Anat. Embryol. 158, 179-191 (1980) Anatomy and Embryology Q by Springer-Verlag 1980

The Placenta of the Pig 1. Finestructural Changes of the Placental Barrier During Pregnancy

Armin E. Friess, 1 Fred Sinowatz, 1 Rita Skolek-Winnisch,2 and W. Tr~iutner 3 t Institute of Anatomy, University Regensburg, Universit/itsstraBe 31, D-8400 Regensburg, Bundesrepublik Deutschland 2 Institute of Histology and Embryology, 3 Institute of Gynecology & Obstetrics, University of Veterinary Medicine, Linke Bahngasse 1 l, A-1030 Wien, Austria

Summary. The finestructural changes of the interareolar porcine placenta during pregnancy are described. After perfusion fixation of the placenta the change in the thickness of the placental barrier from day 30 to day 110 of gestation is much more evident than after immerson fixation as has been used by all former authors. The alterations are due to the indenta- tion of both the trophoblast and uterine epithelium by their corresponding capillary-network. This indentation is limited to the lateral wall and the summit of the chorionic ridges, while at the base the trophoblast as well as the uterine epithelium remains high columnar.

This indicates that in the interareolar porcine placenta, which is rep- resented by the chorionic ridges and the corresponding endometrial folds, at least two different areas with different structure and function may be discerned.

1) The lateral side and the top of the chorionic ridges seem to be predesti- nated for gaseous exchange. The placental barrier in this area is often less than 2 gm.

2) The transport of blood-borne nutrients takes place at the base of the chorionic ridges. This transport seems to be facilitated by an intercellular channel system between the uterine epithelial cells.

Key words: Swine - Fine structure - Placenta barrier.

Introduction

In the pig histological and histochemical studies of the fetal membranes and their development during pregnancy have been performed by several authors (Grosser, 1979; Heuser, 1927; B rambell, 1933; T6ndury, 1944; Wislocky and

For offprints contact: Prof. Dr. Armin E. Friess, Institute of Anatomy, UniversitfitsstraBe 31, D-8400 Regensburg, Federal Republic of Germany

0340-2061/80/0158/0179/$02.60

180 A.E. Friess et al.

Dempsey, 1946; Hitzig, 1949; Amoroso, 1952; Perry and Rowland, 1962; Ash- down and Marrable, 1967; Christie, 1968). According to these authors the blastocyst of the pig goes through a phase of rapid elongation between the 6th and 12th day of gestation. It changes from a sphere of 2 mm in diameter to a long membraneous thread (Perry and Rowland, 1962). The formation of the amnion by folding is complete on day 18. The allantois appears about the 14th of gestation and grows very rapidly. By 17 days it is as long as the embryo itself. On day 19th the mesodermal covering of the allantois makes contact with a small area of the chorion. Allantoic vessels proceed from here into the chorion and by day 30 the chorion is extensively vascularized by allantoic blood vessels (Wislocky and Dempsey, 1946; Hitzig, 1949). Only the extremities of the chorionic sac are not provided with a vascular supply. By day 40 the chorion can be divided into three areas (Fig. 1 a) the large placental zone which occupies the central region of the sac, the laterally adjacent paraplacental zone and the avascular extremeties (Steven and Morriss, 1975).

In the placental zone the chorionallantois shows numerous small folds which interlock with corresponding endometrial folds. (Fig. 1 b) Thus the pig's placenta is a placenta diffusa and according to the classification of Grosser (1909) of the epitheliochorial type. Over the mouths of the uterine glands the allantocho- rion is not attached to the endometrial epithelium but forms regular (Fig. 1 c) or irregular areolae (Brambell, 1933) which first appear on day 30 of pregnancy.

Among the allantochorial placental membranes, the epitheliochorial placentae have been considered to constitute the most complete morphological barriers because the original six layers of tissue elements (Grosset, 1909) inter- vene between maternal and fetal blood streams. Recent ultrastructural studies on the morphology of the epitheliochorial placenta of the mare (Steven and Samuel, 1975; Samuel et al., 1976)however, revealed that the epitheliochorial type is more highly developed and much more complex than was supposed by early workers,

In contrast to the considerable amount of information on the histology of the porcine placenta, only a few ultrastructural studies have been performed (Bjorkman, 1965, 1970, 1973; Crombie, 1972). From these studies we have proceeded to a more detailed investigation on the ultrastructure of the porcine placenta paying special attention to the regional differences. Our first report deals vgith the changes in the fine structure of the placenta barrier in the inter- areolar area from day 30 to the end of term.

