Domestic Sheep
Ovis aries

Order: Artiodactyla
Family: Bovidae

1) General zoological data of species

Domestic sheep have presumably derived from Ovis musimon, the mouflon, a species that is distributed across Mediterranean islands. Domestication is thought to have begun approximately 10,000 years ago. There are six species and numerous subspecies of sheep distributed throughout Asia and North America, but the domestic sheep now occurs worldwide. Because some sheep species and/or subspecies are significantly endangered, they now require protection and have been listed as CITES I species (Nowak, 1999). Longevity varies with species and race. For mouflons, the maximal life span is given as 19-20 years (Puschmann, 1989).

Mossman (1987) differentiated between three types of cotyledons: the "flat" kind of certain deer, the "convex" kind of other deer and giraffes, and the "concave" kind of sheep and caprines. The concave cotyledonary sheep placenta implants primarily upon the uterine caruncles. In between the cotyledons, the chorion is simple and lacks villi. The trophoblast intertwines with the uterine endometrium in the caruncular regions, to form the so-called "placentome". Following death of a pregnant animal it is easy to peel the placenta away from the endometrium, without significant damage to either structure. There is a large allantoic sac filled with urine. Urine is led to it through the allantoic duct and that passes from the dome of the bladder through the umbilical cord. Some urine must also pass through the urethra into the amnionic cavity. Sheep possess multiply branched villi, in contrast to some other ruminants. In reconstructions they appear as folds, rather than the multiply branched thin villi that are seen in primate placentas.

Alexander (1964) sought to explore the relation between fetal weight and cotyledonary weights in Merino sheep gestations; Corredale sheep had slight differences and were not described in detail. He extensively discussed the differences between different breeds, and emphasized the need to be mindful of the existence of wide differences. He suggested that it is necessary to collect normative data for breeds and altitude when studies are undertaken to ascertain influences of altitude etc. on fetal development. The number of cotyledons was poorly related to birth weight, in contrast to their weights. Not all caruncles are used for cotyledonary development and this apparently varies widely. Moreover, it appears that some caruncles disappear with aging. Male fetuses occupy more caruncles than do female fetuses. Alexander separated the cotyledons (without intervening membranes) from the caruncles by traction; after blotting them dry, he weighed them. There was an excellent correlation between fetal weight and cotyledonary mass at different stages of gestation. Male fetuses and cotyledons were slightly heavier. Those of twins were slightly smaller. The average number of uterine caruncles was 73 in Merino sheep (from 64 to 145). Also, the number of caruncles was generally similar in both uterine horns. Older ewes had a slightly heavier cotyledonary mass which varied from 115 to 130 g. Individual cotyledons weighed about 1.6-1.8 g. There were, however, enormous differences in their weights - 0.1 -12 g of fetal cotyledons and 0.1-45 g of complete placentomes. The fetal part of a complete cotyledon was 40% and was darker than the maternal part. Alexander made another important observation. He found that whereas in immature cotyledons the maternal tissue (caruncle) tissue surrounds the cotyledon (which is concave, according to Mossman), towards term the opposite is true. The components then become convex. It is further interesting to contemplate that most of these investigators considered only the cotyledonary weight although it had been long appreciated that the intercotyledonary region serves also as an important exchange (nutritive) region. Perhaps it should be included in the weights when an association with fetal growth is sought. Osgerby et al. (2003) studied the effects of maternal nutrition on the condition of neonates and placenta. They showed convincingly that the type of nutrition significantly influences placental cotyledonary structure as well as fetal condition.

5) Details of barrier structure

The nature of the interface between maternal epithelium and trophoblast was described in the electronmicroscopic study of Lawn et al. (1969). This interface comprises also an area in which maternal blood is extravasated "to supply the fetus with iron". Wimsatt (1950) had studied this region in great detail and it will be discussed more fully below.

The endometrial crypt is lined with epithelium that has a striking microvillous surface. This, in turn, is closely adherent to the trophoblast with which its microvilli interdigitate. The maternal epithelium is continuous, is not interrupted, and it is often multinucleated. The microvilli of trophoblast and endometrium interdigitate diffusely and thus, this placenta is here epitheliochorial (-bichorial to be correct), as was first clearly defined by Ludwig (1962). Because of degeneration of some endometrial epithelium during the course of gestation, areas of syndesmochorial relation exist. Mossman (1987), however, correctly pointed out that there is little vascular supply in those areas and, from a functional viewpoint, they are probably of little importance. Thus, one should consider that the sheep placenta is principally an epitheliochorial one.

