Early development of the horseshoe crab, Limulus polyphemus, L.: 1) ultrastructure of the cortical reaction and amino acid incorporation during egg activation, and 2) ultrastructure and protein synthesis of the extra-embryonic shell
Date of Award
Doctor of Philosophy
Egg activation in the horseshoe crab, Limulus polyphemus L., sets into motion many structural and metabolic changes. During the first 60 min after sperm-egg attachment, the cortical reaction takes place. On the fertilized egg surface the cortical reaction results in the formation of large pits which are formed by the fusion of cortical vesicles with the plasma membrane. Cortical vesicles were grouped into two classes by virtue of their size and position in the cortex of the unfertilized egg. At fertilization the small vesicles fuse with the closely apposed plasma membrane. Large vesicles, which were observed in the lower portion of the cortex of the unfertilized egg, fuse with the plasma membrane, dumping their contents into the perivitelline space and form pits on the fertilized egg surface. Fusion of large vesicles continues for approximately 60 min after fertilization, when the surface becomes smooth again;Parameters of amino acid incorporation and intracellular pH (pH(,i)) were determined for the first 14 hr of development. Intracellular pH remained constant (7.69) from 60 min - 14 hr after fertilization, but amino acid incorporation exhibited a 5-50 fold increase beginning at 8 hr. The increase in incorporation rate was inhibited by artificially lowering the pH(,i) (pH(,i) 6.77) before the increase had occurred. However, amino acid incorporation was not inhibited when pH(,i) was acidified (pH(,i) 6.77) after the increase had occurred. These results indicate that protein synthesis is pH sensitive before the increase in amino acid incorporation, but becomes pH insensitive after the increase in incorporation rate has occurred;The synthesis of a specific set of proteins (extra-embryonic shell (EES) proteins) was investigated during egg activation and later in development. EES proteins were synthesized for the 10 days following fertilization. The highest percentage of total TCA precipitable radioactive counts accumulated during one day occurred during the first 24 hr after fertilization. Of the 17 EES protein bands observed, 15 were synthesized after fertilization. This coupled with calculations of possible synthetic rate of EES proteins during this 10 day period, strongly suggests that the majority of EES proteins are made either during oogenesis or in the unfertilized egg;The EES represents a second protective shell in which the embryo continues to develop after sloughing the egg envelope. The ultrastructure of the EES revealed that it consisted of three discernible layers. The outer third of the EES exhibited large indentations representing a source of preformed surface area utilized when the EES expands;The research detailed in this dissertation indicates that Limulus is an excellent system in which to study egg activation. Future work which is critical to the development of this system into an important research tool is the delineation of nuclear events from fertilization through blastulation and the development of conditions which do not allow for spontaneous activation of eggs immediately after spawning.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Gary Anthony Bannon
Bannon, Gary Anthony, "Early development of the horseshoe crab, Limulus polyphemus, L.: 1) ultrastructure of the cortical reaction and amino acid incorporation during egg activation, and 2) ultrastructure and protein synthesis of the extra-embryonic shell " (1981). Retrospective Theses and Dissertations. 6870.