Material and Methods

The material used in this investigation comprised the placentae of 7 sows (German Land-race) removed on day 30 (two animals), and on days 58, 80, 100 and 110 (two animals) of pregnancy. For fixation the uterus was exposed as in Caesarian section under deep thiopental anesthesia of the sow.

Perfusion was performed via a branch of the uterine artery which supplies one or two ampullae. The perfusion apparatus consisted of a simple plastic reservoir, hung at a height of 170 cm, which was connected to a glass cannula via plastic tubes and a three way vaIve. Perfusion was started by a flush of Hank's BBS, ph 7.4 containing 0.1 procain, followed by an aldehyde fixative. The

" . . . . . P Z

~

,=

e 41

'PL. EPITH.- CHORIALIS

0 B

la

AECS kREOL/F-

Fig. 1, a Schematic diagram showing the different areas of the porcine placenta. Within the placental zone (PZ) the chorionic sac and the uterus are attached to form the placenta diffusa. (/IECS avascular extremities of chorionic sac). The placenta diffusa is of an epitheliochorial type within the interareolar area, as indicated in the rectangle and in Fig. lb . b Porcine placenta, day 30 o f pregnancy. In the interareolar area of the placenta epitheliochorialis the chorion and the endometrial surface interlock with numerous ridges. A chorionic ridge ( ~ ) consists of a base (CB) (for which the term chorionic trough is also used) and of the top (C7). In the depth of the chorionic troughs, the cells of the trophoblast (TR) are high columnar. UE uterine epithelium; MC maternal capillary, x 280. c Shows a histological view of the chorionic areolae (CA), which are formed over the mou th of the uterine glands (UG). x 28()

182 A.E. Friess et al.

fixation solution consisted of 1% paraformaldehyde and 1.25 glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 and 0.2 Dextran 40. After the end of perfusion strips of tissue were cut from different sites of the interareolar area and postfixed for two hours in a fixative solution of the same composition at 4 ~ C. Subsequently the specimens were rinsed several times in 0.1 M cacodylate-buffer, pI:I 7.4, postfixed in I% buffered OsO 4 (Millonig, 1961) dehydrated in a graded series of ethanol and embedded in an Epon/Araldite mixture (Texas). Ultrathin sections were routinely stained with uranyl acetate and lead citrate (Reynolds, 1963). EM photographs were taken using a Siemens 101 or Zeiss EM 10 A electron microscope.

Results

Day 30

Chor ion and endometr ia l surface interlock with numerous low ridges or rugae and corresponding t roughs or fossae (Fig. 1 b). In the depth of the chorionic t roughs the cells o f the t rophoblas t are high columnar. Their average cell height is 40 gm which gradually decreases to 20 gm on the side and on the summit of the ridges. The height of the corresponding uterine epithelium does not show much variation. Its average height is about 20 ~tm. The apical surface of each t rophoblas t cell (Fig. 2 TR) possesses numerous long microvilli which interdigitate with those of the maternal epithelium. The cytoplasm between the nuclei and the submicrovil lous zone is occupied by numerous oval to elon- gated mi tochondr ia with lamellar cristae. Between the mi tochondr ia a great number of small vesicles occur. The nuclei are situated in the basal par t of the t rophoblas t cells. They are rich in euchromatin . Their shape is round to oval, often with a slightly irregular outline. Small nucleoli are occasionally observed. Around the nuclei m a n y short strands o f rough E R and dense bodies of varying size occur. The area beneath the nuclei is often occupied by a n irregular-shaped vacuole which contains light flocculent material. The t ropho- blast cells rest on a well-developed basal lamina. At this early stage of gestation the capillaries are usually separated f rom the epithelial basal lamina by the sur rounding mesenchymal tissue (Fig. lb) . The t rophoblas t cells are closely apposed at their apical lateral borders with well developed tight junctions. The lateral membranes of neighbour ing cells show a high degree o f interdigita- tion.

