Decidualisation in the Prepuberal Rat Uterus N. SANANES* and C. LE GOASCOGNE**
* Laboratoire de Physiologie de la Reproduction, Cnrs ER 122, HBpital de Bichre, F-94270 Kremlin-Bici%re, France.
** Unite de Recherches sur le Metabofisme Moleculaire et la Physio-Pathologie des Steroides, Inserm U 33, Hapital de Bicstre, F-94270 Kremlin-BicBtre, France.
Decidualisation has been experimentally induced in the prepuberal rat. It is morphologically similar to that obtained in the adult. The cytodifferentiation includes the formation of binucleated cells and extensive specialised cellular junctions. Furthermore, blastocysts from adults transferred into uterine horns of immature specimens implant and develop, indicating that the decidual response they induce is functionally comparable to the uterine response at the time of nidation in the fertilised adult. The model of the experimental decidual induction in the immature is used to study the ontogenic processes of uterine maturation. Preliminary observations indicate that the 7- 10-days period might be of significance in the maturation of the rat uterus. Introduction
Profound transformations of the uterus occur in response to the implanting embryo, especially in the endometrium where stromal cells differentiate into decidual cells. Such a decidual reaction can be experimentally induced in adult ovariectomised rats following suitable hormonal preparation. Although the sequence of the events leading to decidualisation is well-documented [ 11, their mechanism remains obscure. In order to get more insight into this transformation, application to the still sexually immature animal of the experimental procedure used in the adult appeared to offer the possibillity of studying (1) the transformation of already differentiated uterine stroma1 cells into decidual cells and (2) the chronology of events giving uterine cells progressively the capacity to respond to environmental (hormonal) stimuli by phenotypic changes. Results show that a typical decidual response can be induced in the 3-week-old rat and that there may be a critical period in the maturation process of the uterus at 7-10 days of age. Furthermore, blastocysts from adult rats, transferred into the uterine horns of 18-day immature animals, implant, develop and provoke a decidual response as in the adult. Methods Animals and Experimental Procedures. Twenty nine immature female rats of the Sprague Dawley strain were used. The experimental
Differentiation 5 , 133-144 (1976)
-
0 by Springer-Verlag 1976
procedure (Fig. 1) that gives the decidual response in the ovariectomised adult (data not shown) has been used. The hormonal preparation for endometrial sensitisation consists of 3 mg progesterone in 0.15 ml sesame oil daily from day 15 to 18, and 0.1 pg oestradiol in 0.1 ml sesame oil on day 18, both hormones being given subcutaneously. Induction of decidualisation is produced by intraluminal instillation of 0.1 ml of sesame oil into one horn on day 19, the other horn serving as control. To allow decidual growth, progesterone is continued daily until day 21. Nine animals were treated according to this procedure and served as controls (group I). Two other groups (Fig. 2) of 9 females each were submitted to the same experimental procedure but, in addition, received subcutaneously either 0.5 pg diethylstilboestrol (DES) in saline solution on days 8 and 9 (group 11), or 3 mg progesterone in sesame oil on days 7, 8 and 9 (group 111). The decidual response is estimated by the weight difference between the oil-instilled horn and the contralateral-control horn. Decidualisation was induced in two animals by blastocysts transferred on day 18 (Fig. 3). The blastocysts were collected by flushing 1 ml of Tyrode solution through horns of adult rats on the 5th day of pregnancy. Two blastocysts were introduced into one recipient horn with a micropipette through the utero-tuberal junction, while sesame oil was instilled in the other horn. Histology. Specimens were fixed in Bouin's solution, dehydrated through graded ethanol series and embedded in paraffin. Five-micron-thick sections were stained in Masson's Trichrome. Histochernistry. Pieces of horns were fixed overnight in ethanol 80% at 4 O C , then rapidly dehydrated through ethanol series, and after benzene impregnation, embedded in paraffin. Alkaline phosphatase activity is revealed on the 10 p sections by incubation for 2 h at 37' C with P-glycerophosphate, according to the Gomori calcium-cobalt method 121. Cytology and Electron Microscopy. Specimens were fixed in 1% osmium tetraoxide for 1 h at 4' C, dehydrated through ethanol series to propylene oxide and embedded in Epon 812.
