespective of the time of the foregoing treatment with 1-MA. These results prove the indispensability of GV contents for inducing the cytoplasm of the maturing starfish oocyte to initiate its own cyclic activity, and suggest that the normal process of cytoplasmic maturation may consist of two phases, i.e., (1) a GV-independent phase initiated by 1-MA treatment, and (2) a second phase initiated by mixing of GV contents with cytoplasm.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

Breakdown of starfish ovarian follicle induced by maturation-promoting factor

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.     

INHIBITION OF GERMINAL VESICLE BREAKDOWN AND POLAR BODY FORMATION BY NICOTINAMIDE IN STARFISH AND SURF CLAM OOCYTES*

Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD⁺was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF.     

Initiation of cleavage in starfish eggs by the injection of triton-treated spermatozoa

Triton X-100-treated spermatozoa were injected into immature (fully grown, germinal vesicle stage) or mature (pronuclear stage) oocytes of the starfish, Asterina pectinifera, to study relation between initiation of cleavage and cortical reaction. Immature oocytes into which Triton X-100-treated spermatozoa were injected were treated with 1-methyladenine. Such immature oocytes initiated cleavage after completion of meiosis without formation of the fertilization membrane. The same results were obtained when Triton X-100-treated spermatozoa were injected into mature oocytes. Control oocytes into which only calcium-free sea water was injected did not cleave. These results indicate that the initiation of cleavage is independent of the cortical reaction but dependent on the existence of spermatozoa (spermatozoon) in the egg cytoplasm.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.     

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-beta-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobic chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 X 10(3) D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.     

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.     

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.     

Role of Contents of the Germinal Vesicle in Male Pronuclear Development and Cleavage of Starfish Oocytes

During 1-methyladenine induced germinal vesicle breakdown, contents of the germinal vesicle of starfish oocytes are mixed with the surrounding cytoplasm. Upon injection of contents of the germinal vesicle from immature (fully grown) oocytes into enucleated and inseminated oocytes, incorporated spermatozoa were not observed to change structurally. Alternatively, after treatment of the above oocytes with 1-methyladenine, sperm asters and male pronuclei were developed and subsequent cleavage was also detected. From these results, it is concluded that both action of 1-methyladenine and participation of contents of the germinal vesicle are indispensable for male pronuclear development and subsequent cleavage.     

Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish

Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.     

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.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.     

Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation.

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.     

Oocyte Competence to Maturation-inducing Hormone. I. Breakdown of Germinal Vesicles of Small Oocytes in Starfish,Asterina pectinifera*

1-Methyladenine (1-MeAde) is the endogenous maturation-inducing substance (MIS) in starfish. However, small oocytes have no competence to 1-MeAde even at the concentration of 10^?5M. Furthermore, when they were injected with cytoplasm of fully-grown (large) and maturing (1-MeAde-treated) oocytes, known to contain maturation-promoting factor (MPF), they did not undergo germinal vesicle breakdown (GVBD). On the other hand, germinal vesicles (GV) of the small oocytes underwent nuclear breakdown when the small oocytes were fused with the large maturing oocytes. Therefore it is concluded that the GV of the small oocytes are capable of undergoing nuclear breakdown in the presence of the sufficient MPF, but that the small oocytes can not amplify the injected MPF. Fused cells displayed particular shape changes during the course of nuclear breakdown of both the large and the small oocytes.     

Inhibition of starfish oocyte maturation by some inhibitors of proteolytic enzymes

Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

Injection of isolated centriolar complex to induce mitosis in starfish eggs

When centriolar complex isolated from starfish spermatozoa was injected into starfish immature oocytes (fully grown, germinal vesicle stage) followed by treating the latter with 1-methyladenine, mitotic events such as condensation and division of chromosomes of the female pronucleus and cytokinesis following completion of meiosis were observed. No cortical reaction detected in the oocytes. Essentially the same was noted for mature oocytes (pronuclear stage) into which the centriolar complex had been injected. The oocytes that had received sperm tail fraction or buffer alone did not initiate cleavage. It would thus appear that sperm centriolar complex is significantly essential to the initiation of cleavage.     

EFFECT OF LOCAL APPLICATION OF 1-METHYLADENINE ON THE SITE OF POLAR BODY FORMATION IN STARFISH OOCYTE*

Mechanism by which the site of polar body formation is determined in starfish oocytes was investigated in relation to the action of 1-methyladenine (1-MeAde). Local staining with Nile Blue of Asterina pectinifera oocytes revealed that there exists a prospective site of polar body formation (PSPBF) on the nearest surface to the position of germinal vesicle. The site of polar body formation was found to shift to some extent from PSPBF toward the area locally applied with 1-MeAde, suggesting that the actual site of polar body formation is not determined yet at the germinal vesicle stage. Oocytes whose germinal vesicles had been shifted by centrifugation from PSPBF to the opposite surface before the commencement of germinal vesicle breakdown (GVBD) (less than 15 min after 1-MeAde treatment), failed to form polar bodies, whereas oocytes centrifuged after commencement of GVBD (20 min after 1-MeAde treatment) did form polar bodies where their fading germinal vesicles had reached by centrifugation. In the oocytes which failed to form polar bodies by centrifugation, an aster was observed near PSPBF of each oocyte. When inseminated, every oocyte treated with 1-MeAde developed normally irrespectively of the mode of polar body formation including the site and the occurrence, and the animal pole of every larva was derived from PSPBF.