chelonian egg shell
DESCRIPTION
a report of physiological egg shellTRANSCRIPT
Structure of the Softshell Turtles
Shell and Tertiary Membranes of Eggs of (Trio n yx spin if e r us) MARY J. PACKARD AND GARY C. PACKARD Department of Zoology and Entomology, Colorado State Uniuersity, Fort Collins, Colorado 80523
ABSTRACT Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very sim- ilar to avian eggshells. The outer crystalline layer is composed of roughly col- umnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip a t its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except a t the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur a t the intersection of four or more shell units.
Studies recently completed in our labora- tory have revealed important physiological similarities between avian eggs and eggs of the softshell turtle Trionyx spini fems (Pack- ard et al., '79). Eggs of Trionyx are rigid and calcareous, as are avian eggs (Becking, '751, and they apparently lose water vapor con- tinuously to the nest environment (Packard et al., '791, as do avian eggs (Rahn and Ar, '74). Although incubating eggs of softshell turtles may absorb liquid water from the substrate (Packard et al., '791, neither avian embryos nor the turtle embryos depend upon the ab- sorption of liquid water to sustain em- bryogenesis (Packard et al., '79; Rahn and Ar, '74). Given these functional similarities be- tween avian eggs and eggs of Trionyx spi- niferus, we felt it important to determine if there also are structural similarities between eggs of softshell turt,les and those of birds.
MATERIALS AND METHODS
Eggs of softshell turtles were collected from natural nests on sand bars in the South Platte River near Crook, Colorado, in June, 1977. These eggs were used in an experiment assess- ing the effect of substrate water potential on water exchanges between incubating eggs and their environment (Packard et al., '79). Shells
J . MORPH. (1979) 159: 131-144
of all eggs from which turtles hatched were saved, and it is these eggshells that formed the bulk of the material used in this study. In addition, we examined eggshells from unhatched eggs; some of these shells were gathered from the vicinity of nests opened by predators, whereas others came from eggs that were incubated but from which no hatchling emerged. Subsequent examination of eggs which did not hatch following incubation in the laboratory revealed that virtually no development had occurred, but it was not possible to determine if these eggs were infertile or whether the embryo had simply died early in development. In any case, shells of unhatched eggs did not differ morphologically irrespective of their origin, and observations on these eggshells are pooled for comparison with shells from hatched eggs. Observations were made on shell fragments and/or shell membranes from a total of 35 eggs.
Most shell fragments were simply air-dried and stored for future use. However, some frag- ments were boiled for five minutes in 5% NaOH to remove the shell membranes and make it possible to examine the inner surface of the shell. The outer shell membrane, which usually is bound to the calcium layer, was pre-
131
132 MARY J. PACKARD AND GARY C. PACKARD
pared for examination by decalcifying pieces of shell in Decal” (Scientific Products) or in concentrated HC1 (for 60 seconds). Both pro- cedures gave similar results. All material treated with NaOH, Decalo, or HCI was rinsed in distilled water and air-dried. These prepa- rations, and those receiving no chemical treat- ment, were placed in a desiccator over flakes of KOH for a minimum of 24 hours prior to preparation for scanning electron microscopy.
After drying, pieces of shell or membrane were mounted flat on aluminum stubs using reflective paint or Scotch@ double-coated tape. Radial fractures were prepared by breaking off small fragments of shell and mounting them on edge. In this manner, i t was possible to examine the radial (cross sectional) face of the shell. Mounted materials were then coated with carbon and gold in a Technics@ hummer and examined with an Hitachia scanning elec- tron microscope (model HHS2-R) operated at an accelerating voltage of 20 kv. Pictures were taken with Polaroid” 105 or 665 film, and prints were made from negatives using standard photographic techniques. Magnifica- tions are not exact, but are within 20% of re- ported values.
The composition of the shell was analyzed using techniques of X-ray diffraction (Cullity, ’56). Pieces of shell were ground to a powder with a mortar and pestle, and the X-ray pat- tern was generated with a General Electric@ XRD-5 diffractometer using copper radiation and a nickel filter (Cullity, ’56).
