Initial vascularisation in the pig placentaI. Demonstration of nonglandular areas by histology and corrosion castsкод для вставкиСкачать
THE ANATOMICAL RECORD 238:177-190 (1994) Initial Vascularisation in the Pig Placenta: 1. Demonstration of Nonglandular Areas by Histology and Corrosion Casts VIBEKE DANTZER AND RUDOLF LEISER Department of Anatomy and Physiology, Royal Veterinary and Agricultural Uniuersity, Frederiksberg C, Denmark (V.D.); Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig-University, 0-6300 Giessen, Germany (R.L.) ABSTRACT The vascular interrelationship of the well-established porcine placenta has previously been described from vascular casts and histology, but not its developmental stages. This study was performed using the same methods on 17 sows of well-known stages of gestation ranging from 9% to 43 days post coitum (p.c.1. At the precontact stage, days 91/2 to 12% P.c., the subepithelial capillaries formed a wide open meshwork of variable diameter, 3-14 pm, without any difference between meso- and antimesometrial side. At the early contact and adhesion stages (days 13 to 18 p.c.1, the first increase in vasculature was seen at the mesometrial side close to the embryonic disc of the very long blastocyst at day 15 P.c., 2 days after the first contact between trophoblast and maternal epithelium was seen. At day 18 P.c., the areas with dense capillaries increased markedly at the mesometrial side with the same parallel organization as seen at day 15 P.c., whereas the antimesometrial side still had a relative loose appearance comparable to the previous stages. At the early placental stages (days 20Vz to 23 P.c.), the capillary bed formed smooth folds, which in some areas at day 20% days developed into smaller folds or prerugae. Here the capillaries changed to convoluted forms with slightly bulbous dilations measuring about 3 0 3 5 pm in diameter. This developmental progress became more elaborate at day 23: capillaries of the low ridges of prerugae formed irregular dilations up to 50 pm in some areas. At this stage the parallel arrangement of the capillary meshwork characteristic of the previous stage was not longer discernable. By days 3 2 4 3 P.c., an increase in microscopic folding was present, and the maternal arterioles could be traced to the top of the ridges, creating the characteristic vascular architecture needed for an efficient exchange of oxygen, carbon dioxide, and nutrients of the basically developed porcine placenta. o 1994 Wiley-Liss, Inc. Key words: Pig placenta, Vascularisation, Scanning electron microscopy, Implantation During placentation the vasculature on the maternal and fetal sides are in continuous development though different characteristics exist for each type of species (Dantzer et al., 1988; Leiser and Koob, 1992). Development of the placental vascular architecture is of considerable importance in influencing the exchange of nutrients, oxygen, and carbon dioxide between mother and fetus (Faber and Thornburg, 1983). Other factors, such as change of the components of the interhemal membrane and prolonged inanition of the mother pig, have a lesser effect on fetal growth (Hard and Anderson, 1982) than does restriction of blood flow (Molina et al., 1985). Microvascular architecture and maternal-fetal blood flow interrelations have been investigated in the pig model between days 35 and 99 of gestation (Leiser and Dantzer, 19881, though the vascular morphology during earlier placental development has not hitherto 0 1994 WILEY-LISS, INC. been described. During porcine gestation uterine blood flow up to 11 days p.c. was found to be similar to the non-pregnant state, whereafter it increased on days 12 and 13 (Ford and Christenson, 1979). Blood flow to those uterine segments in contact with the conceptus was increased in comparison with those segments not in contact (Ford et al., 1982). The ultrastructural anatomy of placentation from days 10 to 19 (Keys and King, 1990) and from days 13 Received March 4, 1993; accepted September 10, 1993. Address reprint requests to Dr. Vibeke Dantzer, Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, Bulowsvej 13, DK-1870 Frederiksberg C, Denmark. 178 V. DANTZER AND R. LEISER Fig. 1. Scanning electron micrograph of endometrial blood vessel cast viewed from the luminal side from day 9% p.c. a: Low magnification of the circular endometrial folds formed by an open and loose capillary network. The bar represents 100 Fm. b Higher magnifica- tion showing the variable diameter of the capillaries of the open and irregular network, giving sight to vessels deeper in the endometrium. The bar represents 50 Fm. to 26 p.c. (Dantzer, 1985) has previously been described, as has embryonic and uterine development during early porcine gestation (Stroband et al., 1986; Stroband and Van der Lende, 1990; Dantzer et al., 1991). The three-dimensional architecture of the exposed complementary maternal and fetal placental