Lawn et al. (1969) paid special attention to the development of the well-known binucleate cells of the ruminant placenta. This cell has spawned an enormous amount of literature. These investigators traced the cells' development from early nuclear division (without cytoplasmic division) to their accumulation of numerous cell organelles. Among these the Golgi complex appeared first and was then succeeded by a large number of granules. In the mature cell, the granules fill the cellular cytoplasm, nearly crowding out all other components. It was also shown that these cells never completely bordered the endometrial epithelium - a rim of cytoplasm always separated the two cell types. Contrary to earlier investigators then, Lawn et al. (1969) correctly identified the binucleate cells as having derived from trophoblast. The organelles, as well as some of the endometrial crypt lining cells, stain intensely with the PAS method. They are now known to contain placental lactogen.

The original classification of the sheep placenta as being an epitheliochorial organ, however, is no longer considered being entirely correct. (Please see also the chapter on Cretan Goat for additional considerations.) Autoradiographic studies by Wooding et al. (1981) probably now supersedes the older information. Relative to its cotyledons, the ovine/caprine placenta must now be considered as a focally syndesmochorial organ. The intercotyledonary regions remain epitheliochorial in the mature placenta, as had been previously determined.

The most controversial aspect perhaps of ungulate placentation has been the origin of the syncytium that lines the crypts of the cotyledons. Similarly, the nature of, or the relation of the syncytium to the binucleated cells has been interpreted differently. Binucleate cells are clearly trophoblastic in origin and they produce the placental lactogen. The autoradiographic study by Wooding et al. (1981) investigated the origin of syncytial cells. It appeared to me that it convincingly showed that the syncytium has a trophoblastic origin and that it is not derived by fusion with endometrial epithelium, as was once thought. Presumably, the binucleate cells initiate the process of fusion and subsequent migration within the trophoblastic layer, and they continue to do so during the entire pregnancy. Moreover, the fact that there are no mitoses in the syncytium, while they do occur in the cellular trophoblast, supports this notion. Thus, the syncytium was considered to develop much like other fusion-induced cells. Wooding (1984) later showed that some fusion with maternal epithelium also produces trinucleate hybrid cells. These cells are quite specific, and secretion of the lactogen-containing granules (perhaps to maintain gestation) was described in his paper. Wooding emphasized that there is no immune reaction as the result of maternal epithelial erosion and this fusion ("hybridization" of cells). This may be an erroneous conclusion, as will be seen shortly.

In addition to the binucleate cells, numerous giant cells are found deep in the placentome. According to Lawn et al. (1969) they originate from the endometrial epithelium in later gestation; more likely they are part of the "syncytium". In addition to these developments, the endothelium of the maternal capillaries becomes markedly hypertrophied.

7) Uteroplacental circulation

In his study of sheep placentation, Wimsatt (1950) injected the fetal artery with colored dyes and thus characterized the villous vascular tree. A single artery injects blood centrally into the cylindrical main stem villus; it then divides, sending a single vessel into the flattened villous ramifications, where a plexiform capillary network lines the surface of the villi with protrusions into the trophoblast. A complex network of venules drains the villous structures, the veins having larger caliber and being less muscular.

Makowski (1968) illustrated the injected maternal/fetal vasculature of individual cotyledons. He found that there was no evidence of a true counter-current blood flow. The maternal arterioles apparently have a sphincter-like arrangement at their bases. This is presumed to dictate the blood flow through the maternal plates. There was no evidence of a neural supply to the vessels.

8) Extraplacental membranes

There are no "free" membranes in the sheep placenta. Between the cotyledons, however, the intercotyledonary chorion is located, which is a relatively simple membrane. It is avillous and covered on the outside with a simple layer of cylindrical trophoblastic epithelium that has a microvillous surface. The trophoblastic epithelial cells have a varied morphology, as is evident in the next photograph. They are pressed against the endometrium by pressure from within the allantoic cavity. In late stages, the exocoelomic space is completely obliterated. The allantoic sac, which is of endodermal origin, fuses with the chorionic sac as early as on the 22nd day of development (Davies & Wimsatt, 1962). It is very large and elongate. Its columnar epithelium possesses only very short microvilli and, in the mature placenta, it is relatively flat. Davies (1952) was perhaps the first to draw attention to the accumulation of large amounts of fructose in the allantoic fluid.

