Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy.код для вставкиСкачать
THE AMERICAN JOURNAL OF ANATOMY 188:221-238 (1990) Microscopic Examination of Porcine Conceptus-Maternal Interface Between Days 10 and 19 of Pregnancy J.L. KEYS AND G.J. KING Department of Animal and Poultry Science, The University of Guelph, Guelph, Ontario, Canada N l G 2 W l ABSTRACT Conceptus-maternal interactions in the pig were examined at days 10, 13, 16, and 19 of pregnancy. Contact between the chorion and uterine epithelium was not preserved on day 10, but extensive contact occurred in 3 of 5 pregnant gilts on day 13 and was related to localized differentiation of the uterine epithelium. Attachment events occurred earlier and more rapidly than previously reported, commencing in the region of the embryonic disc and progressing toward the extremities of the chorionic vesicle, with stages from apposition through attachment occurring simultaneously along a conceptus at day 13 and thereafter. Apical protuberances on maternal epithelial cells and interposing blunt chorionic processes were evident at sites of early maternalconceptus interaction, but were reduced at regions exhibiting microvillous interdigitation. Placentation in the pig represents a developmental process consisting of a continuum of sequential events which occur over a broad time frame rather than at specific discrete times. MATERIALS AND METHODS Nulliparous crossbred (Yorkshire X Landrace) gilts of similar age and weight were observed daily for estrus in the presence of a vasectomized boar. Following two estrous cycles of normal duration (18 to 22 days), animals were randomly assigned for slaughter a t day 10, 13, 16, or 19 (n=Mgroup). Gilts were inseminated with approximately 50 ml of fresh unextended semen on the first day of standing estrus (day 0) and 24 h r later. Uteri from gilts which had been bred but did not contain normally developed blastocysts were discarded. The reproductive tract was removed from each animal immediately following exsanguination and trimmed free of the mesometrium. The right uterine horn was opened along the anti-mesometrial aspect; and two 20 cm segments were excised, pinned endometrial side up in a wax-based steel tray containing 0.2M cacodylate buffer (pH 7.41, and examined under a magnifier-illuminator in order to locate the conceptuses a t day 13 of gestation and later. Flag pins were inserted lateral to the embryonic disc(s). Upon removal of the buffer, the tissue was gently flooded with 3% glutaraldehyde in 0.2M cacodylate buffer and left to fix for 30 min a t room temperature. Endometrial samples were selected randomly from the mesometrial region of pregnant uteri at day 10 INTRODUCTION since the blastocysts were not attached to the mucosa. Despite the essential role the porcine uterus plays in To investigate whether epithelial adaptations for emthe establishment of pregnancy, alterations in the lu- bryonic attachment were localized to regions of trophominal epithelium which occur during preimplantation blast proximity or represented a generalized uterine stages and initial placental development have not been response, 3 mesometrial regions were sampled along widely studied. With few exceptions (Corner, 1921; each of 2 embryos within each gravid uterus at day 13 Stroband et al., 1986), investigators have neglected to and later: 1) immediately adjacent to the embryonic elucidate structural alterations which are pregnancy- disc, 2) approximately 5 cm from the disc with adherent specific by comparing pregnant endometria with tissue membranes, and 3) from sites lacking trophoblastic on corresponding days of the estrous cycle. In addition, contact. Samples were processed for light and electron few morphological studies have been designed to study microscopy as previously described (Keys and King, localized interactions between the pig conceptus and 1989). Twelve sampling sites were examined per anithe endometrium during this critical phase of the re- mal. Height measurements of surface epithelial cells were productive process. Preliminary results indicated that contact between made on 6 consecutive cells in 3 regions on each of 4 porcine trophectoderm and uterine epithelium could slides prepared for light microscopy from each samnot be preserved a t day 10, but attachment had com- pling site, resulting in 432 measurements per animal. menced by day 13 (Keys and King, 1984; Keys et al., The cell-height data were analyzed statistically by one1986). The objectives of this study were to develop a way ANOVA, and treatment means were compared comprehensive morphological description of conceptusmaternal interactions a t days 10, 13, 16, and 19 of pregnancy; to describe epithelial changes accompanying and possibly facilitating the attachment process; Received November 28, 1989. Accepted February 15, 1990. and to look for morphological evidence of a localized Address reprint requests to Dr. G.J. King, Dept. Animal & Poultry influence by the blastocyst on the structure of the ma- Science, The University of Guelph, Guelph, Ontario, CANADA N l G 2w1. ternal epithelium. 0 1990 WILEY-LISS, INC 222 J.L. KEYS AND G.J. KING with data from contemporary cycling animals (Keys and King, 1989) a t equivalent stages using a Scheffe's test. RESULTS Uterine epithelial cell heights on days 10 (20.382 0.37 pm), 13 (22.320.21 pm), and 16 (21.04+0.18 pm) were similar (P<0.05). Luminal epithelial cell height was lower on day 19 of pregnancy (17.6420.21 pm) relative to heights recorded on days 10 through 16 (P<0.05). A comparison of these measurements with epithelial cell heights from cyclic animals (Keys and King, 1989) indicated that the epithelial cells were significantly taller in pregnant gilts on days 10 and 13, similar on day 16, and shorter on day 19 (P<0.05). Structural Features of the Uterine Luminal Epithelium Day 10 The trophectoderm was not preserved in contact with the uterine epithelium in any of the animals slaughtered on day 10. The simple columnar epithelium formed a hillocky array containing a predominance of densely stained, compressed cells with condensed basal nuclei and little glycogen (Fig. 1).The epithelial cells were characterized by electron-dense nuclei and cytoplasm; dense mitochondria; elaborate Golgi; endoplasmic reticulum (ER); a smooth basal plasmalemma and basal lamina; small vesicles below the apical plasmalemma; and irregularly dispersed, long, fine microvilli. Cells were coririected apically by junctional complexes with zonula occludentes. Apical profiles were generally flat to slightly