Although shells from eggs of softshell tu r - tles are similar to those of birds, there are some differences. For this reason, we have used a rather simple terminology to describe our observations (Erben, ’701, and have tried to avoid extensive use of a terminology best suited for descriptions pertaining specifically to avian eggs (Tyler, ’69).
RESULTS
Eggs of softshell turtles are white, brittle, and nearly spherical, averaging 2.5 cm in di- ameter (n = 65; s.d. = 0.13). Analysis by X-ray diffraction revealed tha t the eggshell consists of calcium carbonate in the form of aragonite crystals, as is the case with eggs of other chelonians (Young, ’50; Erben, ’70; Solomon and Baird, ’76). In contrast, avian eggshells are composed of calcite (Erben, ’70). Interior to the crystalline layer are two tertiary egg membranes: the inner shell membrane, adja-
CALCAREOUS
/LAYER OUTER
INNER SHELL MEMBRANE
AIR CELL
Fig. 1 Schematic diagram of an egg of Trionyx spiniferus.
cent to the albumen, and the outer shell mem- brane, adjacent to the crystalline layer (fig. 1). The two membranes are closely apposed to one another and cannot be distinguished visually a s separate entities except a t the air cell. At the air cell, the inner shell membrane separates from the outer shell membrane, and a space forms between them (fig. 1). Since these eggs are nearly spherical, i t is not possi- ble to identify a particular region of the egg a t which the air cell forms. This is in contrast to the situation in most avian eggs, where the air cell forms at the so-called “blunt” pole of the egg (Romijn and Roos, ’38).
Examination of the outer surface of shells of softshell turt le eggs by scanning electron mi- croscopy revealed tha t the eggshell is formed by the close association of individual crys- talline aggregates or shell units. In surface view, the borders of individual shell units are most easily seen at points of intersection adjacent to pores (fig. 3). The pores in these eggshells a re highly irregular in size and shape, making i t difficult to characterize a “typical” pore. (Pores can also be located using a dissecting microscope, where they ap- pear as irregularly-shaped holes in the shell.) In radial or transverse section (fig. 4) i t is pos- sible to see tha t pores through an eggshell pro- vide for a direct connection between the exter- nal environment and the interior of the egg.
We found no evidence of a true cuticle (Board et al., ’77) on the surface of these eggs. A surface film and bits of debris were found to be irregularly distributed on the surface of all eggs examined, and this film occasionally oc-
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS 133
cluded pores (see fig. 4). However, most pores seen in surface view (fig. 3) or in radial frac- tures were free of occluding material. Al- though we are uncertain of the origin of this debris, we suspect that it results from ovidu- cal secretions or from the leaking of extraem- bryonic fluids during hatching.
In addition to revealing that pores pene- trate the thickness of the shell, radial frac- tures also disclosed that each shell unit is composed of fine, needle-like crystallites radi- ating out from a common center (figs. 4, 5). In some cases, the plane of fracture in transverse sections resulted in a near-median cross sec- tion through the shell unit (figs. 4-61 and ex- posed the central or core area of a shell unit. The crystalline matrix of the central area is smoother and more homogeneous than the ma- trix of the rest of the shell unit (fig. 7). Occa- sionally, an amorphous mass of material adhered to the exposed center of a shell unit (fig. 6, left), whereas in other shell units the core area was free of adhering material (fig. 6, right).
In some radial fractures (usually those from unhatched eggs), the outer shell membrane was visible adjacent to the calcium layer (fig. 5 ) , and the shell unit tapered to a somewhat rounded tip a t its base (fig. 4). Note, too, that fibers of the membrane penetrate the central area (fig. 5).
