The Mechanism of Sperm Incorporation and an Analysis of Microvilli and Microfilament Organization in Sea Urchin Eggs Examined Through the Use of Cytochalasin B and Ammonium Chloride.

Abstract

Sperm incorporation in Lytechinus pictus and Strongylocentrotus purpuratus eggs incubated (10 min) and fertilized in the presence of 2 x 10('-6) M cytochalasin B (CB) and 1 and 10 mM ammonium chloride (NH(,4)Cl) occurs in the presence of limited microvilli and microfilament organization and cortical exocytosis. CB inhibition on sperm incorporation is time related and continues to occur long after sperm-egg fusion. In S. purpuratus, at least 30% incorporation is observed in all CB treated eggs. Sperm do not incorporate until 2.5 min after insemination. Maximum incorporation occurs between 7 to 10 min. Percent incorporation approached 60% when eggs were incubated and fertilized in the presence of combined mixtures of CB and NH(,4)Cl. In L. pictus, sperm incorporation is not seen until 30 min postinsemination and declines from a maximum of 18% to 3% at 50 min postinsemination. In all continuous CB samples in which eggs were reincubated in fresh CB following sodium dodecyl sulfate (SDS) addition and dilution for 60 min, sperm did not migrate beyond the egg cortex. Cortical reactions and chromosome condensation were not seen in eggs reincubated in fresh sea water or test media for at least 60 min. The time patterns of percent incorporation suggest that sperm remain in the cytoplasm and do not undergo pronuclei fusion and condensation. Morphological transmission electron microscopic and scanning electron microscopic comparisons of CB treated fertilized whole eggs and egg cortices decorated with heavy meromyosin (HMM) reveal short (0.36 to 0.38 (mu)m) microvilli and disorganized actin microfilaments. Eggs incubated and fertilized in the presence of a mixture of CB and NH(,4)Cl exhibit features transient between CB inhibition and normal responses. At 15 min of insemination, microvilli of CB and NH(,4)Cl treated eggs elongate to 0.63 (mu)m, approximately one-half the length of untreated control eggs at 10 min postinsemination. Comparatively, isolated cortices exhibit some organization of HMM decorated actin complexes. CB inhibition is also seen in the maintenance of high G-actin pools. Comparative analysis of G-actin and F-actin pools as shown by DNA hydrolysis and SDS-polyacrylamide gel electrophoresis reveals increased percent DNAase 1 inhibition by G-actin and relatively low DNA hydrolysis in eggs incubated and fertilized in the presence of CB and/or CB and NH(,4)Cl. Sperm incorporation in the presence of CB and in the absence of an organized microfilament system is suggestive of an alternate mechanism of incorporation.