Achromosomal cleavage of fertilized starfish eggs treated with cyclic guanosine 3′,5′‐monophosphate

Abstract

Cyclic guanosine 3',5'-monophosphate (cGMP) was injected into fertilized starfish eggs in order to study the effects of this cyclic nucleotide on early embryogenesis. cGMP at approximate intracellular concentrations higher than 0.05 mM causes achromosomal cleavage in the presence of 1 mM caffeine, that is, each blastomere is devoid of a nucleus but not spindle and asters. The treated embryos cleave slower than control, never form blastulae, and disintegrate just before blastulation. CAMP, dibutyryl cGMP and dibutyryl CAMP failed to cause achromosomal cleavage. These results suggest that unphysiologically high levels of intracellular cGMP inhibit in vivo DNA synthesis of fertilized starfish eggs.Aphidicolin is an inhibitor of eukaryotic DNA polymerase CY and inhibits replicative DNA synthesis in eukaryotes (lkegami et al., '78; Oguro et al., '79; Wist and Prydz, '79; Huberman, '81). Fertilized echinoderm eggs whose replication is inhibited by this drug cleave achromosomally (Nagano et al., '81; Yamada et al., '85; Hirai et al., '84), i.e., each blastomere is devoid of a nucleus or chromosomes but not spindle and asters. AphidiColin-treated embryos never form blastulae and disintegrate before blastulation.It has been reported that cGMP inhibits the DNA polymerase CY activity of rat liver and yeast (Eckstein, '81). So, we investigated the effects of cGMP on early embryogenesis of the starfish by the microinjection method. The results show that cGMP also caused achromosomal cleavage in fertilized starfish eggs. MATERIALS AND METHODSThe starfish, Asterina pectinifera, were collected during their breeding season at Hashirimizu (Kanagawa) and Asamushi (Aomori) and were kept in laboratory aquaria supplied with running sea water. Maturing oocytes were obtained from ovarian fragments by treatment with M 1-methyladenine (1-MeAde). 1-MeAde (Sigma, USA) was dissolved in distilled water at a concentration of 5 mM and diluted with sea water before use. Spermatozoa were obtained from isolated testis and diluted with sea water. Histidine was added to the sperm sus ension to give a final concentration of lo-' M in order to increase their motility. The oocytes were inseminated in normal sea water 40 min after 1-MeAde treatment and allowed to develop at 20°C. Fertilized eggs were transferred to sea water containing 1 mM caffeine when the first polar body was expelled (65 min after the 1-MeAde treatment). In experiments involving microinjection of reagents into fertilized eggs, injection was carried out in the period between the appearance of the first and the second polar bodies (Fig. 1). The injected oocytes were allowed to develop in the presence of 1 mM caffeine at 20°C. Microinjection was carried out according to the method of Hiramoto ('74). The amount of reagent solutions injected into an oocyte was 100 pl. This volume is approximately 1/40Address reprint requests to