In general, the outer shell membrane is tightly bound to the crystalline matrix of un- hatched eggs, although this attachment can be weakened if the eggshell is thoroughly dried. However, the outer shell membrane does not remain bound to the crystalline layer of hatched eggs. Instead, the outer shell mem- brane separates from the shell sometime dur- ing incubation, carrying with it part of the crystalline layer. As a result, the base of a shell unit from a hatched egg appears flat (fig. 61, rather than rounded (fig. 4). Moreover, an examination of the outer surface of the outer shell membrane from a hatched egg revealed that the surface is obscured by spherical or amorphous masses from which crystallites radiate in all directions (fig. 8). In some cases, these masses resembled those seen a t the base of a shell unit from a hatched egg (fig. 61, whereas in others the spheres seemed to be encased in a crystalline matrix similar to that of the core area of a shell unit (fig. 8). Masses similar to those seen on the surface of the outer shell membrane of hatched eggs were
also observed on the surface of the membrane from unhatched eggs which had been de- calcified (fig. 9). Since decalcification re- moved all visible traces of the calcium layer, there are no crystallites and no crystal- line matrix associated with these structures (fig. 9).
The outer shell membrane can be gently separated from the shell of unhatched eggs after thorough desiccation, but this sort of separation generally leads to little or no dis- ruption in shell structure (fig. 10). On the other hand, the separation of the outer shell membrane that occurs during incubation re- sults in a more extensive disruption in struc- ture (fig. 11). The only region of the shell to which the outer shell membrane remains at - tached throughout incubation is a t the air cell. Removal of the membrane from the air cell (by treatment with NaOH) revealed a sharp transition in the appearance of the shell units a t the margins of the air cell (fig. 12) . The shell units from beneath the air cell are roughly columnar structures tapering to a somewhat conical tip at the base (fig. 12, bot- tom), whereas those shell units from the area adjacent to the air cell appear to have had their tips broken off (fig. 12, top), revealing the central area of each shell unit.
The structure of the inner and outer shell membranes of softshell turtle eggs is superfi- cially similar to that of avian eggs (Bellairs and Boyde, ’69; Becking, ’75). Both mem- branes from eggs of softshell turtles appear to be composed of several layers of fibers, al- though i t was not possible to determine the exact number of layers present. The inner shell membrane generally exhibits a more fibrous structure than does the outer shell membrane (fig. 13). The inner shell membrane was sheared during preparation in figure 13, revealing the presence of two or three layers. On occasion, the outer shell membrane also was sheared during preparation so that one of the inner layers of the membrane was exposed. When this occurred, tips of the shell units could be seen protruding through the mem- brane (fig. 14). Generally, the tips were ob- scured by an overlying layer(s) of the outer shell membrane.
As in avian eggs, the inner surface of the in- ner shell membrane (i.e., that surface facing the embryo and albumen) has an amorphous, structureless appearance, similar to the “lim- iting membrane” described by Bellairs and
134 MARY J. PACKARD AND GARY C. PACKARD
Boyde ('69) in their studies of avian eggshells. This surface also is similar to the inner sur- face of the inner shell membrane from eggs of the turtle Chelonia m y d a s (Solomon and Baird, '76).
DISCUSSION
The process of shell formation in avian eggs is relatively well understood (Tyler, '69) and provides an excellent framework from which to make inferences concerning shell formation in eggs of Trionyx spiniferus.
After formation of the shell membranes in avian eggs, the first evidence of calcification is the appearance of small granules on the outer surface of the outer shell membrane (Fujii and Tamura, '70; Stemberger et al., '77). These small projections are not removed by acid (Fujii and Tamura, '70), indicating that their chemical composition is different from that of the crystalline shell. Moreover, the granules resemble projections remaining on the outer surface of the outer shell mem- brane after decalcification of an intact egg (Fujii and Tamura, '70). I t is generally assumed that these granules are the organic cores which serve as nuclei for the growth of crystalline aggregates (shell units) during shell formation (Simkiss, '67; Tyler, '69).
