Sheep trophoblast in monolayer cell culture

Placenta (1980) 1,209-221

Sheep Trophoblast in Monolayer Cell Culture

D. H. S T E V E N a, K A T H L E E N P. W. N A T H A N I E L S Z ~

A. M A L L O N b &

aDepartment of Anatomy, University of Cambridge, Downing Street, Cambridge. bLaboratory of Fetal Physiology, UCLA School of Medicine, 1000 West Carson Street, Torrance, California 90509, USA. Requests for reprints should be addressed to Dr P. W. Nathanielsz at the UCLA School of Medicine.

INTRODUCTION The placenta of the sheep is cotyledonary in form and epitheliochorial in its histological structure (Ludwig, 1962; Lawn, Chiquoine and Amoroso, 1969). Two types of cell are found within the chorionic epithelium. The majority are the principal or uninucleate cells of the trophoblast, which are cuboidal or columnar in shape and contribute to the microvillous junction at the fetal-maternal interface (Steven, 1975; Boshier and Holloway, 1977). Interspersed at irregular intervals between the principal cells are giant or binucleate cells, which are rounded or oval in outline, usually contain a prominent Golgi apparatus, dilated cisternae of rough endoplasmic reticulum and aggregations of electron-dense membranebound inclusions, and do not normally contribute to the formation of the fetal-maternal interface (Steven, 1975; Boshier and Holloway, 1977; Steven et al, 1978). Binucleate cells appear within the trophoblast at the 16th day of pregnancy (Boshier, 1969) and are thought to be derived from the principal cells of the trophoblast (Assheton, 1906; Wimsatt, 1951). The maternal side of the placental barrier is formed by the uterine epithelium, which in the early stages of pregnancy undergoes a syncytial transformation and thereafter takes the form of thin and contiguous sheets of multinucleate cytoplasm (Lawn, Chiquoine and Amoroso, 1969). Binucleate cells are spared from the destructive changes which, in the short interval between the birth of the fetus and delivery of the afterbirth, overtake the principal cells of the trophoblast (Perry, Heap and Ackland 1975; Steven 1975). Free and undamaged binucleate cells can be recovered from the washings of naturally-delivered fetal membranes, but such cells are heavily contaminated with bacteria, blood and much cell debris. Our early attempts to culture binucleate cells from this source were completely unsuccessful. The report of Dubois, Martal and Djiane (1976), who were the first to show by immunofluorescence techniques that binucleate cells contain ovine placental lactogens (oPL; otherwise known as ovine chorionic somatomammotropin, oCS), encouraged us to seek alternative methods of approach. In this paper we describe the techniques that have enabled us to maintain sheep trophoblast for periods of 12 to 16 weeks in monolayer cell culture. MATERIALS AND M E T H O D S Solutions and reagents The following solutions and reagents were used in our experiments: DMEM. Dulbecco's modified Eagle's medium (Gibco Biocult, Santa Clara, California). This 0143-4004/80/0103-0209 $02.00

