185 Ultrastructure of staghorn coral (Acropora cervicornis) oocytes following spawning and fertilization

Significant work is being undertaken globally on the conservation of coral reefs that are in peril because of climate change, disease pressures, and other threats. Ocean warming can cause coral bleaching that has significant negative effects on coral reproduction including fewer and smaller oocytes following bleaching. The objectives of this study were (1) to elucidate the ultrastructure of coral oocytes to provide a baseline for gamete health that can be used for comparison following events such as coral bleaching, providing information on potential impacts on coral reproduction; and (2) to investigate whether a cortical reaction can be discerned following presumed fertilization. Acropora cervicornis sperm and oocytes, released as gamete bundles, were collected during an annual spawning event in a non-bleaching year and allowed to break down and disperse as oocytes and sperm. Gametes were separated and oocytes washed three times in filtered seawater. Fertilization experiments were conducted with oocytes and sperm from different genotypes, as self-fertilization does not occur in A. cervicornis in the Keys region. Three different genotypes were sampled to generate a pool of gamete bundles (n = 1; 100 μL), unfertilized oocytes (n = 1; 100 μL; 0 min), and putative embryos (n = 1; 100 μL) collected at 2, 4, 6, 20, 30, 40, 50, and 60 min post-fertilization. Samples were fixed in 0.9 mL of 4% glutaraldehyde and stored at 4°C until examination by transmission electron microscopy (TEM). A total of 15 oocytes/putative embryos were examined, and 133 TEM images were reviewed; the images revealed the presence of endoplasmic reticulum and Golgi apparatus for protein and lipid biosynthesis. Oocytes (~530–540 μm in diameter) contained multiple spherical lipid droplets (5- to 7-μm diameter) surrounded by a cortex containing multi-layers of highly electron-dense cortical vesicles (1- to 2-μm diameter) preceding a multi-layer of similar sized moderately electron-dense cortical vesicles. Microvilli extended from the oocyte surface, and the whole was enclosed within a vitelline layer ~2 μm thick. Following presumed fertilization, the cortical vesicles underwent exocytosis releasing exudate. No sperm were visible in the oocyte vicinity at 2–6 min, but multiple sperm were present at the edge of the vitelline layer after 20 min and sperm were similarly observed in the same location at time points of 30–60 min. The surface of the oocyte was initially uniform and regular (0–6 min), but by ≥ 20 min it was more disrupted and irregular, with areas of exudate that contributed to an increasing depth in vitelline layer (5–10 mm) over the next ~60 min. The outer layer of moderately electron-dense vesicles moved outwards toward the edge of the oocyte, releasing contents until they were mostly depleted after ~60 min. Results indicate dynamic secretory events occurred following presumed fertilization that were analogous to the cortical reaction and likely mitigated polyspermy. The presence of multiple spherical lipid droplets and overall size of the oocyte are likely to aid embryo development and might contribute to the success of the development of free-swimming larvae and eventual larval settlement. Future studies will examine oocyte ultrastructure following a bleaching event to determine quantifiable morphometrics. Results described here document for the first time the presumed cortical reaction in fertilized A. cervicornis oocytes occurring between 6 and 20 min. Further investigation of additional time points will narrow down this timing. Understanding basic physiologic reproductive events in A. cervicornis is expected to provide useful information for in situ and ex situ conservation studies.