Between the microvilli of the uterine epithelium (Fig. 2, UE), which seem to be fewer in number than their fetal counterparts , pinocytot ic invaginations of the apical p lasma membrane are frequently seen (Fig. 2, inset ,). Numerous

Fig. 2. Porcine placenta, day 30 of pregnancy. Maternal capillaries (MC) are separated from the uterine epithelium (UE) only by a small layer of connective tissue. TR Trophoblast. x4,200. Inset." The apical surface of the trophoblast cells (TR) shows numerous long microvilli which interdigitate with corresponding microvilli of the uterine epithelium (UE). In the trophoblast many pinocytotic vesicles (,) are seen. • 16,000

Fig. 3. Porcine placenta on day 58 of pregnancy. Capillaries (FC) now deeply indent the fetal epithelium on the lateral side and on the summit of the ridges. Note also the high columnar appearance of the trophoblast cells at the base of the ridges (double arrows), x 760

Pig Placenta: Fine Structural Changes of the PlacentaI Barrier 183

184 A.E. Friess et al.

small vesicles containing moderately electron dense material occur beneath the microvilli. The supranuclear cytoplasm contains a well developed rough ER, often arranged in parallel arrays. A small Golgi apparatus consisting of a few cisternae and vesicles is found supranuclearly. The nuclei of the uterine epithe- lium are oval or elongated. The outline of each nucleus is more irregular and indented than that of the fetal nuclei. Their chromatin is evenly dispersed. Only a small rim of heterochromatin is seen on the inner nuclear membrane. Many nuclei show pronounced nucleoli. The mitochondria are distributed throughout the cells. They are of oval shape with lamellar cristae. The small cytoplasmic area beneath each nucleus contains a few mitochondria and lysoso- mal like dense bodies of varying size. In the basal part of the epithelium the lateral cell membranes of neighbouring cells separate and intercellular channels are established into which fingerlike processes of the cells protrude. Similar convolutions appear in the basal cell membranes. The basal lamina of the uterine epithelium is well defined. The maternal vessels are separated from the epithelium by a small layer of connective tissue. But the abundant network of maternal capillaries is already in close proximity to the uterine epithelium (Fig. 2).

Day 58

On day 58 (Fig. 3) the ultrastructure of the trophoblast cells and the position of the fetal vessels has changed considerably: Capillaries deeply indent the fetal epithelium on the lateral side and on the summit of the ridges. The vessels often appear surrounded on three sides by trophoblastic cells. In the region of indentation the basal lamina of the capillaries usually has fused with that of the trophoblast cells to a single lamina. The overall height of the trophoblast cells decreases from 35 lam at the base of the ridges to 15 lain on the sides and on the tops of the ridges. In areas where capillaries protrude into the epithelium the cell height is reduced to 2 gm or less. (Fig. 4)

Trophoblast cells at the base of the ridges are high columnar and compara- tively narrow. (Fig. 5) Lateral plasma membranes of adjacent cells are highly interdigitated in their apical part. The elongated nuclei are usually situated in the basal part of the cells. A small rim of heterochromatin lines the relatively

Fig. 4. Porcine pIacenta on day 58 of pregnancy. Fetal capillaries (FC) protrude deeply into the trophoblast. The distance between the fetal capillaries and the microvillous contact zone is reduced to 2 ~tm or less. UE uterine epithelium. • 5,000

Fig. 5. Porcine placenta on day 58 of pregnancy. Trophoblast ceils (TR) at the base of the ridges are high columnar. Beneath their nuclei a varying number of electron dense granules are seen (arrows) which sometimes fuse to a single large dense body (asterisk). Maternal capillaries (MC) do not protrude into the uterine epithelium, x 1,600

Fig. 6. Shows the porcine placenta on day 110 at the lateral side of a chorionic ridge. At this late stage of gestation, also maternal capillaries (MC) protrude between uterine epithelial cells. The trophoblast cells (TR) are nearly complete separated by foetal vessels and possess an accumula- tion of dense granula

Pig Placenta: Fine Structural Changes of the Placental Barrier 185

186 A.E. Friess et al.

irregular inner nuclear membrane. Some of the nuclei possess pronounced nu- cleoli. The apical cytoplasm is generally occupied by many mitochondria with transverse cristae. In the basal cytoplasm vacuoles of different size occur. Some of them contain an electron dense flocculent material. Rough ER is mainly concentrated lateral of the nuclei, often arranged in parallel arrays. Under the nuclei a varying number of electron dense granules are seen, which some- times appear to fuse to a single large electron dense body. Trophoblast cells on the side or on the summit of the ridges are of an isoprismatic to flat appearance. These cells are usually much broader than the narrow high columnar cells at the base of the ridges. The supranuclear cytoplasm contains many mitochondria, which are concentrated beneath the microvilli, several cisternae of rough ER and some small vacuoles with clear contents. Some electron dense granules are regularly seen beneath the nuclei. The extended cytoplasm lateral to the nuclei, where the height of the trophoblast cells is reduced to 2 ~tm or less contains some mitochondria, rough ER and many pinocytotic vesicles.

