161
J. Anat. (1976), 121, 1, pp. 161-184 With 32 figures Printed in Great Britain
An ultrastructural study of implantation in the golden hamster I. Loss of the zona pellucida and initial attachment to the uterine epithelium TERRY A. PARKENING* Department of Biology, University of Oregon, Eugene, Oregon 97403
(Accepted 9 May 1975) INTRODUCTION
Ultrastructural studies investigating the implantation process have been conducted on such laboratory mammals as the mouse (Reinius, 1967; Potts, 1968), rat (Enders & Schlafke, 1967; Tachi, Tachi & Lindner, 1970), golden hamster (Young, Whicher & Potts, 1968), and rabbit (Larsen, 1961; Steer, 1971; Enders & Schlafke, 1971). A review has also been prepared on the ultrastructural aspects of mammalian implantation by Potts (1969). In an earlier study on implantation in the golden hamster, Young et al. (1968) examined six blastocysts ranging in age from 82 to 116 hours post-coitum. The purpose of this study was to continue ultrastructural observations on a larger number of specimens in an effort to expand current knowledge about the implantation process in this species. MATERIALS AND METHODS
Golden hamsters (Mesocricetus auratus) were maintained under a constant light cycle of 12 hours light and 12 hours darkness. Virgin females (3-5 months of age) cycled by vaginal discharge were placed with males in the early evening and observed for the onset of behavioural oestrus. Developmental age was expressed from the time of ovulation, which was considered as 8 hours from the beginning of lordosis (Harvey, Yanagimachi & Chang, 1961; Parkening & Soderwall, 1975). Females were checked for spermatozoa the morning following mating. Fifteen females at various times from 3-4 days of pregnancy (post-ovulation) were anaesthetized with sodium pentobarbital and injected with 0-25 ml of 1 0 % pontamine blue via the femoral vein (Orsini, 1963). After allowing 15 minutes for diffusion of the dye, the animals were perfused (1 ml/minute for 10-15 minutes) with 2-5 % glutaraldehyde (phosphate buffered, pH 7*4 at 4 °C) via the abdominal aorta. Some animals were perfused with 3-0 % glutaraldehyde (cacodylate buffered, pH 7-2 at room temperature), but mitochondria were poorly preserved. In animals in which implantation had begun, areas of the uterus exhibiting a faint localization of pontamine bide were dissected out and fixed for an additional 1--2 hours in the appropriate buffered glutaraldehyde. Uteri of animals which failed to exhibit any dye were removed and cut into smaller pieces for further fixation. Transverse sections of uteri * Present address: Worcester Foundat-on for Experimental Biology, Shrewsbury, Massachusetts 01545.
II
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were prepared approximately 3 mm long x 2 mm wide x 2 mm thick, post-fixed in 2X0 % osmium tetroxide for 2 hours, dehydrated in an ethanol series and embedded in Epon-Araldite (Mollenhauer, 1964). Serial sections (1-3 pm) were cut dry using glass knives on a Porter-Blum MT-1 ultramicrotome, floated on water on glass slides, and heated on a slide warmer. Each blastocyst or implantation site was photographed unstained using phase contrast optics and re-embedded for electron microscopy using the technique of Schabtach & Parkening (1974). Silver to gold sections were obtained with a Reichert ultramicrotome, using either glass knives or a diamond knife, floated on water and picked up with either 100 or 150 mesh copper grids containing a formvar film (0'3 % in ethylene dichloride). After staining the sections with lead citrate and uranyl acetate, they were stabilized with a thin coat of evaporated carbon and examined in a Philips 300 electron microscope. A total of 45 blastocysts and implantation sites was examined by light and electron microscopy. Several thick sections were obtained from each specimen; in some instances thin sections were examined from all sections of a single specimen, while in other cases only selected sections of a specimen were examined with the electron microscope. After obtaining 5-6 grids of thin sections for electron microscopy, there was generally enough of the thick section remaining to obtain a 4 um section for light microscopy. These were cut with glass knivcs, floated on water and transferred to glass slides. The 4,pm sections were stained with a 1 000 solution of p-phenylenediamine in 70 ' ethanol (Ledingham & Simpson, 1972) and photographed with phase contrast optics using a yellow filter. OBSERVATIONS
As in other ultrastructural studies which have examined implantation in myomorph rodents (Reinius, 1967; Young et al. 1968; Tachi et al. 1970), blastocysts acquired from the same female are not always at the same stage of embryonic development. Embryonic age becomes less important than the sequence of histological changes occurring within and between the embryo and the uterine epithelium. The descriptions which follow, however, pertain to the vast majority of tissues examined at each stage of pregnancy. Fig. 1. Adjoining trophoblast cells from a blastocyst at 3 days of pregnancy (post-ovulation). A cell of the inner cell mass is visible below the trophoblast cells. PS, perivitelline space; M, mitochondrion; DB, degradation body; L, lipid droplet; P, plaques; BC, blastocyst cavity. x 9100. Inset: thick section (05 /tm) of the blastocyst from which the electron micrograph was taken. x 400. Fig. 2. The nucleus of a trophoblast cell from a blastocyst at 3 days of pregnancy. Note the reticulated nucleoli which contain granular and fibrillar areas. The perivitelline space and the zona pellucida (ZP) are visible at the upper left and a cell of the inner cell mass is present at the lower right. x 6700. Fig. 3. A cell of the inner cell mass with two adjoining trophoblast cells from a blastocyst at 3 days of pregnancy. A large number of plaques (P) are visible in the cytoplasm. x 8400. Inset: enlargement of a plaque illustrating the linked-chain appearance of these structures. x 97 800.
