Initial vascularisation in the pig placentaII. Demonstration of gland and areola-gland subunits by histology and corrosion castsкод для вставкиСкачать
THE ANATOMICAL RECORD 238:326-334 (1994) Initial Vascularisation in the Pig Placenta: II. Demonstration of Gland and Areola-Gland Subunits by Histology and Corrosion Casts RUDOLF LEISER AND VIBEKE DANTZER Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig-University, Giessen, Germany (R.L.); Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C , Denmark (V.D.) ABSTRACT Tissues from 10 pregnant pigs between 9Yz and 43 days post coitum (P.c.) were prepared for histology and vascular corrosion casts to examine the vascularisation of the gland and areola-gland subunits of the early pig placenta. The endometrial vascular networks shown by casts were equal in both glandular and interglandular fields from days 9%to 14-15 p.c. This represents a cyclic stage uninfluenced by the early implanting embryo on day 13 p.c. By day 15 P.c., the first areola formation was observed both histologically and on casts. The maternal areolar and glandular capillary network developed into a widely meshed loose type, in contrast to the interareolar type which had a dense and parallel architecture. In some cases extremely large, crooked, and highly anastomosed maternal capillaries developed between these two networks. This specialized transition is thought to form the base for the ring-like seal formation (RSF) of the areolar periphery. A firm materno-fetal anchor therefore “seals” the areola and its glandular secretory contents from its interareolar surroundings. Therefore, the glandular and maternal areolar vasculatures are clearly discernable from that of the fetal areolar vasculature. The former are associated with the known materno-fetal substance transfer of the porcine areola-gland subunit. This association, however, seems to remain unspecialized in early placentation, as only the regular roundish areola-not the irregular areola-can be distinctly detected. o 1994 WiIey-Liss, Inc. Key words: Pig placenta, Vasculature, Areolar development, Uterine glands, Morphology Porcine endometrial glands are relatively sparse (Perry and Crombie, 1982; Leiser et al., 1988). After initial placentation of the embryo in the uterus at days 13-15 p.c. (Dantzer, 1985; Keys and King, 1990), the area around each of the gland openings becomes the maternal part of a n areola, or maternal areolae. The cavity represents a specialized area of the uterine lumen (Dantzer, 1984, 1986) separating the uterine epithelium from the allantochoriodamniochorion, or fetal areolae. The combined uterine gland together with the maternal and the fetal areolae form the so-called areola-gland subunits of pig placenta (Amoroso, 1952). Glandular secretions accumulate in the areolar cavities, a n area being free of materno-fetal contact. The areolae are macroscopically clearly visible as opaque circular spots in the last half of gestation and are subdivided into regular and irregular areolae receiving histiotrophe secreted by one or several uterine glands respectively (Brambel, 1933; Amoroso, 1952). The first occurrence of areolae is described on histological section related to the central part of pig placenta from day 18 p.c. (Crombie, 1972). Due to glandular secretions the areolae, and the space between the maternal and fetal circulations, represent a specialized subunit for materno-fetal ex0 1994 WILEY-LISS, INC. change (Palludan et al., 1970; Friess et al., 1981; Dantzer, 1986) of larger molecules and ions such as iron (Wislocki and Dempsey, 1946; Buhi et al., 1979; Dantzer and Nielsen, 1984; Dantzer and Leiser, 1992). The development of this capacity may be influenced by the architecture and microstructure of the vascular system of the areola-gland subunit, and therefore knowledge of blood flow interrelationships is important to understand the exchange capacity of this diffuse epitheliochorial placenta (Dantzer et al., 1988). Early development of the areola-gland subunit and the development of the periglandular and areolar blood vasculature of porcine placenta during the initial stages of placentation was investigated by means of Received March 4, 1993; accepted September 10, 1993. Address reprint requests to Dr. Rudolf Leiser, Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig-University, Frankfurter Str. 98, D-35392 Giessen, Germany. This paper is dedicated to the 