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solution contained 4.5 g glucose/l without sodium pyruvate. DPBS. Dulbecco's phosphate buffered saline (Gibco). FBS. Fetal bovine serum obtained from calves at 6 to 9 months of gestation (Gibco). Holding solution A. DPBS containing 0.1 mg/ml Fungizone (Squibb, Santa Monica, California), 0.1 mg/ml Neomycin (Squibb, Princeton, New Jersey) and 0.1 mg/ml Gentamycin (Scherring, Kenilworth, New Jersey). Trypsin solution B. (0.25 per cent w/v) Hanks' balanced salt solution without Ca ++ or Mg § containing 2.5 g trypsin/1 (Gibco). Washing solution C. 100 ml distilled water to which was added 8.0 g NaC1, 4.0 g KC1, 1.0 g dextrose and 0.2 g Na2EDTA. Trypsin solution D. (2.5 per cent w/v) Normal saline containing 25 g trypsin/1 (Gibco). Trypsin solution E. Nine vols washing solution C and 1 vol trypsin solution D. Nutrient mixture F. 100 ml FBS, 1.24 ml 1.0 M Hepe's buffer (Gibco), 5 ml 200 mM lyophilized L-glutamine 29.2mg/ml (Gibco), 1.0ml beef insulin 100i.u./ml (Lilly, Indianapolis, Indiana), 1 ml Fungizone 10 mg/ml and 1 ml Neomycin 10 mg/ml made up to 500 ml with DMEM. Nutrient mixture G. 220 ml FI2 nutrient mixture (Ham), 5 ml 200 mM lyophilized L-glutamine, 220 ml DMEM, 50 ml FBS, 2.5 ml 1.0 M Hepe's buffer, 1 ml Fungizone 10 mg/ml, 1 ml Neomycin 10 mg/ml and 0.5 ml beef insulin 100 i.u./ml. Trypsin solution H. (1.5 per cent w/v) Washing solution C to which was added 15 g trypsin/1. Aseptic r e m o v a l of cotyledons Sixteen pregnant ewes at 120-135 days of gestation were anaesthetized and prepared for surgery by methods previously described (Lowe et al, 1979). One or two cotyledons were removed under aseptic conditions from the antimesometrial wall of the pregnant horn of the uterus. The intact cotyledons were placed into freshly prepared holding solution A at 4 ~ After 2 hours in this solution the cotyledons were manually separated into fetal and maternal components. The fetal villi were washed three times in DMEM at 37 "C while the capsule of the cotyledon and other maternal tissues were discarded.

Preparation of dispersed cultures Washed fetal villi were cut into 0.5-1.0 mm cubes and periodically rinsed with DMEM at 37 ~ until they were as free as possible of erythrocytes and cell debris. The pieces were then placed in a 150 ml dimpled conical flask, which contained 25 ml of trypsin solution D, and which was maintained at 37 ~ on a heated plate. The tissues were thoroughly agitated by magnetic stirring bar for 20 minutes, and after this time were allowed to settle to the bottom of the flask. The supernatant, cloudy with free-floating cells, was then discarded. After transfer to 25 ml trypsin solution B, the tissues were retrypsinized at 37 ~ for three consecutive periods of 20 minutes. The supernatant from the first retrypsinization was always discarded, for the cells in suspension were predominantly erythrocytes and fibroblasts. After the second and third retrypsinizations the supernatant was decanted into 15 ml Corning conical centrifuge tubes (Scientific Products, Irvine, Los Angeles). These contained 750 ltl FBS, which at a concentration of approximately 5 per cent was sufficient to inhibit further trypsinization. After 10 minutes' centrifugation at 2000 r/min at 4 ~ the cells were washed in DMEM at 37 ~ re-suspended in the same medium and centrifuged four more times until the suspension was clear and free from debris. The cells were finally rinsed in nutrient mixture F, filtered