The maternal epithelium in this period of gestation also shows some variation in its cellular height. The average height of uterine epithelial ceils opposite the base of the trophoblast ridges is 25 ~tm, whereas cells opposite to the lateral side and to the summit of the ridges rarely exceed 15 ~tm. The outlines of the nuclei are more regular than their trophoblastic counterparts. Marked nu- cleoli are occasionally seen. A characteristic feature of the ceils is a well devel- oped endoplasmic reticulum consisting of many short rough and some smooth profiles and numerous small vesicles. Also a distinct Golgi apparatus is present. (Fig. 4) Mitochondria, which are generally smaller than those of the trophoblast cells, are randomly distributed throughout the cytoplasm. Dense bodies and myelin figures are frequently observed especially in those uterine epithelial cells opposite the base of the trophoblast ridges. As in earlier stages of pregnancy, the lateral and basal plasma membranes are highly convoluted, and distinct intercellular channels are seen.

Late Pregnancy: Days 100 and 110

During the last third of gestation the indentation of fetal vessels into the tropho- blast proceeds and the height of the trophoblastic cytoplasm overlying the fetal capillaries is further reduced. Also on the maternal side, capillaries project between the uterine epithelial cells (Fig. 6). This, in conjunction with a general thinning of the uterine epithelium and the already mentioned indentation of fetal capillaries into the trophoblast leads to a marked reduction of the trans- placental intervascular distance. At the end of pregnancy the effective placental barrier separating the fetal and maternal bloodstream measures 2 gm or even less (Fig. 7).

The nuclei of the trophoblast cells are generally confined to those parts of the cytoplasm which protrude between neighbouring fetal capillaries. In this area also comparatively large mitochondria with a dense matrix and transverse cristae, cisternae of rER and a small Golgi apparatus, and many small vesicles can also be observed. Beneath the nuclei a varying number of electron dense

Fig. 7. Placental barrier on day 110. At the end of pregnancy the effective placental barrier separating the fetal and maternal bloodstream measures 2 gm or even less. FE fetal endothelium, M E maternal endothelium, M V microvilli, TR trophoblast, UE uterine epithelium, x 32,400

188 A.E. Friess et al.

Fig. 8. Porcine placenta on day 110: Uterine epithelium. A complicated system of intercellular channels is established between adjacent uterine epithelial cells and between the base of the uterine cells and the maternal basal lamina. MC maternal capillary, M V microvillous contact zone, f fenestrae in the maternal endothelium

granules (Fig. 6) occur. The thin cytoplasmic region lateral to the nuclei and above the capillaries consistently contains only pinocytotic vesicles and occasion- ally a few mitochondria. At the base of the fetal microvilli, electron dense material can be seen.

In this stage of pregnancy the height of the uterine epithelium appears markedly reduced. The nuclei of the uterine epithelial cells are round to oval. Some of them possess distinct nucleoli; mitochondria are randomly distributed in the cytoplasm. A well-developed Golgi apparatus is present, usually consisting of numerous tight lamellae. Many cisternae of the endoplasmic reticulum, mainly of the rough-surfaced type, and an abundance of free ribosomes are seen.

In contrast to the fetal side, where neighbouring trophoblast cells are closely apposed, adjacent uterine epithelial cell are separated by a complicated system

Pig Placenta: Fine Structural Changes of the Placental Barrier 189

of intercellular channels (Fig. 5; Fig. 8). These spaces partly run from the basal lamina to the apical tight junctions of the uterine epithelium. These intercellular and also basal channels are very prominent in uterine epithelia at the base of the chorionic ridges, which correspond to the summit of the uterine ridge.