rounded. Intermediate-staining cells were second to the dark cells in prevalence. Palestaining cells with large vesicular nuclei in basal to central positions and a n abundance of cytoplasmic vacuoles were relatively rare. Degenerative cells were rare, and ciliated cells were sparsely scattered throughout the epithelium. Day 13 Contact between the chorion and maternal epithelium was preserved in 3 of 5 animals slaughtered on day 13. The embryonic disc area was most firmly adhered to the uterine mucosa on examination at the gross level, while more distal regions of the chorionic vesicle floated free of the luminal surface. Epithelial cells throughout the uterus exhibited morphology suggestive of increased metabolic activity. The smooth, simple, columnar epithelium in sites which lacked trophoblast contact contained predominantly intermediate- and pale-staining cells. Dark compressed cells were dramatically reduced in prevalence from the previous stage. The nuclei were oriented perpendicular to the basement membrane and displaced from the basal CE d ER G M ME mV T Abbreviations chorionic epithelium dense inclusion endoplasmic reticulum Golgi apparatus mitochondria maternal (uterine luminal) epithelium microvilli trophoblast plasma membrane to occupy central to apical positions by basal vacuoles (Fig. 2). The periphery of the vacuoles stained intensely PAS positive a s did numerous small granules which were distributed throughout the cytoplasm. The apices of cells in these regions were generally flat. Pale-staining cells were often overlain by secretory material and exhibited uneven cell height, domed apices, and occasionally pseudostratification. Degenerative cells undergoing vacuolation occurred frequently in unapposed regions, and cellular debris was observed in the uterine lumen. Several ultrastructural features of the epithelial cells differed from those of day 10 pregnant uteri. The nuclei were larger and more vesicular. Euchromatin was finely dispersed throughout the nucleoplasm, and a few chromatin clumps were associated with the nuclear envelope. Nucleoli were more prominent. The cytoplasm was less condensed and contained dramatically increased basal deposits of glycogen (Fig. 3). This carbohydrate was also distributed a s small inclusions throughout the cytoplasm. Small vesicles and polyribosomes were ubiquitous, and ER composed of many long rough strands and a few smooth profiles was also present. Dense inclusions occurred basally in extremely complex groups (Fig. 3) and were scattered a s larger forms toward the apices of the cells. Numerous pleomorphic mitochondria with electron-lucent matrices and distinct lamellar cristae were concentrated in the apical cytoplasm and adjacent to basal glycogen dep0sit.s (Fig. 3). In most cases, the basal lamina was closely associated with a smooth basal plasmalemma, but protrusions of basal cytoplasm and corresponding folds in the basal lamina were occasionally observed (Fig. 3). Apices were covered with a uniform coat of short blunt microvilli and a very thick, fibrous glycocalyx. Occasionally, secretory vesicles were observed among the microvilli. Embryonic membranes a t day 13 of pregnancy lay in close proximity to the uterine epithelium; and, although they were often separated from the mucosa, they closely followed the contours of the mesometrial endometrium. Gradual but dramatic changes in uterine cell morphology and arrangement were observed upon moving from a n unapposed region (Fig. 2) toward sites of contact with the trophoblast (Figs. 4-8). The cytoplasm stained less intensely and vacuoles accumulated to a greater extent basally, often occupying onethird to one-half of the cell (Fig. 4).Nuclei were larger, more vesicular and their position was more variable (Fig. 5). Small apical vesicles and PAS positive granules were numerous. Cell height was increased, the basement membrane was undulatory, and cell apices were consistently domed (Fig. 6). Where directly overlain or contacted by trophoblast, shallow folding of the stroma was evident; and the epithelium formed knoblike structures, resulting in the appearance of pseudostratification and irregular orientation of cell axes to the basement membrane (Figs. 6,7). The apical portion of each cell was often extended into a protuberance above the level of the terminal junctional complex, over which the trophoblast was closely moulded (Fig. 7). In proximity to the trophoblast, these structures contained pale-staining cytoplasm with a dense band a t the base (Fig. 7). The most advanced stages of attachment occurred Figs. 1-30. Micrographs represent uterine luminal epithelium from pregnant gilts. Fig. 1 . Day 10. Dark compressed cells form a hillocky, simple columnar epithelium. x 550. Flg. 2. Day 13. Epithelial cells which lack contact with the embryonic membranes exhibit intermediate staining properties, vesicularnuclei, large basal vacuoles (arrowheads),and flat apices. x 550. Fig. 3. Day 13. Several cytoplasmic processes (small arrowheads) and folds in the basal lamina (large arrowheads) complicate the basal profile of the epithelium. Electron-dense inclusions, an intraepithelial lymphocyte (L), and basal glycogen deposits tG1) associated with mitochondria are also indicated. EM, x 6900. Figs. 4-8 PLACENTATION IN THE PIG nearest to the embryonic disc, and contact between the embryonic and maternal epithelia was more consistently maintained through processing a t these regions. In such areas, undulations of the stroma and epithelium resulted in a n arrangement suggestive of early placental fold formation (Fig. 8). Regions of cells which were morphologically intermediate to unapposed epithelia and to epithelia a t sites of contact were evident in the two gilts lacking areas of attachment, suggesting that the trophoblast may have previously been apposed but lifted away during processing. The uterine epithelium a t areas in proximity to or directly contacted by the trophoblast exhibited uniformly pale staining. Degenerative forms were very rare and always occurred as isolated individual cells rather than in groups. Both of these observations were in contrast to the features of unapposed regions. Staining with PAS indicated more numerous and ubiquitously distributed granules throughout the epithelium directly associated with the trophoblast (Fig. 9). Near gland openings, in regions of epithelial folding, or where overlain by trophoblast, cellular remnants, diffuse finely granular or flocculent material which was faintly basophilic, and a n electron-dense amorphous phase which stained intensely with