Implantation in the golden hamster
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Pre-attachment Fine structure of the blastocyst Blastocysts examined in situ at 3 days of pregnancy retain their zonae pellucidae and are free within the uterine lumen. The zona pellucida is relatively uniform in thickness at both light and electron microscope levels, measuring approximately 6 #m. During this stage of development, little if any, blastocyst expansion occurs, since a prominent perivitelline space is evident between the trophoblast and the zona pellucida (inset of Fig. 1). Ultrastructurally, short irregular microvilli containing a filamentous core appear over the apical surface of the blastocyst. The basal surface of the trophoblast is somewhat irregular with fewer microvilli. Pinocytotic vesicles located over the surface of the trophoblast are most abundant at the apical surface. Other vesicles of various sizes appear intermittently throughout the cytoplasm; while some are vacant, others contain round particles and can be classified as multivesicular bodies (Sotelo & Porter, 1959). Trophoblast cells, united by junctional complexes at their borders, contain predominantly circular or oval mitochondria with few cristae (Figs. 1-3). The cristae which are present are generally distributed peripherally in an arched arrangement. Occasionally, round lipid droplets and electron-dense, irregularly shaped bodies, referred to as yolk granules (Enders & Schlafke, 1965) or degradation bodies (Enders, 1971), appear in the cytoplasm (Fig. 1). They will be referred to as degradation bodies in this report, although yolk may be associated with these structures. These organelles are generally very electron-dense, either containing a few translucent vesicles and/or granules denser and slightly larger (25 nm) than single ribosomes. These latter particles may represent glycogen, as suggested by Enders & Schlafke(1965). Endoplasmic reticulum is sparse, while single ribosomes and clusters of ribosomes (polyribosomes) are scattered throughout the cells. Golgi complexes are generally located next to the nucleus or at the periphery of the cell. Double-stranded cytoplasmic elements, previously referred to as cytoplasmic whorls (Hadek, 1966), cytoplasmic lamellae (Weakley, 1967, 1968) and yolk plates (Szollosi, 1972) in rodent oocytes, and as fibrous material (Enders & Schlafke, 1965) and plaques (Schlafke & Enders, 1967; Tachi et al. 1970) in rodent zygotes, appear both as individual units and as groups throughout the trophoblast (Fig. 3). Since these structures are different Fig. 4. Inner cell mass and trophoblast cells from a blastocyst at 3 days 4 hours of pregnancy. Degradation bodies (DB), mitochondria (M), lipid droplets (L), autophagic vacuoles (A V), plaques and polyribosomes are found throughout the cytoplasm. ZP, zona pellucida. x 10600. Inset: a thick section (0-5g,im) of the blastocyst from which the electron micrograph was taken. x 400.
Fig. 5. Uterine epithelium of a 3 day pregnant hamster. During this stage of pregnancy blastocysts are still free within the uterine lumen (UL). x 5500. Fig. 6. An autophagic vacuole which has started to form within a cell of the inner cell mass of the blastocyst in Fig. 4. Note the presence of plaques within the vacuole. x 20500. Fig. 7. An autophagic vacuole from an inner cell mass cell of a blastocyst at 3 days of pregnancy. This vacuole has started to separate itself from the surrounding cytoplasm. Autophagic vacuoles increase in number throughout the cells of the inner cell mass as embryonic development proceeds from 3-4 days of pregnancy. x 31900.
