J. Anat. (1977), 123, 3, pp. 601-614 With 18 figures Printed in Great Britain
601
The developmental changes in the placenta of the guinea-pig W. H. TAM AND S. M. BURGESS
Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada
(Accepted 7 May 1976) INTRODUCTION
Although the histology (Davies, Dempsey & Amoroso, 1961 a, b) and histochemistry (Wislocki, Deane & Dempsey, 1946) of the guinea-pig placenta have been studied, little is known about the growth parameters of the various placental tissue components. It was the aim of this investigation to try to locate the site of progesterone synthesis in the guinea-pig placenta by determining the relative growth rates of its component tissues, especially the spongy and labyrinthinetissues, and correlating the results with reported changes in progesterone secretory activity during gestation. MATERIALS AND METHODS
Animals Normal adult guinea-pigs of the English short haired strain, weighing 600-900 g, were used. They were kept at 22-25 °C with 14 hours of light per day in winter, and in natural temperature and lighting conditions during the summer. Food and water were provided ad libitum. At least three oestrous cycles were followed through before any animal was used for experimental purposes. The oestrous cycle was approximately 16 days, and the mean gestation period was 68 days. For the calculation of the gestation period, the day the vaginal plug was observed was counted as day 0.
Surgical procedure Fetectomy and ovariectomywereperformed on day 34 of gestation, and the animals were killed 30 hours later. Anaesthesia was induced first with a subcutaneous injection of Nembutal (36 mg/kg body weight) and then with diethyl ether. When fetectomy was performed, small incisions were made ventrally in the body wall and antimesometrially in the uterine wall. One fetus was removed from each pregnant animal along with its visceral yolk sac and amnion after the allantoic blood vessels had been ligated to minimize blood loss. The placentae with intact fetuses were used for later observations. Ovariectomy was carried out via two dorso-lateral incisions. Histology Tissues were fixed in Bouin's fluid, embedded in paraffin, sectioned at 5 or 8 ,m and stained with haematoxylin and eosin. Measurements of cellular and nuclear 38
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diameters were made with an ocular micrometer. The area of an elliptic cell or nucleus was calculated from the formula Irab, where a and b are the minor and major diameters. The areas of the labyrinthine and spongy zones were estimated by projecting the sections with a microprojector at 24 x linear magnification, tracing the outline of the zones, and measuring the outlined areas with a compensating polar Fig. 2. The allantoic mesoderm first appeared on day 18 as thin sheets of lightly staining labyrinthine tissue (arrows) which included branches of the allantoic blood vessel filled with nucleated fetal red blood cells. The large maternal blood spaces (MBS), characteristic of the early placenta, can be seen with the SZ tissue interspersed between them, otherwise the SZ is morphologically similar to that found in later stages of gestation. Day 18 of gestation. x 80. Fig. 3. The parietal yolk sac endoderm of the placenta on the 18th day is almost entirely composed of ovoid cells with a scanty amount of basophilic cytoplasm. A few columnar cells have made their first appearance. Large maternal blood spaces filled with non-nucleated maternal red blood cells and a few white blood cells can be seen together with the much smaller blood sinusoids (arrows). The large maternal blood spaces soon disappear after day 18. x 269. Fig. 4. Spongy zone at 35 days of pregnancy. The cytotrophoblast (arrows) can be seen on the top and the ISZ at the centre of the section. The blood sinusoids of the SZ give the syncytiotrophoblast its characteristic spongy appearance. LZ tissue can be seen at the lower left corner. x22. Fig. 5. The MSZ (lower half of the picture) is peripheral to the subplacenta where large maternal blood vessels are usually found. The MSZ tissue may penetrate deeper into the placenta by forming the walls of the main branches of these maternal blood vessels. The MSZ is characterized by the large amount of cytoplasm associated with the relatively large nuclei. Compare with the ISZ in the upper half of the picture. The SZ has almost reached its maximal development at this stage of pregnancy. 35 days of gestation. x 22. Fig. 6. Labyrinthine syncytiotrophoblast at 35 days of gestation. The histology, and the amount of LZ tissue, are the same as those in later stages, but the LZ is still not well vascularized. The anastomosing cords of tissue are closely packed and the blood sinusoids are small. Compare with Fig. 7. x 51.
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Table 1. Nuclear and cellular dimensions in the placenta of the guinea-pig. Mean ± S.E. (number of observations). Number Nuclear area of the Pregnancy of interlobular spongy (days) animals zone (/4tm2) 14 17-18 20-21 25-26 30-31 35 40 49-51 61-62 64-69 *
Labyrinthine nuclear area
(/Im2)
Chorionic giant cells _ __ Nuclear area Cell area
(jim2)
(#m2)
3 93-1 ± 3 9 (18) Nil* Nil Nil 4 Nil 100-8±3-2 (24) 1738-1 ±286-4 (8) 419-4±30-5 (9) 5 114-4±9-2 (30) 350±7+20 5 (27) 63-9±3-6 (18) 1267-4±79-2 (27) 3 110-0±6-1 (18) 68-3±4-2 (18) 285-2±32-4 (18) 1195-5± 188-7 (16) 4 79 0± 3 6 (24) 66 4±2+9 (24) 448 3 ± 34-6 (25) 2933 3 ±466-6 (25) 2 82-2+±83 (12) 80-9±3-8 (12) 251-8±22-8 (11) 1075-1±144-9 (18) 3 76-1+4*1 (18) 63-4±4-6 (18) 357-5± 30-7 (14) 1775-4±127-8 (14) 5 71-5±2-8 (30) 74-4±2-9 (24) 572-7±92-9 (24) 96-4±9-7 (24) 2 87-2+7-0 (12) 717+2-7 (12) 1101 ±100 (9) 348-5±16-1 (9) 4 568±25 (24) 116-1 8-0 (15) 642±42 (24) 639-4±45-9 (18) Nil = no measurement was made because these placental zones were not yet developed.
planimeter. The areas of all major blood vessels were always excluded from the measurements. Two measurements from two different regions were always made of one placenta from each animal used. All results were expressed as mean ± S.E. Student's t test was used to determine significant differences, and confidence limits were set at 95 %. For thedetection of lipid inclusions, frozen sections were made at 14 ,um with a cryostat microtome. The lipids were stained with either oil red 0 or Sudan black B, and if the latter was used the section was counterstained with carmalum. RESULTS
Placental and fetal weight The weights of the placentae and fetuses are shown in Figure 1. The greatest increase in placental weight occurred between 30 and 53 days of pregnancy, and the phase of rapid increase in fetal weight was from 45 days to term. Though these two phases overlapped, they did not coincide.