PAS were interposed between the two epithelia (Fig. 9). These characteristics persisted to day 19. Stubby microvilli with a thick fibrillar glycocalyx were uniformly distributed over the apical surfaces of most maternal epithelial cells and scattered diffusely over the trophoblast (Fig. 10). Loss of microvilli and extrusion of apical protuberances from the luminal epithelial cells accompanied interaction with the trophoblast (Figs. 10, 11).Initial apposition between maternal and trophoblastic epithelia was apparently confined to these smooth maternal apices. Attachment appeared to begin with discrete areas of cell-to-cell contact after which the trophoblast cells became moulded closer to a greater portion of the maternal apical protuberances. The cytoplasm contained within such domes was almost devoid of organelles and separated from the more condensed cytoplasm occupying the rest of the cell by a band of microfilaments extending between zonulae adherens a t the lateral cell membranes Fig. 4. Day 13. Distal to an area of trophoblast attachment, the epithelium contains cells which are undergoing pseudostratification and contain extensive basal vacuoles. x 550. Fig. 5. Day 13. An unapposed region closer to the contact site than that shown in Fig. 4 has taller pale-staining cells with large vesicular nuclei, flat cell apices, and an abundance of vacuoles and vesicles. x 550. Fig. 6. Day 13. Immediately adjacent to an area of contact with the trophoblast, the maternal epithelium contains tall, pale-staining cells with large basal vacuoles and rounded apices. The stromal-epithelial interface is undulatory. X 550. Flg. 7. Day 13. The trophoblast is directly apposed to the maternal epithelium and follows its apical contours, closely adhering to the prominent pale apical domes (arrowheads). x 550. Fig. 8. Day 13. An advanced area of attachment near the embryonic disc exhibits stromal-epithelial folds on the maternal side, over which the trophoblast IS applied. x 550. 225 (Fig. 12). The maternal glycocalyx appeared to be thickened and fibers extended between the two epithelia, anchoring them together (Fig. 13). The nature of adhesion was tenuous, consisting of close apposition between the overlying trophoblast and contours of the maternal apical plasmalemma, with broad processes of the former extending between the maternal domes to the level of the junctional complexes (Fig. 14). There was some evidence that microvilli had begun to reform, initiating definitive attachment (Fig. 14). The trophoblast cells a t this stage contained large round mitochondria with fenestrated septate cristae, scattered RER, a n abundance of polyribosomes and electron-dense inclusions similar to those of the maternal epithelium, as well as elaborate networks of microfilaments running parallel to the long axis of the cells (Figs. 12-14). Each trophoblast cell covered the apices of several maternal cells (Fig. 14) and sometimes contained electron-dense amorphous material and cellular remnants. Day 16 Contact between the chorion and uterine epithelium was evident in all of the animals and ranged from apposition to apparently firm attachment, depending on the site examined. A variety of cell morphologies was observed within the uteri of these animals. The features which differed most among the epithelial cells were the amount of glycogen, the degree of apical doming, and the distribution of microvilli. Low simple columnar epithelium exhibited wide oval nuclei occupying almost the entire cell volume, flat apices, and sparse large vacuoles. The apical plasmalemma often lacked microvilli and had shallow folds. In other areas, somewhat taller cells with paler staining properties, more elongated oval nuclei, large basal vacuoles, and large apical projections connected to the cell by narrow stalks were observed. These two morphologies were the most common in unapposed epithelia. A third cell type was very tall and narrow, and up to one-half of the cell was filled with large vacuoles which displaced the nucleus to an apical position (Fig. 15).Vacuoles were also present above the nucleus, and a very low nuc1eus:cytoplasm ratio was evident. The apices of these cells were usually elevated into irregular blebs which were packed with organelles and abundant glycogen and covered by a smooth plasmalemma. The basic ultrastructural features of the maternal epithelial cells were similar to those at the former stage. Cells changed form in regions near preserved attachment, adopting irregular features and giving an impression of disarray (Fig. 16).Nuclei varied in staining intensity, shape, and orientation. The cytoplasm was usually pale and contained more vesicles but smaller basal vacuoles. Cell height varied, but low cells with large apical domes or blebs forming the entire apex were most common (Fig. 16). In proximity to the attached chorion, the epithelium was in even greater disarray (Fig. 17). Mitochondria were distributed throughout the cytoplasm of the cells, and extremely long thin forms were often aligned with the peripheral borders of basal glycogen deposits (Fig. 18).Endoplasmic reticulum, primarily of the rough type, was packed into the apical cytoplasm, sometimes forming a spiral, and long strands wound into the basal cytoplasm, often Fig. 9. Day 13. The uterine epithelial cells are arranged irregularly over shallow stromal folds and contain PAS positive granules. Intensely stained material (asterisk)is interposed between the maternal epithelium and trophoblast. x 480. Fig. 10. Day 13.The maternal epithelium is overlain by the trophoblast. Blunt microvilli cover the domed apices of most of the maternal cells. A bulbous protuberance with a smoother apical plasmalemma is indicated by the asterisk. EM, x 3,000. Ftg. 11. Day 13. Indentations in the trophoblast correspond to the contours of two maternal epithelial protrusions which were probably juxtaposed prior to processing (arrows).EM, x 2,900. Fig. 12. Day 13. A discrete area of close contact is present between the smooth plasmalemma of a maternal epithelial cell and the trophoblast. A band of microfilaments (F)extends between the junctional complexes below the apical dome of the maternal cell. EM, x 11,700. Fig. 13.Day 13. A maternal apical protrusion and the microvilli of an adjacent cell are covered by a thick fibrous glycocalyx. The enclosed area is enlarged in the inset. Inset: Higher magnification of the fibers which extend between the two epithelia. EM, x 10,650; inset, x 15,400. Flg. 14. Day 13. A single trophoblast cell overlies three maternal epithelial cells. The