surfaces from day 20 to day 100 has previously been published (Dantzer, 19841, though no details of vascular development were described. In a study of the endothelial cells of the subepithelial endometrial capillaries from cyclic and pregnant pigs a t days 10-19 after estrus, Keys and King (1988) described increased numbers of fenestrations of capillaries close to the maternal epithelium at day 13 p.c. compared to day 10 p.c. and cyclic stages. The fenestrations occurred independently of adherent fetal membranes, and this observation was supported by a later study (Laforest and King, 1992) of endometrial capillaries on days 13 and 15 after estrus or mating. However neither the location, nor the development of the vascular bed was described. This study was undertaken to elucidate the development of the endometrial microvasculature during the initial stages of placentation. Vascular casts and sections from hard plastic embedding were used from day 9 when tissue is similar to non-pregnant stages (Ford and Christenson, 1979;Keys et al., 1986), and up to day 43 when formation of interdigitating microvilli is still progressing at the periphery of the chorionic sac (Friess et al., 1982). Preliminary observations have previously been reported (Dantzer and Leiser, 1988; Leiser and Dantzer, 1990; Dantzer et al., 1991). MATERIALS AND METHODS Uteri from 17 pregnant Danish Landrace sows were used. The sows were inseminated at 6 am and at 6 pm on day 0 , 12 hours after the first sign of oestrus. The uteri were obtained at 10-11 am on gestational day 9%*, 12%*, 14, 14, 15* (3 animals), 16, 18+ (2 animals), 201/2+, 21, 23+, 26, 32+, 33, and 43+ at an abattoir 4 min after slaughter. Microperfusion fixation was performed on placentas from all stages (Leiser and Dantzer, 19881, using 3% glutaraldehyde in 0.07 M phosphate buffer (Bjorkman et al., 1981) with 3%PVP (polyvinyl pyrrolidone). Uterine horns from the 5 earliest stages were either microperfusion fixed andlor im- EARLY PIG PLACENTA INTERAREOLAR VASCULATURE 179 mersion fixed by gentle installation of the fixative into p.c. the blastocysts were isolated by flushing one of the the uterine lumen, as earlier described (Dantzer, 1985). uterine horns in order to verify pregnancy. The contact areas of blastocysts to the mesometrial The stages marked with an * and + were also used for preparation of vascular casts. At day 9% and day 12% side of the endometrium were identified in well fixed areas by gentle dissection in Ringer chloride solution, whereafter these areas, and part of the corresponding antimesometrial side, were cut into 2 mm pieces and immersion-fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer for a further 3 hours. The pieces were then rinsed in buffer, postfixed in 1%OsO, in 0.1 M cacodylate buffer at 5°C for 2 hours, and subsequently dehydrated and embedded in epon or historesin (Technovit 7100 “Kluzer”) by routine methods. From all stages 2 p.m epon or historesin sections were stained with toluidine blue and studied by light microscopy in order to see variations in the vasculature beneath the maternal epithelium and its relation to the embryonic attachment sides from the same part of the horn. From selected stages up to 100 serial sections were also studied. Parts of the uterine horns (20 cm in length) of the animals marked above with an * as well as placental parts from sows marked with a + were used for blood vessel casts. The uterine horns of sows are long with anastomosing rami cornuales from arteria uterina towards the rami uterini from arteria ovarica, as well as of the arteria vaginalis (Boye, 1956). Anastomosing branches on each side of the injection sites were therefore clamped in order to obtain efficient rinsing and subsequent filling of a given uterine or placental part (Leiser and Dantzer, 1988). For the detailed preparation of vascular casts see Leiser and Kohler (19831, Dantzer et al. (19881, and Leiser and Dantzer (1988). A mixture of methylmethacrylate of Batson no. 17 compound (Polyscience, Warrington, PA) and Sevriton (R) was used as a vessel-filling medium because it produces very accurate vessel casts after polymerization (Risco and Nopanitaya, 1980). At the early placental stages there is a very dense vasculature in the myometrium and the circular mucosal folds, which almost overlap creating an extremely narrow uterine lumen. The specimens for scanning electron microscopy therefore required thorough corrosion by potassium hydroxide solution before water rinsing and processing for scanning electron microscopy. To keep an overview of the surface of these folds, large casts (3.5 x 4 cm2) were first examined in order to select specific areas; thereafter specimens were prepared by sectioning the cast after embedding in 20% gelatine cooled to 5°C (compare with Leiser, 1985). These much smaller pieces were then corroded by po- Fig. 2. Light micrograph of the endometrium from the mesometrial side at day 12% p.c. The