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Comparative ultrastructure of the decidua in pregnancy and pseudopregnancy
Comparative
ultrastructure
of the decidua in
pregnancy and pseudopregnancy MARGARET
W.
RALPH
M.
JENNIFER
A.
JACK
M.
Chicago,
Illinois,
The
ORSINI,
WYNN,
PH.D.*
M.D.**
HARRIS
BULMASH, and Madison,
B.S. Wisconsin
ultrastructural
development of decidua in pregnant hamsters and in deciduomas age are compared during the period of 3 days beginning 5 days and 19 hours after ovulation. At first, the decidua produced in response to the blastocyst resembles in detail that of the deciduoma caused by injection of air into the pseudopregnant uterine horn. Although the decidua in true pregnancy maintains its characteristic ultrastructural features, the first signs of degeneration within the deciduoma are detectable as early as 6 days and 19 hours after ovulation. This tissue may show mitoses and regressive changes simultaneously. By 7% days after ovulation, degeneration of the deciduoma is advanced. Beyond that period, ultrastructural details are obliterated. Thus, by the time that a typical pericellular deposit has formed around the decidua in true pregnancy, the deciduoma has begun to deteriorate. The role of the trophoblast in inducing deposition of this capsular material therefore could not be evaluated in these experiments. The two triggering mechanisms, blastocyst and injection of air, induce a common pathway of decidualitation in the progesterone-stimulated uterus. of equivalent
A L T H o u G H the most extensive studies of decidualization have been carried out in the rat,l* 3 pregnancy and pseudopregnancy in the hamster are accompanied by more nearly identical morphologic changes in ovum and uterus than in other experimental animals2* lo, I2 In the pseudopregnant hamster, the ovum loses its zona pellucida at the
same time after ovulation as does the blastocyst in true pregnancy.gl l1 Pseudoimplantation, furthermore, occurs at a time after ovulation comparable to that of initiation of the first attachment of the blastocyst in pregnancy.lOT l2 When the reproductive tracts of pregnant hamsters and pseudopregnant animals (with decidualization induced by injection of air) of comparable postovulatory ages are cleared in benzyl benzoate and examined by oblique light, the gross similarities are striking.*? 5* 6* 7 In view of the physiologic parallels in pregnancy and pseudopregnancy in the hamster, and the gross and histologic resemblances of the respective deciduas, our primary objective in this study was to ascertain the degree of ultrastructural similarity in the two conditions. To many biologists and most obstetricians the term “decidua” refers specifically to the transformed endometrial stroma of pregnancy. In this paper, however, the broader
From the Department of Obstetrics and Gynecology, University of Illinois at the Medical Center, Chicago, Illinois, and the Department of Anatomy, Universitv of Wisconsin. Madison. Wisconsin. . This investigation was supported in part by Grants HD 04152 and HD 00267 from the United States Public Health Service and Grant M.68.71 from The Population Council. *Present address: Department of Anatomy, Bardeen Medical Laboratories, University of Wisconsin School of Medicine, Madison, Wisconsin. **Reprint requests to Dr. Wynn: Department of Obstetrics and Gynecology, 840 South Wood Street, Chicago, Illinois 60612.
14
Volumt Number
106 1
Ultrastructure
definition suggested by Mossman is employed. In his classic monograph he defines decidua as “. . . a tissue made up of endometrial connective tissue cells which have enlarged and become rounded or polyhedral due to the accumulation of glycogen or lipoids within their cytoplasm, and which occur either in pregnancy, pseudopregnancy,
Fig. 1, 2, 7, 8, 12, and All specimens except that micrographs were prepared.
13. Longitudinal shown in Fig.
of decidua
15
or in artificially or pathologically stimulated deciduomata.“4 Our previous electron microscopic studies of decidua in the guinea pig,13 man,14 and a variety of other animalsI indicated greater ultrastructural complexity of decidua adjacent to trophoblast. Examination of the deciduoma, which lacks contiguous tropho-
sections with the mesometrium 12 were from the hamsters from
toward the top. which the electron
Fig. 1. Pregnant uterus of hamster at 5 days and 19 hours after ovulation. Longitudinal section shows two conceptional swellings, with mesometrium above. Developing embryos (~770~s) at the ectoplacental cone stage lie within a mass of antimesometrial decidua. The decidual cavity in which the embryo lies is connected to the main uterine cavity, which appears as a slit above the decidual swelling. (x12.) Fig. 2. Pseudopregnant uterus at 5 days and 19 hours after ovulation and 48 hours after induction of decidualization by injection of air. Two decidualized masses are shown antimesometrially (arrows). Compare with Fig. 1. (x12.)
16
Orsini
et al.