respective plasmalemmae are closely aligned (arrowheads), and small areas of microvillous interdigitation are present (asterisks).EM, x 15,600. Fig. 15. Day 16. Tall simple columnar cells a r e extensively vacuolated, have apical nuclei and slightly domed apices. x 500. Fig. 16. Day 16. Disarrayed epithelium is evident near a site of attachment. x 500. Fig 17. Day 16. Where overlain by chorion, the maternal epithelial cells have irregularly domed apices and contain nuclei which vary in position and orientation. x 800. Fig. 18. Day 16. Dense inclusions occur a s groups in the basal region and also extend throughout t h e cytoplasm. Long strands of endoplasmic reticulum occur near the lateral membranes (arrowhead). Elongated mitochondria and membrane whorls ( W ) a r e associated with large glycogen accumulations ( G l ) . The basal lamina is closely aligned to the smooth basal plasmalemma ( b l ) .EM, x 9,200. Fig. 19. Day 16. The maternal epithelium forms several knobs ( a r rowheads) around which t h e thick chorion is moulded. A few scattered endoderm or mesenchyme cells a r e found above the chorionic epithelium (El. ~ 5 0 0 . Fig. 20. Day 16. Small dense inclusions lie below the smooth plasmalemma of rounded maternal apices (arrowheads). Several layers of chorionic epithelial cells lie above and contain prominent nucleoli (Nu),finely dispersed euchromatin, abundant endoplasmic reticulum, and lipid inclusions ( L i ) .Microvilli, shown in cross-section, a r e probably of chorionic origin. EM, x 6,100. 230 J.L. KEYS A N D G.J. KING 16 persisted. Abundant membraneous material suggestive of secretory vesicles was distributed over the microvilli. The basal complexes of dense inclusions were more prominent than a t day 16. Glycogen was distributed irregularly throughout the cytoplasm and often filled apical blebs. Membrane whorls were associated with the larger deposits. Degenerative cells were rarely observed. Embryonic membranes a t day 19 of pregnancy were extensively applied over and attached to the folded uterine mucosa. Disarrayed cells with pale apical blebs and spiky or rounded processes were overlain by the chorion. At sites of attachment the maternal cells retained prominent rounded apices and contained small vesicles, as did the overlying chorion (Fig. 25). In some sections, the maternal epithelium appeared to be composed of cuboidal cells, the entire apices of which were raised into wide low domes. There were no signs of degeneration in areas of attachment. Mitotic figures were observed in both the maternal and chorionic cells. The ultrastructure of maternal epithelial cells a t sites of attachment was similar to those a t day 16 of pregnancy. In contrast to unattached regions, basal complexes of dense inclusions were very small. Large dense bodies were scattered throughout the cytoplasm, and small forms were often found below the apical plasmalemma. Apical and basal glycogen deposits were large and often contained membrane whorls. Rough ER was plentiful throughout the cells and was commonly organized in large spirals within the apical cytoplasm. The cytoplasm within the bulbous cell apices was packed with organelles and only a n occasional glycogen deposit (Fig. 26). The chorion was extensively vascularized by blood vessels introduced by the allantois. The structure of the chorionic cells was similar to the previous stage except for increased height and evidence of polarity at advanced sites of attachment. Smooth and rough ER and mitochondria were dispersed close to the fetalmaternal interface. Nuclei tended to be placed distal to the maternal cells. The chorion was backed by connective-tissue cells. At sites where electron-dense secretion was interposed between the two epithelia, the chorionic cells sent long microvilli into this substance and complexes of small dense inclusions were observed along the face of the chorionic cells. Various stages of attachment were observed a t day 19. In many places the chorionic and uterine epithelial cells had lost their microvilli and fibers of glycocalyx extended between the two, or the respective smooth plasmalemmae were intimately apposed (Fig. 26). In other regions, microvillous interdigitation was just beginning and long slender processes from the chorion extended down between the maternal cells (Fig. 26). In areas where microvillous interdigitation was most advanced, chorionic processes were shorter and apical doming on the maternal side was less dramatic (Fig. 27). Three types of modifications a t the placental interface were observed. Regular areolae were commonly observed a s thickenings in the chorioallantois a t uterine gland openings in regions near the embryonic disc Day 19 (Fig. 28). The gland opening contained PAS positive In unattached areas, the variety of regional cell mor- secretory material which was similar in staining propphologies and ultrastructural features described a t day erties to large granules in the overlying chorion (Fig. arranged parallel to the lateral cell membranes (Fig. 18). Cells occupying areas close to or apposed by the chorion contained abundant glycogen and a plethora of organelles. Degenerative cells could not be identified in regions of contact. In some areas, the maternal epithelium formed small mounds or knob-like proliferations around which the chorion was moulded (Fig. 19). Most of the cells contained large vacuoles, sometimes accumulated to the point where the entire cell was vacuolated. Large accumulations of glycogen were present above and below the nucleus. The entire apex of each cell was gently rounded and evenly covered with either very short microvilli or a smooth plasmalemma with a thick, fibrous glycocalyx. Small complexes of dense inclusions occurred a t the basal plasmalemma and similar bodies were scattered just below the apical plasmalemma (Fig. 20). Prominent Golgi complexes often contained electron-dense material. There was evidence of small channels a t the base of some of the microvilli on the maternal side (Fig. 21). The chorionic cells varied in shape from elongated and flat to cuboidal or columnar. Spindle-shaped endoderm or mesenchyme cells were observed over the chorion (Fig. 19), and the well-vascularized yolk sac endoderm was present. Nuclei within chorion cells varied in shape, contained finely dispersed euchromatin and one to two prominent nucleoli, but lacked chromatin at the nuclear envelope (Fig. 22). Round to oval mit,nchondria conhining an electron-lucent, mat,rix and fenestrated lamellar cristae were numerous. Golgi were complex and some cisternae