黑綿羊胎盤.Ovine Placenta Extract

 Shearing ewes at mid-pregnancy is associated with changes in fetal growth and development

D. K. Revell, S. T. Morris, Y. H. Cottam, J. E. Hanna, D. G. Thomas, S. Brown and S. N. McCutcheon


This study investigated the effect of mid-pregnancy shearing (at Day 70 of pregnancy, P70) on herbage intake of grazing single- and twin-bearing ewes, lamb birth weight, and cold resistance of new-born lambs. At pregnancy diagnosis on P50, 30 single-bearing and 30 twin-bearing ewes were allocated either to be shorn at P70 (n = 15 for each pregnancy rank) or to remain unshorn to serve as controls (n = 15 for each pregnancy rank). All ewes were mated over a 3-day period with synchronisation of their oestrus. Herbage intake was measured indirectly from in vitro pasture digestibility and faecal output of grazing ewes, with the use of intra-ruminal chromium slow-release capsules, over six 5-day periods from P64 to P105. The weights of placental and fetal tissues were assessed in a subgroup of 16 ewes at P140 and P141. In the remaining sheep, lamb liveweight at birth and during lactation until weaning at 103 days of age was measured, and cold-resistance of new-born lambs was assessed by measuring summit metabolic rate (SMR) by indirect calorimetry. Ewe liveweight (corrected for fleece weight), condition score, and herbage intake during pregnancy were not affected by shearing treatment. Mid-pregnancy shearing did not affect placental weight, but increased the relative weights (i.e. g/kg liveweight) of fetal thyroid gland and lungs and reduced the relative weight of adrenal glands and heart. The ratio of secondary to primary wool follicles in near-term fetal skin was about 10% higher in offspring of shorn than of unshorn ewes. The metabolic rate of fetal hepatic tissue was increased by mid-pregnancy shearing, particularly in twin fetuses, possibly indicative of an increase in placental transport of nutrients to the fetuses. This conclusion is supported by the greater birth weight (average response 0.5 kg) of lambs born to ewes shorn at mid-pregnancy. Mid-pregnancy shearing also increased the SMR of new-born twin lambs by 16%, but decreased the SMR of singleton lambs by 26%. These results indicate that mid-pregnancy shearing can increase lamb birth weight without increasing ewe herbage intake or placental weight. An increase in the efficiency of nutrient uptake by the placenta is implied, and possible effects on the activity of thermogenic tissues are discussed.

Occurrence of a growth hormone-releasing hormone-like messenger ribonucleic acid and immunoreactive peptide in the sheep placenta

MC Lacroix, H Jammes and G Kann

Growth hormone releasing factor (GHRH) has been described in the rat, mouse and human placentae. This study reports the presence of an immunoreactive GHRH activity (IR-GHRH) in the ovine placenta. This activity was detected by radioimmunoassay from day 50 (D50) until the end of pregnancy. Higher IR-GHRH concentration in placental tissue was observed on days 100 (543 +/- 123 pg/g) and 140 (550 +/- 62 pg/g) and, when compared with the GHRH content of the ovine hypothalamus (1.2 ng/hypothalamus), represents a considerable amount of GHRH per placenta (a mean of 200 ng). Perifused placenta explants released IR-GHRH in vitro at a mean rate of 200 pg/g/h. Depolarization by 55 mM KCl increased the IR-GHRH concentration of the perifusion media 1.7 times over basal values. The elution position of GHRH immunoreactivity in the gel filtration chromatography profiles was the same for placenta and hypothalamus extracts and lay very near to the molecular weight of bovine GHRH. Northern blot hybridization analysis revealed the existence of a placental transcript whose size (0.75 kb) was comparable to the size of the ovine hypothalamus and rat placenta GHRH transcripts. Hybridization signal was observed at each stage studied from D50 until D120 of pregnancy. This study demonstrated the existence of a IR-GHRH peptide in the ovine placenta 


 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).


2)    General gestational data

Keisler (1999) has provided a detailed review of the reproductive parameters of domestic sheep and goats. The reader is also referred to the Chapter on Cretan Goat. That species' reproductive aspects have many similarities. There are about 800 different "breeds" of sheep with many different physical characteristics. Depending on the breed, puberty occurs between 5 and 12 months. Breeding occurs throughout the year in domestic sheep and is somewhat dependent on photoperiods. The duration of estrus is in between 3 and 72 hrs. (average 29 hrs.); it is influenced by the presence of a male. The estrous cycle averages 16 days.

While the length of gestation in wild sheep species varies between 150 and 180 days, the domestic sheep has an average length of gestation of 148 days. Most sheep produce singletons, but some races regularly have twins, even triplets. Singletons weigh about 1.5 kg (Alexander, 1964). The placental weight is difficult to ascertain as the membranes are voluminous and of little importance to fetal growth as such. Kleemann et al. (2001) provided weights around 600 g. They were interested in the relation of placental and fetal sizes after progesterone treatment in early gestation. Whether these weights reflect a direct relation to the provision of nutrients to the fetus can be questioned. Therefore, investigators have weighed what they considered to be the most meaningful weight - that of blotted cotyledons. This is discussed below in detail (Alexander, 1964).