Wimsatt (1964) paid special attention to this region and observed that, soon after implantation, the endometrium of these areas is lost and the trophoblast apposes the connective tissue of the uterus. The placental relation then is temporarily of a syndesmochorial kind. In later stages (after 100 days) the epithelium of the endometrium has regenerated and then the trophoblast has the usual epitheliochorial relation again.

A small amount of acellular debris separates the trophoblast from the endometrial epithelium. The trophoblastic epithelium of these areas also accumulates various electron-dense inclusions in advanced gestation. They are of uncertain nature and not comparable to the lactogen granules. They probably relate to the small extravasations of maternal blood in the "arcades". The intercotyledonary membrane region is also characterized by the "areolae", an old description dating back to Eschricht (reviewed by Ludwig, 1968). Numerous studies have suggested that the areolae are situated above the mouths of several endometrial glands and that uterine "milk" secreted here serves a nutritive function for the fetus. The areolae increase in size with gestation, up to a 3mm diameter, and were beautifully studied by Wimsatt (1964). He showed PAS positive material to increase in the course of gestation at these locations, and found several modifications of the histologic appearance of the trophoblastic epithelium over time. With the secretion of the glands, a "pit" forms and the trophoblastic epithelium becomes focally elevated. Wimsatt also segregated three "epochs" in the development of the areolae, which are not usually discussed further. These are presumably related to an increased nutritive uptake or the production of uterine milk. Ludwig (1968) suggested the term "enteroid" for these areolae, while he called the cotyledonary regions as subserving a "nephropneumoid" function.

Wimsatt (1964) was also the first author to study the vasculature of the intercotyledonary membranes in some detail. Interestingly, here the arteries are located in such a manner that they are situated above the veins, which are closer to the trophoblast. That is the same arrangement as is found in primate placentas. The capillaries are "more copious" in the vicinity of the areolae than elsewhere in the chorion.

9) Trophoblast external to barrier

There is no infiltration of trophoblast beyond the caruncles, and there is no subplacenta.

10) Endometrium

There is no decidua in the ovine uterus but osteopontin is expressed in the endometrial stroma at the site of superficial endometrial erosion (Johnson et al., 2003). This further supports some syndesmochorial nature of this placentation, as it osteopontin expression is absent in pigs. Endometrial glands remain as scattered islands below the cotyledons.

11) Various features

There is no subplacenta.

Progesterone appears to have a significant effect on fetal growth when administered in the first few days of gestation according to Kleemann et al. (2001). They found fetal size and weight to increase with minor effect on placental weights. These authors also made extensive measurements of fetal body parts and of the placental surface and its components that must be read in the original publication.

Numerous studies have addressed the hormonal output of the binucleate trophoblastic cells. Thus, Wooding et al. (1986) showed that 15-20 % of trophoblast are binucleated and contain ovine placental lactogen. Their number decreases shortly before parturition but this decline was prevented by fetal hypophysectomy, indicating fetal control of this population of cells. That this is due to fetal cortisol levels was elegantly shown in their subsequent experiments (Ward et al., 2002) which indicated an inverse correlation of cortisol levels and binucleate cell numbers. Nevertheless, even at very high levels of cortisol, a small number of binucleate cells remained. Miyazaki et al. (2002) studied the development of caprine trophoblast and binucleate cells by in vitro manipulation of a caprine trophoblast cell line.

12) Endocrinology

Keisler (1999) provided detailed information on endocrine signals of ewes. Nine hours after onset of estrus, large amounts of pituitary LH are released. Ovulation occurs 21-26 hours later. Three days following ovulation, progesterone levels can be detected and the uterus produces PGF2 episodically. This leads to the dissolution of the corpus luteum and a fall in progesterone levels. If conception takes place, "corpus luteum rescue" occurs with the first maternal recognition of pregnancy. The conceptus secretes various proteins (interferons) that inhibit PGF2 production. Later in gestation, a variety of pregnancy-specific proteins and placental lactogen are produced. These increase to term; the progesterone secretion by the corpus ceases to be essential after 60 days. The placental production of progesterone is then adequate to maintain pregnancy.