Implantation in the golden hamster
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TERRY A. PARKENING 166 from lamellae as described in ultrastructural studies on other mammalian oocytes (Baker, 1970; Zamboni, 1971) and embryos (Gulyas, 1971, 1972), and because they have not yet been shown to contain yolk, they are referred to as plaques in this report. They exhibit a definite periodicity, appearing as a linked chain, with each link approximately 36 nm long (inset of Fig. 3). The general appearance and arrangement of the plaques indicate they may exist as sheets of material throughout the cytoplasm. Nuclei of the trophoblast cells contain numerous nuclear pores and generally have reticulated nucleoli, containing granular and fibrillar areas, indicative of cells in the process of division (Figs. 2, 3). Cells of the inner cell mass, which differentiate to become the embryo proper, are loosely apposed to one another and the surrounding trophoblast, leaving numerous intercellular spaces. At intervals, short regions of cellular contact occur between the trophoblast and other cells of the inner cell mass, causing an increased cytoplasmic density representative of primitive junctions. Ultrastructurally, the contents of the embryonic cells are similar to those described above, except that plaques appear more uniformly distributed and more abundant than in the trophoblast.
Fine structure of the uterine epithelium The uterine epithelium consists of columnar cells united by apical junctional complexes and desmosomes. Digitiform microvilli with a filamentous core extend over the free surface of the cells (Fig. 5). A zonation of organelles occurs in these cells similar to that described in the rat (Enders & Schlafke, 1967). Numerous mitochondria occur along the apical surface of the epithelial cell, where they are interspersed with polyribosomes. Below the compact region of mitochondria are lipid droplets, Golgi complexes, an occasional lysosome, more mitochondria and a larger number of polyribosomes. The nucleus occupies the lower half of the cell together with some rough endoplasmic reticulum, polyribosomes, and a few mitochondria and lipid droplets. Loss of the zona pellucida The manner in which the zona pellucida is lost in the hamster is of interest because blastocysts flushed from uterine horns with Hanks' solution exhibit a progressive thinning of the zona pellucida (Figs. 12-15). This change occurs rapidly between Fig. 8. Trophoblast cell from a blastocyst at 3 days 4 hours of pregnancy. A remnant of the sperm tail (S) appears within the cytoplasm of the cell. PS, perivitelline space. x 8400. Inset: a thick section (0-5 ,im) of the blastocyst from which Figures 10 and 11 were obtained. The zona pellucida is broken, as some red blood corpuscles adhere to this covering. The blastocyst is from a hamster pregnant for 3 days 6 hours. x 400. Fig. 9. A blastocyst from a young hamster at 3 days 4 hours of pregnancy. The zona pellucida (ZP) is still retained by this blastocyst. BC, blastocyst cavity; M, mitochondrion; DB, degradation body. x 6800. Fig. 10. A trophoblast cell from a blastocyst at 3 days 6 hours of pregnancy. The zona pellucida (ZP) has thinned and is less homogeneous than those surrounding blastocysts at an earlier stage of development. x 7300. Fig. 11. Trophoblast cells in the region of the blastoeyst where the zona pellucida (ZP) has broken (lower portion of blastocyst in the inset of Figure 8). BC, blastocyst cavity. x 6400.
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172 TERRY A. PARKENING During this phase of implantation the trophoblast in some blastocysts is intimately associated with the uterine epithelium, leaving electron-dense deposits of material between the adjoining plasma membranes (Figs. 31, 32). In other blastocysts a narrow layer of filamentous material forms intracellularly along the plasma membrane of the trophoblast. These fine fibrils resemble microfilaments as described by Wessels et al. (1971) and probably represent the initial step in the formation of trophoblastic processes for invasion and phagocytosis of the uterine epithelium. In a few blastocysts the trophoblast has already begun to invade the uterine epithelium. Morphological changes continue to occur in mitochondria within the trophoblast as the organelles are normally cylindrical with an increased number of cristae (Fig. 29). Besides the transition in the outer shape of the mitochondria, lamelliform cristae at 4 days expand to appear almost tubular in some instances. Polyribosomes become more numerous (compare Figs. 25-30), and plaques become almost non-existent. At the time the trophoblast invades the uterine epithelium plaques are completely absent from the embryo. Some degradation bodies within the trophoblast also begin to assume a different shape with various internal densities. In one blastocyst, a portion of a sperm tail similar to that found in an earlier blastocyst appeared in one of the trophoblast cells. Similar changes occur in most mitochondria of the inner cell mass, although in some mitochondria the cristae are not as numerous and the intracristal spaces are not as pronounced as in mitochondria within the trophoblast. Plaques exist in greater numbers than within the trophoblast, with many visible in autophagic vacuoles which become quite abundant throughout the embryonic cells. These plaques disappear when the trophoblast begins to phagocytose the uterine epithelium. During this time many of the degradation bodies enlarge and assume various shapes. At 4 days of pregnancy entodermal cells are first discernible within the inner cell mass. These cells characteristically display abundant strands of granular endoplasmic reticulum whose cisternae contain a moderately dense amorphous material. Other organelles within these cells remain similar to other cells in the inner cell mass. DISCUSSION
The hamster blastocyst undergoes a considerable number of ultrastructural changes between 3 and 4 days of pregnancy (post-ovulation). An important change during this time period is the loss of the zona pellucida. The manner in which this mucopolysaccharide layer is lost in mouse and rat blastocysts is still a subject of controversy (Dickmann, 1969; McLaren, 1969, 1970; Bergstrom, 1972 a, b; Ljungkvist Fig. 20. Cells of the inner cell mass of the blastocyst in Figure 19. Some of the mitochondria are starting to become more elongated, with a greater number of cristae. Degradation bodies (DB) are increased in number compared with previous stages. x 12300. Fig. 21. A trophoblast cell (Tr) making initial contact with the uterine epithelium (UE). From the blastocyst in Figure 19. Note the absence of microvilli over the apical surface of the trophoblast cell. x 8900. Fig. 22. A trophoblast cell from a blastocyst at 3 days 8 hours of pregnancy. The zona pellucida has been lost and the trophoblast (Tr) is starting to make contact with the microvilli of the uterine epithelium (UE). x 9700.