Amer.
blast, in comparison with normal pregnancy of the same postovulatory age thus provides a means of assessing the role of the trophoblast in inducing specific ultrastructural development of the adjacent decidua and its associated extracellular depositsI Material
and
methods
All hamsters selected were of known age and reproductive history. They were virgin prior to initiation of this study, weighed 80 grams or more, and had undergone a minimum of two normal estrous cycles. Females in the pregnant series were mated to males of proved fertility; the presence of sperm in the vagina was confirmed by vaginal cytologic smears. Pseudopregnancy was induced by observed matings with vasectomized males. At 3 days and 19 hours after ovulation, 0.05 ml. of air was injected into one uterine horn of each pseudopregnant animal, the other horn serving as a control. At 5 days and 19 hours, 6 days and 19 hours, and 7 days and 12 hours after ovulation, 0.25 ml. of a 1 per cent solution of Pontamine sky blue in normal saline was injected into the femoral vein of pregnant and pseudopregnant animals. Laparotomy was performed 15 minutes later. Sites of decidualization in both pregnant and pseudopregnant hamsters were selected. Each specimen was covered with 4 per cent phosphate-buffered glutaraldehyde, opened, and washed in the same fixative. Areas in which the orientation was easily recognized were removed for electron microscopic study. Tissues from interconceptional areas of the pregnant hamsters and from the nontraumatized uterine horns of the pseudopregnant animals were similarly removed and fixed.
Fig. tion. cyte Fig.
1, 1970 Gym.
The hamsters were then killed. The remaining portions of the reproductive tracts were removed and fixed in 10 per cent phosphate-buffered formalin for histologic examination. Sections were cut at 8 p and studied after staining by Groat’s method. Glutaraldehyde-fixed tissues were postfixed in phosphate-buffered 1 per cent osmic acid, dehydrated in an ethanolic series followed by propylene oxide, and embedded in Aralditc: 502. After slow polymerization, thick (2 EL) sections were prepared with a Porter-Blum MT-1 ultramicrotome for phase-contrast microscopy. Thin sections representing about 16 areas of each specimen were doubly stained with lead citrate and uranyl acetate and esamined with a Zeiss EM-9A electron microscope. Results
At 5 days and 19 hours after ovulation the ultrastructural features of the decidua and deciduoma shown histologically in Figs. 1 and 2, respectively, were remarkably similar. In both tissues there were numerous ribosomes and a small amount of rough-surfaced endoplasmic reticulum. Mitochondria were perhaps more numerous in the pregnant uterus (Fig. 3) at this stage, although in both tissues they were small and otherwise unremarkable. Deposition of glycogen appeared somewhat more extensive in the pseudopregnant animal (Fig. 4). Golgi membranes and smooth endoplasmic reticulum were fairly well developed in both forms of decidua (Figs. 5 and 6). Tonofilaments and tight junctions were more readily distinguished in the pregnant animal, which appeared to have a larger number of lysoson~~s and more apparently multinucleated cells. This decidua displayed a more typically epi-
3. Electron micrograph of decidua of pregnant Note the grouping of endometrial stromal cells with a process projecting through capillary wall. 4. Deciduoma of pseudopregnant hamster 5 days
cells with extensive deposition of glycogen lent stage of true pregnancy. Compare with
Jammy .J. Obstet.
hamster 5 days and 19 hours after ovulaaround capillary. Arrow indicates a leuko(Neg. 5670. ~4,500.)
and 19 hours after ovulation. The stromal are grouped around the capillary, as in the equivaFig. 3. (Neg. 5730. ~4,500.)
Volume Number
106 1
Figs.
Ultrastructure
3 and 4. For legends
see opposite
page
of
decidua
17
8
@sini
et 01.
Figs. 5 and 6. For legends
Amer.
see opposite
page.
January J. Obstet.
1, 1970 Gynec.
Volume Number
106 1
Ultrastructure
of
decidua
19
Fig. 5. Detail of decidua shown in Fig. 3. Numerous small mitochondria (M), fairly well developed Golgi complexes (Go), many tonofilaments (T), and glycogen are seen. (Neg. 5677. x15,000.) Fig. 6. Detail’of deciduoma shown in Fig. 4. The ultrastructural appearance is quite similar to that of the decidua at the equivalent stage of true pregnancy, but there are fewer tonofilaments, somewhat larger mitochondria, and greater deposition of glycogen. Compare with Fig. 5. (Neg. 5726. ~15,000.)
thelioid pattern, more obvious deposition of pericellular capsular material, and more extensive diapedesis of polymorphonuclear leukocytes. Twenty-four hours later, at 6 days and 19 hours (Fig. 7)) no essential change in ultrastructure of the pregnant decidua was detected, except possibly for deposition of somewhat more glycogen. The pseudopregnant decidua, however, showed distinct changes during this 24 hour period of development (Fig. 8)) having begun to undergo degeneration. Its pattern was more clearly epithelioid; lipid and diffuse glycogen were more obvious. Smooth cytomembranes were more prominent, and much of the endoplasmic reticulum was distended with secretion (Fig. 9). Despite the degeneration, numerous mitotic figures were evident (Fig. 10). Although the epithelium had not changed much during the preceding 24 hours, there were many lysosomes and degenerating organelles. Intercellular connections comprised true desmosomes in addition to tight junctions. The ultrastructural characteristics of the epithelium, except for the greater development of rough endoplasmic reticulum, resembled those of the stroma at this stage of development. Throughout the period of study, the interconceptional areas of the pregnant uterus and the entire control horn of the pseudopregnant animal underwent no essential change. The typical low columnar epithehum had short microvilli, numerous ordinary mitochondria and tortuous plasma membranes, and fairly well developed Golgi complexes. The stromal cells resembled typical fibrocytes with well-developed ergastoplasm and mitochondria (Fig. 11) , At 7% days after ovulation the fine structure of the decidua shown histologically in
Fig. 7. Pregnant uterus at 6 days and 19 hours after ovulation. The relative mass of decidua has become greater, extending through almost the entire basal endometrium. (x12.) Fig. 8. Deciduoma from uterus of pseudopregnant hamster at 6 days and 19 hours after ovulation and 3 days after injection of air. Antimesometrial decidua is vacuolated, resembling that surrounding the decidual cavity in Fig. 7. (x12.)