appeared to contain electron-dense material. Rough endoplasmic reticulum and small cytoplasmic vesicles were extremely abundant. Lipid inclusions were prominent in some chorionic epithelial cells (Figs. 20, 21), a s were electrondense inclusions. Tight junctions connected the cells. Microvilli were scattered across the surface of the chorion cells and were particularly long and plentiful where fetal cytoplasm extended between the maternal domes (Fig. 23). An abundance of small dense vesicles occurred near the interface of the two epithelia, particularly a t the base of the microvilli (Fig. 23). Attachment at this stage involved blunt extensions of chorionic epithelial cytoplasm between the maternal epithelial domes (Figs. 20,21, 23). Fibers from the glycocalyces extended between the two epithelial surfaces. Where microvilli were absent from both epithelial surfaces, the respective cell membranes were closely apposed (Fig. 23). There was evidence of restricted areas of microvillous interdigitation (Fig. 24). In three sites near the embryonic disc, the maternal epithelium was markedly thickened and overlain by a broad region of the chorion which contained large vacuoles. The space between the two epithelia contained PAS-positive secretion, amorphous material, and cellular debris. These structures may have represented early irregular areolae. Staining with PAS indicated that positive granules were distributed throughout the uterine epithelium and chorion and within the f locculent material overlying both. Fig. 21. Day 16. The cells of both the chorionic and maternal epithelium are joined together by tight junctions. The chorion contains lipid inclusions iLi), endoplasmic reticulum, and mitochondria with fenestrated septate cristae. Arrowheads indicate possible channels at the base of the blunt microvilli on the maternal side. EM, x 23,400. Fig. 22. Day 16. Chorionic epithelium lies in direct contact with maternal epithelial cells and contains large round nuclei ( N ) with evenly dispersed euchromatin, a n absence of chromatin a t the nuclear envelope, and large round nucleoli (Nu). Mitochondria containing electron-lucent matrices and crisp cristae are abundant as is rough endoplasmic reticulum and small cytoplasmic vesicles. Golgi complexes are prominent, and some cisternae contain electron-dense material. EM, x 6,000. 232 J.L. KEYS AND G.J. KING Fig. 23. Day 16. Extensions of the chorion are covered by microvilli and extend between the maternal apical domes. Numerous small vesicles are observed along the interface (v). Where denuded of microvilli, the two epithelia are closely aligned (arrowheads). EM, x 18,400. 28). Irregular areolae occurred as much larger st~-iictures formed by irregular mounds of uterine epithelial cells over which lay a space enclosed by markedly vacuolated chorionic epithelium (Fig. 29). Pale basophilic flocculent or solid PAS positive secretion along with cell fragments were found within the enclosed lumen. Finally, this stage was marked by a n extremely complex inward folding of the uterine epithelium to form a branched gland-like structure (Fig. 30). It appeared as though cells similar to chorionic cells in nuclear characteristics, size, and content of PAS positive granules were present within the lumina of these folded structures (Fig. 30). DISCUSSION Significantly greater cell height on days 10 and 13 of pregnancy relative to comparable stages of the cycle was unexpected since a previous publication indicated that differences could not be detected until days 18 to 20 (Corner, 1921). This discrepancy may be explained on the basis of sampling protocol. Corner (1921) and also Crombie (1972) recorded cell height measurements for selected animals at each stage (n = 1or 2) and measured fewer cells than in the current study. Cell height varies substantially between and within individuals, so relatively large samples are necessary to detect moderate differences. In contrast to previous reports, approximately two-thirds of individual cellheight measurements on day 13 and thereafter were taken from areas with preserved chorionic contact. Glycogen and organelles were particularly abundant in the cells a t attachment sites, and many cells possessed bulbous apical protrusions which contributed to epithelial height. An explanation for increased cell height in randomly sampled areas of day 10 pregnant uteri is more dif'ficult since internal ceIl structure was essen- tially similar to that observed on day 10 of the cycle (Keys and King, 1989). This phenomenon, however, may represent a n early pregnancy-associated modification in the luminal epithelium. Mean cell height on day 16 of pregnancy was equivalent to that at days 10 and 13 of pregnancy and day 16 of the cycle. Uterine epithelial cell height was significantly lower in pregnant animals on day 19 than in pregnant gilts on days 10 to 16 or in cycling animals on days 16 and 19 of the estrous cycle, in agreement with a previously published report (Corner, 1921). The lower cell height reflected broad epithelial cells with less rounded apices a t regions of advanced attachment. Corner found no difference between uterine epithelium in non-pregnant and pregnant gilts until day 15, but Stroband et al. (1986) observed ultrastructural modifications on day 12. Clear pregnancy-associated morphological changes were not detectable by day 10 in the current study, but subsequent alterations suggested increased potential for metabolic and secretory activity. Proximity to the chorion a t and after day 13 appeared to be related to further localized differentiation in the structure and arrangement of the adjacent maternal epithelium. The uterine epithelium in pregnant gilts a t days 16 and 19 was markedly different from the pseudostratified epithelium containing cells that varied greatly in staining properties, mitotic figures, and degenerative cells a t equivalent stages of the estrous cycle (Keys and King, 1989). In regions of developing attachment, nuclei were displaced to central or apical positions within the epithelial cells, similar to the shift described between days 14 and 16 of gestation by King et al. (1982). Nuclear displacement was correlated with abundant basal deposits of glycogen and increases in basally located, synthesisrelated organelles, as observed in the glandular epithe- PLACENTATION I N THE PIG lium of the porcine uterus later in gestation (Sinowatz and Friess, 1983) and in the luminal epithelium of women during the luteal phase of the menstrual cycle (Verma, 1983). At day 13, supranuclear and basal accumulations