134
ri P
17
-
o n e (+3 m g /da;
o g e+s t e
16
Days 15
N. Sananes and C. Le Goascogne:
18
19
7 21
20
Sesame oil intraluminatiy instilled ( 0.1 m l )
Hormonal sequence for sensitization of endometrium
8
7
Days
9
10
11
Autopsy
Decidual response
12
13
:I
14
16
15
19
20
t t t t t
Sesame oil intraluminally
- -
: Progesterone ( 3 mg/dayl
17
Autopsy
e I 3 [g/dayj-i
oge i te ro
16
. f A
Fig. 2. Procedure to compare the decidual response after hormonal pretreatment with DES or progesterone between days I and 9
; Control : Diethyl Stilboestrol (0.5pgiday)
rPi
Days 15
17 18
r o g e s t e r o n
-
JI JII
Fig. 1. Procedure for experimental induction of decidualisation
deciduomata
- -
I
22
18
1'
19
20
En' 01pg )
Transfer of 5 days o!d blastocyst from fertilized adult
21
22
I
Autopsy
One-micron-thick sections, cut on an LKB ultramicrotome, were stained in Giemsa for tight microscopy. Ultrathin sections stained in uranyl acetate and lead citrate were observed with a Siemens Elmiscop 101 electron microscope.
Results I . Decidualisation in the Immature Rat
Deciduoma in the 3 week old rat, hormonally prepared according to the sequence shown in Fig. 1, can be induced either by oil instillation in the uterine lumen or transfer of blastocysts from the adult (Fig. 3).
Fig. 3. Procedure for induction of decidualisation by
blastocysts transfer
1. The Decidual Response
When sesame oil is instilled, the decidual response is massive with a regular swelling of the horn throughout its whole length. The diameter of the horn is 4-fold that of the control (Figs. 4a, b), with an average increased weight of 350%. Nevertheless, the magnitude of response varies according to individuals (Fig. 16). Nearly 50% give such a generalised reaction, 20% form 2-4 nodules (increased weight 100-200%) and the remaining 309h give no response. When blastocysts are transferred, the decidual reaction is always restricted to the implantation sites forming nodules.
Decidualisation in the Prepuberal Rat Uterus
135
Fig. 4. Histological comparison between the oil-instilled decidualised horn (b) and the opposite noninstilled control horn (a) AM, antimesometrial; M , mesometrial area. x 25 Fig. 5. An enlargement of part of Fig. 4b to display the histological differencesbetween the mesometrial (M) and antimesometrial (AM) regions. The epithelium (e),still preserved in the mesometrial area where stromal cells are loosely distributed, is flattening and degenerating in contact with the differentiated and compact decidual cells of the A M region. - L, uterine lumen. x 150
136
Figs. 6 and 7. (Legends see page 137)
N. Sananes and C. Le Goascogne:
Decidualisation in thc Prepuberal Rat Uterus
a) Light Microscopy. Two regions can be clearly distinguished on cross sections: the mesometrial and the antimesometrial zones (Fig. 5). Although the transition between these zones is progressive, they show different characteristic patterns in regard to the luminal epithelium and the stromal cells. In the antimesometrial area, epithelial cells show signs of degeneration: they are exremely flat, with little cytoplasm-containing lipids surrounding a clear nucleus. The subjacent stromal cells (Fig. 6 ) are large, tightly packed, with almost no intercellular space and abundant dense basophilic cytoplasm in which frequent lipid droplets are easily identified. They are binucleated and most of the nuclei, which have a reticulated aspect, contain 2-4 prominent nucleoli frequently observed in close contact to the nuclear membrane. These characteristic features progressively disappear towards periphery, and, near the myometrium, cells have the ordinary morphology of fibroblasts. They are small and spindle-shaped, separated by large intercellular space, with only one clear nucleus. Mitotic figures are frequently observed in this area. In the mesometrial zone, the luminal epithelium retains its palissadic aspect with relatively high cells, rich in lipid droplets accumulated beneath the nucleus. Stroma1 cells remain small, mononucleated with prominent intercellular space. Since alkaline phosphatase activity is considered as a good indicator of decidualisation [31, the distribution of the enzyme was histochemically investigated. An intense reaction for alkaline phosphatase activity is noted throughout the stroma of the oil-instilled horn, being more pronounced in the antimesometrial area. No activity at all is found in the luminal epithelium. On the contrary, in the control horn, alkaline phosphatase activity is restricted to the surface epithelium and is absent from the stroma. b) Electron Microscopy. The decidual cells are characterised by two nuclei, specialised junctions between cytoplasmic membranes of adjacent cells and accumulation of filaments within the cytoplasm.