The onset of labor has been of particular interest to investigators. It is mediated via the fetal hypothalamic-pituitary-adrenal axis. Likewise, the placental ability to produce prostaglandins increases towards term. Fetal hypophysectomy or adrenalectomy leads to postmaturity. Fetal ACTH production increases two weeks before parturition with marked increase of cortisol production by the fetus approximately 3-4 days before delivery. There is extensive literature on these investigations, some of which is cited by Keisler (1999), other publications can be found in the summary by Liggins (1969).
Progesterone appears to have a significant effect on fetal growth when administered in the first few days of gestation according to Kleemann et al. (2001). They found fetal size and weight to increase with minor effect on placental weights. These authors also made extensive measurements of fetal body parts and of the placental surface and its components that must be read in the original publication.

Numerous studies have addressed the hormonal output of the binucleate trophoblastic cells. Thus, Wooding et al. (1986) showed that 15-20 % of trophoblast are binucleated and contain ovine placental lactogen. Their number decreases shortly before parturition but this decline was prevented by fetal hypophysectomy, indicating fetal control of this population of cells. That this is due to fetal cortisol levels was elegantly shown in their subsequent experiments (Ward et al., 2002) which indicated an inverse correlation of cortisol levels and binucleate cell numbers. Nevertheless, even at very high levels of cortisol, a small number of binucleate cells remained. Miyazaki et al. (2002) studied the development of caprine trophoblast and binucleate cells by in vitro manipulation of a caprine trophoblast cell line.

13) Genetics

Sheep have 54 chromosomes that have been well delineated in numerous studies. Long & Williams (1980) studied the frequency of chromosomal errors in embryos and unfertilized eggs. They found 4.7% trisomies in 89 fertilized eggs and one mosaic diploid/haploid specimen. Several eggs or embryos had a "cracked" zona pelludica.

Domestic sheep have hybridized with many other taxa (Gray, 1972), especially other ovine forms. Of greatest interest have been the many attempts to hybridize sheep (2n=54) and goat (2n=60). Occasional offspring have been achieved (2n=57) but that has been difficult and hemolytic disease often causes fetal demise. In a large series of experiments, Dent et al. (1971) showed with electronmicroscopy that the trophoblast of the hybrids is normal and implantation appears to proceed normally at first. There begins, however, an extensive accumulation of platelets in the maternal blood vessels and endothelial swelling to the point of occlusion with hemorrhage ensuing between 34 and 38 days. These authors suggested that this is akin to an immunological rejection of the fetal hybrid implant. The hybrids are only exceptionally fertile (Bunch et al., 1976). Dent et al. (1971) indicated that fertilization occurs regularly in such attempts at hybridization but that placentation usually fails before the second month of gestation. The platelet accumulation in such hybrid placentas on day 34 beneath the uterine epithelium of the placentomes is the apparent cause of failure. "The maternal blood vessels showed the presence of large numbers of platelets, either alone or mixed with isolated red blood cells". On day 38 the platelets were very prominent, but absent in normal goat placentas. Subsequently the uterine epithelium became necrotic and hemorrhage occurred focally. They interpreted these lesions as resulting from antigen-antibody interaction, akin to that seen in skin grafts. Embryo transfers of mouflon into sheep, however, are successful (see Figure 2).

Karyotypes of male and female domestic sheep.

14) Immunology

The sheep has been used to study the onset and the intricate nature of early antibody production by the fetus (Silverstein et al., 1963). When the fetus is stimulated with specific antigens, it responds by producing different antibodies at different gestational times. Moreover, skin grafts placed upon fetuses from other fetuses were rejected in a typical manner; it was discovered then, however, that plasma cells did not participate in the rejection process as the fetus was not yet capable to produce them (Silverstein, et al. 1963).

15) Pathological features

An important ovine disease is scrapie, a CNS degenerative disorder that is caused by a variant prion protein. It is so important because, in recent years, infected animal derivatives were fed to cattle with the occurrence of bovine spongiform encephalopathy (BSE). Subsequently, through ingestion of beef, humans have developed a variant of Creutzfeldt-Jakob Disease (vCJD) traced to this source. Houston et al. (2000) showed that blood from a sheep that was fed BSE-infected brain of cattle transmitted this prion to sheep via transfusion of the blood from the challenged sheep.

