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Department of Biology, St. Mary’s College of Maryland, St. Mary’s City, Maryland 20686, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Much of what we know about cephalopod development is drawn from observing embryos in, or dissected from, naturally laid jelly strings or egg capsules (1,2). Although embryos extracted from jelly strings survive in culture for short periods (3), reliable in vitro fertilization and culture would greatly enhance our ability to study the molecular and cellular events of fertilization and development, including pronuclear migration (4), cleavage (5), cytoplasmic movements (6), differentiation, and organogenesis. When in vitro fertilized embryos are cultured in glassware or plastic petri dishes, their development is typically arrested at the early blastoderm stage (<48 h). Embryos fertilized in vitro may be successfully cultured on cushions of 0.2% agarose in Millipore (0.22 µm) filtered seawater (MFSW) (Sigma, Type II) (7), but their chorions do not reliably swell to permit complete development or hatching.
Recently, a considerable advancement in culture methodology was reported for in vitro fertilized embryos of the argentine short-fin squid, Illex argentinus (8). In this study, embryos cultured in plastic petri dishes filled with seawater alone died after 3 days, while those cultured in seawater supplemented with fresh or freeze-dried oviductal gland jelly underwent sufficient chorion expansion to support normal development and hatching. It is worth mentioning that for this to occur, the culture medium was changed at 3-h intervals throughout the 6–7-day culture period (15 °C). In comparison, Loligo pealeii embryos require 20–21 days to develop at 17 °C, and therefore the goal of this study was to develop an alternative culture method for routinely obtaining in vitro fertilized cephalopod hatchlings. This report describes an in vitro fertilization method, in addition to the effects of a commercially available sera and serum protein on chorion expansion and embryonic development. Embryos from some of these cultures undergo significant chorion expansion, develop normally, and after 20–21 days in culture (17 °C) hatch independently.
In vitro fertilization. Clear mature eggs from the oviduct of a cold-anesthetized dissected female were collected and washed several times with well-aerated MFSW. Next, spermatophores (20–30) from the spermatophore duct of a dissected, anesthetized male were placed in 15 ml MFSW in a syracuse dish and the sperm were discharged with slight pressure. To this sperm suspension, approximately 1500–2000 eggs were added in concentrated drops (
100 embryos/drop). After 15 min, the fertilized eggs were washed several times in MFSW to remove sperm and spermatophore casings. Rate of fertilization (typically 85%–95%) was determined following first and second polar body formation (20 min and 1.5 h, respectively) and first cleavage (3.5 h).
Embryo culture. To enhance embryonic development, either horse serum (HS) (Sigma) or bovine serum albumin (BSA) (ICN) was added to the MFSW culture. In each case, 20–40 embryos per dish were cultured in 60-mm plastic petri dishes (Falcon) lined with a cushion of 0.2% agarose (Type II-A, Sigma) in MFSW. Dishes and solutions were changed every other day, and dead or damaged embryos were removed. Control embryos cultured in MFSW alone, although alive, do not undergo chorion expansion and fail to develop normally or hatch. Adding 0.2% or 0.5% HS to the culture resulted in the reduction or loss of the perivitelline space (the area between the embryo and inner chorion surface), possibly due to osmosis. Embryos cultured in lower concentrations of HS/MFSW (0.01%, 0.05%, and 0.1%), grew well initially, underwent some chorion expansion, but failed to complete development or hatch. In striking contrast, embryos from six independent trials cultured in BSA (0.1%, 0.2%, or 0.5% dissolved into MFSW and refiltered) developed normally and underwent gradual chorion expansion from 1600 µm (egg chorion length at fertilization) to measure an average of 1800, 2000, and 3000 µm (100 embryos/concentration), respectively, after 21 days in culture (Fig. 1a, b). Moreover, embryos cultured in 0.5% BSA/MFSW nearly always completed development to hatch independently after 20–21 days in culture (Fig. 1c). Although the majority of embryos (53%–91% from 100 embryos/trial x 6 trials) cultured in 0.5% BSA/MFSW develop normally in a synchronous fashion, and proceed through similar or identical developmental stages when compared to embryos in naturally laid jelly strings, it is important to note that there was variability among these cultures. Perhaps, when the method of Sakai and Brunetti (8) is considered, increasing the frequency of embryo transfer to fresh culture dishes and media might improve the overall percentage of normal embryos observed in BSA/MFSW cultures.
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Recently, homeobox gene clusters were identified from tissues of adult cephalopods (9), and the role of these genes in early neurogenesis is being explored using embryos harvested from jelly strings (10). Culturing squid embryos in vitro creates new opportunities to study and understand cephalopod development. While in vitro fertilization enables one to control parentage, culturing embryos away from their tenacious jelly allows for the study of individual embryos, simplifies stage-specific embryo collection for immunohistochemistry and in situ hybridization analysis, and permits the embryo to be microinjected with lineage-tracing dyes or factors designed to inhibit or enhance development. Moreover, embryo culture enables us to treat cephalopod embryos with chemical agents, such as lithium chloride (11) or retinoids (12), that have been shown to predictably alter development in other organisms, thereby enhancing our ability to work with and understand the embryos of this evolutionarily old and successful group of organisms.
This work was supported by the Marine Biological Laboratory Associates Fellowships, The James A. and Faith Miller Memorial fund, and The Evelyn and Melvin Spiegel Fellowship Fund to Karen Crawford.
Literature Cited
This article has been cited by other articles:
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  P. H. Wadeson and K. Crawford Formation of the Blastoderm and Yolk Syncytial Layer in Early Squid Development Biol. Bull., October 1, 2003; 205(2): 179 - 180. [Full Text] [PDF]
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K. Crawford Lithium Chloride Inhibits Development Along the Animal Vegetal Axis and Anterior Midline of the Squid Embryo Biol. Bull., October 1, 2003; 205(2): 181 - 182. [Full Text] [PDF]
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