The chemical composition of the granules seen during the early stages of shell formation in avian eggs is unknown (Stemberger et al., '77), and no one has yet demonstrated the presence of an organic core on the outer shell membrane of an egg removed from the oviduct prior to the beginning of calcium deposition (Tyler, '69). Nonetheless, the crystallites of an intact shell unit do radiate out from a discrete core which is organic in composition (Terepka, '63b; Simkiss, '67; Tyler, '69). As pointed out by Tyler ('691, the formation of the organic core may be quite rapid, or it may occur simul- taneously with the initiation of calcification, making it unlikely that the naked cores could be detected during studies of shell formation.
In avian eggs, the organic cores are an- chored to the fibers of the outer shell mem- brane (Simons and Wiertz, '63; Simkiss, '67; Bellairs and Boyde, '69) and are eventually surrounded by the crystalline matrix of the shell (Simons and Wiertz, '63; Simkiss, '67; Bellairs and Boyde, '69). The crystallites of calcium carbonate initially grow outward from the core in all directions, growing into the membrane and enclosing fibers of the membrane (Terepka, '63a; Erben, '70) as well
as the organic core (Simons and Wiertz, '63; Simkiss, '67; Tyler, '69). However, growth of crystals toward the membrane is eventually inhibited (Simkiss, '671, so that the bulk of growth occurs laterally and outward (Simkiss, '67; Tyler, '69). Ultimately, in an avian egg, the borders of the individual shell units meet at their lateral margins, and this contact be- tween shell units limits growth in the lateral direction. As the shell increases in thickness, the individuality of the shell units is obscured, and in radial (or transverse) section it is dif- ficult to trace an individual shell unit through the entire thickness of the shell (Tullett et al., ' 75 ; Board et al., '77).
Based on our observations of the structure of eggshells of Trionyx spiniferus, we infer that a similar process of shell formation oc- curs in this species. Presumably, the small masses remaining on the outer surface of the outer shell membrane after decalcification of an intact eggshell (fig. 9) served as the centers of crystallization during calcification of the shell and are analogous to the organic cores of avian eggs (Tyler, '69). We assume that the amorphous masses occasionally seen a t the base of a shell unit in transverse section (fig. 6) and those seen on the surface of the outer shell membrane when it detaches from the crystalline layer (fig. 8) are comparable to the cores seen after decalcification.
As in avian eggs, crystal growth apparently occurs in all directions initially. Eventually, however, the rate of growth away from the outer shell membrane outstrips that occurring toward the membrane, and the shell unit tends to grow laterally and outward (fig. 2). In contrast to the situation in avian eggs, how- ever, each shell unit in an eggshell of a soft- shell turtle retains its individuality through the thickness of the shell, making it possible to identify an individual shell unit in trans- verse section (figs. 4-6). Thus, as pointed out by Erben ('701, the entire shell of a turtle egg corresponds to the so-called mammillary layer of an avian egg (Tyler, '69; Erben, '70).
Fibers of the shell membrane penetrate the core area of shell units in softshell turtle eggs (fig. 51, and the tips of the shell units grow into a layer (or layers) of the outer shell mem- brane (fig. 14). These observations suggest that, as in avian eggs, the crystalline matrix surrounds the core and some of the fibers of the outer shell membrane. Since the core may remain attached to the outer shell membrane when it separates from the shell proper (fig.
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS 135
Fig. 2 Schematic diagram of a shell unit from an egg of Trionyz spiniferus. As we envision it, after for- mation of the shell membranes, the central core of a shell unit is secreted onto the outer surface of the outer shell membrane. This core then serves as a nucleus for the initiation of crystal growth. The primary direction of growth is here represented by the length of the arrows representing the crystallites of calcium carbonate. However, no proportionality of size is intended.
81, we assume that. the core is anchored to the fibers of the outer shell membrane, as it is in avian eggs. Presumably, then, the cores seen on the surface of the outer shell membrane of hatched eggs (fig. 8) or a t the base of a shell unit of hatched eggs (fig. 6) would occupy the core area (figs. 4, 5, 7, 11) in an intact shell unit. This interpretation is strengthened by the observation that cores seen on the outer shell membrane often are encased in a crys- talline matrix similar in appearance to that of the core area of a shell unit (figs. 7, 8).