Development of the spongy zone syncytiotrophoblast The spongy zone (SZ) comprises the cytotrophoblast, the interlobular sub-zone (ISZ) and the mesometrial sub-zone (MSZ). At day 14 the cytotrophoblast was represented by a single layer of relatively small nuclei (668 + 300 #m2, n = 18), associated with a scanty amount of basophilic cytoplasm, at the fetal pole of the placental disc. The ISZ was situated immediately below the cytotrophoblast, and was composed of approximately two rows of larger nuclei (Table 1) which, together with their cytoplasm, extended the entire sagittal section of the placenta. The MSZ constituted a loose network around the large maternal blood spaces in the maternal half of the placenta. At this stage the two sub-zones of the SZ and the cytotrophoblast were approximately equal in size. The SZ was vascularized by relatively few large maternal blood spaces (Fig. 2); but at day 18 it was well-vascularized with typical
605
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Table 2. Cross sectional area of spongy and labyrinthine zones in sagittal sections ofguinea-pig placenta. Mean ± S.E. (number of observations). Number Pregnancy of (days) animals
Spongy zone area ,
A
cm2 x 10-2
Percent*
Labyrinthine zone area
__,_ cm2 x 10-2
__
_
Percent*
4 25-26 10-36±0-90 (8) 6296±580 (8) 6-54±1-31 (8) 3704±580 (8) 30-31 4 20-42+0-13 (8) 1522+1-29 (8) 5735±302 (8) 42-66±3-02 (8) 35 3 17-88 + 2 55 (6) 45 52±4 40 (6) 20-98 +2-31 (6) 54-49±4A40 (6) 40 3 22-87+ 1-82 (6) 36-44± 1-87 (6) 39-64+2 50 (6) 63 56± 1-87 (6) 49-51 6 31-63±325 (12) 29-39±235 (12) 74-64±350(12) 70-66±2-34 (12) 61-64 3 20 00± 2 97 (6) 20 86± 3 06 (6) 76 94± 5 96 (6) 79-15 ± 3-06 (6) 3 66-69 14-76+ 1-88 (6) 14-53 + 1-59 (6) 87-52+ 8-01 (6) 85 47± 1- 59 (6) * The results are expressed as a percentage of the total area of the spongy and labyrinthine zones.
blood sinusoids (Figs. 3, 4). The SZ increased in size mainly by mitosis in the ISZ. Mitotic figures were observed occasionally from days 14-21 in the ISZ, but reached a peak of 16-24 per sagittal section between days 30 and 35. A few mitotic figures were also observed in the cytotrophoblast from 14-35 days. No mitotic activity was ever observed in the MSZ. Mitosis was not seen in the SZ after the 40th day. As a result of these differences in mitotic activity, the ISZ made up to 70 %, and the cytotrophoblast 20 %, of the SZ from day 20 to term. The MSZ formed only 10% of the SZ from 20-50 days. From then on, the MSZ decreased in size and, just before parturition, it was sometimes difficult to locate. The nuclei in the SZ differed in size in the different sub-zones. From day 14-26 the nuclei of the ISZ were significantly larger than those found during the remainder of the gestation period (Table 1). After day 30, perhaps as a result of intensive mitotic activity, nuclear size in the ISZ was significantly smaller, but remained constant until 62 days of gestation. This was followed by another significant decrease in size from day 64 to just before parturition. Between 14 and 25 days the nuclei of the cytotrophoblast (73 4 ± 22 ,Um2, n = 90) were smaller than those of the ISZ. However, as they did not show a corresponding decrease in size, most of them became indistinguishable from those of the ISZ from mid-pregnancy onwards. The nuclei of the MSZ were the largest in the SZ. Their average area was 165-8 ± 13 0 ,#m2 (n = 18) on day 14; this increasedto a significant peak of 271-9 ± 10-3 ,/m2 (n = 24) at mid-pregnancy, and then declined gradually to a significant low of 1367 ± 13-9 m2 (n =18) just before parturition. The amount of cytoplasm was scanty throughout the SZ on day 14, but was abundant in the ISZ and MSZ during the rest of the gestation period (Figs. 4, 5). The relative amounts of SZ and labyrinthine zone tissues were estimated by calculating the areas of the zones. It was on day 25 that the significantly smallest SZ area was recorded (Table 2). Concomitant with the wave of mitosis, the SZ significantly increased in size on day 30 and, except the peak during days 49-51, there was no other significant change in the size of the SZ up to day 64. After day 64 the SZ area decreased significantly to a low that almost approached that of day 25.
606
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Development of the labyrinthine zone syncytiotrophoblast The allantoic mesoderm first appeared in the placental disc on day 18 (Fig. 2). The isolated patches of labyrinthine tissue did not develop into recognizable lobules until day 30, and even then their blood supply was still relatively poor (Fig. 6). The first typical mature form of the labyrinthine zone (LZ) was only attained at day 40, when the syncytiotrophoblast was arranged into radiating, anastomosing cords and was well vascularized (Fig. 7). Mitotic figures were observed at a frequency of 2-5 per sagittal section between 20-25 days, and reached a maximum of 50-58 per section between 35-40 days (Fig. 8). Occasional mitotic figures could still be seen during the remainder of the gestation period. The growth of the LZ was rapid during 25-50 days of gestation (Table 2). After this time there was no further significant growth of the LZ. There was no consistent pattern of change in the size of the nuclei (Table 1) and, in contrast with the SZ nuclei, there was no significant decrease in the LZ nuclear size during the final phase of gestation.
Development of the chorionic giant cells The giant cells were arranged on day 17 as two small groups just peripheral to the subplacenta (Fig. 9). Occasional mitotic figures could be observed during this period. By the 35th day the layer attained its maximal development, reaching from the subplacenta to the point where the visceral yolk sac was attached to the placental disc. After day 35 necrotic changes began to appear. The nuclei in some cells became crenated and the cytoplasm began to resemble a loose network owing to the appearance of numerous large vacuoles (Fig. 10). These necrotic changes spread to more cells and reached a peak from day 50 to term. The cells at the periphery of the subplacenta were always the largest of the layer, and only the dimensions of these cells were measured and given in Table 1. Development of the parietal yolk sac endoderm The 14-18 day endodermal cells were small, ovoid and their longitudinal axis was tangential to the placental disc (Fig. 3). The transformation into mature endoderm took place during day 18-21 when the endodermal cells at the rim of the placental disc became columnar and acquired a much larger amount of cytoplasm. However, Fig. 7. The labyrinthine syncytiotrophoblast on the 49th day has numerous large, straight blood sinusoids interspersed between the tissue cords. This arrangement is typical of the LZ from 40 days of pregnancy to term. x 51. Fig. 8. Mitotic figures observed in the LZ at day 35 of gestation. x 660. Fig. 9. Day 21 of gestation. Although the chorionic giant cells (GC) have begun to spread to other regions of the placenta, most of them are still aggregated around the periphery of the subplacenta. The MSZ can be seen at the top of the picture. x 166. Fig. 10. Chorionic giant cells on the 35th day. A double row of these large cells is found peripheral to the subplacenta and underneath the MSZ. These cells have reached their maximal development at this stage of gestation, and large vacuoles have already begun to appear. x 48. Fig. 11. Parietal yolk sac endoderm on the 35th day. The columnar endodermal cells are thrown into alternating thicker and thinner regions. A layer of peripheral giant cells is sandwiched between the endodermal cells at the top and the SZ syncytiotrophoblast at the bottom. x 78.