Discussion

Biochemical and physiological studies show that different transport mechanisms exist to overcome the placental barrier, depending on the molecular weight and chemical composition of the substance to be transferred. The main transfer mechanisms are simple diffusion, facilitated diffusion, active transport and trans- port via micropinocytotic processes (Sperhake, 1971). Oxygen and carbon diox- ide pass across the placental membranes by simple diffusion and the rate of transfer depends on the difference of concentrations, on the surface area available for transfer and on the physical thickness of the placental membranes (Villee, 1965). The present study shows that the porcine placenta remains epitheliochorial in arrangement throughout gestation, but considerable changes in the ultrastruc- ture of the trophoblast and the uterine epithelium as well as in the position of the placental capillaries can be observed. Even in the early stages of gestation marked differences exist in the cellular height of trophoblast cells at the base of the chorionic ridges as compared to that of cells on the lateral side or summit of the ridges. The different appearance of the trophoblast cells in these locations is accompanied and accentuated by different behaviour of the fetal capillaries. Whereas capillaries soon start to indent the trophoblast on the lateral side and on the summit of the ridges - on day 60 trophoblast cells are frequently seen to be enclosed on three sides by capillaries - they do not protrude into the chorionic epithelium at the base of the ridges. The basal lamina of the indenting capillaries on the fetal side generally fuses with that of the trophoblastic epithelium to a single lamina. By contrast, the basal lamina of the maternal capillaries and uterine epithelium are maintained as separate structures throughout gestation. The progressive indentation of capillaries on both sides of the placenta causes a marked reduction of the intervascular distance. At the end of pregnancy the distance between fetal and maternal capillaries is often less than 2 gm. Indentation of capillaries into the trophoblast has also been reported for other species (Steven and Samuel, 1975). Samuel et al. (1976) reported a reduction of the placental barrier in the epitheliochorial placenta of the mare. By day 200 the tissue separating the fetal and maternal vascular system is about 12 gm thick. This barrier is reduced during the last two months of gestation to 2.5 gin. Our measurements revealed a much thinner barrier in the pig than that of the mare and other species. The diffusion distance is therefore also smaller than in other epitheliochorial placentae.

Similar to the situation in the mentioned species, in the porcine placenta indentating capillaries are always separated by a basal lamina from the epithelial cells and are never really "intraepithelial" as was claimed in some earlier light microscopical studies (Goldstein, 1926).

190 A.E. Friess et al.

The indentation of both the trophoblast and uterine epithelium by their corresponding capillary-network not only shortens the distance between fetal and maternal bloodstream but also reduces the amount of actively respiring placental tissue. As those parts of the cells which contribute to the placental barrier do not contain many organelles, oxygen may be transferred from the maternal to the fetal blood with minimal uptake by the intervening tissue. Among the epitheliochorial placentae of domestic animals this special feature of the placental barrier was observed in the mare, cow and ewe (Steven u. Morriss, 1975) but does not seem to be as much developed as in the pig.

The chorionic troughs are lined by a high columnar epithelium throughout the course of gestation. The corresponding uterine epithelium remains also comparatively high, and no indentation by protruding capillaries could be observed on either side. Histochemical investigations (Wislocky and Dempsey, 1946; Christie, 1968) demonstrated that the activity of alkaline phosphatase was confined to the brush border of the columnar cells of the chorionic throughs. In no other location of the trophoblast was alkaline phosphatase activity found at any stage of gestation. Our ultrastructural results show many vacuoles of different size in the supranuclear cytoplasm of the columnar cell of the chorionic troughs, some of them containing dense flocculent material. Beneath the nuclei of these cells many dense lysosomal - like granules occur which frequently fuse to a single dark dense-body. In our opinion the columnar cells of the trophoblast of the chorionic troughs take up and metabolize less diffusible material, which is transmitted by the corresponding uterine epithelium. The base of the chorionic ridges which now comprise trophoblast and uterine epithe- lium together therefore do not take part in that placental barrier which is responsible for gaseous exchange. The base seems to be the preferred site for the transport of blood borne nutrients.

The development of intercellular spaces in the fetal and maternal epithelium which is confined to the area of the chorionic troughs, supports this opinion. The formation of an intercellular channel system appears first in the uterine epitehlium and in a later stage of pregnancy also in the trophoblast. Although no continuity between the maternal and fetal intercellular channels exists the high number of pinocytotic vesicles in the fetal epithelium facilitates the transport from mother to fetus. Our preliminary tracer studies with horse-radish peroxidase (HRP) injected into the fetal circulation also supports this direction of transport. HRP is only found in the intercellular spaces of the trophoblast and no uptake into pinocytotic vesicles could be found.