60th birthday of Prof. Dr. J . Frewein, Veterinary Anatomical Institute, Zurich, Switzerland. EARLY AREOLA-GLAND VASCULATURE OF PIG PLACENTA semithin sections and scanning electron microscopy of corrosion casts. A corresponding study of areolar vascularisation in the near-term placenta has been presented using the same methods (Dantzer and Leiser, 1993b). Early Contact (Apposition) and Adhesion Stages-Days to 18p.c. 327 13 Uteri from 11 pregnant sows at days g1/2, 121/2, 13, 15, 18, 20Y2, 23, 32, 33, 35*, and 43 p.c. were obtained at a n abattoir within 4 min after slaughter. Gestational age was well known a t all stages from the time of natural insemination with the exception of day 35 when this was estimated using crown-rump length and weight (De Villiers et al., 1958; Marrable, 1971). Endometrium or placenta was fixed by intrauterine immersion fixation up to day 15, or later by microperfusion, then dehydrated and embedded in epon or historesin (Technovit 7100 “Kulzer”). Routine methods were employed as described earlier in detail by Dantzer (1986) and by Leiser and Dantzer (1988). Semithin sections were cut from selected blocks of all stages, and stained with toluidine blue for light microscopy. From all gestational stages (except the one marked with *) the uterine walls were used to make vascular casts of the maternal placental endometrium. For detailed preparation of vascular casts see Leiser and Kohler (1983),Leiser and Dantzer (1988), and Dantzer and Leiser (1993a). Vascular changes (as demonstrated by corrosion casts) were not visible on day 13 px., the time when implantation of the embryo occurs close to the embryonic disc (Dantzer, 1985). However by day 15 P.c., the stage when more intimate contact took place with the formation of interdigitating microvilli (Dantzer, 1985), the first occurrence of a n areola was seen histologically (Fig. 2a). The endometrial gland mouth and the overlying chorion distinctly enclosed darkly stained embryotrophe (uterine secretion) typical for the areola. As compared with the precontact stage, subepithelial capillaries were now larger, more numerous, and were nearer to the surface epithelium; towards the glandular epithelium, capillaries remained sparse with a distinct distance to the epithelium (Fig. 2a). Vascularisation of the chorion was not evident at this stage (day 15 P.c.; Fig. 2a). The vascular cast at this stage demonstrated wider recess-like gland openings at the endometrial surface a s compared to the precontact stage (compare with Figs. 2b and lb). The capillary network of the gland openings still had a rather loosely meshed capillary arrangement, whereas the capillary network of the interglandular endometrium had changed to a dense and parallel arranged pattern. The interglandular capillaries had a uniform diameter though the glandular capillaries remained irregular as before. RESULTS Early Placentation StageDays 20% to 23 p.c. MATERIALS A N D METHODS Precontact Stage of the Embryo-Days 9% to 12% p.c. Histologically, the gland openings of the pig endometrium were sparse and narrow (Fig. la). Subepithelial capillaries, located in the stratum compactum beneath the superficial and glandular epithelium, were relatively numerous but had a distinctly visible distance from the epithelium. A few arterioles and venules, averaging about three times the diameter of capillaries (Fig. la), represented the blood vessels of the deeper endometrial stroma (stratum reticulare) with loosely arranged connective tissue. As visible in casts (Fig. lb), the endometrial surface was formed by a two-dimensional widely meshed network of the above described subepithelial capillaries. It follows the undulating pattern of plicae, whereas along the tubular glands it forms a peritubular network reaching deep into the endometrium. Gland openings appeared as narrow recesses in this network. In these recesses the mesh size of network was similar in character to that of the interglandular endometrium. In both areas capillaries were irregularly shaped and crooked with no prevailing orientation, having a variable diameter. Arterioles and venules from the deep layer of the endometrium supplied this capillary network in a randomly branching pattern (Fig. lc). The subepithelial capillary network of the gland openings extended along the tubular glands forming the peritubular, loosely meshed network of capillaries predominantly oriented across the