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174 TERRY A. PARKENING & Nilsson, 1974). The three principal ideas on its elimination are: (1) dissolution by a lytic factor which is derived from either the blastocyst or uterus, (2) 'hatching', i.e. the rupture of a portion of the zona which allows the blastocyst to escape, leaving a virtually intact zona pellucida, and (3) a combination of lysis and 'hatching'. In the present study the thinning zona pellucida, with its subsequent expansion in blastocysts flushed from uterine horns, is similar to that previously described by Orsini (1963) for golden hamsters. This probably occurs because there is some shrinkage of the blastocyst, causing an increased volume in the perivitelline space, and because the zona pellucida as it becomes less homogeneous increases its permeability - causing osmotic changes. Whatever the cause, it is an artefact, because blastocysts examined in utero fail to demonstrate this feature. Orsini (1965) also suggested that the zona of the hamster blastocyst was lost by dissolution brought about by a uterine lysin. The present study supports the idea that a lytic factor does act, at least partially, in eliminating the zona pellucida. A similar conclusion was also recently advanced by Ljungkvist & Nilsson (1974) in an ultrastructural study of implanting rat blastocysts. From the outer appearance of the zonae pellucidae in hamster blastocysts it appears likely such a lysin originates from the uterus. Perhaps a proteinase similar to the one isolated from mouse uterine fluid influences shedding of the zona pellucida (Pinsker, Sacco & Mintz, 1974). Occasionally in the rat (Enders & Schlafke, 1967; Ljungkvist & Nilsson, 1974) and routinely in the ferret (Enders & Schlafke, 1972) remnants of the zona exist between the blastocyst and uterine epithelium during early implantation, whereas in the hamster the zona pellucida is absent before blastocysts become enclosed by the uterine folds. Parr (1973) suggested that micropinocytosis may be a means whereby thinning of the zona pellucida takes place in the guinea-pig. In the hamster, micropinocytotic vesicles are most abundant in the late blastocyst stage, but they are never numerous and are more likely concerned with ingestion of materials from the luminal fluid. Mouse, rat and rabbit blastocysts, still retaining their zonae pellucidae, incorporate radioactively labelled amino acids (Manes & Daniel, 1969; Weitlauf, 1971) and protein tracers (Enders, 1971; Hastings, Enders & Schlafke, 1972; Hastings & Enders, 1974) from the uterine lumen. Blastocyst expansion is considered the cause of thinning of the zona pellucida in the mouse (Bergstrom, 1972b) and rabbit (Enders, 1971). In the hamster some expansion appears to occur, but the trophoblast is never directly apposed to the zona pellucida in all regions of the blastocyst. In one blastocyst the zona pellucida was broken; whether this occurred naturally or as a result of specimen preparation is not known. No trophoblastic 'knobs' or 'buds', as described in the mouse (Bergstrom, 1972b) and hamster (Young et al. 1968), are to be seen projecting from the trophoblast into the zona pellucida. Perhaps neither blastocyst expansion nor Fig. 23. An electron microscope montage of a blastocyst enclosed by uterine epithelium (UE) at 3j days of pregnancy. Cells of the inner cell mass (ICM) contain numerous plaques, mitochondria, lipid droplets, degradation bodies and polyribosomes at this stage of pregnancy. Two of the trophoblast cells (Tr) are more electron-dense at the mesometrial side of the uterus, and may be degenerating. x 6800. Inset: a thick section (0 5 ,um) of the blastocyst from which the montage was taken. x 400.