137 The subepithelial cells are in close contact and form a compact area. Numerous and extensive junctions (Fig. 10) of different types are observed: maculae adherentes and tight junctions in which the outer lamellae of the cytoplasmic membranes are fused (Fig. 11). Towards the periphery, cellular junctions are less numerous and of reduced size, while intercellular spaces still persist with bundles of collagen fibrils. In the cytoplasm, most organelles are perinuclear and the accumulation of filamentous material is peripheral (Fig. 7). Mitochondria are numerous and large, with distinct cristae and relatively clear matrix. The cisternae of the rough-surfaced endoplasmic reticulum are loaded with tiny fibrils and free polysomes are abundant (Fig. 8). The Golgi apparatus does not look very active. Lipid droplets with irregular contour are scattered among these cytoplasmic organelles. The filamentous areas (Fig. 9) are almost entirely devoid of organelles except a few polysomes. They are composed of packed filaments 100 A thick and of indefinite length running in different plane sections. Two equally large and regularly shaped nuclei separated by a thin layer of cytoplasm occupy the centre of the cell (Fig. 12). They show very small clumps of condensed chromatin disposed along the lamina densa lining the inner nuclear membrane and interrupted at the level of the nuclear pores. Condensed chromatin is also associated with the nucleoli, while in the remaining nucleoplasm, uncondensed chromatin is evenly distributed. Prominent nucleoli, frequently attached to the nuclear membrane (Fig. 13), are characterised by densely packed nucleolonemas and intermingled fibrillar and granular components. A structure that might be compared to the coiled body 141 is frequently observed, so that it may be considered as a common nuclear organelle in these cells (Fig. 12). It has a thread-like appearance and stains as densely as the nucleolonemas in the nucleoli. Its average size is 0.5 p. The coiled thread is formed of minute fibrils and, in the clear zone that surrounds it, chromatin fibres are observed, establishing connections between this organelle and the chromatin network (Fig. 14).
Fig. 6. Antimesometrial zone. On this semithin Epon section stained in Giemsa, most of the cells are binucleated with prominent nucleoli. Several mitosis indicated by arrows are observed among them. x 1000
2. Implantation and Differentiation of Transferred Blastocysts
Fig. 7. Electron micrograph of the same region as Fig. 6 showing the ultrastructural characteristics of the decidual cells. Cytoplasmic organelles are located around the 2 nuclei (N). The light peripheral zone is filled with fdamentous material v). Cells are connected by cytoplasmic junctions (arrows). Cb, coiled body; n, nucleolus. x 4800
Free blastocysts, collected from the uterine fluid of adult rats on the 5th day after fertilisation and transferred into the uterine lumen of 18-day-old immature rats, hormonally prepared as indicated in Fig. 3, implant and differentiate typically.