gland axis (Figs. lc,d,e). A few arterioles and venules, apparently originating from the stratum reticulare (see above), supplied this periglandular network (Fig. Id; compare also with Fig. 3b,c). At this stage the maternal areolae remained relatively small with funnel-shaped gland openings, as seen from casts of the subepithelial capillary network (Fig. 3a). The casts demonstrated how the capillary network keeps its loose arrangement: quite different from the dense parallel interareolar capillary network seen 5 days earlier (compare with Figs. 3a and 2b). However, between the gland mouth and the interareolar region, a more distinct change of vessel character was created by newly formed extremely large and crooked capillaries (Fig. 3a)-the beginning of rim formation (compare with Figs. 3a and 4c,d). The capillary network of the glands was dense when compared to the precontact stage (Fig. lc), but without any prevailing orientation of the crooked capillaries. Distinct arterioles and venules (without anastomoses) supplied this peritubular capillary system, the latter at different levels (Figs. 3b,c). Chorionic vessels (which can only be demonstrated histologically) formed a flat and extremely widely meshed subtrophoblastic capillary system; this is because the chorionic villi of the fetal areolae were barely developed a s compared to later stages (Fig. 4a). Initial Stage of Basic Placental DevelopmentDays 32 to 43 p.c. At day 32 p.c. the areolae measured up to 1 mm in diameter, just visible macroscopically through the allantochorion or amniochorion. The maternal areolae, as shown by histology in Figure 4a, were flat around the gland opening, whereas on the fetal areola corrugations of the allantochorion had developed as a kind of primitive chorionic villous or Fig. 1. EARLY AREOLA-GLAND VASCULATURE OF PIG PLACENTA Fig. 2. Implantation-related vasculature on uterine gland mouth and surroundings. a: Histology of a n early areola (A) enclosing dark uterine milk from day 15p.c. bridged by the trophoblast of chorion (T). To the right an endometrial invagination with close contact to the interareolar trophoblast. Subepithelial endometrial capillaries are more numerous and located closer to the epithelium in the interglandular region (arrows)than in the areas of gland mouths (arrowheads). ~~ Fig. 1. Vasculature of uterine gland at precontact stage of embryo. a: Histological overview of a typical gland mouth being extremely narrow, day 9.5 p.c. In the endometrial stratum compactum, the subepithelial capillaries of endometrial surface and gland tubule (arrowheads) are not conspicuous and distinctly separated from the epithelium. Arterioles and venules (*) are seen in stratum reticulare, which is relatively poor of vessels and cells. Cilia1 cells (arrows). The bar represents 100 pm. b Corrosion cast of a gland mouth and its surroundings seen from the uterine cavity, day 12.5 p.c. The subepithelial blood capillaries (compare to Fig. la) form a widely meshed network around the narrow recess to the gland opening (arrow). Capillaries are irregularly shaped and crooked without prevailing orientation. The bar represents 100 pm. c: Overview of cracked endometrial blood vessel cast from day 12.5 p.c. The two-dimensional, undulating capillary network of endometrial surface (ES) narrows into a gland mouth (arrow) where it turns over to the twisted periglandular network, which is visible only in its deeper part. The network is drained by a system of randomly branching venules (VI)and veins (V) which for the superficial part of endometrium are distinctly larger than for the peritubular part. The bar represents 200 pm. d Detail of peritubular network from Figure lc. The crooked and loosely anastomosed capillaries give appearance of “unoriented wrapping” of the space of gland tubule (white dashed line). Arterioles (Al) and venules (Vl) supplying this peritubular network do not show any direct connection to nearby located large vessels (V) being tributary to the superficial network. The bar represents 200 pm. e: Cross section of a periglandular network which is solidly bound in a “circular tube” nearby the gland mouth. The bar represents 50 pm. 329 Chorionic vessels are not developed yet. The bar represents 100 pm. b Capillary cast of gland mouth and adjacent interglandular region, day 15 p.c. Uneven crooked capillaries form the widely meshed network of distinct gland mouth area, whereas