capillaries (MC) are related to the uterine epithelium as seen in cyclic diestrous stage, with some distance both to the maternal epithelium (ME) and among themselves. The bar represents 50 pm. Fig. 3. Scanning electron micrograph of vascular cast, at day 12V2 P.c., from an area comparable to Figure 2. The capillary network still has an open form and some capillaries show short and dilated segments, which were not seen at the previous stage. The bar represents 10 pm. 180 V. DANTZER AND R. LEISER Fig. 4. Light micrographs from day 15 p.c. a: The endometrium from the antimesometrial side with extremely columnar epithelium (ME). The underlaying capillary bed (MC) has not yet developed and is comparable to the mesometrial side at day 12% p.c. seen in Figure 2. The bar represents 50 pm. b Endometrium from the mesometrial side with the maternal epithelium (ME) in contact with the trophoblast (T) of the fetal membranes. At this side, the capillaries (MC) have changed markedly, as they have become larger and closer to each other. Notice their variable size and oval shape. The connective tissue is rather poor in cellular elements. The bar represents 50 pm. Fig. 5. Scanning electron micrograph of endometrial blood vessel cast from the mesometrial side at day 15 p.c. a: The low magnification on parts of the top of endometrial folds demonstrates how the capillary network is still mainly loose, but lined with areas where the network is much denser (**I. The bar represents 0.5 mm. b Higher magnification of the area marked in Figure 5a showing the transition from a part with a very well-developed and dense capillary network (DC) to the slightly more open network seen around (OC). The bar represents 50 pm. tassium hydroxide as above before final examination in the scanning electron microscope. Corrosion casts The vascular casts from day 9% p.c. showed a very widely meshed and irregular network of capillaries, covering large irregular endometrial folds and arRESULTS ranged in a circular pattern in the uterine horn (Fig. Precontact Stage-Days 9% to 12%p.c. la). The caDillaries had a variable diameter (3 to 14 pm). There-was no prevailing orientation of the capilHistology laries in relation to the circular endometrial folds. The The capillaries were seen a t irregular intervals be- network was so open that the larger vessels of the subneath the maternal epithelium a t day 9?hand 12%P.c., jacent lamina propria and submucosa were demonwith a distance of 2-8 epithelial cells (10 to 40 pm) strated through the meshes (Figs. la,b, 14). between them. In most cases there is some distance At day 12% P.c., the vasculature showed no major between the capillaries and the epithelium (Fig. 2). No difference from the previous stage, though the diameapparent morphological differences were found be- ters of the capillaries were within the same range and tween the mesometrial and antimesometrial sides of looked more voluminous due to local swellings (Fig. 3). uterine horns. Mast cells with metachromatically At these two early stages, there were no differences in stained granules are frequently observed in the rather morphology between vessels from the mesometrial and the antimesometrial side. cell-rich stroma. EARLY PIG PLACENTA INTERAREOLAR VASCULATURE Fig. 6. Scanning electron micrograph of blood vessel casts at the mesometrial side a t day 15 p.c. a: Detail of a fractured cast seen from the side. The arterioles (Al) and the venules (V1) reaching the subepithelial capillary bed (top and right) seem not to have any preference for the origin a t the top or at the base of the smooth endometrial folds. Early Contact and Adhesion Stages-Days 13 to 18 p.c. Macroscopic observation At days 13 and 15 P.c., it was not possible to determine the position of the embryo when inspecting the intact uterine horn, whereas the dilations representing the embryos in their amniotic sacs were clearly visible at days 16 to 18 p.c. The freshly opened uteri had a fine hyperemic line at the mesometrial side at day 13 P.c., most clearly seen close to the embryonic disc. At day 15 p.c. there was a double-hyperemic line. Histology At day 15 P.c., there was a marked increase in vascularisation of the mesometrial side in contact to the blastocyst (Fig. 4b). This is in contrast to the antimesometrial side (Fig. 4a), which looked like the previous stages described above. The capillaries of the mesometrial side were densely packed beneath the epithelium, 181 Luminal side (L).The bar represents 100 pm. b: Detail of the capillary network from the mesometrial side seen from the uterine lumen. The capillaries are larger in diameter than at the previous stages and form a close meshwork with a distinct parallel arrangement. Notice a few thin anastomoses (triangles). The bar represents 10 pm. leaving only a small space between the capillary wall and the base of the maternal epithelium. The diameter of the capillaries varied from 5 to 30 pm. The