Fig. 12 differed in the various parts of the uterus. In the antimesometrial portion, a typical pavementlike pattern was obvious. Mitoses in the capillaries, numerous typical small mitochondria, and tight junctions were found. Endoplasmic reticulum underwent no further development and glycogen was not so
20
Orsini
et al.
Fig. 9. The
decidualized cells, connected by tight junctions (I), are surrounded by amorphous extracellular material (arroru). They remain adjacent to endometrial capillaries (C). The cytoplasm contains much lipid (L) and glycogen (Cl). S omewhat elongated mitochondria (M) are in close relation to distended endoplasmic reticulum (ER), which contains a moderately electron-dense material. (Neg. 6034. x12,500.)
Fig. 10. Deciduoma
6 days and 19 hours after ovulation. Mitotic figures (arrour) are obvious, although early signs of degeneration have already appeared elsewhere in the tissue. The pattern has become more typically epithelioid. Details are shown in Fig. 9. (Neg. 6017. ~4,500.) Fig. 11. Unstimulated control horn of hamster bearing deciduoma shown in Fig. 10 in opposite horn. The tissue comprises typical unstimulated endometrial epithelium (E) and stroma consisting of many ordinary fibrocytes (F). (Neg. 6028. ~4,500.)
Volume Number
106 1
Figs. 10 and 11. For legends
Ultrastructure
see opposite
pags.
of decidua
2
22
Orsini
et al.
apparent in proportion to the total mass of tissue. In the mesometrial portion, more glycogen was deposited and more apparently multinucleated elements were found. In the lateral, or orthomesometrial, decidua, the ultrastructural appearance was intermediate, in general resembling the antimesometrial portion except for the greater number of
January J. Obstet.
Amer.
1, 1970 Gynec.
lysosomes and lipid granules. At this stage, however, differences between pregnant and pseudopregnant deciduas were striking. The deciduoma had undergone extensive degeneration (Fig. 13). Unlike the pregnant decidua (Fig. 14)) the deciduoma demonstrated free lipid, breakdown of cell membranes, crenulation of nuclei, and rupture of mitochondria (Fig. 15). Beyond this stage decidual ultrastructure in pseudopregnancy was too badly damaged to permit identification
of specific
features.
Comment
In pseudopregnancy, at the time corresponding to implantation, in the unstimulated uterus the zona-free ovum evokes a uterine reaction detected by intravenously injected Pontamine sky blue.lOv I2 Sections taken
through
the
uterus
at
this
time
reveal
the free ovum lying in the antimesometrial portion of the uterine cavity, in contact with and compressing the epithelium. No true decidua forms, but luminal changes and localized edema are associated with these areas. Except for the absence of cleavage of the ovum, such areas are entirely comparable to the
site
of
initial
attachment
of
the
blasto-
cyst.
Fig. 12. Longitudinal section of normal pregnant uterus at 7 days and 9 hours after ovulation. This animal was not used in the electron microscopic studies, but the comparable site of interest is shown to greater advantage here. P.A.S. staining brings out the opacity and glycogen at the mesometrial side of the decidual cavity. (x12.) Fig. 13. Deciduoma at 7 days and 12 hours after ovulation. This pseudopregnant hamster had received an injection of air 4 days previously. A small round deciduoma (arrow) projects into the uterine cavity. Contact at the mesometrial side is decreased. Compare with Fig. 12. (x12.)
Fig. 14. Decidua
Response to Pontamine sky blue continues in association with implantation and development of the conceptus. In pseudopregnancy, however, unless decidualization has been evoked, the reaction is transient. The ova can be recovered throughout pseudopregnancy, however, and luminal changes continue to mark their sites in cleared uteri of later stages.lOp I2 Injection of air at the sensitive period just prior to and coincident with implantation (3 days and 9 hours to 4 days after ovulation), however, can induce deciduomas, which are often discrete and which resemble in their
at 7 days and 12 hours after ovulation. The epithelioid pattern persists. Tight junctions and numerous small mitochondria create the appearance of typical mature decidua. Signs of degeneration are absent. (Neg. 8241. ~4,500.) Fig. 15. Deciduoma at 7 days and 12 hours after ovulation. Unlike the decidua shown in Fig. 14, the deciduoma at this stage has undergone almost complete degeneration. (Neg. 8249.
x4,500.)
Volume Number
106 1
Figs. 14 and 15. For legends
UltraStructure
see opposite
#age
of detidua
23
24
Orsini
et al.