outside of it, they are larger, uniform in diameter, and densely meshed as well as parallelly oriented. The bar represents 50 pm. papillary structure. Capillaries located beneath the columnar endometrial epithelium of gland tubules and areolae were sparse, narrow, and distinct from the epithelium. By contrast, large subepithelial capillaries were present in the interareolar region, but not in a n “intraepithelial” location (Goldstein, 1926; Amoroso, 1952) as is usual a t later stages of development (Dantzer and Leiser, 1993a). Capillaries of the fetal areola were rather scarce. These contained reticulocytes and did not reach the high-columnar trophoblast. These capillaries did not differ from those surrounding the fetal areola in the interareolar region. As visible on the maternal vessel cast (Fig. 4b), some areolae did not appear round due to the relief from the neighboring placental folds, but were regular. This type of areola dominated development at this stage; irregular areolae could not be observed, either by histology or by vessel casts. However, the close location of regular areolae, a s seen in Figure 4b, may later expand and fuse, thereby forming a n irregular areola. Maternal regular areolae in casts, like the histology (see above), were also flattened, or shallow, as compared to the earlier stages (compare Fig. 4b with 3a). 330 R. LEISER AND V. DANTZER Fig. 3. Vascular corrosions of uterine glands and surroundings at early placentation stage, day 20.5 p.c. a: Funnel-shaped glandular mouth (G) with a n inner loosely meshed network of subepithelial capillaries of early maternal areola and a surrounding densely meshed network of large and parallelly oriented capillaries-the interareolar region. Extremely large and crooked capillaries (*) signify the beginning of rim formation. The bar represents 100 pm. b Cracked cast, viewed obliquely from the myometrial side. The distinct “glandular” tube formed by the periglandular network, is apparently drained by one venous system (GV, arrows). Supplying vessels for superficial capillary network (above) generally are more venous in character (V) and large and crooked; in contrast, the few arterial vessels (A) are thin as well as slightly bending. The bar represents 200 pm. c: Peritubular network, tilted and magnified from Figure 3b. The network, characterized by crooked capillaries of no prevailing orientation, is drained by venules originating from different endometrial levels (arrows). The network at lower right is missing because of cracked edge of cast. The bar represents 100 pm. The subepithelial capillaries formed a widely meshed, two-dimensional network without elevations, whereas the interareolar capillary network was densely meshed, forming ridges and troughs (Fig. 4b). An abrupt rim bordering the maternal areola was formed at this stage. However, the capillaries of the areolae were continuous with, and extended in diameter a t the rim towards, the interareolar network (Fig. 4c,d). The details of the areolar capillaries were similar to previous stages, i.e., crooked, intensely ramified, and variable in diameter. The gland openings seen in the cast were smaller than in the earlier stages, though more visible and mainly located in the center of the areola EARLY AREOLA-GLAND VASCULATURE OF PIG PLACENTA (Fig. 4b,c). The fetal part of the areolae could not be demonstrated using vessel casts on day 32 p.c. due to the very fine and fragile nature of the fetal vascular system. DISCUSSION Preimplantative and Implantation-Related Phenomena of Endometrial Areolar Vasculature From day 9Ih to 14/15 P.c., the porcine endometrial vasculature, especially the subepithelial capillary network remains in a morphological stage similar to that of the nonpregnant when studied by light and electron microscopy (compare with Leiser et al., 1988). This vascular system here is uniform with no morphological differences between the interglandular region and the narrow recess-like gland mouths, nor between these mouths and the peritubular network of the glands. Therefore, the endometrial vascular morphology in early pig gestation is not influenced by maternal recognition of pregnancy (Ford, 1985; Dantzer and Leiser, 1993a), even though Ford and Christenson (1979) a s well as Stice et al. (1987) observed a n increase of blood flow to the uterus after day 11 px., and a n accumulation of fenestrations and caveolae were shown on