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J. Anat. (1976), 121, 1, pp. 161-184 With 32 figures Printed in Great Britain
An ultrastructural study of implantation in the golden hamster I. Loss of the zona pellucida and initial attachment to the uterine epithelium TERRY A. PARKENING* Department of Biology, University of Oregon, Eugene, Oregon 97403
(Accepted 9 May 1975) INTRODUCTION
Ultrastructural studies investigating the implantation process have been conducted on such laboratory mammals as the mouse (Reinius, 1967; Potts, 1968), rat (Enders & Schlafke, 1967; Tachi, Tachi & Lindner, 1970), golden hamster (Young, Whicher & Potts, 1968), and rabbit (Larsen, 1961; Steer, 1971; Enders & Schlafke, 1971). A review has also been prepared on the ultrastructural aspects of mammalian implantation by Potts (1969). In an earlier study on implantation in the golden hamster, Young et al. (1968) examined six blastocysts ranging in age from 82 to 116 hours post-coitum. The purpose of this study was to continue ultrastructural observations on a larger number of specimens in an effort to expand current knowledge about the implantation process in this species. MATERIALS AND METHODS
Golden hamsters (Mesocricetus auratus) were maintained under a constant light cycle of 12 hours light and 12 hours darkness. Virgin females (3-5 months of age) cycled by vaginal discharge were placed with males in the early evening and observed for the onset of behavioural oestrus. Developmental age was expressed from the time of ovulation, which was considered as 8 hours from the beginning of lordosis (Harvey, Yanagimachi & Chang, 1961; Parkening & Soderwall, 1975). Females were checked for spermatozoa the morning following mating. Fifteen females at various times from 3-4 days of pregnancy (post-ovulation) were anaesthetized with sodium pentobarbital and injected with 0-25 ml of 1 0 % pontamine blue via the femoral vein (Orsini, 1963). After allowing 15 minutes for diffusion of the dye, the animals were perfused (1 ml/minute for 10-15 minutes) with 2-5 % glutaraldehyde (phosphate buffered, pH 7*4 at 4 °C) via the abdominal aorta. Some animals were perfused with 3-0 % glutaraldehyde (cacodylate buffered, pH 7-2 at room temperature), but mitochondria were poorly preserved. In animals in which implantation had begun, areas of the uterus exhibiting a faint localization of pontamine bide were dissected out and fixed for an additional 1--2 hours in the appropriate buffered glutaraldehyde. Uteri of animals which failed to exhibit any dye were removed and cut into smaller pieces for further fixation. Transverse sections of uteri * Present address: Worcester Foundat-on for Experimental Biology, Shrewsbury, Massachusetts 01545.
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were prepared approximately 3 mm long x 2 mm wide x 2 mm thick, post-fixed in 2X0 % osmium tetroxide for 2 hours, dehydrated in an ethanol series and embedded in Epon-Araldite (Mollenhauer, 1964). Serial sections (1-3 pm) were cut dry using glass knives on a Porter-Blum MT-1 ultramicrotome, floated on water on glass slides, and heated on a slide warmer. Each blastocyst or implantation site was photographed unstained using phase contrast optics and re-embedded for electron microscopy using the technique of Schabtach & Parkening (1974). Silver to gold sections were obtained with a Reichert ultramicrotome, using either glass knives or a diamond knife, floated on water and picked up with either 100 or 150 mesh copper grids containing a formvar film (0'3 % in ethylene dichloride). After staining the sections with lead citrate and uranyl acetate, they were stabilized with a thin coat of evaporated carbon and examined in a Philips 300 electron microscope. A total of 45 blastocysts and implantation sites was examined by light and electron microscopy. Several thick sections were obtained from each specimen; in some instances thin sections were examined from all sections of a single specimen, while in other cases only selected sections of a specimen were examined with the electron microscope. After obtaining 5-6 grids of thin sections for electron microscopy, there was generally enough of the thick section remaining to obtain a 4 um section for light microscopy. These were cut with glass knivcs, floated on water and transferred to glass slides. The 4,pm sections were stained with a 1 000 solution of p-phenylenediamine in 70 ' ethanol (Ledingham & Simpson, 1972) and photographed with phase contrast optics using a yellow filter. OBSERVATIONS
As in other ultrastructural studies which have examined implantation in myomorph rodents (Reinius, 1967; Young et al. 1968; Tachi et al. 1970), blastocysts acquired from the same female are not always at the same stage of embryonic development. Embryonic age becomes less important than the sequence of histological changes occurring within and between the embryo and the uterine epithelium. The descriptions which follow, however, pertain to the vast majority of tissues examined at each stage of pregnancy. Fig. 1. Adjoining trophoblast cells from a blastocyst at 3 days of pregnancy (post-ovulation). A cell of the inner cell mass is visible below the trophoblast cells. PS, perivitelline space; M, mitochondrion; DB, degradation body; L, lipid droplet; P, plaques; BC, blastocyst cavity. x 9100. Inset: thick section (05 /tm) of the blastocyst from which the electron micrograph was taken. x 400. Fig. 2. The nucleus of a trophoblast cell from a blastocyst at 3 days of pregnancy. Note the reticulated nucleoli which contain granular and fibrillar areas. The perivitelline space and the zona pellucida (ZP) are visible at the upper left and a cell of the inner cell mass is present at the lower right. x 6700. Fig. 3. A cell of the inner cell mass with two adjoining trophoblast cells from a blastocyst at 3 days of pregnancy. A large number of plaques (P) are visible in the cytoplasm. x 8400. Inset: enlargement of a plaque illustrating the linked-chain appearance of these structures. x 97 800.