Figs. 8-11. (Legends see page 140)
Figs. 12-14. (Legends see page 140)
140
Four days after blastocyst transfer, the recipient uterine horns show nodules at the implantation sites corresponding to those observed in the uterus of adults on day 9 of normal pregnancy. Serial histological cross sections reveal that each swelling is the result of decidual growth induced by an individual blastocyst (Fig. 15). The characteristics of this decidual reaction are in every respect comparable to those described above. Implantation occurs eccentrically in an antimesometrial crypt, the uterine lumen is closed and the development of the embryo is similar to that in the adult pregnant rat. Besides the ectoplacental cone and the trophoblastic cells, primary ectoderm, endoderm and mesoderm are differentiated and limit four cavities: ectoplacental, exocoelomic, amniotic and yolk sac. The two endodermal sheets lining the yolk sac cavity are themselves already distinct: large proximal endoderm cells and distal endoderm composed of small scattered cells along the Reichert membrane. Outside and along this membrane, giant trophoblastic cells migrate in contact with the decidual cells. Intense mitotic activity is seen in the ectodermic region surrounding the amniotic cavity where the embryo is to be formed.
N. Sananes and C. Le Goascogne:
II. Alteration of Uterine Responsiveness The frequency and magnitude (increased weight) of the decidual response induced by oil in the 3-week immature rat as described above, are modified by hormonal treatments performed between days 7 and 9. The response is completely abolished by progesterone pretreatment, while DES seems to increase it. As seen in Fig. 16, no response is obtained after pretreatment with progesterone. After DES, it seems that the response is amplified since the frequency of generalised deciduoma or nodule formation is increased as compared to the controls (not treated between days 7 and 9). The action of DES is also indicated by the increased size of the decidual cells in the subepithelial zone. Electron microscopy reveals that the cytoplasmic organelles are more numerous than in the control, especially the rough-surfaced endoplasmic reticulum (Fig. 19). In addition to abundant filamentous material, glycogen ( a and3! , forms) is also accumulated (Fig. 18). Cytoplasmic junctions are particularly well developed (Fig. 17).
Discussion Fig. 8. Cytoplasmic organelles in decidual cells. Amidst the numerous mitochondria (M) and the well-developed rough-surfaced endoplasmic reticulum (ER),free polysomes (p), several lysosomes (Ly) and a few lipid droplets (Lz]are observed. filamentous material. x 12,000 Fig. 9. The filamentous peripheral area is formed by accumulation of 100 A thick tangled filaments. N , nucleus; p , polysomes. x 40,000 Fig. 10. Cytodifferentiation of specialised junctions. Notice the alternation of the tight junctions (TJ) and maculae adherentes (MA). x 40.000 Fig. 11. Detail of a tight junction (TJ) in between two maculae adherentes (MA). The pentalaminar aspect results from the fusion of the external leaflets of the plasma membranes. x 120,000 Fig. 12. Typical nucleus of a decidual cell. The chromatin is evenly
dispersed in the nucleoplasm except at the periphery where it forms small clumps of condensed chromatin (H, heterochromatin) along the lamina densa (L)lining the inner nuclear membrane. The nucleolus (n) is large. A coiled body (Cb)is present. Arrows point to cytoplasmic junctions. Collagen fibrils (c) are seen in the intercellular spaces. x 12,000 Fig. 13. A large nucleolus attached to the nuclear membrane. Its fibrillar (F)and granular (0 components are intermingled. Notice persistance of the lamina densa (L),H , heterochromatin. x 40,000 Fig. 14. A coiled body. The dense coiled thread structure seems to be formed by tiny fibrils. This organelle is connected by chromatin fibers that are easily visible in the clear surrounding zone (arrow)
Decidualisation, normally occurring in the adult rat at the time of blastocyst implantation, requires a hormonal preparation of the uterus by a well-established progesterone-oestradiol sequence 151. The decidual response can also be experimentally induced in the castrated adult rat by oil instillation in animals given progesterone and oestradiol in such a way as to reproduce the physiological hormonal sequence. The fact that it was possible to provoke decidualisation on day 19 indicates that the uterus, at this stage of development, is not only equipped with the hormone receptors but that the complex interactions of oestradiol and progesterone may also take place, as well as in adults, at the level of epithelial-stroma1 interrelationship. Indeed, although the mechanism of decidualisation is far from being completely understood, it has been shown in the castrated adult rat that the mitogenic action of oestradiol on the epithelium is redirected towards the stroma after preparation with progesterone [6, 7, 81. Prenidatory release of oestrogen is supposed to sensitize the epithelium to blastocyst contact. In fact, oil or other substances I91 intraluminally instilled cannot induce decidualisation unless the progesterone-prepared castrated adult is given oestradiol. On the other hand, traumatism of the uterine horn, with mechanical alteration of both epithelium and stroma, does not require oestrogen but