Amer.
developmental pattern the decidua of normal pregnancy. 7p 8 The mass of decidualized tissue in pseudopregnancy may be less than, equal to, or greater than that in true pregnancy. By 24 hours after stimulation, clearing of such deciduomas in benzyl benzoate reveals opaque white shields, comparable to those found at normal implantation sites at equivalent stages after ovulation. Subsequent development of the decidua is similar in pregnancy and pseudopregnancy in that the pattern of opacity, which reflects to a large extent the deposition of glycogen, changes in a definite sequence in both tissues. Gross enlargement in pregnancy and pseudopregnancy with decidualization is evident by the sixth day (5 days and 12 hours after ovulation) . The opacity, representing decidua and glycogen, extends from its original antimesometrial position in the endometrium to reach a peak, in proportional mass, by 6 days and 12 hours. At this time a central nonopaque cavity appears within both conceptus and deciduoma. Sections of these areas can be shown to represent the zone occupied by the developing ectoplacental cone in the pregnant animal. A comparable clear space develops in the deciduoma in pseudopregnancy. At the same time, sheaths begin to appear about the arterial vessels in the mesometrial endometrium in both pregnancy and pseudopregnancy. By 7 days and 12 hours, the opacity is more pronounced at the mesometrial side and it begins to regress in the antimesometrial endometrium. Degeneration may begin within the deciduoma at some time during the seventh to eighth day after ovulation in pseudopregnancy. The extent and rapidity of the regression, however, are somewhat variable. Degeneration is characterized by loss or diminution of the Pontamine sky blue reaction, necrosis within the decidualized tissue, and finally, separation of the deciduoma as a result of undercutting at
the mesometrial and antimesometrial borders until it lies free within the uterine cavity. At 5 days and 19 hours after ovulation, whether the stimulus to decidualization is the blastocyst or the traumatic injection of air, the ultrastructural effects on the endometrium are almost identical. By 24 hours later, the decidua of true pregnancy has undergone no essential change. That in the deciduoma, however, has deposited much more lipid and glycogen and shows signs of degeneration despite the obvious growth in other areas, as evidenced by mitoses. By 7f/2 days the two structures appear quite different. The pregnant decidua has formed a typical epithelioid pattern with organellar characteristics common to the decidua of other species. The deciduoma, however, is markedly degenerated with lipid, crenulated nuclei, and rupture of cytomembranes and organelles. The early degeneration of the deciduomas is undoubtedly a result of the temporal limits of pseudopregnancy. Corpora lutea of pseudopregnancy reach their developmental peak during the seventh day after ovulation; heat recurs on the evening of the eighth or ninth day after that initiating pseudopregnancy. Ovulation recurs even though necrotic masses of deciduomas may still be present within the uterine lumen. In pseudopregnancy the decidua reflects this life-span, for it begins its involution at a stage prior to formation of the extensive pericellular deposit that accompanies decidualization within the pregnant uterine horn at the same postovulatory age. The close resemblance in developmental patterns of decidualization (grossly, histologically, and ultrastructurally) from the two triggering mechanisms (blastocyst and injection of air) suggest a common pathway of action in the progesterone-stimulated uterus and a similarity of reaction.
REFERENCES
1.
DeFeo, V. M., editor:
J.: Decidualization, Cellular Biology
in of
Wynn, R. the Uterus,
January 1, 1970 J. Obstet. Gynec.
2.
New York, Inc., Chap. Foster, G.
1967, 8. A.,
Orsini,
Appleton-Century-Crofts, M.
W.,
and
Strong,
Volume Number
106 1
F. M.: Proc. Sot. Exper. Biol. & Med. 113: 262, 1963. 3. Jollie, W. P., and Bencosme, S. A.: Am. J. Anat. 116: 217. 1965. 4. Mossman, H. %‘.: Contrib. Embryol. 26: 129, 1937. 5. Orsini, M. W.: J. Reprod. & Fertil. 3: 283, 1962. 6. Orsini, M. W.: J. Reprod. & Fertil. 3: 288, 1962. 7. Orsini, M. W.: J. Endocrinol. 28: 119, 1963. M. W.: J. Reprod. & Fertil. 5: 323, 8. Orsini, 1963. 9. Orsini, M. W.: Morphological Evidence on the Intrauterine Career of the Ovum, in Enders, A. Cl., editor: Delayed Implantation,
Ultrastructure
10.
11. 12.
13. 14. 15. 16.
Chicago, Press, pp. Orsini, M. on Animal semination. Orsini. M. Orsinij M. stenholme, Stages of Churchill. Wynn, R: Wynn, R. 832, 1967. Wvnn. R. Wynn: R. 723, 1969.
of decidua
25
1963, The University of Chicago 155-170. W.: Fifth International Congress Reproduction and Artificial In7: 309, 1964. W.: 1. Anat. 99: 922. 1965. W.: “General Discussion, in WolG. E. W., editor: Preimplantation Pregnancy, London, 1965, J. A. Ltd.. no. 162-167. M.:‘FertiI. & Steril. 16: 16, 1965. M.: AM. J. OBST. & GYNEC. 97: M.: M.:
Obst. AM.
& Gvnec. J. 0;s~.