largest capillaries were most numerous, whereas the very smallest were scattered between them. The larger capillaries were not round but oval, with their long axis perpendicular to the maternal epithelium (Fig. 4b). Occasionally the endothelium seemed to be close to the maternal epithelium. At day 18P.c., the capillaries had become slightly larger than at day 15 P.c., showing less variation in size, although the oval form was still very pronounced. A small distance to the maternal epithelium remained for most of the Capillaries. Corrosion casts At day 15 P.c., the endometrial capillary network outlined irregular rather broad folds oriented circularly to the longitudinal axis of the uterus (Fig. 5). At 182 V. DANTZER AND R. LEISER a: At the mesometrial side the folds become more numerous or sub- divided as evident on a presumably developing cleft (arrow). The capillaries have increased in diameter compared to the previous stage and compose a very closed parallelly arranged network with many connections (*) between the parallel capillaries of almost the same diameter. The bar represents 100 pm. b At the antimesometrial side the capillaries still form an open network, which is more developed than at day 12%P.c., with a tendency to parallel arrangement across the length of the circular folds (the edge of one is seen to the left). The bar represents 100 pm. the mesometrial side, besides the generally loosely meshed network, there was a marked increase in capillary density. This was often seen as two parallel lines or areas of more densely packed capillaries on both sides of a central area with somewhat larger meshes (Fig. 5a). This represents a transition stage. The loosely packed network showed very irregular meshes with a wide variation in the diameter of the capillaries, which also had a bulbous appearance. The capillaries in the dense areas formed a parallel pattern perpendicular to the circular endometrial folds (Figs. 5a,b, 6b, 14). The majority of the capillaries here having a diameter of 15-25 p,m were closely related and connected with many thick as well as few thin branches (Fig. 6b). The precapillary arterioles and the postcapillary venules, seen a t the cut edge of the cast (Fig. 6a), showed no preference in location towards the endometrial folds, and exhibited a smooth surface formed by the capillary network. The vascular casts of the antimesometrial side a t day 15 p.c. formed a wide open irregular network, comparable to the earlier stages. At day 18 P.c., the areas with a dense capillary network had increased markedly at the mesometrial side. The tight network was clearly organized in the same parallel way as described for the dense capillary network seen at day 15 p.c. (Figs. 7,8a); the antimesometrial side still had a relatively loose appearance, with some tendency towards a parallel arrangement of the Fig. 7.Scanning electron micrograph of cast from the mesometrial side at day 18p.c. The capillary bed now forms a dense layer outlining the smooth endometrial folds. The capillaries are oriented in a parallel manner across to the direction of the endometrial folds. The arrow marks location of arteriole or venule reaching the capillary network. The bar represents 100 pm. Fig. 8. Details of the capillary bed of the endometrium at day 18 p.c. EARLY PIG PLACENTA INTERAREOLAR VASCULATURE Fig. 9.Light micrograph from day 20% p.c. of gestation. Fetal capillaries (FC)) with hemocytoblasts are seen close to the trophoblast (T). Maternal capillaries (MC) of the endometrium are very wide and form impressions (arrows) into the uterine epithelium (ME). The bar represents 50 pm. Fig. 10. Overview scanning electron microscopic picture, 2OYz days P.c., showing the transition from the rather large and smooth endometrial folds on the bottom (arrows) to an increase in surface area by a finer subdivision of microscopic folds or prerugae (**). The bar represents 0.5 mm. capillaries (Fig. 8b). The vascular architecture at the mesometrial side then developed into low smooth microscopic folds (Figs. 7, 8a), oriented in the same direction as the large irregular circular folds mentioned earlier (Fig. 5a, 14).The arterial and venous supply of the capillary network was not yet visible at either the top or base of these newly formed microscopic folds. 