Structure of Shell Membranes and Water Permeability in Eggs of the Chinese Soft-Shelled Turtle Pelodiscus Sinensis (Reptilia: Trionychidae)
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Current Herpetology 23 (1):1-6, June 2004 (c)2004 by The Herpetological Society of Japan Structure of Shell Membranes and Water Permeability in Eggs of the Chinese Soft-shelled Turtle Pelodiscus sinensis (Reptilia: Trionychidae) NORIO YOSHIZAKI1*, OSAMU DOI2, AND NORIHIKO UTO3 1Department of Biological Diversity, Faculty of Agriculture, Gifu University, Gifu 501-1193, JAPAN 2Department of Biological Resources and Production, Faculty of Agriculture, Gifu University, Gifu 501-1193, JAPAN 3Department of Biology, School of Medicine, Hamamatsu University, Hamamatsu 431-3192, JAPAN Abstract: The shell membrane of a Chinese soft-shelled turtle egg acts as a bag enclosing egg white and water. The main body of the shell membrane is a meshwork of fibers, and is organized into an inner and an outer membrane. The limiting membrane demarcates the shell membrane at the interface with the egg white. Transmission electron microscopy revealed that the limiting mem- brane has a width of 362nm and consists of dense materials. There were many canals that run perpendicularly or obliquely to the plane of the membrane. Scanning electron microscopy showed many holes, 20nm in size, on the inner surface of the membrane. The water permeability of the shell membrane was 5nl/mm2 per min. Neither the ultrastructure nor water permeability changed throughout the incubation period of 54days. No significant difference was observed between membranes of the embryonic side and yolk side of individual eggs. These results are compared with those of corresponding observations of avian eggs. Key words: Chinese soft-shelled turtle; Shell membrane; Limiting membrane; Ultrastructure; Water permeability a component of the shell membrane which INTORODUCTION retains the water in the yolk and egg white in For eggs that are laid on land, it is important the early stages of development. However, at that a certain amount of water be maintained the late stages of development, avian embryos around the embryos to protect them from need to take up calcium from the eggshell for drying out. In addition to the outermost egg- bone formation (Johnstone and Comar, 1955; shell, avian eggs possess a limiting membrane, Simkiss, 1961; Ono and Wakasugi, 1984) and need to dry out for initiation of breathing. To * Corresponding author . Tel: +81-58-293-2853; achieve this, avian embryos produce a thin Fax: +81-58-293-2853; limiting membrane with an as yet unknown E-mail address: [email protected] agent, thus enabling acceleration of water 2 Current Herpetol. 23 (1) 2004 permeation, and the parent birds turn the apparatus, HCP-1 (Hitachi, Japan), coated eggs to expand the area of the limiting with a layer of gold in an IB-3 ion coater membrane that contacts the agent-producing (Eiko, Japan), and examined under a model S- cells (Yoshizaki and Saito, 2002). 4300 scanning electron microscope (Hitachi, Oviparous reptiles spawn eggs in a wide Japan). range of environments, from wet habitats to For the determination of water permeability, dry land. It is not clear how the water balance the shell membrane was treated as reported inside the egg is controlled and how calcium is previously (Yoshizaki and Saito, 2002). Briefly, taken up from the eggshell (Packard et al., the shell membrane was removed from the 1984) or the environment. Most studies in this emptied eggshell by treatment with 0.5N HCl area have focused on the structure of the outer for 3min. After being washed repeatedly eggshell (Solomon and Baird, 1976; Packard with distilled water, the shell membrane was and Packard, 1979; Packard, 1980; Packard et attached, with its limiting membrane facing al., 1979, 1982, 1984; Kitimasak et al., 2003). inwards, to the lower end of a glass tube The present study shows the structure and (5.5mm in diameter) with a cyanoacrylate nature of the shell membranes in commercially glue, AlonAlpha (Konishi Co., Japan). The available eggs of the Chinese soft-shelled glass tube was filled with 1.5ml of distilled turtle (Pelodiscus sinensis), and compares water and then covered at its upper end with a them with those in Japanese quail (Coturnix paraffin film in which a pinhole (0.5mm in japonica) eggs. diameter) was made. These preparations were set in an incubator at 39C with 60% humidity for 24h. The incubation temperature adopted MATERIALS AND METHODS was determined to compare the results with Fertilized eggs of Chinese soft-shelled turtles those for quails (Yoshizaki and Saito, 2002). were purchased from a dealer in Hamamatsu The amount of water that evaporated through during July and August. These eggs were the membrane was determined by measuring embedded in styrene foam boxes filled with the distance the meniscus moved. wood shavings to which