29: 644. &
GYN&
1967. 103:
ultrastructure
of the decidua in
pregnancy and pseudopregnancy MARGARET
W.
RALPH
M.
JENNIFER
A.
JACK
M.
Chicago,
Illinois,
The
ORSINI,
WYNN,
PH.D.*
M.D.**
HARRIS
BULMASH, and Madison,
B.S. Wisconsin
ultrastructural
development of decidua in pregnant hamsters and in deciduomas age are compared during the period of 3 days beginning 5 days and 19 hours after ovulation. At first, the decidua produced in response to the blastocyst resembles in detail that of the deciduoma caused by injection of air into the pseudopregnant uterine horn. Although the decidua in true pregnancy maintains its characteristic ultrastructural features, the first signs of degeneration within the deciduoma are detectable as early as 6 days and 19 hours after ovulation. This tissue may show mitoses and regressive changes simultaneously. By 7% days after ovulation, degeneration of the deciduoma is advanced. Beyond that period, ultrastructural details are obliterated. Thus, by the time that a typical pericellular deposit has formed around the decidua in true pregnancy, the deciduoma has begun to deteriorate. The role of the trophoblast in inducing deposition of this capsular material therefore could not be evaluated in these experiments. The two triggering mechanisms, blastocyst and injection of air, induce a common pathway of decidualitation in the progesterone-stimulated uterus. of equivalent
A L T H o u G H the most extensive studies of decidualization have been carried out in the rat,l* 3 pregnancy and pseudopregnancy in the hamster are accompanied by more nearly identical morphologic changes in ovum and uterus than in other experimental animals2* lo, I2 In the pseudopregnant hamster, the ovum loses its zona pellucida at the
same time after ovulation as does the blastocyst in true pregnancy.gl l1 Pseudoimplantation, furthermore, occurs at a time after ovulation comparable to that of initiation of the first attachment of the blastocyst in pregnancy.lOT l2 When the reproductive tracts of pregnant hamsters and pseudopregnant animals (with decidualization induced by injection of air) of comparable postovulatory ages are cleared in benzyl benzoate and examined by oblique light, the gross similarities are striking.*? 5* 6* 7 In view of the physiologic parallels in pregnancy and pseudopregnancy in the hamster, and the gross and histologic resemblances of the respective deciduas, our primary objective in this study was to ascertain the degree of ultrastructural similarity in the two conditions. To many biologists and most obstetricians the term “decidua” refers specifically to the transformed endometrial stroma of pregnancy. In this paper, however, the broader
From the Department of Obstetrics and Gynecology, University of Illinois at the Medical Center, Chicago, Illinois, and the Department of Anatomy, Universitv of Wisconsin. Madison. Wisconsin. . This investigation was supported in part by Grants HD 04152 and HD 00267 from the United States Public Health Service and Grant M.68.71 from The Population Council. *Present address: Department of Anatomy, Bardeen Medical Laboratories, University of Wisconsin School of Medicine, Madison, Wisconsin. **Reprint requests to Dr. Wynn: Department of Obstetrics and Gynecology, 840 South Wood Street, Chicago, Illinois 60612.
14
Volumt Number
106 1
Ultrastructure
definition suggested by Mossman is employed. In his classic monograph he defines decidua as “. . . a tissue made up of endometrial connective tissue cells which have enlarged and become rounded or polyhedral due to the accumulation of glycogen or lipoids within their cytoplasm, and which occur either in pregnancy, pseudopregnancy,
Fig. 1, 2, 7, 8, 12, and All specimens except that micrographs were prepared.
13. Longitudinal shown in Fig.
of decidua
15
or in artificially or pathologically stimulated deciduomata.“4 Our previous electron microscopic studies of decidua in the guinea pig,13 man,14 and a variety of other animalsI indicated greater ultrastructural complexity of decidua adjacent to trophoblast. Examination of the deciduoma, which lacks contiguous tropho-
sections with the mesometrium 12 were from the hamsters from
toward the top. which the electron
Fig. 1. Pregnant uterus of hamster at 5 days and 19 hours after ovulation. Longitudinal section shows two conceptional swellings, with mesometrium above. Developing embryos (~770~s) at the ectoplacental cone stage lie within a mass of antimesometrial decidua. The decidual cavity in which the embryo lies is connected to the main uterine cavity, which appears as a slit above the decidual swelling. (x12.) Fig. 2. Pseudopregnant uterus at 5 days and 19 hours after ovulation and 48 hours after induction of decidualization by injection of air. Two decidualized masses are shown antimesometrially (arrows). Compare with Fig. 1. (x12.)
16
Orsini
et al.