Decidualisation in the Prepuberal Rat Uterus
14 1
Fig. 15. Implanted embryo in the uterus of a 3-week-old rat. AM, antimesometrial zone; dE, distal endoderm; EC, ectoplacental cav-
ity; EM, embryo; EP, ectoplacental cone; EX, exocoelomic cavity; L, uterine lumen; pE, proximal endoderm; Rm,Reichert membrane; YS, yolk-sac cavity. x 125
only progesterone to provoke deciduoma formation as obtained also in the prepuberal rat [lo]. Thus it can be concluded that the uterus of the 3week-old rat is competent to give a well-integrated expression of growth and differentiation, similar to the mature organ, long before sexual maturity which is achieved between days 35 and 31. Another aspect of the stromal-epithelium interrela-
tionship is well illustrated in the course of evolution of epithelial cells in contact with the differentiating mesenchymal cells. In the mesometrial region of the uterus, the luminal epithelium retains its morphology as well as the stromal cells. On the contrary, in the antimesometrial zone, epithelial cells in contact with decidualising ones degenerate. This is a good example of a short-range interaction between different cell types and it could serve
142
N. Sananes and C. Le Goascogne
n
E t$
75-
m
C 0 P
v)
a,
U
350 >.
0 C a, 3 U
a,
25
0
--
Generalized
2-4 nodules ._
Magnitude of Responses Fig. 16. Comparison of the effects of progesterone (P)or diethylstilboestrol (DES) pretreatments on the decidual response. C , control not pretreated
as a model for the study of the importance of epithelialmesenchymal interactions that intervene in a variety of morphogenetic processes. A characeristic aspect of the decidualisation is the progressive spreading of stromal cell differentiation from the subepithelial zone towards periphery. This transformation comprises the cytodifferentiation of cellular junctions including tight junctions, where transfer of substances from cell to cell is supposed to take place, and junctions of the desmosome type (maculae adherentes), ensuring mechanical adhesion between cells. These contacts suggest some kind of close communication in the cell-to-cell interactions of which the functional aspect in this tissue is still a matter of speculation 1111. Differentiation of decidua involves the formation of binucleated and polyploid cells. It has been shown in the mouse that binucleated cells do not result from cell fusion 1123 and that in the rat, formation of binucleated cells precedes polyploidy [ 13 1. Binucleated cells are transformed into polyploid ones during mitosis by fusion at metaphase of chromosomes from the two simultaneously dividing nuclei. The significance of such an
evolution towards polyploidisation, which also occurs in the regenerating liver, remains obscure. Some hormonal-dependent ultrastructural modifications of the uterine epithelial cell nuclei occur in the prepuberal rat between days 8 and 10. The number of nuclear bodies [141 that appear at that time is increased by DES and reduced by progesterone (Le Goascogne, C. and Baulieu, E. E., unpubhshed). Correlation of these observations with several other hormonally related changes occurring at the same time, lead to the suggestion that the 8-10 day period might be critical in the maturation process of the uterus (Le Goascogne, C . and Baulieu, E. E., unpublished). If this were true, disturbance of this maturation could be responsible for altered responses of the organ to physiological solicitations at a later period of development. Results show that the decidual response under present experimental conditions is completely inhibited after progesterone injection on days 7, 8 and 9. Furthermore, it seems that DES treatment during the same period can induce a slight amplification of the decidual reaction. These results corroborate the assumption that the 8- 10 day period may be of particular significance in the maturation of the rat uterus. Implantation and development of the transferred blastocyst indicate that the decidual reaction obtained in the prepuberal rat, is morphologically comparable to that experimentally induced in the adult [151 and even functionally similar. In the present experiments, further development of the embryo and of the foetus has not been studied. Transfer of blastocysts to the immature offers new possibilities to investigate the mechanism of nidation. Acknowledgements: The authors are thankful to E.E. Baulieu and A. Psychoyos for continuous interest and critical reading of the manuscript. They are also grateful to Francine Delahaye for expert technical assistance, to Christiane Barrier for preparing the illustrations and to Daniele Prod’homme for the typing of the manuscript.