In conclusion, it should be emphasized that in the interareolar part of the porcine placenta at least two different areas with obviously different structure and function may be discerned:

1) On the summit and lateral side of the ridges protruding capillaries reduce the placental barrier during pregnancy to less than 2 lam. This area seems to be predestinated for the exchange of freely diffusible substances, especially for the gaseous exchange.

2) In the chorionic troughs with their high columnar epithelium the transport of less diffusible material takes place. By morphological and histochemical criteria it seems that the direction of transport from mother to the fetus in

Pig Placenta: Fine Structural Changes of the Placental Barrier 191

favoured. Tracer and ultracytochemical studies already in progress should help to clarify this point.

Acknowledgement. The authors are greatly indebted to Prof. Dr. K. Arbeiter for the supply of pregnant sows and for his great interest in our placenta project.

References

Amoroso, E.C.: Placentation: In Parkes Marshall's Physiology of Reproduction. 3rd ed., Vol. 2, pp. 127-311 London: Longman Green 1952

Ashdown, R.R., Marrable, A.W. : The development of the embryonic membranes in the pig: observa- tions on afterbirths. Res. vet. Sci. 11,227 233 (1970)

Bjorkman, N.: On the fine structure of the porcine placental barrier. Acta anat. 62, 334-342 (1965)

Bjorkman, N.: An atlas of placental fine structure. London: Bailliere, Tindall and Conell, 1970 Bjorkman, N.: Fine structure of the fetal-maternal area of exchange in the epitheliochorial and

endotheliochorial types of placentation, Acta anat. 86, Supp. 1, 1-22 (1973) Brambell, C.E. : Allantochorionic differentiation of pig. Am. J. Anat. 52, 397-459 (1933) Christie, G.A.: Histochemistry of the placenta of the pig. Histochemie 12, 208 221 (1968) Crombie, P.R.: The ultrastructure of the pig's placenta throughout pregnancy. P h . D . Thesis

University of Cambridge 1972 Goldstein, S.R. : A note of the vascular relations and areolae in the placenta of the pig. Anat. Rec.

34, 25-36 (1926) Grosser, O.: Vergleichende Anatomie und Entwicklungsgeschichte der Eih~iute und der Placenta.

Wien: Braumiiller, 1909 Heuser, C.H. : A study of the implantation of the ovum of the pig from the stage of the bilaminar

blastocyst to the completion of the fetal membranes. Contrib. Embryol. Carneg. Inst. 19, 229-243 1927

Hitzig, W.H.: fiber die Entwicklung der Schweineplacenta. Acta anat. 7, 33-81 1949 Millonig, G.: Advantages of a phosphate buffer for OsO4 solutions in fixation. J. appl. Physiol,

32, 1637-1645 1961 Perry, J.S., Rowland, I.W.: Early pregnancy in the pig. J. Reprod. Fert. 4, 175-188 (1962) Reynolds, E.S. : The use of lead citrate at high ph as an electron-opaque stain in electron microscopy.

J. Cell Biol. 17, 208-212 (1963) Samuel, C.A., Allen, W.R., Steven, D.H.: Studies on the equine placenta. II. Ultrastructure of

the placental barrier. J. Reprd. Fert. 48, 257-264 (1976) Silver, M., Steven, D.H., Comline, R.S.: Placental exchange and morphology in ruminants and

the mare. In: Foetal and Neonatal Physiology, pp. 245-271. (R.S. Comline, K.W. Cross, G.S. Dawes, and P.W. Nathanielsz. Eds.) Cambridge University Press (1973)

Sperhake, B.: Zur Durchl/issigkeit der Placenta des Schweines - Eine Literaturstudie. Inaug. Diss. Hannover, 1971 (1971)

Steven, D.H., Morriss, G.: Development of the foetal membranes. In: Comparative Placentation. Ed. D.H. Steven. Londen, New York, San Francisco: Academic press, (1975)

Steven, D.H., Samuel, C.A.: Anatomy of the placental barrier in the mare. J. Reprod. Fert. Suppl. 23, 579-582 (1975)

T6ndury, G.: Zum Feinbau des Chorion-epithels der Schweineplacenta. Rev. suisse de Zool. 51, 369-376 (1944)

Villee, C.A.: Placental transfer of drugs. Ann. N.A. Acad. Sci. 123, 237 244 (1965) Wislocky, G.B., Dempsey, E.W.: Histochemical reactions of the placenta of the pig. Am. J. Anat.

78, 181-225 (1946)

Accepted October 7, 1979