183 Fig. 11. Details of the capillary bed from day 20% p.c. a: Mesometrial side with development of prerugae (left). The very dense capillary bed is more convoluted than seen at the previous stages, but a parallel arrangement is still visible. The capillary diameter has increased during the last two days bulging to “intraepithelial” locations (compare with the marked impressions in Figure 9). The bar represents 50 pm. b At the antimesometrial side the capillary network of the smooth endometrial folds has retained the open form. The bar represents 100 Fm. Early Placental Stage-Days 20% to 23 px. Histology At day 20% P.c., the maternal capillaries increased in diameter at the mesometrial side often making a slight impression into the maternal epithelium (Fig. 9). At this stage, the fetal side of the placenta in the same area was similarly vascularised, specially close to 184 V. DANTZER AND R. LEISER Fig. 12. Demonstration of different degrees of endometrial blood vessel development on casts at day 23 p.c. a: This detail shows the transition in capillary development from the pattern seen a t day 20Y2 p.c. (compare with Fig. l l a ) to the more developed stages seen in Figures 12b,c. Notice the beginning of capillary enlargements (*) at the top of the prerugae. The bar represents 100 pm. b Low magnification of endometrial cast from a very well-developed area. The capillary network now forming prerugae to distinct parallel ridges with a bulging appearance, due to dilated loops (*). The bar represents 100 pm. c: Slightly higher magnification demonstrating the pattern of these dilated capillaries (*) from an area not quite as well developed as the one seen in Figure 12b. The capillaries at the top of the rugae provide them with an undulating surface. The bar represents 100 pm. observed at the mesometrial side with a few capillaries reaching the same size in diameter, namely 30-35 pm, as seen at the mesometrial side. When seen from the myometrial side, the branching of venules and arterioles can be followed, but without any preference between the top of the prerugae or the fossae between them. At day 23 P.c., this developmental process could be followed from a pattern comparable to the previous stage, but with a further increase in capillary enlargement (Fig. 12a) to other areas, where the process of vascular development progressed dramatically forming irregular dilations of the capillaries of the low ridges of Corrosion casts prerugae (Fig. 12c). In some areas this pattern became At day 20Y2 P.c., the capillary network at the me- even more elaborate (Fig. 12b), because the capillaries sometrial side formed smooth folds, becoming more increased in diameter to about 50 pm; those capillaries elaborate towards smaller folds, or prerugae, in some only measured about 40 pm in diameter a t the dilated areas (Fig. 10). In the smooth folds the capillaries cre- parts of the more regular rugae. Hence it was almost ated a very close meshwork with a parallel arrange- impossible, in those dilated parts (Figs. 12b,c), to disment perpendicular to the longitudinal axis of the cern the parallel pattern seen at the earlier stages of folds, roughly comparable to the first dense network development. seen a t day 15 p.c. However in the prerugae, the capinitial Stage of Basic Placental Developmentillaries changed to convoluted forms (Figs. 10, l l a , 14) Days 32 to 43 p.c. with slightly bulbous dilations measuring about 30-35 pm in diameter. The interconnections between these Histology At day 32 P.c., the placenta increased its microscopic capillaries generally showed the same size, but were also as small as 5 pm. At the antimesometrial side, the foldings compared to the previous stages, though the capillary network was still rather loose (Fig. l l b ) . The depth of the fossae was less, than at later stages of capillaries had a greater variability in diameter than gestation. The capillaries, also called “intraepithelial the embryo. Small microscopic folds, prerugae, were now developed in some parts. At the antimesometrial side, the capillary network was less well developed than at the mesometrial side, although it showed more variations as compared with the previous stages. At day 23 P.c., the microscopic folding had increased followed by a well-developed capillary network beneath the maternal epithelium at the mesometrial side close to the embryo. At the fetal side the vascularisation had increased, too. This development did not yet include the antimesometrial side or paraembryonic parts of the endometrium. EARLY PIG PLACENTA INTERAREOLAR VASCULATURE 185 Flg. 13.Vascular casts from day 32 p.c. a: Endometrial cast viewed from the luminal side. The ridges (R) have become more pronounced forming fossae (F) between them, where the moderately dense network of capillaries is clearly seen. The tops of the ridges are undulated and provided with some dilated capillaries (9.An arteriole (AI) can be followed running from the myometrial side to the top of a ridge. The bar represents 50 pm. b The branching of arterioles (Al) and a venule (V1)can be followed at the myometrial side of the endometrial capillary network. The arteriofes reach to the ridges (not visible here) by entering between the densely meshed network (arrows) of large capillaries forming the row-shaped fossae. The venule arises at the bottom of a fossa ( + 1. The bar represents 50 pm. capillaries,” were indented into the epithelium. The epithelium was thinned on the fetal side at the top and upper sides of the fetal ridges, and a t the bottom and sides of the complementary maternal fossae. This