As shell formation proceeds to completion in avian eggs, there may be an incomplete meet- ing of the borders of intersecting shell units, resulting in the formation of a space or pore between them (Becking, '75; Tullett, '75; Board et al., '77). Pores generally occur a t the intersection of four or more shell units in avian eggs (Tullett, '75; Fox, '761, and a simi- lar situation obtains in eggs of Trionyx spiniferus. With respect to avian (and chelo- nian) eggs, i t is not known what causes the discontinuities that lead to pore formation (Tyler, '69). However, in both avian and che- lonian eggs, i t is through these openings in the shell that gas and water exchanges are ef- fected.
The pores of an avian egg provide for a con- tinuous loss of water from the egg throughout
incubation (Rahn and Ar, '74). Indeed, an avian egg must lose a certain amount of water during incubation to insure formation of a large air cell (Romanoff and Romanoff, '49). Avian embryos then pip the inner shell mem- brane over the air cell and use the air cell as a source of air to inflate their lungs in the hours prior to hatching (Romanoff and Romanoff, '49). Eggs of Trionyx spiniferus, on the other hand, tolerate a relatively wide range of water loss during incubation (Packard et al., '79). Although most eggs examined in this study formed an air cell, some of the air cells were extraordinarily small. We closely examined the shell membranes of all eggs from which hatchlings emerged and could find no evi- dence to suggest that embryos of Trionyx spiniferus preferentially pip the inner shell membrane over the air cell. These observa- tions support the contention that embryonic softshell turtles do not use the air cell as a source of air to inflate the lungs prior to hatching.
Sometime during development in avian eggs, during the later stages of incubation, the outer shell membrane separates from the crystalline matrix except at the air cell, where the outer shell membrane remains firmly at- tached to the shell (Tyler and Simkiss, '59; Terepka, '63b). Since the tip of a shell unit
136 MARY J. PACKARD AND GARY C. PACKARD
grows into the outer shell membrane and encloses fibers of the membrane (Bellairs and Boyde, '69; Erben, '70; Fujii, '741, some of the shell unit remains attached to the membrane when i t separates from the shell (Erben, '70; Fujii, '74). The shell units from which the tips have been removed in this manner show a cen- tral depression (Tyler and Simkiss, '59; Erben, '70; Fujii, '74). and adhering to the surface of the outer shell membrane are roughened gran- ules tha t presumably were liberated from the shell unit when the outer shell membrane sep- arated from the shell proper (Tyler and Simkiss, '59; Terepka, '63b; Erben, '70; Fujii, '74). (Although i t is convenient for us to speak of the outer shell membrane as separating from the shell, this generality is only partially correct. Actually, i t is the tip of a shell unit tha t separates from the rest of a shell unit, and i t is unclear whether the outer shell mem- brane is a passive participant in this process or whether shrinkage and drying of the membrane during incubation facilitates this separation.)
In any case, it is generally assumed tha t re- sorption of calcium from the inner surface of the shell by a n avian embryo weakens the at- tachment between the tip of a shell unit and the rest of the shell unit, leading to a separa- tion of the tip from the shell unit (Bellairs and Boyde, '691. This interpretation is supported by the observation tha t the shell units remain intact at the air cell (Tyler and Simkiss, '59), which is the only region of the inner surface of the shell where there is not an intimate con- tact between the embryo, via the chorioallan- toic membrane, and the shell (Simkiss, '671, and thus, the only region from which calcium is not removed. Moreover, a t about the time the embryo begins to resorb calcium from the shell (Johnston and Comar, '551, the ectoder- ma1 layer of the chorioallantois becomes high- ly differentiated (Coleman and Terepka, '72a) and begins actively to transport calcium (Gar- rison and Terepka, '72). Presumably, dissolu- tion of the shell is accomplished by carbonic acid formed from water and carbon dioxide (Dawes, '751, and the calcium liberated during this process is actively transported across the chorioallantoic membrane. The calcium may be transported in certain specialized cells (Coleman and Terepka, '72a,b) or in the ex- tracellular space (Crooks e t al., '76; Saleuddin et al., '76). Current evidence supports either interpretation.
