Setsuro Hirai

Fast polyspermy block and activation potential: Correlated changes during oocyte maturation of a starfish☆

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.

ROLE OF FOLLICLE CELLS IN MATURATION OF STARFISH OOCYTES

The role of the follicles in oocyte maturation was studied with the starfish, Asterina pectinifera. Follicles, obtained from isolated oocytes with follicular envelopes, produced maturation-inducing substance (MIS) when incubated in sea water containing gonad-stimulating substance (GSS). Isolated oocytes without follicular envelopes were found to have no such capacity to produce MIS even in the presence of GSS. MIS produced by follicles under the influence of GSS was extracted with 95% ethanol and purified on a Sephadex G-15 column. This MIS was identified as 1-methyladenine with thin layer chromatography, showing that the follicles can produce 1-methyladenine under the influence of GSS. MIS was also produced by follicles when they were incubated in sea water containing 1-methyladenosine in the absence of GSS. This indicates that I-methyladenosine ribohydrolase, which splits l-methyladenosine to 1-methyladenine and ribose, is present in these follicles. The capacity of follicles to produce MIS under the influence of GSS was found to depend on the age of oocytes from which the follicles were isolated; follicles taken from young oocytes failed to produce MIS even in the presence of GSS. 1-methyladenosine ribohydrolase was found to be present even in young follicles, although its activity was lower than that of the follicles of full-grown oocytes.

Achromosomal cleavage of fertilized starfish eggs in the presence of aphidicolin

Abstract Development of fertilized eggs of starfish, Asterina pectinifera , was studied in the presence of aphidicolin, a specific inhibitor of eukaryotic DNA synthesis. Fertilized eggs continued to cleave eight or nine times in the presence of aphidicolin, although the delay of development was observed and the size of blastomeres was not equal. The nucleus and chromosomes were undetectable in blastomeres of embryos developed in the presence of aphidicolin, although the amphiaster was clearly visible in the cytoplasm. DNA synthesis of such embryos was nearly completely suppressed. These morphological and biochemical lines of evidence indicate that the above cleavage is independent of chromosomal replication. The stage when achromosomal divisions cease and embryos begin to die corresponds to the onset of blastulation, suggesting that information derived from the embryo genome is required for blastula formation in this species.

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.

Cytoplasmic Maturity Revealed by the Structural Changes in Incorporated Spermatozoon during the Course of Starfish Oocyte Maturation

Immature oocytes of the starfish, Asterina pectinifera, are polyspermic. Spermatozoa can enter immature oocytes upon insemination, but the changes associated with the fertilization process in oocytes matured with 1‐methyladenine (1‐MeAde), such as the formation of aster and pronucleus, were not observed. After immature oocytes, previously inseminated, were matured with 1‐MeAde, the formation of the sperm monaster was observed during germinal vesicle breakdown (GVBD). Amphiasters and pronuclei were formed after the formation of the second polar body. The acquisition by oocytes of the capacity to undergo the normal process of fertilization, therefore, occurs during the course of oocyte maturation. After injection of the cytoplasm of maturing oocytes into inseminated immature oocytes, the formation of aster and pronucleus was observed, suggesting that maturation‐promoting factor (MPF) may be involved in establishing the cytoplasmic conditions (cytoplasmic maturity) necessary for the fertilization process to occur. In contrast, when enucleated, inseminated halves of immature oocytes were treated with 1‐MeAde, only monasters were formed, while in the nucleated halves both amphiasters and sperm pronuclei were formed. Thus, germinal vesicle material is required for the formation of amphiaster and sperm pronucleus but not for the formation of monaster. It is possible that the amount of MPF produced in enucleated halves was sufficient only for the formation of the monaster but not for the formation of the amphiaster and pronucleus, since it has been previously established that germinal vesicle material is necessary for the amplification of MPF. The formation of the monaster in the enucleated halves at a time corresponding to GVBD in nucleated controls suggests that the amount of MPF needed for this event is rather small. For the induction of subsequent fertilization process, large amounts of MPF may be required to establish the necessary cytoplasmic conditions, although other possible role of nuclear material is not excluded.

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.

SITE OF 1-METHYLADENINE RECEPTORS IN THE MATURATION OF STARFISH OOCYTES

Maturation of vitelline coat‐free (VCF) oocytes of the starfish, Asterina pectinifera, was studied. When the oocytes, the vitelline coats of which were elevated by adding the ionophorc A‐23187, were forced through two sheets of copper mesh, the vitelline coats were completely removed from the oocytes. Although some of the VCF oocytes underwent germinal vesicle breakdown following this mechanical treatment, most of them retained the normal germinal vesicles. These VCF immature oocytes underwent breakdown of germinal vesicles after addition of 1‐methyladenine (1‐MA). Dose‐response curves of VCF oocytes to 1‐MA were similar to those of normal oocytes. These results indicate that 1‐MA reacts with the plasma membrane and that the presence of the vitelline coat is not prerequisite for inducing oocyte maturation.

Bioelectric responses of sea urchin eggs inseminated with oyster spermatozoa: a sperm evoked potential without egg activation.

Multiple oyster spermatozoa can enter sea urchin eggs with or often without fertilization membrane formation (Osanai and Kyozuka, 1982). In the present work, electrical responses of sea urchin (Temnopleurus hardwicki) eggs inseminated with oyster (Crassostrea gigas) sperm were examined and correlated to the failure of monospermy and egg activation. With diluted sperm, a transient depolarization of the membrane with a constant pattern appeared repeatedly and discretely, and the depolarizations (sperm evoked potentials, SEPs) were not associated with fertilization membrane elevation. With dense sperm, the SEPs occurred consecutively, and sometimes an assembled consecutive depolarization was followed by an activation potential associated with cortical granule discharge. When the membrane potential was artificially held at positive levels, the frequency of SEPs was strongly suppressed but not completely blocked. The present results indicate that an individual heterologous spermatozoon neither produces a depolarization sufficient to block additional sperm entry, nor stimulates egg activation, and that simultaneous entries of multiple heterologous spermatozoa, as possibly reflected by the assembled consecutive depolarizations, induce cortical granule discharge and egg activation.