16) Physiological data

The sheep has been used for numerous physiological studies; many are designed to better understand human prenatal development. Thus, Barron (1951) reviewed in some detail the transfer of oxygen. He and others have studied this transfer at high altitude and compared it with data found at sea level. Liggins (1969) reviewed the endocrine signals for the onset of labor and parturition. Kleemann et al. (2001) showed that progesterone administration in very early pregnancy enhances fetal growth. Alexander (1964) removed up to 84 caruncles from Merino sheep prior to mating in order to assess the impact on placentation. While fertility was not affected, the ewes in which sixty or more caruncles had been removed delivered prematurely and the lambs had reduced birth weights. The caruncle removal was compensated by an increased size of the cotyledons. Worthington et al. (1981) carried out similar reductions with electrocautery. They found a similar effect on fetal growth. Creasy et al. (1972) embolized fetal cotyledons with microspheres between days 96 and 125 and found significant reduction in fetal size. The affected cotyledons showed hyalinization and necrosis with marked reduction in size. This was followed by similar experiments of Duncan et al. (2000). These investigators examined the consequences of interference with placentation on fetal brain development. They identified significant damage to the white matter of the CNS in the growth-restricted lambs. There are many other, similar studies that probe fetal CNS development by restricting placental performance. For instance, Dawes & Mott (1964) altered oxygen supply to fetal lambs and studied their responses. Keunen et al. (1997) found no CNS damage with cord occlusion of up to ten minutes, but Ball et al. (1994) found that seizures took place when there was severe cord occlusion for 90 minutes. Ikeda et al. (1998) sought to explain the CNS lesions after cord occlusion by brain lipid peroxidation. Mallard et al. (1992) found primarily hippocampal lesions after occlusion, while Clapp et al. (1988) found mostly white matter damage after intermittent occlusion of the cord's vessels. Gardner et al. (2002) used sheep placentation to study the effect of periodic cord occlusion. They found that the number of caruncles did not decrease, but their weights decreased and appearances changed.

Gilbert et al. (1996) showed that technetium crossed the ovine placenta more rapidly than the smaller sodium ion. Ovine urine production was studied by Ross, et al. (1988) and by many other investigators who are listed by these authors. Ross et al. found in this carefully executed protocol (days after surgical canulations, standing ewes, 4-hour experiment, careful electrolyte measurements) that the amount of allantoic fluid was greater than that of amniotic fluid; that fetal urination is greater into the allantoic sac (through urachus) than the amnionic sac (through urethra); that transmembranous flow probably exists; that lung fluid participates in amnionic fluid composition and swallowing in disposing of it. These are difficult experiments whose interpretation is complicated by the presence of two large fluid-filled sacs in the ovine placenta. They are not directly transferable to human amnionic fluid metabolism for that reason. With Barron we catheterized the urachus in an instrumented sheep that was suspended in a water bath. We found that urine production decreased markedly over a few hours when all urine from the urachus was collected externally. Its osmolality also increased and much fructose was contained in the urine. Urination increased rapidly when water was injected into the allantoic cavity. We conjectured that the water was rapidly absorbed by the allantoic circulation and rehydrated the water-deprived fetus. Matsumoto et al. (2000) ligated the fetal esophagus and urachus and were unable to produce polyhydramnios, as had been their aim. This suggested to them that transmembranous transport must be large.

More recently, Daneshmand et al. (2003) investigated the regulation of amnionic fluid volume in sheep and suggested that vascular endothelial growth factor increases intramembranous absorption by microvesicular transport.

17) Other resources

Cell strains of various sheep species are available from CRES at the Zoological Society of San Diego.

18) Other remarks - What additional Information is needed?

Relatively few noninfectious placental lesions have been described in sheep. More knowledge is also needed on umbilical cords. Mossman (1987) pointed out that more information is needed on the insertion of the cords of multiple gestations. Because there is still some uncertainty as to the true origin of the syncytial giant cells, additional studies are warranted.

Acknowledgement

Most of the animal photographs in these chapters come from the Zoological Society of San Diego. I appreciate also very much the help of the pathologists at the San Diego Zoo.

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