Figs. 17-19. Effects of DES injected on days 8 and 9, on the ultrastructure of decidual cells. Fig. 17. A large decidual cell with numerous cytoplasmic organelles and extremely well developed cellular junctions. x 4800 Fig. 18. Glycogen storage. The bulk of the glycogen is in the form of aggregates ( a rosettes) of the individual p particles. x 38,000 Fig. 19. Detail of the abundant rough endoplasmic reticulum (ER) and of the almost continuous cytoplasmic junctions. Li,lipid droplets; N , nucleus. x 16,000
Figs. 17-19. (Legends see page 142)
144
References 1. De Feo, V. J.: In: Cellular Biology of the Uterus, Wynn, R. M. (ed.), p. 191. Amsterdam: North Holland Publishing Co. 1967 2. Gomori, G.: In: Microscopic Histochernistry. Univ. Chicago Press, 1952 3. Finn, C. A., Hinchliffe, J. R.: J. Reprod. Fertil. 8, 331, 1964 4. Monneron, A., Bernhard, W.: J. Ultrastruct. Res. 21, 266, 1969 5. Psychoyos, A.: In: Vitamins and Hormones. Hams, R. S., Munson, P. L., Diczfalusy, E., Glover, J. (eds.), Vol. XXXI, p. 201. New York: Academic Press 1973 6.Martin, L., Finn, C. A.: J. Endocr. 41, 363, 1968 7.Clark, B. F.: J. Endocr. 50, 527, 1971
N. Sananes and C. Le Goascogne
8. Tachi, C., Tachi, S., Lindner, €1. R.: J. Reprod. Fertil. 31, 59, 1972 9. Psychoyos, A.: In: Adv. Reprod. Physiol., McLaren, A. (ed.), Vol. 11, p. 257, London: Logos Press 1967 10. Madjerek, Z. S.: Acta Morphol. Neerl. Scand. 10, 259, 1972 1 1 . Finn, C. A., Lawn, A. M.: J. Ultrastruct. Res. 20, 321, 1967 12. Ansell, J. D., Barlow, P. W., McLaren, A.: J. Ernbryol. exp. Morph. 31, 223, 1974 13. Dupont, H., Duluc, A. J., Mayer, G.: C. R . Acad. Sci. (Paris) 212, 2360, 1971 14. Dupuy-Coin, A. M., Kalifat, S. R., Bouteille, M.: J. Ultrastruct. Res. 38, 174, 1972 15. Jollie, P. W., Bencosme, S . A,: Am. J. Anat. 116, 217, 1965
* Laboratoire de Physiologie de la Reproduction, Cnrs ER 122, HBpital de Bichre, F-94270 Kremlin-Bici%re, France.
** Unite de Recherches sur le Metabofisme Moleculaire et la Physio-Pathologie des Steroides, Inserm U 33, Hapital de Bicstre, F-94270 Kremlin-BicBtre, France.