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164
Pre-attachment Fine structure of the blastocyst Blastocysts examined in situ at 3 days of pregnancy retain their zonae pellucidae and are free within the uterine lumen. The zona pellucida is relatively uniform in thickness at both light and electron microscope levels, measuring approximately 6 #m. During this stage of development, little if any, blastocyst expansion occurs, since a prominent perivitelline space is evident between the trophoblast and the zona pellucida (inset of Fig. 1). Ultrastructurally, short irregular microvilli containing a filamentous core appear over the apical surface of the blastocyst. The basal surface of the trophoblast is somewhat irregular with fewer microvilli. Pinocytotic vesicles located over the surface of the trophoblast are most abundant at the apical surface. Other vesicles of various sizes appear intermittently throughout the cytoplasm; while some are vacant, others contain round particles and can be classified as multivesicular bodies (Sotelo & Porter, 1959). Trophoblast cells, united by junctional complexes at their borders, contain predominantly circular or oval mitochondria with few cristae (Figs. 1-3). The cristae which are present are generally distributed peripherally in an arched arrangement. Occasionally, round lipid droplets and electron-dense, irregularly shaped bodies, referred to as yolk granules (Enders & Schlafke, 1965) or degradation bodies (Enders, 1971), appear in the cytoplasm (Fig. 1). They will be referred to as degradation bodies in this report, although yolk may be associated with these structures. These organelles are generally very electron-dense, either containing a few translucent vesicles and/or granules denser and slightly larger (25 nm) than single ribosomes. These latter particles may represent glycogen, as suggested by Enders & Schlafke(1965). Endoplasmic reticulum is sparse, while single ribosomes and clusters of ribosomes (polyribosomes) are scattered throughout the cells. Golgi complexes are generally located next to the nucleus or at the periphery of the cell. Double-stranded cytoplasmic elements, previously referred to as cytoplasmic whorls (Hadek, 1966), cytoplasmic lamellae (Weakley, 1967, 1968) and yolk plates (Szollosi, 1972) in rodent oocytes, and as fibrous material (Enders & Schlafke, 1965) and plaques (Schlafke & Enders, 1967; Tachi et al. 1970) in rodent zygotes, appear both as individual units and as groups throughout the trophoblast (Fig. 3). Since these structures are different Fig. 4. Inner cell mass and trophoblast cells from a blastocyst at 3 days 4 hours of pregnancy. Degradation bodies (DB), mitochondria (M), lipid droplets (L), autophagic vacuoles (A V), plaques and polyribosomes are found throughout the cytoplasm. ZP, zona pellucida. x 10600. Inset: a thick section (0-5g,im) of the blastocyst from which the electron micrograph was taken. x 400.
Fig. 5. Uterine epithelium of a 3 day pregnant hamster. During this stage of pregnancy blastocysts are still free within the uterine lumen (UL). x 5500. Fig. 6. An autophagic vacuole which has started to form within a cell of the inner cell mass of the blastocyst in Fig. 4. Note the presence of plaques within the vacuole. x 20500. Fig. 7. An autophagic vacuole from an inner cell mass cell of a blastocyst at 3 days of pregnancy. This vacuole has started to separate itself from the surrounding cytoplasm. Autophagic vacuoles increase in number throughout the cells of the inner cell mass as embryonic development proceeds from 3-4 days of pregnancy. x 31900.