A similar separation of the outer shell mem-
brane from the shell apparently occurs in soft- shell turt le eggs during incubation, since, a t hatching, the crystalline matrix falls away from the outer shell membrane. Moreover, the appearance of the shell units at the inner sur- face of the shell and the corresponding appear- ance of the outer surface of the outer shell membrane strongly suggest tha t the separa- tion of the shell membrane from the shell is quite similar to tha t described for avian eggs. Our interpretation is t ha t the spherical mass- es seen on the surface of the outer shell mem- brane are the central cores and tha t they (and part of the crystalline matrix of the shell unit) are pulled from the shell unit when the mem- brane separates from the shell. Presumably, the central depression seen in shell units from hatched eggs (figs. 11, 12 top) once contained the core, and it is this core plus the associated crystals of the tip tha t adhere to the surface of the outer shell membrane (fig. 8) . We believe tha t the similarities between these eggs and those of birds are too striking to warrant an entirely different interpretation.
Although we do not know if embryos of Trionyx spiniferus remove calcium from the inner surface of the shell for incorporation into embryonic tissues, we infer from the structural similarities with avian eggs tha t they must. The outer shell membrane can be pulled away from the shell of an unhatched egg if the egg is stored over a desiccant first. However, such mechanical separation of the membrane from the shell is neither as com- plete nor as striking as tha t occurring nat- urally during incubation (figs. 10, 111, sug- gesting tha t some change takes place in the shell during development of the embryo. Our interpretation is strengthened by the observa- tion tha t embryos of other chelonians are known to remove calcium from the shell dur- ing incubation (Bustard et al., '691. Whether or not there also a re changes in the chorioal- lantoic membrane tha t are correlated with the onset of calcium resorption in softshell turt le eggs remains to be determined.
In conclusion, eggs of Trionyx spiniferus are very similar morphologically to avian eggs. Trionyx eggs are hard and rigid, as are avian eggs; shells a r e comprised of a n outer, calcareous layer bounded on the inner surface by two shell membranes, as are avian eggs; pores form at the intersection of four or more shell units, as in avian eggs; and the process of shell formation seems to be similar to tha t oc- curring in avian eggs. Moreover, Trionyx eggs
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS 137
exhibit structural changes during incubation similar to those occurring in avian eggs a s a result of resorption of calcium from the shell. These structural changes suggest tha t em- bryos of Trionyx spiniferus also resorb calcium from the shell, just as do avian embryos.
ACKNOWLEDGMENTS
We thank Marvin Gardner of t he Tamarack Ranch Wildlife Area, near Crook, Colorado, for providing us with access to the collecting site. D. Winder of the Department of Physics at Colorado State University helped us to gather and interpret data in the X-ray diffraction analysis of eggshells. M. Stringer of the De- partment of Anatomy offered valuable advice concerning scanning electron microscopy; and we thank the Department of Anatomy for the use of their facilities for electron microscopy. G. Happ of t he Department of Zoology and Entomology provided access to darkroom facilities and equipment, and offered helpful advice on the preparation of plates. S. Stack (Department of Botany and Plant Pathology) and C. R. Tracy (Department of Zoology and Entomology) critically reviewed drafts of this paper. The manuscript was expertly typed by M. Wright. Our research has been supported, in part, by grants from the National Science Foundation (DEB 75-18179) and from the BRSG Committee a t Colorado State Univer- sity.
LITERA.TURE CITED
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Board, R. G., S. G. Tullett and H. R. Perrott 1977 An arbi- trary classification of the pore systems in avian eggshells. J. Zool., 182: 251-265.
Bustard, H. R., K. Simkiss and N. K. Jenkins 1969 Some analyses of artificially incubated eggs and hatchlings of green and loggerhead sea turtles. J. Zool., 158: 311-315.
Coleman, J. R., and A. R . Terepka 1972a Fine structural changes associated with the onset of calcium, sodium and water transport by the chick chorioallantoic membrane. J. Membrane Biol., 7: 111.127. - 1972b Electron probe analysis of the calcium
distribution in cells of the embryonic chick chorioallan- toic membrane. 11. Demonstration of intracellular loca- tion during active transcellular transport. J. Histochem. Cytochem., 20: 414-424.
Crooks, R. J., C. P. M. Kyriakides and K. Simkiss 1976 Routes of calcium movement across the chick chorioal- lantois. Quart. J. Exp. Physiol., 61: 265-274.
Cullity, €3. D. 1956 Elements of X-ray Diffraction. Addison-Wesley Publishing Co., Inc., Reading, Massachu- setts.
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Erben, H. K. 1970 Ultrastrukturen und Mineralisation rezenter und fossiler Eischalen bei Vogeln und Reptilien. Biomineralisation, 1: 1-66.
Fox, G. A. 1976 Eggshell quality: its ecological and phys- iological significance in a DDE-contaminated common tern population. Wilson Bull., 88: 459-477.
Fujii, S. 1974 Further morphological studies on the for- mation and structure of hen’s eggshell by scanning elec- tron microscopy. J. Fac. Fish. Anim. Husb. Hiroshima Univ., 13: 29-56.
Fujii, S., and T. Tamura 1970 Scanning electron microsco- py of shell formation in hen’s eggs. J. Fac. Fish. Anim. Husb. Hiroshima Univ., 9: 65-80.
Garrison, J. C., and A. R. Terepka 1972 Calcium-stimulated respiration and active calcium transport in the isolated chick chorioallantoic membrane. J. Membrane Biol., 7. 128.145.
Johnston, P. M., and C. L. Comar 1955 Distribution and contribution of calcium from the albumen, yolk and shell to the developing chick embryo. Amer. J. Physiol., 183: 365-370.
Packard, G. C . . T. L. Taigen, T. J. Boardman, M. J. Packard and C. R. Tracy 1979 Changes in mass of softshell turtle (Trionyx spiniferus) eggs incubated on substrates differ- ing in water potential. Herpetologwa, in press.
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Romijn, C., and J. Roos 1938 The air space of the hen’s egg and its changes during the period of incubation. J. Physiol., 94: 365-379.
Saleuddin, A. S. M., C. P. M. Kyriakides, A. Peacock and K. Simkiss 1976 Physiological and ultrastructural aspects of ion movements across the chorioallantois. Comp. Biochem. Physiol., 54A: 7-12.
Simkiss, K. 1967 Calcium in Reproductive Physiology. A Comparative Study of Vertebrates. Reinhold Publishing Corp., New York.
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Stemberger, B. H., W. J. Mueller and R. M. Leach, Jr . 1977 Microscopic study of the initial stages of eggshell calcifi- cation. Poultry Sci., 56: 537-543.
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d w
ith
an
arro
w. N
ote
that
eac
h sh
ell
unit
is
inta
ct a
nd r
ound
ed a
t it
s ba
se.
X
216.
Nea
r-m
edia
n cr
oss
sect
ion
thro
ugh
a sh
ell
unit
fro
m a
n un
hatc
hed
egg.
The
out
er s
hell
mem
bran
e is
cl
earl
y vi
sibl
e at
the
bas
e of
the
she
ll u
nit.
Not
e th
at t
he m
embr
ane
exte
nds
into
the
cent
ral
area
. x
352.
Nea
r-m
edia
n cr
oss
sect
ion
thro
ugh
two
shel
l un
its
from
a h
atch
ed e
gg.