Amer.
blast, in comparison with normal pregnancy of the same postovulatory age thus provides a means of assessing the role of the trophoblast in inducing specific ultrastructural development of the adjacent decidua and its associated extracellular depositsI Material
and
methods
All hamsters selected were of known age and reproductive history. They were virgin prior to initiation of this study, weighed 80 grams or more, and had undergone a minimum of two normal estrous cycles. Females in the pregnant series were mated to males of proved fertility; the presence of sperm in the vagina was confirmed by vaginal cytologic smears. Pseudopregnancy was induced by observed matings with vasectomized males. At 3 days and 19 hours after ovulation, 0.05 ml. of air was injected into one uterine horn of each pseudopregnant animal, the other horn serving as a control. At 5 days and 19 hours, 6 days and 19 hours, and 7 days and 12 hours after ovulation, 0.25 ml. of a 1 per cent solution of Pontamine sky blue in normal saline was injected into the femoral vein of pregnant and pseudopregnant animals. Laparotomy was performed 15 minutes later. Sites of decidualization in both pregnant and pseudopregnant hamsters were selected. Each specimen was covered with 4 per cent phosphate-buffered glutaraldehyde, opened, and washed in the same fixative. Areas in which the orientation was easily recognized were removed for electron microscopic study. Tissues from interconceptional areas of the pregnant hamsters and from the nontraumatized uterine horns of the pseudopregnant animals were similarly removed and fixed.
Fig. tion. cyte Fig.
1, 1970 Gym.
The hamsters were then killed. The remaining portions of the reproductive tracts were removed and fixed in 10 per cent phosphate-buffered formalin for histologic examination. Sections were cut at 8 p and studied after staining by Groat’s method. Glutaraldehyde-fixed tissues were postfixed in phosphate-buffered 1 per cent osmic acid, dehydrated in an ethanolic series followed by propylene oxide, and embedded in Aralditc: 502. After slow polymerization, thick (2 EL) sections were prepared with a Porter-Blum MT-1 ultramicrotome for phase-contrast microscopy. Thin sections representing about 16 areas of each specimen were doubly stained with lead citrate and uranyl acetate and esamined with a Zeiss EM-9A electron microscope. Results
At 5 days and 19 hours after ovulation the ultrastructural features of the decidua and deciduoma shown histologically in Figs. 1 and 2, respectively, were remarkably similar. In both tissues there were numerous ribosomes and a small amount of rough-surfaced endoplasmic reticulum. Mitochondria were perhaps more numerous in the pregnant uterus (Fig. 3) at this stage, although in both tissues they were small and otherwise unremarkable. Deposition of glycogen appeared somewhat more extensive in the pseudopregnant animal (Fig. 4). Golgi membranes and smooth endoplasmic reticulum were fairly well developed in both forms of decidua (Figs. 5 and 6). Tonofilaments and tight junctions were more readily distinguished in the pregnant animal, which appeared to have a larger number of lysoson~~s and more apparently multinucleated cells. This decidua displayed a more typically epi-
3. Electron micrograph of decidua of pregnant Note the grouping of endometrial stromal cells with a process projecting through capillary wall. 4. Deciduoma of pseudopregnant hamster 5 days
cells with extensive deposition of glycogen lent stage of true pregnancy. Compare with
Jammy .J. Obstet.
hamster 5 days and 19 hours after ovulaaround capillary. Arrow indicates a leuko(Neg. 5670. ~4,500.)
and 19 hours after ovulation. The stromal are grouped around the capillary, as in the equivaFig. 3. (Neg. 5730. ~4,500.)
Volume Number
106 1
Figs.
Ultrastructure
3 and 4. For legends
see opposite
page
of
decidua
17
8
@sini
et 01.
Figs. 5 and 6. For legends
Amer.
see opposite
page.
January J. Obstet.
1, 1970 Gynec.
Volume Number
106 1
Ultrastructure
of
decidua
19
Fig. 5. Detail of decidua shown in Fig. 3. Numerous small mitochondria (M), fairly well developed Golgi complexes (Go), many tonofilaments (T), and glycogen are seen. (Neg. 5677. x15,000.) Fig. 6. Detail’of deciduoma shown in Fig. 4. The ultrastructural appearance is quite similar to that of the decidua at the equivalent stage of true pregnancy, but there are fewer tonofilaments, somewhat larger mitochondria, and greater deposition of glycogen. Compare with Fig. 5. (Neg. 5726. ~15,000.)
thelioid pattern, more obvious deposition of pericellular capsular material, and more extensive diapedesis of polymorphonuclear leukocytes. Twenty-four hours later, at 6 days and 19 hours (Fig. 7)) no essential change in ultrastructure of the pregnant decidua was detected, except possibly for deposition of somewhat more glycogen. The pseudopregnant decidua, however, showed distinct changes during this 24 hour period of development (Fig. 8)) having begun to undergo degeneration. Its pattern was more clearly epithelioid; lipid and diffuse glycogen were more obvious. Smooth cytomembranes were more prominent, and much of the endoplasmic reticulum was distended with secretion (Fig. 9). Despite the degeneration, numerous mitotic figures were evident (Fig. 10). Although the epithelium had not changed much during the preceding 24 hours, there were many lysosomes and degenerating organelles. Intercellular connections comprised true desmosomes in addition to tight junctions. The ultrastructural characteristics of the epithelium, except for the greater development of rough endoplasmic reticulum, resembled those of the stroma at this stage of development. Throughout the period of study, the interconceptional areas of the pregnant uterus and the entire control horn of the pseudopregnant animal underwent no essential change. The typical low columnar epithehum had short microvilli, numerous ordinary mitochondria and tortuous plasma membranes, and fairly well developed Golgi complexes. The stromal cells resembled typical fibrocytes with well-developed ergastoplasm and mitochondria (Fig. 11) , At 7% days after ovulation the fine structure of the decidua shown histologically in
Fig. 7. Pregnant uterus at 6 days and 19 hours after ovulation. The relative mass of decidua has become greater, extending through almost the entire basal endometrium. (x12.) Fig. 8. Deciduoma from uterus of pseudopregnant hamster at 6 days and 19 hours after ovulation and 3 days after injection of air. Antimesometrial decidua is vacuolated, resembling that surrounding the decidual cavity in Fig. 7. (x12.)