the capillary endothelium adjacent to the endometrial epithelium after day 13 p.c. (Keys et al., 1986; Keys and King, 1988) when implantation begins (Dantzer, 1985). Already at day 15 P.c., the first occurrence of a primitive areola was observed, in contrast to earlier observations where Crombie (1972) described it at day 18, as did Keys and King (1990) at day 19 p.c. It is formed when the chorion reaches over the glandular mouth without contacting the endometrial epithelium; creating a secretion-filled space or areolar cavity (see below; Dantzer, 1986). In the maternal areola, the subepithelial capillaries are located close to the epithelium, slightly larger and more numerous than those capillaries found at gland openings of the previous stage. Hence, being a better base for substance transfer and secretory activity, these findings are in accordance with the observed ultrastructural characteristics of the epithelium around the gland mouth (Perry and Crombie, 1982). Early Placentation-RelatedVascular Adaptations in Creating Areola-Gland Subunits From days 15 to 32 P.c., the primitive areola (see above) develops into a “minute white circular disc with a prominent peripheral thickening” (Friess et al., 19811, which, as observed in our study, grows up to 1 mm in diameter. It then comprises the typical requisites of the areola-gland subunit, which are: (1) the maternal areola, a shallow or cup-shaped depression of endometrial wall around a small gland opening; (2) the fetal areola located opposite the maternal areola as a dome-shaped allanto(amnio)chorionic wall with a n unfolded, but villous allantolamniochorion; (3)the areolar cavity located between the maternal and fetal areolae-a remnant of uterine cavity (Goldstein, 1926) communicating with the glandular lumen and filled by uterine glandular secretory products; (4) the ring-like seal formation (RSF, Dantzer and Leiser, 1993b) where maternal and fetal areolar walls are in contact through a ring-formed convexity and a n elevated ring, respectively (Friess et al., 1981; Dantzer, 1984). 331 The vasculature of the ring-like seal formation of areolae can be observed developing from about days 20-30 p.c. Capillaries from the maternal subepithelial network first grow in size and become crooked in a zone between the gland mouth and the interglandular region. Later they form complex loops and anastomoses, creating a rim which clearly delineates the periphery of the areola. As described for the interglandular region during implantation, hormonal effects from the blastocyst, as well as the physical contact with the uterine epithelium (Dantzer, 1985; Keys and King, 1990; Dantzer et al., 1991a), may trigger angiogenesis (Hudlicka and Tyler, 1986)and capillary growth. However, this does not explain the excessive growth observed a t the rim capillary complex of the areolae. Other factors may interact between extracellular matrix and endothelial cells, e.g., fibroblast growth factors which regulate growth locally (Ingber and Folkman, 1989). In addition, when the capillary complex of the rim has grown to a certain size, its relatively voluminous vessels not only facilitate blood flow between the maternal areolar and interareolar capillary networks but also attract and accumulate blood, favoring its surroundings to a place of enhanced nutrition. In addition to promotion of materno-fetal substance transfer, this arrangement will increase trophoblast contact and anchoring onto the periareolar epithelium, creating the ring-like seal formation of areola. The process of sealing of the areola-gland subunit is obviously very important to retain the secretory products inside the rather small zone of areola and to hinder a dissipation of these products into the interareolar region. Assuming the latter, the dissipated secretory products specific for the areolae could separate the microvillous interdigitation between trophoblast and endometrial epithelium, thereby weakening the placental anchoring. This would be crucial in the pig, with a diffuse folded epithelio-chorial placenta representing one of the less firm materno-fetal connections (review by Dantzer, 1986). Thus a strict sealing of the areolagland subunit from the interglandular region is essential to the different functional units for, respectively, hemotrophic and histiotrophic materno-fetal transfer. General growth of the placenta during pregnancy will influence areolar