development progressed during gestation becoming clearly discernable 11 days later, a t day 43 p.c. (compare Figs. 2 and 3 in Friess et al., 1982). a basic principle during the gestation period with a continuous placental development, as previously described and illustrated from days 43 to 109 (Leiser and Dantzer, 1988). Corrosion casts At day 32 P.c., the capillary network of the endometrium was well developed, with parallel ridges, or rugae, producing an undulating upper profile (Fig. 13a). Some of the capillaries were dilated at the top of these undulations to a diameter of 35-40 pm. The diameter of the capillaries in the fossae averaged 22 pm with some small interconnections of 5 to 8 pm (Fig. 14). When viewed from the myometrial side, the arterioles could now be followed towards the top of the ridges where they continued into the endometrial capillary network (Fig. 131. The venules from this network arose close to the bottom of the fossae (Fig. 13b). The distribution of the arterioles to the top, and of the venules arising a t the base, of the endometrial ridges (first seen clearly at day 32 p.c. and described above) remained as DISCUSSION This study describes, for the first time, the development and architecture of the endometrial microvasculature of the diffuse folded epitheliochorial pig placenta, both before and during the initial stages of placentation. ~ o r p ~Related o l ~ to~~ ~ e v e l o p ~ of en~ Vasculature/Conceptus Interrelationship The initial contact between the long (1-1.5 m) porcine blastocysts and the endometrium is restricted to the mesometrial side (Perry and Rowlands, 1962). Here the development of the epithelioc~orial placenta begins a t days 13-14 p.c. a t an area close to the embryonic disc at the mesometrial side (Dantzer, 1985). The contact progresses during initial placentation and subsequent developmental stages from the mesometrial side to the antimesometrial side and also towards the distal ends of the blastocyst (Friess et al., 1982; Dantzer, V. DANTZER AND R. LEISER 186 Schematic drawing of early porcine placental stage-related development of vessel ktagesl) and ime schedule PRECONTACT OF EMBRYO 9.5 to 12.5 days p.c. large irregular EARLY CONTACT AND ADHESION OF EMBRYO 13 to 18 days p.c. large irregular + decreasing size smooth --+ jize and form2) lateral relief) )f endometrial olds C: rather small, variable diameter CM: open, irregular, loose C: irregular, generally large, strictly parallel orientation CM: dense, cleftlike openings Vlorphology If :apillaries (C:) and capillary meshwork (CM:I3 14 Refer t o the most developed endometrial areas at the mesometrial side Fig. 14. Schematic drawing summarizing events during initial placentation in the pig (continued on next page). 1985; Keys and King, 1990; Stroband and Van der Lende, 1990; Dantzer et al., 1991). The main events during contact are: (1)protrusion of epithelial proliferations of the endometrium enclosed by chorionic caps which immobilize and anchor the blastocyst at day 13 P.c., followed by a close apposition between the apical plasma membrane of maternal epithelium and trophoblast at day 14 p.c. and ( 2 ) the development of interdigitating microvilli-adhesion first seen at day 15 p.c. of this diffuse epitheliochorial placenta type (Dantzer, 1985).The vasculature at day 15 p.c. forms areas at the mesometrial side with a marked increase in capillary density and size. This development in vasculature, therefore, happens ll/z to 2 days after the anchoring effect is established and only 1 day after the first occurrence of close apposition, thus indicating a specific local effect due to the presence of the blastocyst. A comparable delay of endometrial vascular development has been reported in the rabbit during implantation (Leiser and Beier, 1988).The subsequent extensions of the vas- 187 EARLY PIG PLACENTA INTERAREOLAR VASCULATURE ~ tasted endometrial folds and corresponding capillary meshworks EARLY PLACENTATION 2 0 . 5 to 23 days p.c. INITIAL BASIC PLACENTAL DEVELOPMENI 32 to 43 days p.c. early: small, numerous, smooth to irregular (prerugae) later: small ridges with bulbous tops narrow ridges (often with bulbous tops) and caveous fossae in between C: 2 - top of ridges: bulbous, undulated, largc convoluted with some parallelity, large and slightly bulbous dilations CM: very dense c--------( Scale for 100 pm - fossae: flattenend, randomly oriented, large 2M: dense on ridges, moderately dense in fossae 3) , 4 Scale for 100 pm cular development follow the same pattern as seen for or ridges. At day 32 p.c. the basic vascular pattern of the establishment of the materno-fetal epithelial con- the mature porcine placenta has been created. The vascular development into low ridges described from mactact, but with slight delay. From day 15 there is an increase in the uterine di- erated tissue exposing the basal lamina of maternal ameter due to the development of the fetal membranes, epithelium (Dantzer et al., 1991) seems to precede the and it is at this stage that the increased number of formation of microscopic endometrial folds, when obcapillaries are first seen. There is a remarkable in- served from the uterine or fetal side of exposed placencrease in capillary density from day 15 to day 18 P.c., tal surfaces of the endometrium (Dantzer, 1984). This becoming much more elaborate at day 20% p.c. At day indicates that the changes in the endometrium, i.e., 23 P.c., a dynamic change in capillary diameter has formation of the first ridges and rugae, occur as a contaken place together with an increase in the endome- sequence of the vascular development described here. In an early publication Perry and Rowlands (1962) trial surface area by the formation of microscopic folds 188 V. DANTZER AND R. LEISER observed a hyperemic line in the endometrium a t the mesometrial side at day 13 P.c., similar to where the initial contact between endometrium and the long blastocysts is first established (Dantzer, 1985). We made similar macroscopic observations, but in addition we revealed a double hyperemic line along the mesometrial side a t day 15 and 16 p.c. corresponding to the observation from overview vascular casts (Fig. 5a). Here the mesometrial side a t day 15 p.c. often showed a mid-zone with a slightly looser central network than the dense capillary network a t each side, thus apparently being identical to the double hyperemic lines. In parallel with the occurrence of these hyperemic lines, a fluorescence was first demonstrated using Evans blue (Keys et al., 1986), and later revealed to be an autofluorescence phenomenon (Keys et al., 1989). This autof luorescence consists of two types, namely bright green and red. The green was confined to the endometrium in contact with embryonic membranes and became randomized a t days 16-18 p.c. whereas the red fluorescence continued to increase in intensity and area up to day 29. By day 32 p.c. it was gone. Therefore, the red fluorescence corresponds in position to the lateral limits of direct contact between the embryonic membranes and the endometrium and seems to proceed or follow the development of the dense capillary network described in the present investigation. Whether the fluorescence is due to increased vascular permeability as suggested by Keys and King (19881, or if it represents a substance which influences vascularisation by other means, remains to be clarified. Angiogenesis is affected by many different factors both chemical, mechanical, and their combinations (Hudlicka and Tyler, 1986). Edema will diminish the compactness of the tissue to facilitate capillary growth, creating conditions for faster angiogenesis and threedimensional organisation. During the initial stages of placentation there are several events which favour this concept: the porcine endometrium develops different degrees of edema during the cyclic stages (Leiser et al., 1988), becoming even more pronounced a t days 13 and 15 p.c. (Laforest and King, 1992) as also observed in the present study. This physiological edema is induced by estrogens (Geissinger et al., 1979) and seems to preceed the development of the dense capillary network, which develops close to the luminal epithelium at the mesometrial side and close to the embryo a t day 15 P.c., as described. Mast cell-like granulocytes were seen during estrus (Stroband et al., 1986) and we observed granula-loaded mast cells in the endometrium a t days 9% and 12Y2 P.c., but only few or not recognisable at later stages of gestation. Due to the fact that mast cells may contain the well-known autocoids histamine, prostaglandin D2, and leukotrienes, their containment of these substances contribute to vascularisation and angiogenesis by inducing vasodilation and increased vascular permeability as well as interacting with endothelial cells, growth factors, and angiogenin (Norrby et al., 1986; Folkman and Klagsbrun, 1987; Cocchiara et al., 1988). stages. However, in the present morphological study of the endometrium there are no major changes in the capillary architecture between day 9% and day 12% p.c. of gestation, as both stages are characterized by a wide irregular network with the same variations in diameter. According to these authors the blood flow a t day 14 p.c. decreased markedly to a level slightly higher than that of day 11p.c. and hence stayed almost constant up to day 19 P.c.; thereafter a dramatic increase in blood flow was demonstrated up to day 30 p.c. The increase in total uterine blood flow a t day 11-13 p.c. may be due to myometrial activity during blastocyst migration (Ford et al., 1982; Thilander et al., 19901, whereas the second increase in the blood flow, 19-30 days P.c., can be related to the development of the endometrial microvasculature described in the present investigation. This is because the gradual development of the endometrial capillaries, from the mesometrial side to the antimesometrial side, takes 5 days or more as described above. The first vascular changes are seen a t day 15 and a t the antimesometrial side at day 20Y2 p.c. when compared to the earlier stages indifferent to cyclic stages. Therefore, the increase in uterine blood flow after day 19 p.c. correlates well with the development in endometrial microvasculature . Hormones Estrogens play, together with the endometrial synthesis of prostaglandins, an important role for the regulation of luteolysis and thereby cycle and placentation (Zavy et al., 1980; Bazer et al., 1984; Mondschein et al., 1985). The development in endometrial vasculature and the variations in blood flow pointed out above correlates also with the synthesis of estrogens by blastocysts. The pig blastocysts synthesize estrogens from day 10-11 p.c. (Perry et al., 1973; Heap et al., 1979; Flint et al., 1979; Gadsby et al., 1980; Geisert et al., 1982; King and Ackerley, 19851, whereafter blastocyst elongation (Geisert et al., 1982) and spacing within the two uterine horns takes place (Dziuk et al., 1964; Dhindsa et al., 1967). This will give an increase in blood flow to the myometrium but will not influence endometrial blood flow very much since no changes were observed in the microvasculature. At day 14 there is a decline in estrogen production whereafter there is a second rise of estrogens from day 16 up to day 30 p.c. (Gadsby et al., 1980; Dantzer and Svenstrup, 1986). However this synthesis of estrogens is not uniformly distributed, as in vitro cultures of different segments of the pig blastocysts from days 14-16-18 p.c. showed that the region enclosing the embryonic disc had the highest production of estrogens (Bate and King, 1988). Estrogens seem therefore to act locally as it is the same region where the initial contact between the blastocyst and the endometrium is established (Dantzer, 1984) and where, with a delay of 1-2 days, the first increase in capillary density occurs as shown in the present investigation. Vascular-Stromal Interrelationship Blood Flow Total blood flow to the porcine uterus was investigated by Ford and Christenson (1979). It increases 3 to 4-fold by day 12-13 p.c. of gestation compared to cyclic Interaction between endothelial cells and the extracellular matrix may serve to regulate capillary development and architecture (Ryan and Barnhill, 1983; Yasunaga et al., 1989; Merwin et al., 1990). Experiments EARLY PIG PLACENTA INTERAREOLAR VASCULATURE indicate that the extracellular matrix components may act locally to regulate growth and pattern. Fibroblast growth factor-stimulated endothelial cells may "switch between growth, differentiation, and involution modes during angiogenesis by altering the adhesiveness or mechanical integrity of their extracellular matrix (Ingber and Folkman, 1989). In addition insulin-like growth factor 1 has recently been shown to be localized in the vessel wall during initial placentation in the pig (Persson et al., 1993). Hudlicka and Tyler (1986)referred that intravascular pressure would not change the capillaries as their diameter is supposed to be rather rigid. However, a higher blood pressure would increase blood flow and subsequently capillary growth, because wall tension and stress seem to be important factors for stimulation of endothelial growth. This may be implicated in the development of the vascular architecture of pig placenta as greater curvatures occur where preexisting vessels bend (Waxman, 1981)or branch. An increase in both blood flow, and flow-oriented stress will act as a viscous drag on the luminal surface of the vascular endothelium, being higher a t the outer sides of vessel curvatures and around branching points (Nerem, 1981; Waxman, 1981). This will give greater growth at the convex curvatures of bendings, which may explain some of the morphological features seen during initial vascular development of porcine placentation. When the architectural changes of the vasculature are considered in this respect the initial growth at day 15 P.c., and later the growth a t the top of the ridges as well as the dilations into sinusoidal structures a t the arterioral side of the capillary bed (Figs. 12, 141, can be understood. The revealed vascular development may therefore reflect a local induced interaction between estrogens, proteins secreted by the blastocyst, endometrial production of prostaglandins, factors for granular depletion of mast cells, and blood flow oriented stress a t the luminal side of the endothelium: all events which aim to stimulate angiogenesis and vascular changes during implantation and early placentation in the pig. 189 Cocchiara, R., G. Albeggiani, G. Di Trapani, A. Azzolina, N. Lampiasi, G. Cervello, and D. Geraci 1988 Dispersal of rat uterine mast cells and their functional response to an embryo-derived histamine releasing factor: A possible model for embryo implantation. J . Reprod. 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