3) Implantation

Within three to four days, the fertilized ovum reaches the uterus as a morula. The blastocyst develops by day 6 and sheds its zona pellucida by day 8 or 9. The blastocyst enlarges rapidly after the tenth day (Assheton, 1906). It becomes "sticky" and, on about day 17-18, it attaches with cytoplasmic protrusions to the endometrial surface. Naturally, this attachment is principally at the caruncles of the endometrium. The placenta remains diffuse for the first month or so after its mesometrial attachment (King et al., 1982). Lawn et al. (1969) did an electronmicroscopic study of goat and sheep placentas. Their youngest sheep gestation was at 42 days of pregnancy; the cotyledons were then just beginning to develop a villous structure. The trophoblast was apposed to an intact endometrium that had the appearance of protein secretion. Microvilli were abundant and occasional binucleated trophoblastic cells were present. Interdigitation of the cells progressed thereafter and the endometrial epithelium began to transform. Moreover, multinucleated giant cells became apparent. The syncytial cytoplasm then also acquires characteristic cytoplasmic granules.


Uterine caruncles of immature common waterbuck (Kobus kob) with normal endometrium in between. (Courtesy, Dr. P. Hradecky).


3)    General characteristics of placenta

The sheep placenta is a polycotyledonary placenta with 70 to 100 cotyledons. Number and size of cotyledons varies widely with maternal age, strain of sheep and perhaps it depends on yet some other factors. The cotyledons are the fetal portions of the placentomes. The latter term, placentome, additionally encompasses the maternal caruncle.

In between the cotyledons is the intercotyledonary chorion, which has an additional and different absorptive function. It is covered by simple trophoblast and modified only over the mouths of endometrial glands. These regions are referred to as the "areolae". In between the areolae there are the "arcades" with focal hematomas and absorption of blood by trophoblast.

After the initial expansion of the blastocyst at about day 10, the elongated chorionic sac fills both of the uterine horns. It is entirely covered with trophoblast. This large sac contains a large, elongated allantoic cavity that apposes the chorion to the end of the outer chorionic sac and apposes the amnion directly over its central portions. The yolk sac is short-lived and plays no major role in ovine placentation and it cannot be found at term.

A bighorn sheep placenta received in May, 2004 weighed 300g, measured 83 cm in longest dimension, had 51 small concave cotyledons and a 27 cm long umbilical cord with four vessels.


Diagram of opened sheep placenta. The largest compartment is the allantoic sac. Inside is the smaller amnionic cavity with the fetus. Dark spots are the placentomes - cotyledons.

Immature sheep placentome. The pale areas are villous tissue; in between villi are the maternal sheaths.


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.

Immature sheep placenta with loosely constructed villi and thin region of maternal tissue, "plates".


Immature sheep placenta with loosely constructed villi and thin region of maternal tissue, "plates".

Implantation site of immature sheep placenta. Note the pink maternal tissue tongues with blood vessels penetrate between the villi.


Branching villus with trophoblast cover (T) adjoining the maternal epithelium.


Branching fetal villus with fetal blood vessel covered by trophoblast (see binucleate cell at right). The maternal epithelium seems to be focally broken.

Mature villous tissue with binucleate cells at arrowheads. The maternal tissue is adjacent but very difficult to identify. Many giant cells.


Surface of cotyledon with large maternal hematoma and adjacent pigment-laden trophoblast (mouflon).

Surface of placenta with massive pigment accumulation in trophoblast. This is derived from former maternal hemorrhages.


6) 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.

7) Extraplacental membranes


Diagram of the intercotyledonary membranes attached to the uterus.


"Internal" membranes of sheep placenta with the amnion above. Beneath it is the allantoic membrane. Note that it is the allantoic chorion that contains the blood vessels. The amnionic connective tissue is avascular. 

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.


Section of the intercotyledonary membranes. The trophoblast below is apposed to the uterine epithelium. It has a microvillous surface. This membrane lies outside the allantoic sac, which is shown above. The allantoic sac has a thick epithelial lining that is of endodermal origin, but it has only very short microvilli. Both membranes have a vasculature.

Trophoblastic lining of the outside of the intercotyledonary membrane with pronounced microvillous surface. Note the pleomorphism of epithelial cells. 

The structure of the ovine amnion has been studied by Bautzmann & Schroder (1955). Their investigations of the amnion from birds and reptiles had disclosed the presence of a smooth musculature in the amnion. This is absent in sheep and other mammals. The ovine amnionic epithelium is flat and single-layered, with occasional "warts" or verrucae. They are composed of squamous proliferations and are similar to the verrucae of squamous metaplasia in the amnionic epithelium of the umbilical cord. These authors described in detail also the collagenous fibers and the glassy membrane of the amnion. These layers were further delineated in the detailed studies of human placental membranes by Bourne (1962). Additional studies on these epithelial proliferations in hoofed animals and whales were published by Naaktgeboren & Zwillenberg (1961). They found them in essentially all hoofed animals and whales with the exception of the pig. In sheep, they were initially white but became yellow as gestation advanced. The allantoic sac also contains irregular quantities of brownish debris, the hippomanes. 

8) 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.