Fig. 12 differed in the various parts of the uterus. In the antimesometrial portion, a typical pavementlike pattern was obvious. Mitoses in the capillaries, numerous typical small mitochondria, and tight junctions were found. Endoplasmic reticulum underwent no further development and glycogen was not so
20
Orsini
et al.
Fig. 9. The
decidualized cells, connected by tight junctions (I), are surrounded by amorphous extracellular material (arroru). They remain adjacent to endometrial capillaries (C). The cytoplasm contains much lipid (L) and glycogen (Cl). S omewhat elongated mitochondria (M) are in close relation to distended endoplasmic reticulum (ER), which contains a moderately electron-dense material. (Neg. 6034. x12,500.)
Fig. 10. Deciduoma
6 days and 19 hours after ovulation. Mitotic figures (arrour) are obvious, although early signs of degeneration have already appeared elsewhere in the tissue. The pattern has become more typically epithelioid. Details are shown in Fig. 9. (Neg. 6017. ~4,500.) Fig. 11. Unstimulated control horn of hamster bearing deciduoma shown in Fig. 10 in opposite horn. The tissue comprises typical unstimulated endometrial epithelium (E) and stroma consisting of many ordinary fibrocytes (F). (Neg. 6028. ~4,500.)
Volume Number
106 1
Figs. 10 and 11. For legends
Ultrastructure
see opposite
pags.
of decidua
2
22
Orsini
et al.
apparent in proportion to the total mass of tissue. In the mesometrial portion, more glycogen was deposited and more apparently multinucleated elements were found. In the lateral, or orthomesometrial, decidua, the ultrastructural appearance was intermediate, in general resembling the antimesometrial portion except for the greater number of
January J. Obstet.
Amer.
1, 1970 Gynec.
lysosomes and lipid granules. At this stage, however, differences between pregnant and pseudopregnant deciduas were striking. The deciduoma had undergone extensive degeneration (Fig. 13). Unlike the pregnant decidua (Fig. 14)) the deciduoma demonstrated free lipid, breakdown of cell membranes, crenulation of nuclei, and rupture of mitochondria (Fig. 15). Beyond this stage decidual ultrastructure in pseudopregnancy was too badly damaged to permit identification
of specific
features.
Comment
In pseudopregnancy, at the time corresponding to implantation, in the unstimulated uterus the zona-free ovum evokes a uterine reaction detected by intravenously injected Pontamine sky blue.lOv I2 Sections taken
through
the
uterus
at
this
time
reveal
the free ovum lying in the antimesometrial portion of the uterine cavity, in contact with and compressing the epithelium. No true decidua forms, but luminal changes and localized edema are associated with these areas. Except for the absence of cleavage of the ovum, such areas are entirely comparable to the
site
of
initial
attachment
of
the
blasto-
cyst.
Fig. 12. Longitudinal section of normal pregnant uterus at 7 days and 9 hours after ovulation. This animal was not used in the electron microscopic studies, but the comparable site of interest is shown to greater advantage here. P.A.S. staining brings out the opacity and glycogen at the mesometrial side of the decidual cavity. (x12.) Fig. 13. Deciduoma at 7 days and 12 hours after ovulation. This pseudopregnant hamster had received an injection of air 4 days previously. A small round deciduoma (arrow) projects into the uterine cavity. Contact at the mesometrial side is decreased. Compare with Fig. 12. (x12.)
Fig. 14. Decidua
Response to Pontamine sky blue continues in association with implantation and development of the conceptus. In pseudopregnancy, however, unless decidualization has been evoked, the reaction is transient. The ova can be recovered throughout pseudopregnancy, however, and luminal changes continue to mark their sites in cleared uteri of later stages.lOp I2 Injection of air at the sensitive period just prior to and coincident with implantation (3 days and 9 hours to 4 days after ovulation), however, can induce deciduomas, which are often discrete and which resemble in their
at 7 days and 12 hours after ovulation. The epithelioid pattern persists. Tight junctions and numerous small mitochondria create the appearance of typical mature decidua. Signs of degeneration are absent. (Neg. 8241. ~4,500.) Fig. 15. Deciduoma at 7 days and 12 hours after ovulation. Unlike the decidua shown in Fig. 14, the deciduoma at this stage has undergone almost complete degeneration. (Neg. 8249.
x4,500.)
Volume Number
106 1
Figs. 14 and 15. For legends
UltraStructure
see opposite
#age
of detidua
23
24
Orsini
et al.