of glycogen were greater in pregnant gilts than in cycling animals, and were largest a t areas of attachment. Dantzer (1985) and Stroband e t al. (1986) have also described massive accumulations of glycogen after day 12 of pregnancy. In contrast to cells a t day 19 of the cycle in which glycogen was absent (Keys and King, 19891, cells in pregnant uteri on this day had abundant reserves of this carbohydrate distributed irregularly throughout the cells and in large basal and apical deposits. Association of smooth ER, membrane whorls, and elongated mitochondria with this carbohydrate a t days 16 and 19 suggested active glycogenolysis. Pig conceptuses produce estrogens (Perry et al., 1973), and experimental evidence in other species indicates that this steroid is capable of increasing the rate of uptake of glucose (Szego and Roberts, 1953; Bitman et al., 1965; Roskoski and Steiner, 1967) and glycogen accumulation by uterine epithelial cells (Boettinger, 1946; Walass, 1952) a s well as the rate of efflux from the uterus (Roskoski and Steiner, 1967). The localized effect of the chorion on accumulation of this carbohydrate at day 13 suggests that a n embryonic signal may direct its deposition. Injection of cycling gilts with estradiol valerate on days 11to 13 was followed by accumulation of glycogen wit,hin the uterine luminal epithelium on day 13; intraluminal instillation of estradiol-17P between days 10 and 13 produced a similar result (Keys and King, 1988). In pregnant gilts, maternal or embryonic signals must not only induce deposition of this carbohydrate prior to and during placental establishment, but also regulate the enzymes involved in glycogenolysis, making glucose available as a n energy substrate for embryonic and placental development. Total uterine fluid glucose increases dramatically from day 12 to days 16 and 18 in pregnant gilts, but remains a t consistently low levels throughout the cycle (Zavy et al., 1982). Dantzer (1984) has hypothesized that glycogen can readily be utilized by the embryo in the early stages of pregnancy, whereas a n extensive lysosomal system in the older placenta is necessary to digest maternal macromolecules to make them suitable for use by the conceptuses. During days 13 to 19 of gestation, dense inclusions formed more prominent basal complexes and extended in greater numbers throughout the cytoplasm than in cycling animals. Similar granules in porcine uterine epithelial cells during early gestation have been observed previously (Dantzer, 1985; Stroband et al., 1986) and are associated with secretion (Crombie, 1972; Dantzer e t al., 1981; Friess et al., 1981). The amorphous, PAS positive secretion interposed between the endometrial folds or chorion and uterine mucosa was similar in density to the dense intracellular granules. Furthermore, PAS positive granules which were not removed following diastase treatment corresponded in distribution to these inclusions, indicating that they may contain acidic glycoproteins. Their incidence was markedly increased and they ranged in distribution from the basal area to cell apices in areas of direct trophoblast contact. Basha et al. (1980) reported 233 that protein secretion by porcine endometria was elevated in tissue underlying a conceptus, suggesting a localized action of the blastocyst in promoting secretory protein synthesis andlor release. Similar granules were also prominent in trophoblast cells a t all stages. Although these inclusions were not directly observed to undergo exocytosis, the change from the strictly basal location to apical positions during early gestation suggested that they may have migrated towards the apex of the epithelial cell for release into the lumen and subsequent uptake by the trophoblast. Sinowatz and Friess (1983) indicated that, although these granules stained with PAS and contained iron, some but not all contained acid phosphatase activity, suggesting that two types of granules may be present. The morphology of these structures in the preparations under study indicated that they could also have been lysosomes which were generated during the cycle and early pregnancy and remained dormant during the time of this study. Lysosomes have been described within the luminal uterine epithelium in a number of species (for review, see Wood, 1970) including the maternal epithelium at the interareolar region of the mature porcine placenta (Friess et al., 1980; Bjorkman et al., 1981; Dantzer, 1984). The membrane whorls occasionally observed throughout the cycle and early pregnancy were probably residual bodies. The large membrane-bound pockets of degenerative material observed a t day 19 of the cycle (Keys and King, 1989) had associated PAS positive staining areas arid resembled the giant lysosomes present during the proliferative stage of the human menstrual cycle (Cavazos and Lucas, 1970). Both the residual bodies and giant lysosome-like structures must develop from primary lysosomes, which are morphologically similar to the dense granules. During early gestation, lysosomes could be produced in increased numbers for the development of a n extensive lysosome system which Dantzer (1984) has described in the mature porcine placenta. At day 13 of pregnancy, secretory vesicles were often present subjacent to the apical plasmalemma and amongst the microvilli, similar to those described by Dantzer (1985) a t day 15. Small vesicles are released from the apical cytoplasm of glandular epithelial cells a t day 12 in response to elongation and to estradiol production by the blastocyst (Geisert et al., 1982a). A similar mechanism operating a t the level of the luminal epithelium could explain the paucity of vesicles directly underlying the apical plasmalemma a t day 13 of pregnancy, in contrast to the abundance of apical vesicles observed at day 10 in some regions. Patterns of protein synthesis are compatible with morphological signs of increased metabolic activity during early pregnancy. Geisert et al. (1982a) reported that the total protein content of uterine flushings at day 12 of pregnancy is dramatically increased over day 12 of the cycle. Where electron-dense material was interposed between the two epithelia, particularly at regions where chorionic processes penetrated between the maternal apical domes, long chorionic microvilli were indented into this substance a t days 16 and 19 of pregnancy. At the base of the microvillous border, small channels and networks of small dense vesicles were observed along the face of the chorionic cells. Similar endocytotic ves- 234 J.L. KEYS AND G.J. KING