Implantation in the golden hamster
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TERRY A. PARKENING 166 from lamellae as described in ultrastructural studies on other mammalian oocytes (Baker, 1970; Zamboni, 1971) and embryos (Gulyas, 1971, 1972), and because they have not yet been shown to contain yolk, they are referred to as plaques in this report. They exhibit a definite periodicity, appearing as a linked chain, with each link approximately 36 nm long (inset of Fig. 3). The general appearance and arrangement of the plaques indicate they may exist as sheets of material throughout the cytoplasm. Nuclei of the trophoblast cells contain numerous nuclear pores and generally have reticulated nucleoli, containing granular and fibrillar areas, indicative of cells in the process of division (Figs. 2, 3). Cells of the inner cell mass, which differentiate to become the embryo proper, are loosely apposed to one another and the surrounding trophoblast, leaving numerous intercellular spaces. At intervals, short regions of cellular contact occur between the trophoblast and other cells of the inner cell mass, causing an increased cytoplasmic density representative of primitive junctions. Ultrastructurally, the contents of the embryonic cells are similar to those described above, except that plaques appear more uniformly distributed and more abundant than in the trophoblast.
Fine structure of the uterine epithelium The uterine epithelium consists of columnar cells united by apical junctional complexes and desmosomes. Digitiform microvilli with a filamentous core extend over the free surface of the cells (Fig. 5). A zonation of organelles occurs in these cells similar to that described in the rat (Enders & Schlafke, 1967). Numerous mitochondria occur along the apical surface of the epithelial cell, where they are interspersed with polyribosomes. Below the compact region of mitochondria are lipid droplets, Golgi complexes, an occasional lysosome, more mitochondria and a larger number of polyribosomes. The nucleus occupies the lower half of the cell together with some rough endoplasmic reticulum, polyribosomes, and a few mitochondria and lipid droplets. Loss of the zona pellucida The manner in which the zona pellucida is lost in the hamster is of interest because blastocysts flushed from uterine horns with Hanks' solution exhibit a progressive thinning of the zona pellucida (Figs. 12-15). This change occurs rapidly between Fig. 8. Trophoblast cell from a blastocyst at 3 days 4 hours of pregnancy. A remnant of the sperm tail (S) appears within the cytoplasm of the cell. PS, perivitelline space. x 8400. Inset: a thick section (0-5 ,im) of the blastocyst from which Figures 10 and 11 were obtained. The zona pellucida is broken, as some red blood corpuscles adhere to this covering. The blastocyst is from a hamster pregnant for 3 days 6 hours. x 400. Fig. 9. A blastocyst from a young hamster at 3 days 4 hours of pregnancy. The zona pellucida (ZP) is still retained by this blastocyst. BC, blastocyst cavity; M, mitochondrion; DB, degradation body. x 6800. Fig. 10. A trophoblast cell from a blastocyst at 3 days 6 hours of pregnancy. The zona pellucida (ZP) has thinned and is less homogeneous than those surrounding blastocysts at an earlier stage of development. x 7300. Fig. 11. Trophoblast cells in the region of the blastoeyst where the zona pellucida (ZP) has broken (lower portion of blastocyst in the inset of Figure 8). BC, blastocyst cavity. x 6400.
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172 TERRY A. PARKENING During this phase of implantation the trophoblast in some blastocysts is intimately associated with the uterine epithelium, leaving electron-dense deposits of material between the adjoining plasma membranes (Figs. 31, 32). In other blastocysts a narrow layer of filamentous material forms intracellularly along the plasma membrane of the trophoblast. These fine fibrils resemble microfilaments as described by Wessels et al. (1971) and probably represent the initial step in the formation of trophoblastic processes for invasion and phagocytosis of the uterine epithelium. In a few blastocysts the trophoblast has already begun to invade the uterine epithelium. Morphological changes continue to occur in mitochondria within the trophoblast as the organelles are normally cylindrical with an increased number of cristae (Fig. 29). Besides the transition in the outer shape of the mitochondria, lamelliform cristae at 4 days expand to appear almost tubular in some instances. Polyribosomes become more numerous (compare Figs. 25-30), and plaques become almost non-existent. At the time the trophoblast invades the uterine epithelium plaques are completely absent from the embryo. Some degradation bodies within the trophoblast also begin to assume a different shape with various internal densities. In one blastocyst, a portion of a sperm tail similar to that found in an earlier blastocyst appeared in one of the trophoblast cells. Similar changes occur in most mitochondria of the inner cell mass, although in some mitochondria the cristae are not as numerous and the intracristal spaces are not as pronounced as in mitochondria within the trophoblast. Plaques exist in greater numbers than within the trophoblast, with many visible in autophagic vacuoles which become quite abundant throughout the embryonic cells. These plaques disappear when the trophoblast begins to phagocytose the uterine epithelium. During this time many of the degradation bodies enlarge and assume various shapes. At 4 days of pregnancy entodermal cells are first discernible within the inner cell mass. These cells characteristically display abundant strands of granular endoplasmic reticulum whose cisternae contain a moderately dense amorphous material. Other organelles within these cells remain similar to other cells in the inner cell mass. DISCUSSION
The hamster blastocyst undergoes a considerable number of ultrastructural changes between 3 and 4 days of pregnancy (post-ovulation). An important change during this time period is the loss of the zona pellucida. The manner in which this mucopolysaccharide layer is lost in mouse and rat blastocysts is still a subject of controversy (Dickmann, 1969; McLaren, 1969, 1970; Bergstrom, 1972 a, b; Ljungkvist Fig. 20. Cells of the inner cell mass of the blastocyst in Figure 19. Some of the mitochondria are starting to become more elongated, with a greater number of cristae. Degradation bodies (DB) are increased in number compared with previous stages. x 12300. Fig. 21. A trophoblast cell (Tr) making initial contact with the uterine epithelium (UE). From the blastocyst in Figure 19. Note the absence of microvilli over the apical surface of the trophoblast cell. x 8900. Fig. 22. A trophoblast cell from a blastocyst at 3 days 8 hours of pregnancy. The zona pellucida has been lost and the trophoblast (Tr) is starting to make contact with the microvilli of the uterine epithelium (UE). x 9700.