Amer.
developmental pattern the decidua of normal pregnancy. 7p 8 The mass of decidualized tissue in pseudopregnancy may be less than, equal to, or greater than that in true pregnancy. By 24 hours after stimulation, clearing of such deciduomas in benzyl benzoate reveals opaque white shields, comparable to those found at normal implantation sites at equivalent stages after ovulation. Subsequent development of the decidua is similar in pregnancy and pseudopregnancy in that the pattern of opacity, which reflects to a large extent the deposition of glycogen, changes in a definite sequence in both tissues. Gross enlargement in pregnancy and pseudopregnancy with decidualization is evident by the sixth day (5 days and 12 hours after ovulation) . The opacity, representing decidua and glycogen, extends from its original antimesometrial position in the endometrium to reach a peak, in proportional mass, by 6 days and 12 hours. At this time a central nonopaque cavity appears within both conceptus and deciduoma. Sections of these areas can be shown to represent the zone occupied by the developing ectoplacental cone in the pregnant animal. A comparable clear space develops in the deciduoma in pseudopregnancy. At the same time, sheaths begin to appear about the arterial vessels in the mesometrial endometrium in both pregnancy and pseudopregnancy. By 7 days and 12 hours, the opacity is more pronounced at the mesometrial side and it begins to regress in the antimesometrial endometrium. Degeneration may begin within the deciduoma at some time during the seventh to eighth day after ovulation in pseudopregnancy. The extent and rapidity of the regression, however, are somewhat variable. Degeneration is characterized by loss or diminution of the Pontamine sky blue reaction, necrosis within the decidualized tissue, and finally, separation of the deciduoma as a result of undercutting at
the mesometrial and antimesometrial borders until it lies free within the uterine cavity. At 5 days and 19 hours after ovulation, whether the stimulus to decidualization is the blastocyst or the traumatic injection of air, the ultrastructural effects on the endometrium are almost identical. By 24 hours later, the decidua of true pregnancy has undergone no essential change. That in the deciduoma, however, has deposited much more lipid and glycogen and shows signs of degeneration despite the obvious growth in other areas, as evidenced by mitoses. By 7f/2 days the two structures appear quite different. The pregnant decidua has formed a typical epithelioid pattern with organellar characteristics common to the decidua of other species. The deciduoma, however, is markedly degenerated with lipid, crenulated nuclei, and rupture of cytomembranes and organelles. The early degeneration of the deciduomas is undoubtedly a result of the temporal limits of pseudopregnancy. Corpora lutea of pseudopregnancy reach their developmental peak during the seventh day after ovulation; heat recurs on the evening of the eighth or ninth day after that initiating pseudopregnancy. Ovulation recurs even though necrotic masses of deciduomas may still be present within the uterine lumen. In pseudopregnancy the decidua reflects this life-span, for it begins its involution at a stage prior to formation of the extensive pericellular deposit that accompanies decidualization within the pregnant uterine horn at the same postovulatory age. The close resemblance in developmental patterns of decidualization (grossly, histologically, and ultrastructurally) from the two triggering mechanisms (blastocyst and injection of air) suggest a common pathway of action in the progesterone-stimulated uterus and a similarity of reaction.
REFERENCES
1.
DeFeo, V. M., editor:
J.: Decidualization, Cellular Biology
in of
Wynn, R. the Uterus,
January 1, 1970 J. Obstet. Gynec.
2.
New York, Inc., Chap. Foster, G.
1967, 8. A.,
Orsini,
Appleton-Century-Crofts, M.
W.,
and
Strong,
Volume Number
106 1
F. M.: Proc. Sot. Exper. Biol. & Med. 113: 262, 1963. 3. Jollie, W. P., and Bencosme, S. A.: Am. J. Anat. 116: 217. 1965. 4. Mossman, H. %‘.: Contrib. Embryol. 26: 129, 1937. 5. Orsini, M. W.: J. Reprod. & Fertil. 3: 283, 1962. 6. Orsini, M. W.: J. Reprod. & Fertil. 3: 288, 1962. 7. Orsini, M. W.: J. Endocrinol. 28: 119, 1963. M. W.: J. Reprod. & Fertil. 5: 323, 8. Orsini, 1963. 9. Orsini, M. W.: Morphological Evidence on the Intrauterine Career of the Ovum, in Enders, A. Cl., editor: Delayed Implantation,
Ultrastructure
10.
11. 12.
13. 14. 15. 16.
Chicago, Press, pp. Orsini, M. on Animal semination. Orsini. M. Orsinij M. stenholme, Stages of Churchill. Wynn, R: Wynn, R. 832, 1967. Wvnn. R. Wynn: R. 723, 1969.
of decidua
25
1963, The University of Chicago 155-170. W.: Fifth International Congress Reproduction and Artificial In7: 309, 1964. W.: 1. Anat. 99: 922. 1965. W.: “General Discussion, in WolG. E. W., editor: Preimplantation Pregnancy, London, 1965, J. A. Ltd.. no. 162-167. M.:‘FertiI. & Steril. 16: 16, 1965. M.: AM. J. OBST. & GYNEC. 97: M.: M.:
Obst. AM.
& Gvnec. J. 0;s~.
29: 644. &
GYN&
1967. 103: