Masters Thesis

Partial Characterization of the Chorion and Choriolytic Enzyme of the California Grunion (leuresthes Tenuis).

Adult California grunion, Leuresthes tenuis, spawn on sandy beaches during spring high tides. Embryos develop within the sand enclosed in the chorion, which must be strong enough to protect the embryo but also allow rapid hatching. Grunion embryos are competent to hatch at 8 days post-fertilization (dpf) at 20°C, but require agitation in seawater to hatch, and can delay hatching up to 30 dpf. I hypothesized that chorions of fertilized eggs would require more force to break than chorions of unfertilized eggs because of hardening of the chorion at fertilization, and that chorions would require more force to break during normal incubation (10 dpf) than after extended incubation (28 dpf) due to degradation over time. Gametes were collected from grunion in March-July 2013- 2015. Eggs were fertilized and embryos were incubated in the laboratory at 20°C for up to 30 dpf. Mean crushing force (N) did not differ significantly between unfertilized and fertilized eggs except for 1 of 9 collection dates when it was significantly greater in fertilized eggs. Thus, the hardness of grunion chorions apparently does not increase at fertilization. Mean crushing force did not differ significantly between 10 dpf and 28 dpf but decreased significantly over the 28 days of incubation in 7 of 14 clutches. I used hydrophobic interaction column chromatography to partially purify the choriolytic enzyme, which allows the embryo to hatch from the chorion, and enzyme kinetics parameters were estimated. The Vmax was 0.045 ± 0.003 ΔA280 min-1 mg-1 protein and the Km value was 0.652 ± 0.111 mg casein ml-1 .

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