formation, e.g., a s seen by the increase in areolar diameter, and by the stretching effect of the maternal areolar capillary network which progressively widens in mesh size. However, it can be hypothesized that the areolae being balloon, round, or lentoid also may imply a n interior forming force of the areolae, probably a pressure originating from continuously voluminous accumulation of secretory products of the uterine gland. This could be explained by the fact that (1)there is evidence of increasing secretory activity along the lengthening gland tubules during early pregnancy (Perry and Crombie, 1982; Sinowatz and Friess, 19831, and (2) the areolar cavity, which being well sealed to prevent dissipation of its contents into the interareolar region (see also above), is a rather small reservoir for the secretory substances related to the whole gland as a secretion producer. The maternal areolar epithelium per se cannot add much to this pressure: it seems not to produce much secretion due to its rather inconspicuous cytological form and small surface area (Friess et al., 1981; Dantzer and Nielsen, Fig. 4. EARLY AREOLA-GLAND VASCULATURE OF PIG PLACENTA 1984; Keys and King, 1990). Moreover, the supplying capillary network is sparse, widely meshed, and has no “intraepithelial capillaries.” This makes it a poor base for secretory activity, which is in contrast to the interareolar region where the capillary network is densely meshed to sustain the need for hemogenic substance transfer (Friess et al., 1980; Dantzer, 1986; Leiser and Dantzer, 1988; Dantzer and Leiser, 1993a). Origin of Regular and Irregular Types of Areola 333 (review by Brambel, 1933; Perry, 1981; Dantzer, 1986). It is suggested that they initially develop from nearbylocated gland mouths hidden in extraordinarily complex folds. Such locations close to the gland mouth, or to complexly folded regions, may hamper the peripheral ring formation necessary for a proper seal. Curing subsequent expansion and growth this would lead to fusion between two or more primary regular areolae forming one irregular areola. This early irregular areola may thereafter change completely (Dantzer and Leiser, 1993b) and have differing functions as compared with the regular areola, e.g., less iron (Bielanska-Osuchowska e t al., 1987) and other enzymes (Skolek-Winnisch e t al., 1985; Dantzer et al., 1991b). The areolae investigated in this study comprise the regular type, appearing the first time in a primitive form (see above) on the mesometrial side of the endometrium soon after implantation at day 15 p.c. Areolar development, then, in correspondence with establishment of the materno-fetal contact and adhesion (King Diverse and Retarded Development of Areolae Versus the lnterareolar Region et al., 1982; Dantzer, 19851, extends topographically Why are the vascular adaptations during early pregfrom the mesometrial to the antimesometrial side of the endometrium and towards the periphery of the em- nancy more and more retarded in the developing areola bryonic sac (compare with Dantzer and Leiser, 1993a). compared to those in the interglandular region? OesThe irregular areola was first observed by Keys and trogen production of pig blastocyst (review by Bazer et King (1990) at day 19 p.c. and described in detail by al., 1986) plus the physical contact of the implantation Brambel (1933) on a 10 mm embryo corresponding to ready blastocyst into the endometrial epithelium may day 22 p.c. (after Marrable, 1971). In our study up to trigger this tissue to a swift remodelling, as observed in day 43 of pregnancy, irregular areolae were observed the interglandular epithelium beginning at implanta(compare with Figure 2b in Dantzer, 1984); however, tion on day 13 p.c. (Dantzer, 1985; Keys and King, they were scarce (compare Brambel, 1933; Tsutsumi, 1990; Dantzer et al., 1991a). Some influence or stimu1962; Dantzer, 1986) and represent a n undeveloped lation from trophoblast contact may penetrate into stage not yet typical for the later stages of irregular deeper endometrial layers, but depending on distance areolae (Dantzer and Leiser, 199313). When fully and different quality of tissue, this may take time; grown, irregular areolae are characterized by fifteen namely, remodelling of interglandular or interareolar times the diameter of the regular areolae and are as- vasculature which begins with a delay of l?h days at sociated with two or more