608
W. H. TAM AND S. M. BURGESS
Table 3. Nuclear and cellular dimensions in the yolk sac endoderm of guinea-pigs. Mean ± S.E. (number of observations). Parietal endoderm Number
Pregnancy of (days) animals 14 17-18 20-21 25-26 30-31 35 40 49-51 61-62 64-69
2 4 5 3 4 2 3 5 2 3
Visceral endoderm
A___A
Nuclear area
Cell height
Nuclear area
Cell height
(Am2)
(#tm2) 14-3±0-8 (6) 13-5 ±07 (21) 14-0±0-7 (30) 17-5±1-2 (18) 47 9± 3-7 (22)
(Gm2)
(Gm2)
78-8±7-6 (12) 77-6± 3-2 (24) 58-1±2-3 (30) 65-4±3-0 (18) 63-6± 3-7 (12) * 51-5±2-3 (6) 63-4±2-4 (24)
189±1-0 (12) 21 -4±07 (24) 20 5±0 8 (30) 24-0±0-8 (18) 25-1±0-4 (12)
89-6± 55 (6) 68-5±4-4 (24) 75 0±2-9 (30) 75-6±3-9 (18) 88-0±4-6 (22) 82 5 ± 55 (12) 71-9±2-8 (19) 889±+3-5 (30) 66-9±4-4(12) 66-3 ± 3-4 (18) *
34-8 ± 1-4 (12) 35 8 ±1-5 (19) 30-3± 1-4 (30) 31-1+1 0(12) 27-8 ±2-0 (18) 38&5±1-5 (6) Results not available.
27-3 ±1-7 (6) 27-9±0-9 (24) 31-3 ± 3-2 (6)
Table 4. The occurrence of lipids in the placenta of the guinea-pig. The scale is from negative (-) through slightly (+) to densely lipoidal (+ + + ++). Spongy zone
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Giant cells
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+++ ++++
Fig. 12. The endodermal cells at the sinus terminalis form a monolayer, and their nuclei are situated at the lower half of the cell. Some large vacuoles can be seen, but vacuolation is not extensive at this stage. 25 days of pregnancy. x 269. Fig. 13. Small lipid droplets (arrows) are found after the 17th day of gestation in the entire ISZ. Oil red 0. 50 days of pregnancy. x 709. Fig. 14. The lipid droplets (arrows) in the LZ are larger than those in the SZ. Oil red 0. 51 days of pregnancy. x 496. Fig. 15. At S0days of gestation the giant cells are filled with large lipid droplets. Oil red 0. x 620. Fig. 16. Lipids in parietal yolk sac endodermal cells at 51 days of gestation. Oil red 0. x 300. Fig. 17. Large lipid globules found in the visceral yolk sac endoderm at 50 days of pregnancy. Oil red 0. x 278. Fig. 18. Degenerating LZ 30 hours after fetectomy at 34 days of pregnancy. x 186.
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611 the endodermal cells in all other areas of the parietal yolk sac remained ovoid and small throughout gestation. Occasional mitotic figures were observed throughout the entire endoderm between 18-35 days of gestation. The cell heights and nuclear areas of the columnar cells are given in Table 3. Possibly as a result of endodermal hyperplasia and hypertrophy, and the slow growth rate of the placenta during most of this period (Fig. 1), the columnar cells were thrown into a series of thickenings during day 35-40 (Fig. 11). This peculiar arrangement was soon lost, and after day 50 numerous large vacuoles gradually developed around the nuclei. Development of the visceral yolk sac endoderm The columnar endodermal cells around the sinus terminalis (Fig. 12) were the only cells in this membrane that showed changes as gestation progressed. Though cell height increased throughout gestation, cytoplasm was lost as a result of extensive vacuolation, and nuclear size decreased significantly during the last few days of gestation (Table 3).
Lipids Some placentae were stained for lipids (Table 4). After the 17th day both the cytotrophoblast and ISZ cytoplasm contained lipid deposited in the form of extremely fine droplets evenly distributed in the cytoplasm (Fig. 13). The lipid droplets in the cytoplasm of the LZ were larger than those of the SZ (Fig. 14). No lipid was located in the giant cells in 17th and 25th day placentae, but in the specimens at 50-51 days the giant cells resembled 'bags' of large lipid globules (Fig. 15). The lipid deposits in the columnar parietal and visceral endodermal cells corresponded to the vacuoles of the wax sections (Figs. 16, 17). The other cells in the parietal and visceral endoderm were always slightly lipoidal and showed no change in fat content throughout pregnancy. Fetectomy and ovariectomy The results of fetectomy and ovariectomy are tabulated in Table 5. Although nuclear size in the ISZ of intact and fetectomized placentae decreased as a result of operational shock (groups 2a, b), nuclear size upon ovariectomy (groups 3 a, b) increased by about 14 % over the intact control. As the result of hypertrophy the amount of SZ tissue increased by 94-30 % over that of the intact control upon ovariectomy and fetectomy. Operational shock and fetectomy probably had no effect on the giant cells, but ovariectomy alone induced an initial increase (nucleus 22 %, cell 35 %), and ovariectomy together with fetectomy brought about a further increase in nuclear (60 %) and cell size (96 %). Fetectomy resulted in degenerative changes in the LZ (Fig. 18). Observations not shown included placental weight, nuclear size and cell height of the parietal endodermal cells. There was a placental weight increase (13 %) in the ovariectomized and fetectomized animals, but this was not statistically significant when compared with the control. The nuclear size and cell height of the parietal endodermal cells were always significantly reduced as a result of fetectomy. Mitotic figures in all regions were also counted and they fell within the range given above for the intact animals.