moisture was occa- Data were analyzed with two-way nested sionally added, and incubated at 30C, as analysis of variance (ANOVA). Statements of recommended by Tokita and Kuratani (2001). significance were based on P≦0.01. Development of the eggs measured in days of incubation and embryonic development at RESULTS each day was confirmed according to the normal table of Tokita and Kuratani (2001). Water permeability of the shell membrane After embryos and egg whites were removed, The water permeability of the shell mem- the shells and shell membranes were immersed brane was measured based on the evaporation in 2.5% glutaraldehyde in 0.1M cacodylate of distilled water through membranes from buffer at pH7.2. The isolated shell membranes eggs incubated for 0, 6, 8, 23, 28, 31, and were further fixed in the same solution for 3h. 54days. The average water permeability was The specimens were rinsed in the buffer, 5nl/mm2 per min.There were no significant postfixed with 1% osmium tetroxide in the differences in this parameter among mem- same buffer for 1h, dehydrated in acetone, branes regardless of the incubation period and embedded in epoxy resin for transmission (F(6,6)=5.54; P=0.028) (Fig. 1). The differ- electron microscopy. Thin sections were stained ence was not significant, either, between with uranyl acetate and lead citrate. For membranes facing the embryo proper (embry- scanning electron microscopy, specimens were onic side) and those facing the yolk (yolk side) dehydrated in acetone, dried in a critical point (F(1,6)=0.735; P=0.424) (Fig. 1). YOSHIZAKI ET AL. -SHELL MEMBRANE OF TURTLE EGG 3 canals in the limiting membrane, which run perpendicularly or obliquely to the plane of the membrane (Fig. 3D). When observed from the inside with the scanning electron micro- scope, the membrane was seen to have many holes (Fig. 2D). The largest holes were 20nm in diameter. This ultrastructural feature was observed in all eggs regardless of incubation period. DISCUSSION FIG. 1. The width of limiting membranes and The thickness of the limiting membrane of the water permeability of shell membranes on the quail eggs gradually decreased from 74nm to embryonic (open symbols) and yolk (closed 35nm with development. Conversely, water symbols) sides in eggs of the Chinese soft-shelled turtle during incubation. Values are means of five permeability through the shell membrane increased from 4 to 5nl/mm2 per min, to 9nl/ experiments. Vertical bars denote ranges of SD. mm2 per min at 39C (Yoshizaki and Saito, 2002). These changes in the Aves are thought Morphological observations of the shell to satisfy the requirements for gas exchange membrane and for the mobilization of calcium from the The shell membrane which is located between eggshell. In unturned eggs, both the decrease the calcareous shell and the albumen had a in the thickness of limiting membranes and the thickness of about 100μm (Fig. 2A). It was increase in water permeability are delayed on organized into an inner shell membrane, the yolk side. Egg turning, which is needed in adjacent to the albumen, and an outer shell most avian eggs for normal embryonic devel- membrane, adjacent to the mineral layer. The opment to take place, is understood to expand main body of the shell membrane was a the area of the limiting membrane in contact meshwork of intersecting fibers (Fig. 2B, C) with the enzyme-producing cells, thus enabling consisting of two components in cross section the thickness of the membrane to be suffi- (Fig. 3A, C), the medulla and cortex, which ciently and equally reduced over the whole randomly run in the plane of the membrane. surface (Yoshizaki and Saito, 2002). The fibers in the outer portion of the inner The rigid-shelled eggs of chelonians, such membrane fused via their cortex, thus forming as those of the Chinese soft-shelled turtle, a flat shape (Fig. 2B, 3B). At its interface with provide a great deal of resistance to the the egg white, the shell membrane was demar- movement of water into and out of eggs cated by a layer of dense material called the (Packard et al., 1979), and as a result, such limiting membrane (Fig. 3C). We measured eggs are virtually insensitive to variations in the thickness of the limiting membrane of eggs the hydric environment (Packard et al., 1982). on days 1, 6, 23, 31, and 54 of incubation. The However, water exchange through the shell average thickness was 362nm. No significant membrane might occur inside the shell, since differences were observed in this measure- such turtles may use the shell as a source of ment among the different incubation periods calcium during embryogenesis (Packard et al., (F(4,4)=0.940; P=0.523), nor between mem- 1984; Ewert, 1985). The shell membrane of branes of the embryonic side and the yolk side the turtle, despite having a limiting membrane of individual eggs (F(1,4)=0.364; P=0.579) that is more than four times thicker than that (Fig.