Figs. 24-26 PLACENTATION IN THE PIG icles involved in the absorption of uterine secretion were described in more mature porcine placentas (Perry, 1969; Friess et al., 1981; Dantzer e t al., 1981), and endocytotic activity was demonstrated by uptake of peroxidase and ferritin in these regions (Stroband et al., 1984). The results of this investigation indicate that attachment events in the pig commence in the region of the embryonic disc as suggested previously (King, 1983; Dantzer, 1985) and progress outward toward the chorionic extremities, a s in the cow (Leiser, 1975; Wathes and Wooding, 1980; King e t al., 1982). Attachment events were initiated earlier and advanced more rapidly than previously reported, with all stages of the “implantation” process from apposition through attachment occurring simultaneously along a conceptus a t day 13 and thereafter. By day 13, all pregnant gilts exhibited localized modifications of the maternal epithelium regardless of whether contact with the chorion was preserved and, although timing of ovulation was not precise, this process undoubtedly began earlier. The rapidly progressive nature of placentation in the pig explains the broad range in temporal estimates for apposition and adhesion, from day 14 (Dantzer, 1985) to day 18 (Perry, 1981), as the presence and nature of fetal-maternal contact is dependent upon proximity of sampling sites to the embryonic disc. Although microvillous interdigitation was previously thought to begin at day 18 (Amoroso, 1952; Crombie, 1972), Dantzer (1985) reported that this relationship was present by day 15 to 16. The samples examined in the present study indicated that i t is certainly present a t day 16 and began formation as early a s day 13 in the region of the embryonic disc. Loss of microvilli accompanied formation of maternal apical protuberances, providing a smooth surface for initial discrete cell-to-cell contacts, after which the trophoblast cells became apposed to a greater portion of the maternal domes to the level of the junctional complexes. The formation of rounded protuberances on porcine uterine epithelial cells was described by early researchers (Robinson, 1904; Assheton, 1906; Heuser, 1927). Corner (1921) first suggested t h a t the frayed or rounded protuberances on maternal epithelial cells were modified to provide a roughened surface for anchoring or immobilization of the blastocyst. In the present study, the small knob-like proliferations of maternal epithelium to which the chorion was applied and ~~ ~ Fig. 24. Day 16. Extensions of chorionic epithelium between mater- nal, microvilli may represent the onset of definitive attachment (arrowheads). EM, x 18,300 Fig. 25. Day 19. Tall pale-staining chorionic cells overlie maternal epithelial cells which have domed apices and contain an abundance of small pale vacuoles. The fetal side is vascularized by the fused allantois, as indicated by capillaries (bv). x 450. Fig. 26. Day 19. The electron-lucent chorionic epithelium extends between the apical processes of the maternal epithelium. Microvillous interdigitation has begun in some regions (arrowheads), while the smooth fetal and maternal plasma membranes are closely aligned elsewhere. The apical protuberances contain abundant rough endoplasmic reticulum, mitochondria, prominent Golgi, and dense inclusions. EM, x 11,700. 235 the shallow placental folds which were initially observed on day 13,probably acted a s a n anchoring mechanism in securing early attachment. Regions exhibiting various phases of the attachment process were present a t both day 16 and day 19; and where microvillous interdigitation was developing or present, the chorionic processes were much reduced, and apical doming on the lower broader maternal epithelial cells was less dramatic. These domes become less conspicuous a s gestation proceeds (King e t al., 1982). During the course of this study the chorionic processes never extended below the level of the junctional complexes of the maternal epithelium, in accordance with other reports (Heuser, 1927; Crombie, 1972; King et al., 1982; Dantzer, 19851, although they have been reported to extend through the epithelial layer (Robinson, 1904; Amoroso, 1952). Folding of the endometrium to form a branched configuration penetrated by chorionic cells on day 19 represents a unique observation in the pig and was suggestive of a n early microcotyledon as described in the mare (Steven and Morriss, 1975). Its significance is unknown, although the area for absorption of material from the uterine epithelium and stability of attachment would be increased by its presence. Crombie (1972) noted that numerous short regions of intercellular junctions were present between porcine trophoblastic and uterine luminal epithelia on day 18. She referred to these a s macula occludens and stated that the opposing cell niembraiies were fused along their outer membrane leaflets. However, it is clear from her discussion that she was referring to gap junctions or nexi, in which the apposed membranes are actually separated by a space of 2 to 3 nm. This type of junction is associated with rapid ionic and small molecule transport a s well a s electrostatic coupling and is found in cardiac muscle and liver, tissues in which communication between constituent cells is essential. A close apposition resembling a tight junction was depicted by Perry et al. (1976) between the chorion and uterine epithelium in a porcine placenta a t day 17. Junctional complexes and primitive punctate desmosomes have been observed during implantation in mammals forming hemo- and endotheliochorial placentae (Enders and Schlafke, 1969; Finn, 1983). However, Dantzer (1985) failed to find evidence of these junctional complexes in the porcine placenta, in accordance with other investigations on epitheliochorial placentation (Bjorkman, 1973; Leiser, 1975; Wathes and Wooding, 1980; Friess et al., 1980; Guillomot et al., 1981; Dantzer et al., 1981; Wooding et al., 1982; Guillomot and Guay, 1982). In regions where the two membranes were devoid of microvilli and most closely associated in the specimens examined in this study, a space of 9 to 20 nm was present. This range indicates that these junctions were not gap junctions. However, some of these areas could be referred to a s zonula adherens, since this type of adhesive junction is characterized by a space of 20 nm between the respective plasma membranes. Changes in the smoothness of the basal plasmalemma and basal lamina and lateral cell membranes were probably related to redistribution phenomena associated with reorganization of the epithelial sheet. Folding in the basal area could accommodate increased surface area for interaction with the stroma or for the Fig. 27. Day 19. An advanced stage of attachment is indicated by microvillous interdigitation and projection of shallow chorionic processes between the maternal epithelial cells. EM, x 11,400. Fig. 28. Day 19. A thickened region of the chorion overlies a uterine gland opening (g).Inset: PAS-positive granules within the chorionic cells (arrowheads). PAS stain, x 200; inset, x 450. Fig. 29. Day 19. Irregular arrangement of the maternal epithelium and vacuolated chorionic cells a t an irregular areola. x 350. Fig. 30. Day 19. The epithelium lining a duct-like structure is similar in appearance to the maternal epithelium. The darker material in the lumen is cellular and may be of chorionic origin. x 300. 