612
W. H. TAM AND S. M. BURGESS DISCUSSION
Of the two major zones in the placental disc the SZ, because of its mode of development, the histological changes within it, the occurrence of lipids and the effects of ovariectomy upon it, is a more likely candidate for progesterone production than the LZ. The SZ was well established by day 18, when it was supplied with blood sinusoids similar to those found in later stages. From 25-30 days this zone increased in size and after that maintained its size almost until parturition, when even the ISZ nuclei dwindled in size (Tables 1, 2). It has been reported that the placenta of the guinea-pig secretes progesterone as early as 15-16 days of gestation (Illingworth & Deanesly, 1972; Illingworth & Challis, 1973), and that placental progesterone alone is sufficient to maintain gestation from days 18-21 onwards (Deanesly, 1963). At this early stage the SZ is the only tissue in the placenta that is sufficiently large and well established to fulfil this role. If this endocrine activity of the placenta is to be maintained up to term, as suggested by Heap & Deanesly (1966) and Illingworth & Challis (1973), the SZ must be in an active phase and of sufficient size to perform this function. The occurrence of fine lipid droplets in the SZ is like that of other steroid-producing cells (Lofts, Phillips & Tam, 1966, 1971). It is possible that the enlargement of the SZ and its nuclei (Table 5) in ovariectomized and fetectomized animals at 35 days of gestation was a compensatory hypertrophy after the still significant ovarian source of progesterone (Illingworth, Perry, Ackland & Burton, 1973) had been removed. The slow development of the LZ makes it an unlikely candidate for progesterone secretion. The large lipid droplets are also uncharacteristic of steroid-secreting cells. The nutritional (Enders, 1965) and absorptive (Hard, 1946) roles of the LZ seem to be reflected in the parallelism between the development of the fetus (Fig. 1) and the LZ (Table 2) during days 30-50, and not between fetal and gross placental weights. The degeneration of the LZ on fetectomy is to be expected, as almost all its blood supply is fetal (Kaufmann, 1969). Resembling the early development of the SZ, the chorionic giant cell layer was well established by the 25th day and it reached maximal development by about day 35. However, the complete absence of lipid at day 25 (Table 4), when the giant cells were most active by histological criteria, is not comparable with other steroid-producing cells (Deane, 1958; Lofts et al. 1966, 1971; Tam, Phillips & Lofts, 1969, 1972). The dramatic decrease in cell and nuclear sizes, the necrosis observed after day 40 (Table 1) and the heavy fat deposits at days 50 and 51 do not suggest active steroid production (Lofts et al. 1966, 1971; Tam et al. 1969, 1972). This early inactivity also does not support the possibility that the giant cells are responsible for progesterone synthesis as it has been reported that the guinea-pig placenta secretes high levels of progesterone almost until parturition (Heap & Deanesly, 1966; Illingworth & Challis, 1973). The attainment of maximal development of the giant cells (Table 1) at a time when the SZ is still undergoing development in intact animals (Table 2), and the synchronous hypertrophy of giant cells and SZ in ovariectomized and fetectomized animals (Table 5), do seem to indicate that the growth of the SZ depends on the activity of the giant cells, In this light, it is possible that the chorionic giant cells secrete trophic factor(s) which stimulate the SZ, as suggested by Davies et al. (1961 a). Our observations indicate that the development of the parietal and visceral yolk
Guinea-pig placenta
613 sac endoderm cannot be correlated with the reported endocrine activity of the placenta. Their development is either too late, or their lipid deposits are too extensive, for them to be regarded as active steroid-producing cells (Table 4 & Figs. 16, 17). SUMMARY
The placental disc and the yolk sac endoderm of intact guinea-pigs from day 14 of gestation to term, and of ovariectomized and fetectomized animals at 35 days of pregnancy, were observed by histological means. The chorionic giant cells were the first to attain maximal development (between days 17-35) and they had begun to degenerate by day 40. The spongy zone syncytiotrophoblast was well established and vascularized by day 18, and the maximal size of the zone was maintained between days 30 and 64. The spongy zone then dwindled in size just before parturition. Labyrinthine tissue appeared on day 18, but rapid growth and complete vascularization was not attained until as late as day 40. However, its continued increase in size almost up to term closely paralleled the growth of the fetus. If the placenta secretes progesterone from day 18 to parturition, the spongy zone is the only tissue sufficiently well established to carry out this activity throughout this period. The compensatory hypertrophy of the spongy zone syncytiotrophoblast in 35 day pregnant animals after ovariectomy and fetectomy supports the hypothesis that the spongy tissue secretes progesterone.
This work was supported by a grant (No. A6792) awarded to W. H. Tam by the National Research Council of Canada. REFERENCES
DAVIES, J., DEMPSEY, E. W. & AMoROSO, E. C. (1961 a). The subplacenta of the guinea-pig: development, histology and histochemistry. Journal of Anatomy 95, 457-473. DAVIES, J., DEMPSEY, E. W. & AMOROSO, E. C. (1961 b). The subplacenta of the guinea-pig: an electron microscopic study. Journal of Anatomy 95, 311-324. DEANE, H. W. (1958). Intracellular lipids: their detection and significance. In Frontiers in Cytology (ed. S. L. Palay), pp. 227-263. New Haven: Yale University Press. DEANESLY, R. (1963). Early embryonic growth and progestagen function in ovariectomized guinea-pigs. Journal of Reproduction and Fertility 6, 143-152. ENDERS, A. C. (1965). A comparative study of the fine structure of the trophoblast in several hemochorial placentas. American Journal of Anatomy 11, 629-68. HARD, W. L. (1946). A histochemical and quantitative study of phosphatase in the placenta and foetal membranes of the guinea pig. American Journal of Anatomy 78, 47-77. HEAP, R. B. & DEANESLY, R. (1966). Progesterone in systemic blood and placentae of intact and ovariectomized guinea-pig. Journal ofEndocrinology 34, 417-423. ILLINGWORTH, D. V. & CHALLIS, J. R. G. (1973). Concentrations of oestrogens and progesterone in the plasma of ovariectomized and ovariectomized norgestrel-treated pregnant guinea-pigs. Journal of Reproduction and Fertility 34, 289-296. ILLINGWORTH, D. V. & DEANESLY, R. (1972). Maintenance of pregnancy by synthetic progesterone in guinea-pigs ovariectomized before implantation; progesterone-binding protein and placental progesterone secretion. Journal of Endocrinology 54, 435 444. ILLINGWORTH, D. V., PERRY, J. S., ACKLAND, N. & BURTON, A. M. (1973). The maintenance of pregnancy, and parturition, in guinea-pigs hypophysectomized within 4 days of mating. Journal of Endocrinology 59, 163-169. KAUFMANN, P. (1969). Die Meerschweinchenplacenta und ihre Entwicklung. Zeitschrift fur Anatomie und Entwicklungsgeschichte 129, 83-101.
W. H. TAM AND S. M. BURGESS 614 LoFTs, B., PHILLIPS, J. G. & TAM, W. H. (1966). Seasonal changes in the testis of the cobra, Naja naja (Linn.). General and Comparative Endocrinology 6, 466-475. LOFTS, B., PHILLIPS, J. G. & TAM, W. H. (1971). Seasonal changes in the histology of the adrenal gland of the cobra, Naja naja. General and Comparative Endocrinology 16, 121-131. TAM, W. H., PHILLIPS, J. G. & LoFrs, B. (1969). Seasonal changes in the in vitro production of testicular androgens by the cobra (Naja naja Linn.). General and Comparative Endocrinology 13, 117-125. TAM, W. H., PHILLIPS, J. G. & LoiTs, B. (1972). Seasonal changes in the secretory activity of the adrenal gland of the cobra (Naja naja Linn.). General and Comparative Endocrinology 19, 218-224. WISLOCKI, G. B., DEANE, W. H. & DEMPSEY, E. W. (1946). The histochemistry of the rodent's placenta. American Journal of Anatomy 78, 281-346.