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through sterile gauze or muslin, and re-suspended in 10 to 50 ml of nutrient mixture F according to the density of the cells recovered. Three 5000/11 samples of this suspension were removed for counting in a Neubauer chamber. Erythrocytes were lysed by adding each sample to 4.5 ml of 1 per cent acetic acid. Known volumes of this acidified suspension were mixed with equal volumes of 1 per cent trypan blue, a procedure which ensured that non-viable cells with damaged membranes were excluded from the count. T h e concentration of the original suspension was then adjusted to a value of 1 x 106 cells/ml. Cells were plated out in two different ways: 3 ml of 1 x 106 cells/ml were added either to 90 mm Coming Petri dishes or to 25 ml C o m i n g tissue culture flasks. Flasks and dishes were placed in a gassing incubator and maintained at 37 ~ under 8 per cent CO 2 in air for 24 hours. At the end of this period the supematant was gently decanted without centrifugation to prevent the accumulation of toxic products from dead or dying cells. The normal viability was 85 to 90 per cent. Viable cells were adherent to the culture vessels after 8 to 10 hours. Subcultures were made as soon as the cells of the monolayer became confluent: at this stage no space was left in the vessels for further growth. The medium was decanted and the cultures were washed with 5 ml of washing solution C at 37 ~ for two consecutive periods of 5 minutes each. The cultures were then flooded with 5 ml trypsin solution E, which was allowed to act for 10 minutes at 37 ~ Loosening of cells from their attachment.to the vessels was assisted by fluid gently pipetted over them. The trypsinized suspension was transfen'ed to C o m i n g conical tubes, which contained sufficient FBS to give a final concentration of approximately 2 per cent: it was then centrifuged at 2 0 0 0 r / m i n for 10 to 15 minutes. The subsequent procedure was the same as that for primary dispersed cultures after the second or third retrypsinization. Secondary cultures were seeded at 106 or 107 cells per vessel and were again confluent after 7 days. Preparation ofexplant cultures from chopped tissue fragments Ten to 20 cubes of tissue prepared as above from washed fetal villi were placed in 25 ml Coming tissue culture flasks, which contained approximately 1.5 ml of nutrient mixture F. This was sufficient to cover the bottom of the flask, but did not totally immerse the tissue fragments. Contact with the base of the flask was essential for the establishment of the culture. Explants incubated at 37 ~ under 8 per cent CO 2 in air were established after 24 to 72 hours. It was unnecessary and inadvisable to disturb them during this period, but after 72 hours the cultures were washed with nutrient mixture F and unattached pieces of tissue were removed. The medium was then changed to 3 ml of fresh nutrient mixture F and incubation was continued until the 5th to 7th day, when the substrate was changed to nutrient mixture G. The original explants were removed at the 10th to 14th day of incubation, when the first subculture was usually required. T h e medium was changed every 3 to 5 days after the first subculture, or at more frequent intervals if suggested by the indicator. Preparation of explant cultures from pretrypsinized chopped tissue fragments This procedure was carried out in a similar manner to the previous technique, except that the explants were trypsinized before incubation in culture flasks. Tissue fragments from the third retrypsinization of the dispersion method were sometimes used: alternatively, more coarsely chopped fragments of washed fetal villi were stirred at 37 ~ for 10 minutes in trypsin solution D and 15 minutes in trypsin solution E. The trypsinized fragments were then washed, cut into smaller pieces and treated as described above.

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The yield of epithelial cells from primary explant cultures was usually greater than that obtained from the dispersed cell system. Staining of cell cultures for histological examination The medium was decanted and the cultures were washed with DPBS at 37 ~ for two 10-minute periods. The DPBS was replaced by 95 per cent alcohol, which was allowed to act for 5 to 10 minutes. The cultures were then flooded for 10 to 20 minutes with a 1 per cent solution of Jenner's stain at room temperature. After excess stain had been removed by a 5-minute wash with distilled water, the cultures were allowed to dry. A Wild Heerbrugg inverted microscope was used for photography and histological examination.

Preparation of cells for electron microscopy Cells were removed from the cultures by the subculturing procedure already described. They were washed three times in DMEM at 37 ~ and centrifuged at 2000 r/min. Cell pellets recovered from the centrifuge tubes were placed in approximately ten times their volume of cacodylate-buffered fixative at 4 ~ and of pH 7.2: this solution contained 1.0 per cent glutaraldehyde, 0.5 per cent paraformaldehyde and 5 per cent sucrose. Cells were dispersed and agitated during this 15-20 minute period to encourage complete fixation. After further centrifugation the fixative was replaced by 0.1 M cacodylate buffer, pH 7.4, containing 5 per cent sucrose. The buffer was then changed twice at 10-minute intervals. The cells were again centrifuged and the pellets were postfixed in 1 per cent osmic acid for 30 minutes. The pellets were then embedded in agar according to the method of Ryter and Kellenberger (1958), dehydrated in acetone and embedded in TAAB resin (TAAB Laboratories, Reading, UK). Ultra-thin sections for transmission electron microscopy were stained with lead citrate and uranyl acetate and examined'either with a Phillips 300 or an AEI EM6B electron microscope.

Measurement of ovine placental lactogen The concentrations of oPL in culture media were measured by specific double antibody radioimmunoassay according to the method of Gluckman et al (1979).

RESULTS Ovine trophoblast grown from dispersed and explanted primary cultures was maintained in viable condition for periods of 12 to 16 weeks. Sixteen animals were used to establish 15 sets of primary cultures of 15 to 20 flasks per set. oPL could be detected in the medium of wellestablished cultures towards the end of the first week of incubation and rose to relatively high concentrations as the cultures developed.

Dispersed p r i m a r y cultures In dispersed primary cultures the seeded cells showed little sign of activity for the first 8 hours, after which time they settled and attached themselves to the bottom of the plastic containers. At 12 hours the cells had lost the spherical shape which followed the process of trypsinization and two main types could be identified. Long multipolar fibroblasts formed overgrowing layers that were more than one cell thick, while the smaller and more rounded epithelial cells remained relatively inactive. At this stage most of the fibroblasts could be removed by a 5-minute wash with 5 ml trypsin solution H, At 24 to 48 hours the epithelial cells had begun to multiply. From this time onwards individual cells grew larger, and vacuoles and dense droplets were discernible in the cytoplasm.

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Figure 1. Monolayercultures of 135 day sheep trophoblast (a) in the third week of incubation and (b) in the fifth weekof incubation. Binucleatecellshave been outlined in black.

At 72 to 96 hours the medium was changed to nutrient mixture G, for at this time the cells seemed less dependent on insulin and fetal serum and appeared to respond to a more enriched substrate. Subcultures were made when the cells became confluent between the fourth and seventh day of incubation. The change of medium enhanced the rate of cell multiplication and appeared to stimulate the production of droplets and vacuoles within the cytoplasm. Binucleate cells seeded in small numbers in the primary cultures could be identified under phase contrast by their nuclei, their large size and their multiple cytoplasmic inclusions. Such cells began to degenerate during the second week of incubation and did not usually survive beyond the 14th day. Newly-formed binucleate cells could be identified clearly at the outer edges of the monolayer by the 14th day of incubation, by which time they formed about 10 per cent of the total cell population. From the third to fourth week about 35 to 40 per cent of the epithelial cells in culture contained two distinct nuclei (Figure la). By the fifth to sixth week of culture the cytoplasm of uninucleate and binucleate cells contained many large clear vacuoles (Figure lb). Explanted p r i m a r y cultures In explanted untrypsinized primary cultures there was an initial outgrowth of fibroblasts and epithelial cells during the first 3 to 4 days. Some cells were shed from the explant into the culture medium, but such cells rarely established themselves and were removed by decanting the medium. Subsequent outgrowth from the explant was relatively slow, so that the first subculture was not usually required until the 10th to the 14th day. Trypsinized primary explant cultures established themselves on the second to third day of incubation and the outgrowth usually became confluent by the seventh to tenth day.

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Table I. Details of maintenance of cultures and oPL concentrations in media.

Gestational age of placenta at time of culture (days)

Duration of culture at time of oPL measurement (days)

oPL in culture medium (ng/ml)

Dispersion 1

120

Dispersion 2

120

Dispersion 3

130

Explant 1

120

5 15 28 35 5 35 7 28 7 21

86 286 > 10000 4651 < 80 4484 98 2236 < 80 2294

Total length of maintenance of culture (days)

42 50 64 36

2.0pm Figure 2. Uninucleate cell from a monolayer culture of 135 day sheep trophoblast in the second week of incubation. Mierovillous processes extend from the peripheral cell membrane. A few mitochondria and some strands of rough endoplasmic reticulum are visible in the cytoplasm.

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2.0 um m

Figure 3. Binucleate cell from a monolayer culture of 135 day sheep trophoblast in the second week of incubation. There are no microvillous projections from the cell membrane.

Fibroblasts could more easily be removed from trypsinized than from untrypsinized primary explant cultures.

Measurement of oPL in m e d i a from dispersed and explanted cell cultures oPL was measured in the media from three dispersed and one explanted culture. The results are summarized in Table 1. Ultrastructural characteristics of cultured trophoblast During the second week of incubation, cells cultured from 135 day trophoblast showed a gradation in ultrastructural appearance from uninucleate cells with microvillous borders (Figure 2) to binucleate cells with smooth limiting membranes (Figure 3). The binucleate cells contained small dilated cisternae of rough endoplasmic reticulum, a prominent Golgi apparatus and membrane-bound inclusions from 0.3 to 0.5/~m in diameter. The finely granular contents of these inclusions were similar in appearance to the contents of the cisternae of the rough endoplasmic reticulum (Figure 4). Numerous polyribosomes contributed to the electron density of the cytoplasm. Very different in appearance were a few large binucleate cells in various stages of degeneration. These cells resembled binucleate cells of sheep trophoblast in vivo in the last

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Figure 4. Part of a binucleate cell from a monolayer culture of 135 day sheep trophoblast in the second week of incubation. This region of the cytoplasm contains Golgi membranes (G), rough endoplasmic reticulum (RER) and membrane-bound inclusions (mbg). The granular contents of the membrane-bound inclusions are similar in appearance to the granules contained within the cisternae of rough endoplasmic reticulum, m, mitochondria; N, nucleus of binucleate cell.

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Figure 5. Uninucleate cell from a monolayer culture of 135 day trophoblast in the sixth week of incubation. The cytoplasm contains clear vacuoles and a scattering of dark droplets. There are numerous microvillousprojections from the cell membrane. third of gestation (see Figures 2 and 7 in Steven, 1975), and had almost certainly been seeded in the primary cultures. During the sixth week of incubation, cells cultured from 135 day trophoblast again showed a gradation in ultrastructural appearance from small dark cells with microvillous borders to larger paler cells with a smooth outer membrane. The dark cells had a densely granular cytoplasm which contained a scattering of electron-dense droplets and clear irregular vacuoles

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Figure 6. Large pale cell from a monolayerculture of 135 day sheep trophoblast in the sixth week of incubation. Clear vacuoules and dark membrane-boundinclusions are limited to the central region of the cytoplasm. The smooth peripheral membraneforms pseudopodialprojections(ps), which resemblethose of mature binucleatecells followingfetal pituitarystalk section.Compare Figure3 in Stevenet al, 1978.

of various shapes and sizes (Figure 5). By contrast, the cytoplasm of the larger cells was filamentous rather than granular in consistency (Figure 6). Small circular profiles of rough endoplasmic reticulum, large pale vacuoles and numerous spherical electron-dense bodies of up to 0.5 g m in diameter were located in the darker and more central regions of the cytoplasm. The electron dense inclusions contained a core of finely granular material, which in some sections appeared to be bounded by a thin dark membrane. Nuclei were often eccentrically

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placed, and the chromatin was evenly dispersed within them. It was not possible to determine from ultra-thin sections whether the large pale cells contained one or two nuclei.

DISCUSSION Placental cotyledons at the time of removal from the pregnant uterus are extremely difficult to separate into fetal and maternal components (Steven, 1973) unless the microvillous attachment at the maternal-fetal interface has previously been loosened by infusion of cortisol into the fetus (Jack et al, 1975). We found in our experiments that the fetal-maternal attachment in cotyledons from normal uninfused animals could be loosened by 2 hours' immersion in DPBS at 4 ~ The antibiotics added to this medium appeared to be critical to the establishment of successful monolayer cultures, for we found that penicillin or streptomycin inhibited cell growth, as did contact with glass surfaces. Once monolayers had been established in plastic containers we had few further difficulties, provided that subcultures were made whenever the cells became confluent. We found that the rate of growth could be partially controlled by the composition of the culture medium. Thus, in established cultures a change from nutrient mixture G to nutrient mixture F slowed down the development of the monolayer, while the reverse procedure accelerated the rate of growth. We are reasonably certain that our cultures were uncontaminated by epithelial cells of maternal origin, for early in gestation the uterine epithelium within the cotyledon undergoes a syncytial change (Lawn, Chiquoin and Amoroso, 1969) and thereafter takes the form of large contiguous sheets of multinucleate cytoplasm (Steven et al, 1978). No syncytial tissues were detected in any of the monolayers, nor in the samples examined before the seeding of primary dispersed cultures. We interpret our experimental results as follows. First, it is unlikely that the binucleate cells that appear in monolayers of cultured trophoblast during the second week of incubation are derived from mature binucleate cells seeded in the primary cultures, for the latter are larger in size, contain many cytoplasmic inclusions and do not normally survive beyond the 14th day. Secondly, we suspect that binucleate cells are formed by nuclear division of the uninucleate cells of the trophoblast, since during the second week of incubation there is a gradation in ultrastructural appearance from the uninucleate to the binucleate condition. This is in accordance with the views of Assheton (1906), Wimsatt (1951) and Boshier and Holloway (1977), who studied binucleate cells in cotyledonary tissues at various stages of normal pregnancy. Thirdly, our preliminary measurements of oPL in the medium of rapidly growing cultures suggest that there may be a correlation between oPL production and the numbers of binucleate cells present in the monolayer. Fourthly, our results do not preclude the possibility that oPL may be released into the culture medium by uninucleate cells that are in the process of transition to the binucleate form. oPL immunofluorescence has been attributed by Reddy and Watkins (1978) to both uninucleate and binucleate cells of the trophoblast between 100 and 130 days of normal pregnancy, yet their micrographs suggest that much of the immunofluorescence not contained within binucleate cells is located in maternal syncytial tissue. On the other hand the immunofluorescence studies of Martal, Djiane and Dubois (1977) clearly show that at the 120th day of gestation fetal oPL is not exclusively restricted to binucleate cells. The ultrastructural localization of oPL by immunocytochemistry may help to resolve some of these problems of interpretation. It is well established that parturition in the sheep can be suppressed by surgical interruption of the hypothalamo-pituitary-adrenal-placental pathway of the single fetus

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(Liggins, Kennedy and Holm, 1967; Drost and Holm, 1968; Liggins et al, 1973), yet the interrelationships of pituitary, adrenal and placenta in the control of parturition are still poorly understood. There is now some evidence to suggest that experimental interference with the fetal pituitary or adrenal in the last third of pregnancy is also followed by changes in placental structure, and particularly by changes in the anatomical relationships of placental binucleate cells (Barnes et al, 1976; Steven et al, 1978). Monolayer cultures of sheep trophoblast may well provide a simple and relatively inexpensive experimental system for the study of the origin and subsequent development of binucleate cells, and for the elucidation of some of the mechanisms involved in placental hormone production.

SUMMARY Methods are described for the preparation and maintenance of ovine trophoblast in monolayer cell culture, and for the preparation of cultured cells for ultrastructural examination. Monolayers grown from dispersed and explanted primary cultures were maintained in consecutive subcultures for periods of 12 to 16 weeks. Towards the end of the first week of incubation ovine placental lactogen could be detected in the culture medium: it rose to relatively high concentrations as the cultures developed. In the second week of incubation small binucleate cells were seen in cultures prepared for electron microscopy. By the 14th day larger binucleate cells were visible at the outer edges of the developing monolayer, and by the third to fourth week 35 to 40 per cent of the cells in culture were in binucleate form. Mature binucleate cells seeded in primary dispersed cultures did not survive beyond the 14th day. The results suggest that (a) the binudeate cells which first appear in monolayers of cultured trophoblast during the second week of incubation are formed by nuclear division of uninucleate cells and not from mature binucleate cells seeded in primary dispersed cultures; and (b) there may be a correlation between the numbers of binucleate cells present in the monolayer and the rate of production of ovine placental lactogen. Monolayer cultures of sheep trophoblast may well provide a useful and relatively inexpensive experimental system for the study of binucleate cells and the mechanisms of placental hormone production.

ACKNOWLEDGEMENTS We are grateful to Dr P. D. Gluckman, Department of Pediatrics, University of California, San Francisco, who so generously carried out the assays for oPL in our culture media. We thank Mr IC W. Thurley, Mr W. C. Mouel and Mr Jeremy Skepper (E. M. Unit, Cambridge University Department of Anatomy), Mr J. F. Crane and Mr C. J. Burton (Audio-visual Aids Unit, Cambridge University Department of Anatomy), Mr Ronald Tarris (HarborUCLA Medical Center) and Mrs J. Reynes (Imperial Cancer Research Fund) for their valuable and skilled assistance at various stages of the project. The work was supported by the Agricultural Research Council and by the National Institute of Health (Grant No. HD 11483-02). REFERENCES Assheton, R. (1906) The morphology of the ungulate placenta, particularly the development of that organ in the sheep, and notes upon the placenta of the elephant and hyrax. Philosophical Transactions of the Royal Society, Series B, 198, 143-244. Barnes, R.J., Comline, R. S., Silver, M. & Steven, D. H. (1976) Ultrastructural changes in the placenta of the ewe after foetal hypophysectomyor adrenalectomy.Journal of Physiology, 263,173-174P. Boshier, D. P. (1969) A histological and histochemical examination of implantation and early placentome formation in sheep.Journal of Reproduction and Fertility, 19, 51-61.. Boshier, D. P. & Holloway, H. (1977) The sheep trophohlast and placental function: an ultrastructural study. JournalofAnatoray, 124, 287-298.

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Drost, M. & Holm, L. W. (1968) Prolonged gestation in ewes after foetal adrenalectomy. Journal of Endocrinology, 40, 293-296. Dubois, M., Martal, J. & Djiane, J. (1976) Immunofluorescence localisation of placental lactogen. In Proceedingsof the 5th International Congressof Endocrinology, Hamburg, p. 25. Briihlsche Universit/its druckerei, Giessen. Gluckman, P. D., Kaplan, S. L., Rudolph, A. M. & Grumbach, M. M. (1979) Hormone ontogeny in the ovine fetus II. Ovine chorionic somatomammotrophin (OCS) in mid and late gestation in the fetal and maternal circulation. Endocrinology, 104, 1828-1833. Jack, P. M. B., Nathanielsz, P. W., Thomas, A. L. & Steven, D. H. (1975) Ultrastructural changes in the placenta of the ewe following foetal infusion of cortisol. Quarterly Journal of Experimental Physiology, 60, 171-179. Lawn, A. M., Chiquoine, A. D. & Amoroso, E. C. (1969) The development of the placenta in the sheep and goat: an electron microscope study.Journal of Anatomy, 105,557-578. Liggins, G. C., Kennedy, P. C. & Holm, L. W. (1967) Failure of the initiation of parturition after electrocoagulation of the pituitary of the fetal lamb. American Journal of Obstetricsand Gynecology, 98, 1080-1086. Liggins, G. C., Fairclough, R. J., Grieves, S. A., Kendall, J. Z. & Knox, B. S. (1973) The mechanism of initiation of parturition in the ewe. Recent Progressin Hormone Research, 29, 11-159. Lowe, K. C., Beck, N. F. G., McNaughton, D. C., Jansen, C. J. M., Thomas, A. L., Nathanielsz, P. W., Mallon, K. A. & Steven, D. H. (1979) Ultrastructural changes in the placenta of the ewe after long term infusion of 2-bromo-~-ergocryptine (CB154) into mother or fetus. Quarterly Journal of Experimental Physiology, 64, 253-262. Ludwig, K. S. (1962) Zur Feinstruktur der materno fetalen Verbindung des Schafes (Ovis aries, L.). Experientia, 18, 212-213. Martal, J., Djiane, J. & Dubois, M. P. (1977) Localisation of immunofluorescence of ovine placental lactogen. Cell and TissueResearch, 184, 427-433. Perry, J. S., Heap, R. B. & Ackland, N. (1975) The ultrastructure of the sheep placenta around the time of parturition. Journal of Anatomy, 120, 561-570. Reddy, S. & Watkins, W. B. (1978) Immunofluorescence localisation of ovine placental lactogen. Journal of Reproduction and Fertility, 52, 173-174. Ryter, A. & Kellenberger, E. (1958) l~tude au microscope ~lectronique de plasmas contenant de l'acide d6soxyribonucldique, Zeitschriftfiir Naturforschung, 13, 597. Cited by Glauert, A. M. (1958) In PracticalMethods in Electron Microscopy(Ed.) Glauert, A. M. Volume 3, pp. 95-96. Amsterdam, Oxford: North-Holland. Steven, D. H. (1973) Placental separation in the ewe.Journal of Physiology, 233, 10-12P. Steven, D. H. (1975) Separation of the placenta in the ewe: an ultrastructural study. Quarterly Journal of Experimental Physiology, 60, 37-44. Steven, D. H., Bass, F., Jansen, C. J. M., Kranc, E. J., Mallon, K., Samuel, C. A. & Nathanielsz, P. W. (1978) Ultrastructural changes in the placenta of the ewe after fetal pituitary stalk section. Quarterly Journal of Experimental Physiology, 63, 221-229. Wimsatt, W. A. (1951) Observations on the morphogenesis, cytochemistry and significance of the binucleate giant cells of the placenta of ruminants. AmericanJournal of Anatomy, 89, 233-282.