237 PLACENTATION IN THE PIG uptake of raw materials for secretory activity. Basal cytoplasmic extensions have been described in many developing systems, including the bovine uterine gland (Atkinson et al., 19841, and are thought to mediate exchange of signals with the underlying stroma which directs differentiation. Assheton (1906) stated that luminal epithelial cells became degenerative a t day 15 of gestation, but this was refuted by Corner (1921). Regions in close proximity to the blastocyst were devoid of cells undergoing degeneration a t all of the stages examined in the present study. Areas within the same uterus which lacked contact with the chorion commonly contained cells undergoing diffuse degeneration and contributing to the cellular component of the histotroph. In contrast, Dantzer (1985) reported degenerating cells frequently associated with the tops of the epithelial villi, although she did not define her criteria for classification. It is not always possible to identify degenerative cells with certainty, and they are rapidly removed from the epithelium. However, omission of cells which are involved in anchoring the chorion a t this crucial stage does not make sense from a functional standpoint. Furthermore, there is a need for rapid expansion of the epithelial sheet to accommodate placental fold formation. It is of interest that death of uterine epithelial cells is reduced or prevented by estradiol treatment in the mouse (Martin et al., 1973, 1976; Finn and Publicover, 1981), hamster (Sandow et al., 19791, and rabbit (Conti e t al., 1984). Local concentrations of estradiol at sites of attachment would be elevated during placental establishment in the pig, and its local action could explain a regional reduction in cell death. Elaborate networks of microfilaments running parallel to the long axis of the trophectoderm cells were present on day 13, as noted by Crombie (1972) and Geisert et al. (1982a). Smooth endoplasmic reticulum was prominent and no doubt related to steroidogenesis. The chorion appeared to phagocytose dead cells and cellular debris, a property common to mammalian blastocysts (Finn and Lawn, 1968; Enders and Schlafke, 1969; Dent, 1973; Wathes and Wooding, 1980) and previously reported in the pig (Dantzer e t al., 1981; Stroband et al., 1984). The dense cisternae of Golgi apparatus may have been associated with formation of lysosomes which are common to the trophoblast of many species a t implantation (Anderson and Hoffman, 1984; Stroband et al., 1984). The dense inclusions/PAS positive granules may have been lysosomes, which are thought to aid in digestion of engulfed uterine secretion to satisfy the requirements of the developing conceptus. Prominent lipid inclusions at this stage probably served a s a n accessory energy source and a pool for steroid precursors. The appearance of RER was related to demands for structural proteins. Modification of the intracellular structure and possibly the extracellular coat of the maternal epithelium occurred by day 13 of gestation in the pig, increasing the synthetic and secretory capacity of this tissue and preparing it for attachment of the embryo. The results of this investigation indicate that the alterations may be directed, a t least in part, by (an) embryonic signal(s1 acting on the adjacent endometrium in a localized manner. Local effects of intraluminal estrogens have been implicated in several crucial events during early preg- nancy in pigs, including increased uterine blood flow (Ford and Christenson, 19791, altered distribution of uterine prostaglandins (Bazer and Thatcher, 1977), modified endometrial protein synthesis and secretion (Geisert et al., 1982a,b), preferential stimulation of progesterone production by the ipsilateral ovary (Ford et al., 19821, and alterations in luminal histotrophic components (Zavy et al., 1980, 1982; Geisert et al., 1982a). It is tempting to suggest a key role for this steroid in early blastocyst-endometrial interactions. The distribution pattern of estrogens in the blastocyst at day 12 corresponds well with the localized and progressive nature of structural changes in the maternal epithelium. A large number of cells in the trophectoderm produce estradiol-17P near the embryonic disc, and the number decreases progressively distally (King and Ackerley, 1985; Bate and King, 1988). Any connection between estrogens and localized chorionic influences on the maternal epithelium remains speculative since it is not yet possible to attribute alterations to specific embryonic products. A need clearly exists to resolve the nature and relative importance of direct embryonic signals involved in attachment and placental development. Understanding the structure-function relationships of placentation has possibly been hindered by the desire to attribute definite dates to specific events based on the use of limited samples from static points. The sampling approach utilized in this study indicated that all stages of placental development including tenuous microvillous interdigitation were present on days 13 through 19. Placentation in the pig is a process consisting of a continuum of sequential events which occur over a broad time frame and not a t specific discrete times. Unfortunately, it seems as though a n obvious characteristic of placentation, that i t is a developmental process, has often been overlooked. ACKNOWLEDGMENTS We gratefully acknowledge the technical contributions of Barbara Atkinson, Dominic Bellissimo, and Douglas Wey. Research funds were provided by the Ontario Ministry of Agriculture and Food and the Natural Science and Engineering Research Council of Canada. 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