Not
e th
at t
he b
ase
of e
ach
shel
l un
it i
s fl
at. r
athe
r th
an r
ound
ed.
x 35
2.
5 6
m -1
139
PLA
TE
2
EX
PLA
NA
TIO
N
OF
FIG
UR
ES
7 H
ighe
r m
agni
fica
tion
vie
w o
f ce
ntra
l ar
ea o
f a
shel
l un
it.
X
880.
8 O
uter
sur
face
of
oute
r sh
ell
mem
bran
e fr
om a
hat
ched
egg
sho
win
g tw
o co
res
and
the
crys
tall
ites
ass
oci-
at
ed w
ith
them
. X
43
2.
9 O
uter
sur
face
of
oute
r sh
ell
mem
bran
e fr
om a
n eg
g de
calc
ifie
d in
HC1
for
60
seco
nds.
The
sur
face
of
the
mem
bran
e is
dot
ted
wit
h th
e co
res
that
ord
inar
ily
wou
ld o
ccup
y th
e ce
ntra
l ar
ea o
f in
tact
she
ll u
nits
. X
88
.
Inne
r su
rfac
e of
she
ll f
rom
an
unha
tche
d eg
g. O
uter
she
ll m
embr
ane
deta
ched
fro
m s
hell
afte
r de
sicc
a-
tion
, hut
thi
s de
tach
men
t le
d to
lit
tle
disr
upti
on i
n sh
ell
stru
ctur
e. X
35
2.
10
STR
UC
TU
RE
OF
SOFT
SHE
LL
T
UR
TL
E
EG
GSH
EL
LS
Mar
y J.
Pac
kard
an
d G
ary
C.
Pack
ard
PLA
TE
2
c
Q
PLA
TE
3
EX
PLA
NA
TIO
N
OF
FIG
UR
ES
11
Inne
r su
rfac
e of
she
ll f
rom
hat
ched
egg
. Sep
arat
ion
of o
uter
she
ll m
embr
ane
from
she
ll d
urin
g in
cuba
- tio
n le
d to
a d
isru
ptio
n in
she
ll s
truc
ture
. The
se s
hell
uni
ts a
re n
ot i
ntac
t (c
ompa
re w
ith
fig.
10). T
he
tips
sep
arat
ed f
rom
the
res
t of
the
she
ll u
nits
, and
the
cen
tral
are
a ha
s be
en e
xpos
ed a
s a
resu
lt.
x 43
2.
12
Tra
nsit
ion
zone
fro
m i
nner
sur
face
of
shel
l of
a h
atch
ed e
gg.
The
are
a at
the
bot
tom
of
the
pict
ure
is
from
ben
eath
the
air
cel
l w
here
as t
hat
at t
he to
p is
fro
m a
n ar
ea a
djac
ent
to, h
ut n
ot i
nclu
ded
in, t
he a
ir
cell.
She
ll f
ragm
ent
was
tre
ated
wit
h N
aOH
to
rem
ove
the
oute
r sh
ell
mem
bran
e fr
om t
he a
ir c
ell.
x 56
.
13
Inne
r sh
ell
mem
bran
e sh
owin
g tw
o (o
r 3)
fib
rous
lay
ers.
x
432.
14
Out
er s
hell
mem
bran
e fr
om t
he a
ir c
ell
of a
hat
ched
egg
. A l
ayer
k) o
f th
e m
embr
ane
was
she
ared
aw
ay
duri
ng p
repa
rati
on, r
evea
ling
the
tip
of
a sh
ell
unit
. T
he o
uter
she
ll m
embr
ane
is l
ess
fibr
ous
than
the
in
ner
shel
l m
embr
ane
and
rese
mbl
es a
ret
icul
ate
mat
. x
432.
STR
UC
TU
RE
OF
SO
FTSH
EL
L T
UR
TL
E
EG
GSH
EL
LS
Mar
y J
Pack
ard
and
Gar
y C
. Pa
ckar
d PL
AT
E 3