Ultrastructural observations on the shell membrane of the North American opossum (Didelphis virginiana).код для вставкиСкачать
THE ANATOMICAL RECORD 207:335-338 (1983) Ultrastructural Observations on the Shell Membrane of the North American Opossum (Didelphis virginiana) WILLIAM J. KRAUSE AND J. HARRY CUTTS Department ofAnatomy, School of Medicine, University of MissouriColumbia, Columbia, MO 65212 ABSTRACT Each developing opossum embryo is surrounded by a shell membrane which completely separates embryonic and maternal tissues. During the eighth and ninth prenatal days, the embryos together with their limiting shell membranes float freely within the uterine lumen, surrounded only by the secretions of the uterus. The shell membrane is transparent, nonelastic, tough, and capable of extreme deformation. It consists of a mat of interwoven fibers which vary in external diameter, are electron dense, and show no apparent substructure. The morphology and arrangement of component fibers are similar throughout the width of the shell membrane. After ovulation, the metatherian egg remains in the oviduct for about 24 hr before it enters the uterus; during this short transit period, the egg is fertilized by a single spermatozoon. Although the sperm are paired, both in the epididymis and in the female reproductive tract of Didelphis (Biggers and Delamater, 1965; Krause and Cutts, 1979) the sperm-pairs always separate prior to fertilization (McCrady, 1938). A corona radiata is not present and the metatherian egg initially is surrounded only by a zona pellucida. Following fertilization, a second layer, consisting primarily of acid mucopolysaccharides, is laid down around the zona pellucida. This second layer is the product of glandular secretions of the oviduct (McCrady, 1938; Tyndale-Biscoe, 1975).As the fertilized ovum passes along the remainder of the oviduct, spermatozoa may become embedded within the substance of this layer. In Dasyurus viw errinus, a n Australian marsupial, the second layer may measure 1.5-8.0 pm in thickness. A similar coat of mucopolysaccharide has been described about the ovum of the rabbit (Tyndale-Biscoe, 1975). The metatherian egg, unlike that of the eutherian, becomes surrounded by a third layer, the shell membrane, which also is thought to be produced by secretory cells in the distal oviduct (Hartman, 1916; Anderson, 1928). In the Australian form, Trichosurus vulpecula, the shell membrane is reported to be keratinous in nature, and nonelastic but 0 1983 ALAN R. LISS, INC. capable of extreme attenuation (Tyndale-Biscoe, 1975).It is said to be resistant to enzyme digestion (Hughes, 1977)and apparently does not act as a barrier for the passage of most large molecules to and from the surrounding uterine fluid (Hughes and Shorey, 1971). In most marsupials it persists between the forming fetal membranes and the maternal uterine epithelium until late in gestation. The ultrastructure of the shell membrane has not been reported previously in Didelphis virginiana or any other didelphid marsupial. Even in the Australian forms, the structure of the shell membrane has been described only in passing, primarily during the very early stages of development. The present study reports the light-, scanning-, and transmission electron microscopic observations of the shell membrane in the North American opossum late in the gestation period. MATERIALS AND METHODS Fifteen embryos were collected, the approximate ages of which were determined from timed pregnancies. Wild female opossums, carrying litters, were stripped of their pouch young and placed in breeding pens with continuous access to males. The females usually reenter into estrus within a week after removal of the young and a sperm-positive date was obtained by examining vaginal smears ~~ Received March 14,1983; accepted June 2, 1983. 336 W.J. KRAUSE AND J.H. CUTTS a t 24-hr intervals. The embryos were collected during the last third of the eighth, and first third of the ninth prenatal day. Three adult female opossums were killed by ether anesthesia and the uteri removed and opened. The embryos appeared as clear spheres floating freely within the uterine lumen, and were recovered intact by tipping the uterine contents into fixative. The specimens were fixed for 4 hr a t room temperature, in 3.0% glutaraldehyde buffered in 0.1 M phosphate to a pH of 7.4, after which the tissues were transferred to 0.1 M phosphate buffer for 2 hr, then osmicated for 2 hr in 1.0% osmium tetroxide in 0.1 M phosphate. After washing in the phosphate buffer and dehydrating through a series of graded ethanol solutions, specimens for scanning electron microscopy were critically point dried by liquid COz substitution in a Bomar critical point dryer, mounted on spinner stubs, and coated with gold to a depth of 20 nm in a Polaron SEM coating unit. The specimens were viewed in a JOEL-35 scanning electron microscope operated a t 20 kV. Tissues for transmission electron microscopy were dehydrated through ethanol solutions, cleared in propylene oxide, and infiltrated with and embedded in Epon 812. Thin sections of this material, mounted on uncoated grids and stained with uranyl acetate and lead citrate, were examined in a Phillips 300 electron microscope operated a t 60 kV. Thick sections (0.3-2.0 pm) of the Eponembedded material were examined by light microscopy after staining with toluidine blue. RESULTS Each uterus contained several transparent, fluid-filled spheres approximately 6.5 mm in diameter, floating freely within the secretion of the uterine lumen. Following fixation, a developing embryo could be seen on each sphere by direct observation (Fig. 1).All three germ layers were present and from five to eight somites could be seen. The forming brain consisted of closed cavities but the developing spinal cord remained open. The shell membrane around each embryonic sphere is transparent during life and following fixation. During mechanical manipulation it appears nonelastic but is tough and capable of extreme deformation. When the external surface of the shell membrane is examined at low magnification with the scanning electron microscope, it appears smooth, but at increased magnification it is seen to consist of a mat of interwoven branching fibers that vary in diameter (Fig. 2). With the light microscope, the shell membrane appears as a thin, homogeneous band with no apparent substructure (Fig. 3). However, ultrastructurally, the shell membrane consists of numerous, interwoven electrondense fibers (Fig. 4)that appear to be homogeneous and show no evidence of smaller subunits. DISCUSSION In marsupial species with short gestation periods, the shell membrane surrounds the developing embryo for most of the prenatal period and, even in those species with a much longer gestation (macropodids), it persists for a t least two-thirds of the prenatal period, completely separating embryonic and maternal tissues (Hughes, 1974; Tyndale-Biscoe, 1975). In Didelphis the shell membrane remains intact through the 10th prenatal day (Renfree, 1975) and some portions may persist until near term (McCrady, 1938). During the period when the embryos are surrounded by a n intact shell membrane, they lie free within the uterine lumen and are thought to be nurtured by secretions from the uterus. The nutrients make their way to the growing embryo by passing through the surrounding shell membrane. Renfree (1975) found the uterine secretions of Didelphis to be rich in proteins, principally albumins, prealbumins, and one or two P-globulins. The shell membrane of Trichosurus vulpecula is permeable to both peroxidase and ferritin, although some filtration of the latter does occur (Hughes and Shorey, 1971,1973). Such observations support the idea of free passage of nutrients and wastes to and from the developing embryo. In the shell membranes of three Australian marsupials-the bandicoots, Isoodon macrourus and Perameles nasuta, and the brush-tailed opossum, Trichosurus vulpecula (Hughes, 1977; Lyne and Hollis, 1977)the electron-dense, fibrous material appears to be tightly packed rather than in the form of a series of loose fibers, as observed in Didelphis. The close packing seen in the Australian forms may reflect the very early stages of development examined in these species. The electron-dense material in the shell membrane of marsupials appears similar to that present in the egg shells of the two monotremes, the platypus and echidna (Hughes et al., 1975; Hughes, 1977). The shell membranes of the domestic fowl and some reptilian forms (Solomon and Tippett, 1983) appear similar in structure both to metatherians and prototherians. The avian shell membrane consists of a n open mesh- Fig. 1. A fixed embryo from an opossum uterus taken early during the ninth post-sperm-positive day. Note that the developing embryo is part of a n embryonic sphere. The shell membrane is transparent and, although present, cannot be seen in this photomicrograph. The region of the farming brain, spinal cord, and somites are visible. x25. Fig. 2. The external surface of the shell membrane consists of a mat of interwoven fibers which vary considerably in diameter. x 11,000. Fig. 3. The shell membrane appears as a thin homogeneous band (arrows) when viewed in section at the light microscopic level. Epon 812-toluidine blue. X250. Fig. 4. At the ultrastructural level the shell membrane is seen to consist of numerous electron-dense fibers without apparent substructure. The external surface (E) of the shell membrane lies t o the left of the photomicrograph. x 15,000. 338 W.J. KRAUSE AND J.H. CUTTS work of electron-dense proteinaceous (keratinous) fibers (Simons and Wiertz, 1963; Bellairs and Boyde, 1969; Kaplan and Siegmund, 1973; Creger et al., 1976) and is the product of secretions from glands in the oviduct (Breen and Bruyn, 1969; Wyburn et al., 1973) as is the case both for marsupials and monotremes. In birds, however, the events are complicated somewhat in that calcification of the egg shell begins just before the egg enters the uterus. Permeability of the shell membranes has been thought to be of primary importance in the evolution of viviparity (Hughes, 1977) since it would permit transfer of nutrients from the uterine secretions to the embryo and eliminate dependence upon contained yolk material. Such a change in the means by which the embryo is nourished during development may have permitted the reduction in size of the egg. Similarly, Hughes (1977) has hypothesized that the loss of the shell membrane in eutherian species may be associated with the early invasive properties of the chorion, thus permitting the intimate association between maternal and fetal vascular systems during placentation. ACKNOWLEDGMENTS This work was supported in part by a grant from the J.B. Reynolds Foundation. LITERATURE CITED Anderson, D.H. (1928)Comparative anatomy of the tubouterine junction. Histology and physiology in the sow. Am. J. Anat., 42: 255-305. Bellairs. R.. and A. Bovde (1969) Scanning " electron microscopy of the shell membranes of the hen's egg. Z. Zellforsch., 96t237-249. Biggers, J.D., and E.D. Delamater (1965) Marsupial spermatozoa pairing in the epididymis of American forms. Nature, 206:402-404. Breen, P.C., and P.H. Bruyn (1969)The fine structure of the secretory cells of the uterus (shell gland) of the chicken. J. Morohol.. 128t35-66. Creger, C.R., H. Phillips, and J.T. Scott (1976)Formation of egg shell. 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