openings of uterine glands day 15 p.c. (Dantzer and Leiser, 1993a). In the areola, however, glandular secretory products in the areolar cavity prevent contact between the trophoblast and the endometrial epithelium around the glandular mouth. This secretion, therefore, forms a “cushion” enclosing Fig. 4. Vasculature of areolae at beginning basic placental develop- many different substances between trophoblast and ment, day 32 p.c. a: Histological overview of an areola enclosing rather homogenous content (*) being secreted from the tubular uter- maternal epithelium (see Sinowatz and Friess, 1983; ine gland (arrow). Capillaries beneath the columnar endometrial ep- Dantzer and Nielsen, 19841, which may decrease, diithelium of the gland and the areola are few, small as well as dis- vert, or even neutralize, the influence of trophoblast tinctly separated from the epithelium; however, in the interareolar stimulation with a subsequently slower and different region a t both sides (direction of arrowheads from the solid lines), development of the maternal areolar epithelium and they are large and closely bordering the epithelium. Capillaries of the its nearby vasculature compared to the interareolar fetal areola are sparse, sometimes containing reticulocytes and not reaching close to the high columnar trophoblast. This trophoblast region. Hence, since chorionic vessels and connective contains less dark infranuclear inclusions (double arrow) and shows tissue develop after day 17 P.c., trophoblast alone supranuclear bleb-like vacuoles, different than the interareolar region. As a whole, the areolar trophoblast borders 3 to 4 primitive villi seems to be essential for changes of the endometrial tissue, namely vascularisation, during porcine implan(or papillary formations) ( + ) of the allanto(amnio)chorion. Endometrial venules (V). The bar represents 100 pm. b Overview of subep- tation and early placentation. ithelial capillary network of endometrium showing two maternal areolae (left and right) which as depressions are surrounded by an, in part, vigorously plicated interareolar region. Note the conspicuous widely meshed and almost flattened network of the areolae. The bar represents 100 pm. c: Maternal areola magnified from Figure 4b. The capillaries of its network, as in earlier stages, are crooked, variable in diameter, and intensely ramified in a random orientation; however, towards the gland opening in the center of the areola (arrow), the capillaries become spiral and vanish into the peritubular network. The rim formation is abrupt and partly overhanging the peripheral border of maternal areola (*). The capillary network of the rim is extremely narrowly meshed and folded by ridges and troughs, which radiate towards the interareolar region. The bar represents 100 pm. d: Detail of areolar rim formation. There is a clear continuation between small capillaries of the network of maternal areola (widely meshed, right), and large capillaries of rim and surrounding interareolar region (narrowly meshed, left). However, arteriolar and venular limbs of capillaries have a distinct origin of their own field, as shown by a prevenular vessel in the areola (arrow) and several “ridgeal vessels” in the interareola (*I. The bar represents 50 pm. ACKNOWLEDGMENTS This study was supported by Grant 13-4224 from the Danish Agricultural and Veterinary Research Council. We thank Prof. Peter Kaufmann, Department of Anatomy 11, RWTH Aachen (Germany) for use of the scanning electron microscope, Mrs. Inge Bjerring and Mrs. Sigrid Kettner for their outstanding technical assistance, and John Kingdom, Department of Obstetrics and Gynaecology, University of Glasgow, Scotland, for assistance with preparation of the manuscript. LITERATURE CITED Amoroso, E.C. 1952 Placentation. In: Marshall’s Physiology of Reproduction, vol. 2. A S . Parkes, ed. Longmans Green, London, pp. 127-311. 334 R. LEISER AND V. DANTZER Bazer, F.W., J.L. Vallet, R.M. Roberts, D.C. Sharp, and W.W. Thatcher 1986 Role of conceptus secretory products in establishment of pregnancy. J . Reprod. Fertil., 76r841-850. Bielanska-Osuchowska, Z., A. Sadowski, and A. Malinowska 1987 Distribution of iron in pig placenta (Sus scrofa d0m.L.). Folia Histochem. Cytobiol., 24t29-44. Brambel, C.E. 1933 Allantochorionic differentiations of the pig studied morphologically and histochemically. Am. J. 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