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174 TERRY A. PARKENING & Nilsson, 1974). The three principal ideas on its elimination are: (1) dissolution by a lytic factor which is derived from either the blastocyst or uterus, (2) 'hatching', i.e. the rupture of a portion of the zona which allows the blastocyst to escape, leaving a virtually intact zona pellucida, and (3) a combination of lysis and 'hatching'. In the present study the thinning zona pellucida, with its subsequent expansion in blastocysts flushed from uterine horns, is similar to that previously described by Orsini (1963) for golden hamsters. This probably occurs because there is some shrinkage of the blastocyst, causing an increased volume in the perivitelline space, and because the zona pellucida as it becomes less homogeneous increases its permeability - causing osmotic changes. Whatever the cause, it is an artefact, because blastocysts examined in utero fail to demonstrate this feature. Orsini (1965) also suggested that the zona of the hamster blastocyst was lost by dissolution brought about by a uterine lysin. The present study supports the idea that a lytic factor does act, at least partially, in eliminating the zona pellucida. A similar conclusion was also recently advanced by Ljungkvist & Nilsson (1974) in an ultrastructural study of implanting rat blastocysts. From the outer appearance of the zonae pellucidae in hamster blastocysts it appears likely such a lysin originates from the uterus. Perhaps a proteinase similar to the one isolated from mouse uterine fluid influences shedding of the zona pellucida (Pinsker, Sacco & Mintz, 1974). Occasionally in the rat (Enders & Schlafke, 1967; Ljungkvist & Nilsson, 1974) and routinely in the ferret (Enders & Schlafke, 1972) remnants of the zona exist between the blastocyst and uterine epithelium during early implantation, whereas in the hamster the zona pellucida is absent before blastocysts become enclosed by the uterine folds. Parr (1973) suggested that micropinocytosis may be a means whereby thinning of the zona pellucida takes place in the guinea-pig. In the hamster, micropinocytotic vesicles are most abundant in the late blastocyst stage, but they are never numerous and are more likely concerned with ingestion of materials from the luminal fluid. Mouse, rat and rabbit blastocysts, still retaining their zonae pellucidae, incorporate radioactively labelled amino acids (Manes & Daniel, 1969; Weitlauf, 1971) and protein tracers (Enders, 1971; Hastings, Enders & Schlafke, 1972; Hastings & Enders, 1974) from the uterine lumen. Blastocyst expansion is considered the cause of thinning of the zona pellucida in the mouse (Bergstrom, 1972b) and rabbit (Enders, 1971). In the hamster some expansion appears to occur, but the trophoblast is never directly apposed to the zona pellucida in all regions of the blastocyst. In one blastocyst the zona pellucida was broken; whether this occurred naturally or as a result of specimen preparation is not known. No trophoblastic 'knobs' or 'buds', as described in the mouse (Bergstrom, 1972b) and hamster (Young et al. 1968), are to be seen projecting from the trophoblast into the zona pellucida. Perhaps neither blastocyst expansion nor Fig. 23. An electron microscope montage of a blastocyst enclosed by uterine epithelium (UE) at 3j days of pregnancy. Cells of the inner cell mass (ICM) contain numerous plaques, mitochondria, lipid droplets, degradation bodies and polyribosomes at this stage of pregnancy. Two of the trophoblast cells (Tr) are more electron-dense at the mesometrial side of the uterus, and may be degenerating. x 6800. Inset: a thick section (0 5 ,um) of the blastocyst from which the montage was taken. x 400.
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