601
The developmental changes in the placenta of the guinea-pig W. H. TAM AND S. M. BURGESS
Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada
(Accepted 7 May 1976) INTRODUCTION
Although the histology (Davies, Dempsey & Amoroso, 1961 a, b) and histochemistry (Wislocki, Deane & Dempsey, 1946) of the guinea-pig placenta have been studied, little is known about the growth parameters of the various placental tissue components. It was the aim of this investigation to try to locate the site of progesterone synthesis in the guinea-pig placenta by determining the relative growth rates of its component tissues, especially the spongy and labyrinthinetissues, and correlating the results with reported changes in progesterone secretory activity during gestation. MATERIALS AND METHODS
Animals Normal adult guinea-pigs of the English short haired strain, weighing 600-900 g, were used. They were kept at 22-25 °C with 14 hours of light per day in winter, and in natural temperature and lighting conditions during the summer. Food and water were provided ad libitum. At least three oestrous cycles were followed through before any animal was used for experimental purposes. The oestrous cycle was approximately 16 days, and the mean gestation period was 68 days. For the calculation of the gestation period, the day the vaginal plug was observed was counted as day 0.
Surgical procedure Fetectomy and ovariectomywereperformed on day 34 of gestation, and the animals were killed 30 hours later. Anaesthesia was induced first with a subcutaneous injection of Nembutal (36 mg/kg body weight) and then with diethyl ether. When fetectomy was performed, small incisions were made ventrally in the body wall and antimesometrially in the uterine wall. One fetus was removed from each pregnant animal along with its visceral yolk sac and amnion after the allantoic blood vessels had been ligated to minimize blood loss. The placentae with intact fetuses were used for later observations. Ovariectomy was carried out via two dorso-lateral incisions. Histology Tissues were fixed in Bouin's fluid, embedded in paraffin, sectioned at 5 or 8 ,m and stained with haematoxylin and eosin. Measurements of cellular and nuclear 38
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diameters were made with an ocular micrometer. The area of an elliptic cell or nucleus was calculated from the formula Irab, where a and b are the minor and major diameters. The areas of the labyrinthine and spongy zones were estimated by projecting the sections with a microprojector at 24 x linear magnification, tracing the outline of the zones, and measuring the outlined areas with a compensating polar Fig. 2. The allantoic mesoderm first appeared on day 18 as thin sheets of lightly staining labyrinthine tissue (arrows) which included branches of the allantoic blood vessel filled with nucleated fetal red blood cells. The large maternal blood spaces (MBS), characteristic of the early placenta, can be seen with the SZ tissue interspersed between them, otherwise the SZ is morphologically similar to that found in later stages of gestation. Day 18 of gestation. x 80. Fig. 3. The parietal yolk sac endoderm of the placenta on the 18th day is almost entirely composed of ovoid cells with a scanty amount of basophilic cytoplasm. A few columnar cells have made their first appearance. Large maternal blood spaces filled with non-nucleated maternal red blood cells and a few white blood cells can be seen together with the much smaller blood sinusoids (arrows). The large maternal blood spaces soon disappear after day 18. x 269. Fig. 4. Spongy zone at 35 days of pregnancy. The cytotrophoblast (arrows) can be seen on the top and the ISZ at the centre of the section. The blood sinusoids of the SZ give the syncytiotrophoblast its characteristic spongy appearance. LZ tissue can be seen at the lower left corner. x22. Fig. 5. The MSZ (lower half of the picture) is peripheral to the subplacenta where large maternal blood vessels are usually found. The MSZ tissue may penetrate deeper into the placenta by forming the walls of the main branches of these maternal blood vessels. The MSZ is characterized by the large amount of cytoplasm associated with the relatively large nuclei. Compare with the ISZ in the upper half of the picture. The SZ has almost reached its maximal development at this stage of pregnancy. 35 days of gestation. x 22. Fig. 6. Labyrinthine syncytiotrophoblast at 35 days of gestation. The histology, and the amount of LZ tissue, are the same as those in later stages, but the LZ is still not well vascularized. The anastomosing cords of tissue are closely packed and the blood sinusoids are small. Compare with Fig. 7. x 51.
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Table 1. Nuclear and cellular dimensions in the placenta of the guinea-pig. Mean ± S.E. (number of observations). Number Nuclear area of the Pregnancy of interlobular spongy (days) animals zone (/4tm2) 14 17-18 20-21 25-26 30-31 35 40 49-51 61-62 64-69 *
Labyrinthine nuclear area
(/Im2)
Chorionic giant cells _ __ Nuclear area Cell area
(jim2)
(#m2)
3 93-1 ± 3 9 (18) Nil* Nil Nil 4 Nil 100-8±3-2 (24) 1738-1 ±286-4 (8) 419-4±30-5 (9) 5 114-4±9-2 (30) 350±7+20 5 (27) 63-9±3-6 (18) 1267-4±79-2 (27) 3 110-0±6-1 (18) 68-3±4-2 (18) 285-2±32-4 (18) 1195-5± 188-7 (16) 4 79 0± 3 6 (24) 66 4±2+9 (24) 448 3 ± 34-6 (25) 2933 3 ±466-6 (25) 2 82-2+±83 (12) 80-9±3-8 (12) 251-8±22-8 (11) 1075-1±144-9 (18) 3 76-1+4*1 (18) 63-4±4-6 (18) 357-5± 30-7 (14) 1775-4±127-8 (14) 5 71-5±2-8 (30) 74-4±2-9 (24) 572-7±92-9 (24) 96-4±9-7 (24) 2 87-2+7-0 (12) 717+2-7 (12) 1101 ±100 (9) 348-5±16-1 (9) 4 568±25 (24) 116-1 8-0 (15) 642±42 (24) 639-4±45-9 (18) Nil = no measurement was made because these placental zones were not yet developed.
planimeter. The areas of all major blood vessels were always excluded from the measurements. Two measurements from two different regions were always made of one placenta from each animal used. All results were expressed as mean ± S.E. Student's t test was used to determine significant differences, and confidence limits were set at 95 %. For thedetection of lipid inclusions, frozen sections were made at 14 ,um with a cryostat microtome. The lipids were stained with either oil red 0 or Sudan black B, and if the latter was used the section was counterstained with carmalum. RESULTS
Placental and fetal weight The weights of the placentae and fetuses are shown in Figure 1. The greatest increase in placental weight occurred between 30 and 53 days of pregnancy, and the phase of rapid increase in fetal weight was from 45 days to term. Though these two phases overlapped, they did not coincide.
Development of the spongy zone syncytiotrophoblast The spongy zone (SZ) comprises the cytotrophoblast, the interlobular sub-zone (ISZ) and the mesometrial sub-zone (MSZ). At day 14 the cytotrophoblast was represented by a single layer of relatively small nuclei (668 + 300 #m2, n = 18), associated with a scanty amount of basophilic cytoplasm, at the fetal pole of the placental disc. The ISZ was situated immediately below the cytotrophoblast, and was composed of approximately two rows of larger nuclei (Table 1) which, together with their cytoplasm, extended the entire sagittal section of the placenta. The MSZ constituted a loose network around the large maternal blood spaces in the maternal half of the placenta. At this stage the two sub-zones of the SZ and the cytotrophoblast were approximately equal in size. The SZ was vascularized by relatively few large maternal blood spaces (Fig. 2); but at day 18 it was well-vascularized with typical
605
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Table 2. Cross sectional area of spongy and labyrinthine zones in sagittal sections ofguinea-pig placenta. Mean ± S.E. (number of observations). Number Pregnancy of (days) animals
Spongy zone area ,
A
cm2 x 10-2
Percent*
Labyrinthine zone area
__,_ cm2 x 10-2
__
_
Percent*
4 25-26 10-36±0-90 (8) 6296±580 (8) 6-54±1-31 (8) 3704±580 (8) 30-31 4 20-42+0-13 (8) 1522+1-29 (8) 5735±302 (8) 42-66±3-02 (8) 35 3 17-88 + 2 55 (6) 45 52±4 40 (6) 20-98 +2-31 (6) 54-49±4A40 (6) 40 3 22-87+ 1-82 (6) 36-44± 1-87 (6) 39-64+2 50 (6) 63 56± 1-87 (6) 49-51 6 31-63±325 (12) 29-39±235 (12) 74-64±350(12) 70-66±2-34 (12) 61-64 3 20 00± 2 97 (6) 20 86± 3 06 (6) 76 94± 5 96 (6) 79-15 ± 3-06 (6) 3 66-69 14-76+ 1-88 (6) 14-53 + 1-59 (6) 87-52+ 8-01 (6) 85 47± 1- 59 (6) * The results are expressed as a percentage of the total area of the spongy and labyrinthine zones.
blood sinusoids (Figs. 3, 4). The SZ increased in size mainly by mitosis in the ISZ. Mitotic figures were observed occasionally from days 14-21 in the ISZ, but reached a peak of 16-24 per sagittal section between days 30 and 35. A few mitotic figures were also observed in the cytotrophoblast from 14-35 days. No mitotic activity was ever observed in the MSZ. Mitosis was not seen in the SZ after the 40th day. As a result of these differences in mitotic activity, the ISZ made up to 70 %, and the cytotrophoblast 20 %, of the SZ from day 20 to term. The MSZ formed only 10% of the SZ from 20-50 days. From then on, the MSZ decreased in size and, just before parturition, it was sometimes difficult to locate. The nuclei in the SZ differed in size in the different sub-zones. From day 14-26 the nuclei of the ISZ were significantly larger than those found during the remainder of the gestation period (Table 1). After day 30, perhaps as a result of intensive mitotic activity, nuclear size in the ISZ was significantly smaller, but remained constant until 62 days of gestation. This was followed by another significant decrease in size from day 64 to just before parturition. Between 14 and 25 days the nuclei of the cytotrophoblast (73 4 ± 22 ,Um2, n = 90) were smaller than those of the ISZ. However, as they did not show a corresponding decrease in size, most of them became indistinguishable from those of the ISZ from mid-pregnancy onwards. The nuclei of the MSZ were the largest in the SZ. Their average area was 165-8 ± 13 0 ,#m2 (n = 18) on day 14; this increasedto a significant peak of 271-9 ± 10-3 ,/m2 (n = 24) at mid-pregnancy, and then declined gradually to a significant low of 1367 ± 13-9 m2 (n =18) just before parturition. The amount of cytoplasm was scanty throughout the SZ on day 14, but was abundant in the ISZ and MSZ during the rest of the gestation period (Figs. 4, 5). The relative amounts of SZ and labyrinthine zone tissues were estimated by calculating the areas of the zones. It was on day 25 that the significantly smallest SZ area was recorded (Table 2). Concomitant with the wave of mitosis, the SZ significantly increased in size on day 30 and, except the peak during days 49-51, there was no other significant change in the size of the SZ up to day 64. After day 64 the SZ area decreased significantly to a low that almost approached that of day 25.
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Development of the labyrinthine zone syncytiotrophoblast The allantoic mesoderm first appeared in the placental disc on day 18 (Fig. 2). The isolated patches of labyrinthine tissue did not develop into recognizable lobules until day 30, and even then their blood supply was still relatively poor (Fig. 6). The first typical mature form of the labyrinthine zone (LZ) was only attained at day 40, when the syncytiotrophoblast was arranged into radiating, anastomosing cords and was well vascularized (Fig. 7). Mitotic figures were observed at a frequency of 2-5 per sagittal section between 20-25 days, and reached a maximum of 50-58 per section between 35-40 days (Fig. 8). Occasional mitotic figures could still be seen during the remainder of the gestation period. The growth of the LZ was rapid during 25-50 days of gestation (Table 2). After this time there was no further significant growth of the LZ. There was no consistent pattern of change in the size of the nuclei (Table 1) and, in contrast with the SZ nuclei, there was no significant decrease in the LZ nuclear size during the final phase of gestation.
Development of the chorionic giant cells The giant cells were arranged on day 17 as two small groups just peripheral to the subplacenta (Fig. 9). Occasional mitotic figures could be observed during this period. By the 35th day the layer attained its maximal development, reaching from the subplacenta to the point where the visceral yolk sac was attached to the placental disc. After day 35 necrotic changes began to appear. The nuclei in some cells became crenated and the cytoplasm began to resemble a loose network owing to the appearance of numerous large vacuoles (Fig. 10). These necrotic changes spread to more cells and reached a peak from day 50 to term. The cells at the periphery of the subplacenta were always the largest of the layer, and only the dimensions of these cells were measured and given in Table 1. Development of the parietal yolk sac endoderm The 14-18 day endodermal cells were small, ovoid and their longitudinal axis was tangential to the placental disc (Fig. 3). The transformation into mature endoderm took place during day 18-21 when the endodermal cells at the rim of the placental disc became columnar and acquired a much larger amount of cytoplasm. However, Fig. 7. The labyrinthine syncytiotrophoblast on the 49th day has numerous large, straight blood sinusoids interspersed between the tissue cords. This arrangement is typical of the LZ from 40 days of pregnancy to term. x 51. Fig. 8. Mitotic figures observed in the LZ at day 35 of gestation. x 660. Fig. 9. Day 21 of gestation. Although the chorionic giant cells (GC) have begun to spread to other regions of the placenta, most of them are still aggregated around the periphery of the subplacenta. The MSZ can be seen at the top of the picture. x 166. Fig. 10. Chorionic giant cells on the 35th day. A double row of these large cells is found peripheral to the subplacenta and underneath the MSZ. These cells have reached their maximal development at this stage of gestation, and large vacuoles have already begun to appear. x 48. Fig. 11. Parietal yolk sac endoderm on the 35th day. The columnar endodermal cells are thrown into alternating thicker and thinner regions. A layer of peripheral giant cells is sandwiched between the endodermal cells at the top and the SZ syncytiotrophoblast at the bottom. x 78.
608
W. H. TAM AND S. M. BURGESS
Table 3. Nuclear and cellular dimensions in the yolk sac endoderm of guinea-pigs. Mean ± S.E. (number of observations). Parietal endoderm Number
Pregnancy of (days) animals 14 17-18 20-21 25-26 30-31 35 40 49-51 61-62 64-69
2 4 5 3 4 2 3 5 2 3
Visceral endoderm
A___A
Nuclear area
Cell height
Nuclear area
Cell height
(Am2)
(#tm2) 14-3±0-8 (6) 13-5 ±07 (21) 14-0±0-7 (30) 17-5±1-2 (18) 47 9± 3-7 (22)
(Gm2)
(Gm2)
78-8±7-6 (12) 77-6± 3-2 (24) 58-1±2-3 (30) 65-4±3-0 (18) 63-6± 3-7 (12) * 51-5±2-3 (6) 63-4±2-4 (24)
189±1-0 (12) 21 -4±07 (24) 20 5±0 8 (30) 24-0±0-8 (18) 25-1±0-4 (12)
89-6± 55 (6) 68-5±4-4 (24) 75 0±2-9 (30) 75-6±3-9 (18) 88-0±4-6 (22) 82 5 ± 55 (12) 71-9±2-8 (19) 889±+3-5 (30) 66-9±4-4(12) 66-3 ± 3-4 (18) *
34-8 ± 1-4 (12) 35 8 ±1-5 (19) 30-3± 1-4 (30) 31-1+1 0(12) 27-8 ±2-0 (18) 38&5±1-5 (6) Results not available.
27-3 ±1-7 (6) 27-9±0-9 (24) 31-3 ± 3-2 (6)
Table 4. The occurrence of lipids in the placenta of the guinea-pig. The scale is from negative (-) through slightly (+) to densely lipoidal (+ + + ++). Spongy zone
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Fig. 12. The endodermal cells at the sinus terminalis form a monolayer, and their nuclei are situated at the lower half of the cell. Some large vacuoles can be seen, but vacuolation is not extensive at this stage. 25 days of pregnancy. x 269. Fig. 13. Small lipid droplets (arrows) are found after the 17th day of gestation in the entire ISZ. Oil red 0. 50 days of pregnancy. x 709. Fig. 14. The lipid droplets (arrows) in the LZ are larger than those in the SZ. Oil red 0. 51 days of pregnancy. x 496. Fig. 15. At S0days of gestation the giant cells are filled with large lipid droplets. Oil red 0. x 620. Fig. 16. Lipids in parietal yolk sac endodermal cells at 51 days of gestation. Oil red 0. x 300. Fig. 17. Large lipid globules found in the visceral yolk sac endoderm at 50 days of pregnancy. Oil red 0. x 278. Fig. 18. Degenerating LZ 30 hours after fetectomy at 34 days of pregnancy. x 186.
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611 the endodermal cells in all other areas of the parietal yolk sac remained ovoid and small throughout gestation. Occasional mitotic figures were observed throughout the entire endoderm between 18-35 days of gestation. The cell heights and nuclear areas of the columnar cells are given in Table 3. Possibly as a result of endodermal hyperplasia and hypertrophy, and the slow growth rate of the placenta during most of this period (Fig. 1), the columnar cells were thrown into a series of thickenings during day 35-40 (Fig. 11). This peculiar arrangement was soon lost, and after day 50 numerous large vacuoles gradually developed around the nuclei. Development of the visceral yolk sac endoderm The columnar endodermal cells around the sinus terminalis (Fig. 12) were the only cells in this membrane that showed changes as gestation progressed. Though cell height increased throughout gestation, cytoplasm was lost as a result of extensive vacuolation, and nuclear size decreased significantly during the last few days of gestation (Table 3).
Lipids Some placentae were stained for lipids (Table 4). After the 17th day both the cytotrophoblast and ISZ cytoplasm contained lipid deposited in the form of extremely fine droplets evenly distributed in the cytoplasm (Fig. 13). The lipid droplets in the cytoplasm of the LZ were larger than those of the SZ (Fig. 14). No lipid was located in the giant cells in 17th and 25th day placentae, but in the specimens at 50-51 days the giant cells resembled 'bags' of large lipid globules (Fig. 15). The lipid deposits in the columnar parietal and visceral endodermal cells corresponded to the vacuoles of the wax sections (Figs. 16, 17). The other cells in the parietal and visceral endoderm were always slightly lipoidal and showed no change in fat content throughout pregnancy. Fetectomy and ovariectomy The results of fetectomy and ovariectomy are tabulated in Table 5. Although nuclear size in the ISZ of intact and fetectomized placentae decreased as a result of operational shock (groups 2a, b), nuclear size upon ovariectomy (groups 3 a, b) increased by about 14 % over the intact control. As the result of hypertrophy the amount of SZ tissue increased by 94-30 % over that of the intact control upon ovariectomy and fetectomy. Operational shock and fetectomy probably had no effect on the giant cells, but ovariectomy alone induced an initial increase (nucleus 22 %, cell 35 %), and ovariectomy together with fetectomy brought about a further increase in nuclear (60 %) and cell size (96 %). Fetectomy resulted in degenerative changes in the LZ (Fig. 18). Observations not shown included placental weight, nuclear size and cell height of the parietal endodermal cells. There was a placental weight increase (13 %) in the ovariectomized and fetectomized animals, but this was not statistically significant when compared with the control. The nuclear size and cell height of the parietal endodermal cells were always significantly reduced as a result of fetectomy. Mitotic figures in all regions were also counted and they fell within the range given above for the intact animals.
612
W. H. TAM AND S. M. BURGESS DISCUSSION
Of the two major zones in the placental disc the SZ, because of its mode of development, the histological changes within it, the occurrence of lipids and the effects of ovariectomy upon it, is a more likely candidate for progesterone production than the LZ. The SZ was well established by day 18, when it was supplied with blood sinusoids similar to those found in later stages. From 25-30 days this zone increased in size and after that maintained its size almost until parturition, when even the ISZ nuclei dwindled in size (Tables 1, 2). It has been reported that the placenta of the guinea-pig secretes progesterone as early as 15-16 days of gestation (Illingworth & Deanesly, 1972; Illingworth & Challis, 1973), and that placental progesterone alone is sufficient to maintain gestation from days 18-21 onwards (Deanesly, 1963). At this early stage the SZ is the only tissue in the placenta that is sufficiently large and well established to fulfil this role. If this endocrine activity of the placenta is to be maintained up to term, as suggested by Heap & Deanesly (1966) and Illingworth & Challis (1973), the SZ must be in an active phase and of sufficient size to perform this function. The occurrence of fine lipid droplets in the SZ is like that of other steroid-producing cells (Lofts, Phillips & Tam, 1966, 1971). It is possible that the enlargement of the SZ and its nuclei (Table 5) in ovariectomized and fetectomized animals at 35 days of gestation was a compensatory hypertrophy after the still significant ovarian source of progesterone (Illingworth, Perry, Ackland & Burton, 1973) had been removed. The slow development of the LZ makes it an unlikely candidate for progesterone secretion. The large lipid droplets are also uncharacteristic of steroid-secreting cells. The nutritional (Enders, 1965) and absorptive (Hard, 1946) roles of the LZ seem to be reflected in the parallelism between the development of the fetus (Fig. 1) and the LZ (Table 2) during days 30-50, and not between fetal and gross placental weights. The degeneration of the LZ on fetectomy is to be expected, as almost all its blood supply is fetal (Kaufmann, 1969). Resembling the early development of the SZ, the chorionic giant cell layer was well established by the 25th day and it reached maximal development by about day 35. However, the complete absence of lipid at day 25 (Table 4), when the giant cells were most active by histological criteria, is not comparable with other steroid-producing cells (Deane, 1958; Lofts et al. 1966, 1971; Tam, Phillips & Lofts, 1969, 1972). The dramatic decrease in cell and nuclear sizes, the necrosis observed after day 40 (Table 1) and the heavy fat deposits at days 50 and 51 do not suggest active steroid production (Lofts et al. 1966, 1971; Tam et al. 1969, 1972). This early inactivity also does not support the possibility that the giant cells are responsible for progesterone synthesis as it has been reported that the guinea-pig placenta secretes high levels of progesterone almost until parturition (Heap & Deanesly, 1966; Illingworth & Challis, 1973). The attainment of maximal development of the giant cells (Table 1) at a time when the SZ is still undergoing development in intact animals (Table 2), and the synchronous hypertrophy of giant cells and SZ in ovariectomized and fetectomized animals (Table 5), do seem to indicate that the growth of the SZ depends on the activity of the giant cells, In this light, it is possible that the chorionic giant cells secrete trophic factor(s) which stimulate the SZ, as suggested by Davies et al. (1961 a). Our observations indicate that the development of the parietal and visceral yolk
Guinea-pig placenta
613 sac endoderm cannot be correlated with the reported endocrine activity of the placenta. Their development is either too late, or their lipid deposits are too extensive, for them to be regarded as active steroid-producing cells (Table 4 & Figs. 16, 17). SUMMARY
The placental disc and the yolk sac endoderm of intact guinea-pigs from day 14 of gestation to term, and of ovariectomized and fetectomized animals at 35 days of pregnancy, were observed by histological means. The chorionic giant cells were the first to attain maximal development (between days 17-35) and they had begun to degenerate by day 40. The spongy zone syncytiotrophoblast was well established and vascularized by day 18, and the maximal size of the zone was maintained between days 30 and 64. The spongy zone then dwindled in size just before parturition. Labyrinthine tissue appeared on day 18, but rapid growth and complete vascularization was not attained until as late as day 40. However, its continued increase in size almost up to term closely paralleled the growth of the fetus. If the placenta secretes progesterone from day 18 to parturition, the spongy zone is the only tissue sufficiently well established to carry out this activity throughout this period. The compensatory hypertrophy of the spongy zone syncytiotrophoblast in 35 day pregnant animals after ovariectomy and fetectomy supports the hypothesis that the spongy tissue secretes progesterone.
This work was supported by a grant (No. A6792) awarded to W. H. Tam by the National Research Council of Canada. REFERENCES
DAVIES, J., DEMPSEY, E. W. & AMoROSO, E. C. (1961 a). The subplacenta of the guinea-pig: development, histology and histochemistry. Journal of Anatomy 95, 457-473. DAVIES, J., DEMPSEY, E. W. & AMOROSO, E. C. (1961 b). The subplacenta of the guinea-pig: an electron microscopic study. Journal of Anatomy 95, 311-324. DEANE, H. W. (1958). Intracellular lipids: their detection and significance. In Frontiers in Cytology (ed. S. L. Palay), pp. 227-263. New Haven: Yale University Press. DEANESLY, R. (1963). Early embryonic growth and progestagen function in ovariectomized guinea-pigs. Journal of Reproduction and Fertility 6, 143-152. ENDERS, A. C. (1965). A comparative study of the fine structure of the trophoblast in several hemochorial placentas. American Journal of Anatomy 11, 629-68. HARD, W. L. (1946). A histochemical and quantitative study of phosphatase in the placenta and foetal membranes of the guinea pig. American Journal of Anatomy 78, 47-77. HEAP, R. B. & DEANESLY, R. (1966). Progesterone in systemic blood and placentae of intact and ovariectomized guinea-pig. Journal ofEndocrinology 34, 417-423. ILLINGWORTH, D. V. & CHALLIS, J. R. G. (1973). Concentrations of oestrogens and progesterone in the plasma of ovariectomized and ovariectomized norgestrel-treated pregnant guinea-pigs. Journal of Reproduction and Fertility 34, 289-296. ILLINGWORTH, D. V. & DEANESLY, R. (1972). Maintenance of pregnancy by synthetic progesterone in guinea-pigs ovariectomized before implantation; progesterone-binding protein and placental progesterone secretion. Journal of Endocrinology 54, 435 444. ILLINGWORTH, D. V., PERRY, J. S., ACKLAND, N. & BURTON, A. M. (1973). The maintenance of pregnancy, and parturition, in guinea-pigs hypophysectomized within 4 days of mating. Journal of Endocrinology 59, 163-169. KAUFMANN, P. (1969). Die Meerschweinchenplacenta und ihre Entwicklung. Zeitschrift fur Anatomie und Entwicklungsgeschichte 129, 83-101.
W. H. TAM AND S. M. BURGESS 614 LoFTs, B., PHILLIPS, J. G. & TAM, W. H. (1966). Seasonal changes in the testis of the cobra, Naja naja (Linn.). General and Comparative Endocrinology 6, 466-475. LOFTS, B., PHILLIPS, J. G. & TAM, W. H. (1971). Seasonal changes in the histology of the adrenal gland of the cobra, Naja naja. General and Comparative Endocrinology 16, 121-131. TAM, W. H., PHILLIPS, J. G. & LoFrs, B. (1969). Seasonal changes in the in vitro production of testicular androgens by the cobra (Naja naja Linn.). General and Comparative Endocrinology 13, 117-125. TAM, W. H., PHILLIPS, J. G. & LoiTs, B. (1972). Seasonal changes in the secretory activity of the adrenal gland of the cobra (Naja naja Linn.). General and Comparative Endocrinology 19, 218-224. WISLOCKI, G. B., DEANE, W. H. & DEMPSEY, E. W. (1946). The histochemistry of the rodent's placenta. American Journal of Anatomy 78, 281-346.