Decidualisation has been experimentally induced in the prepuberal rat. It is morphologically similar to that obtained in the adult. The cytodifferentiation includes the formation of binucleated cells and extensive specialised cellular junctions. Furthermore, blastocysts from adults transferred into uterine horns of immature specimens implant and develop, indicating that the decidual response they induce is functionally comparable to the uterine response at the time of nidation in the fertilised adult. The model of the experimental decidual induction in the immature is used to study the ontogenic processes of uterine maturation. Preliminary observations indicate that the 7- 10-days period might be of significance in the maturation of the rat uterus. Introduction
Profound transformations of the uterus occur in response to the implanting embryo, especially in the endometrium where stromal cells differentiate into decidual cells. Such a decidual reaction can be experimentally induced in adult ovariectomised rats following suitable hormonal preparation. Although the sequence of the events leading to decidualisation is well-documented [ 11, their mechanism remains obscure. In order to get more insight into this transformation, application to the still sexually immature animal of the experimental procedure used in the adult appeared to offer the possibillity of studying (1) the transformation of already differentiated uterine stroma1 cells into decidual cells and (2) the chronology of events giving uterine cells progressively the capacity to respond to environmental (hormonal) stimuli by phenotypic changes. Results show that a typical decidual response can be induced in the 3-week-old rat and that there may be a critical period in the maturation process of the uterus at 7-10 days of age. Furthermore, blastocysts from adult rats, transferred into the uterine horns of 18-day immature animals, implant, develop and provoke a decidual response as in the adult. Methods Animals and Experimental Procedures. Twenty nine immature female rats of the Sprague Dawley strain were used. The experimental
Differentiation 5 , 133-144 (1976)
-
0 by Springer-Verlag 1976
procedure (Fig. 1) that gives the decidual response in the ovariectomised adult (data not shown) has been used. The hormonal preparation for endometrial sensitisation consists of 3 mg progesterone in 0.15 ml sesame oil daily from day 15 to 18, and 0.1 pg oestradiol in 0.1 ml sesame oil on day 18, both hormones being given subcutaneously. Induction of decidualisation is produced by intraluminal instillation of 0.1 ml of sesame oil into one horn on day 19, the other horn serving as control. To allow decidual growth, progesterone is continued daily until day 21. Nine animals were treated according to this procedure and served as controls (group I). Two other groups (Fig. 2) of 9 females each were submitted to the same experimental procedure but, in addition, received subcutaneously either 0.5 pg diethylstilboestrol (DES) in saline solution on days 8 and 9 (group 11), or 3 mg progesterone in sesame oil on days 7, 8 and 9 (group 111). The decidual response is estimated by the weight difference between the oil-instilled horn and the contralateral-control horn. Decidualisation was induced in two animals by blastocysts transferred on day 18 (Fig. 3). The blastocysts were collected by flushing 1 ml of Tyrode solution through horns of adult rats on the 5th day of pregnancy. Two blastocysts were introduced into one recipient horn with a micropipette through the utero-tuberal junction, while sesame oil was instilled in the other horn. Histology. Specimens were fixed in Bouin's solution, dehydrated through graded ethanol series and embedded in paraffin. Five-micron-thick sections were stained in Masson's Trichrome. Histochernistry. Pieces of horns were fixed overnight in ethanol 80% at 4 O C , then rapidly dehydrated through ethanol series, and after benzene impregnation, embedded in paraffin. Alkaline phosphatase activity is revealed on the 10 p sections by incubation for 2 h at 37' C with P-glycerophosphate, according to the Gomori calcium-cobalt method 121. Cytology and Electron Microscopy. Specimens were fixed in 1% osmium tetraoxide for 1 h at 4' C, dehydrated through ethanol series to propylene oxide and embedded in Epon 812.
134
ri P
17
-
o n e (+3 m g /da;
o g e+s t e
16
Days 15
N. Sananes and C. Le Goascogne:
18
19
7 21
20
Sesame oil intraluminatiy instilled ( 0.1 m l )
Hormonal sequence for sensitization of endometrium
8
7
Days
9
10
11
Autopsy
Decidual response
12
13
:I
14
16
15
19
20
t t t t t
Sesame oil intraluminally
- -
: Progesterone ( 3 mg/dayl
17
Autopsy
e I 3 [g/dayj-i
oge i te ro
16
. f A
Fig. 2. Procedure to compare the decidual response after hormonal pretreatment with DES or progesterone between days I and 9
; Control : Diethyl Stilboestrol (0.5pgiday)
rPi
Days 15
17 18
r o g e s t e r o n
-
JI JII
Fig. 1. Procedure for experimental induction of decidualisation
deciduomata
- -
I
22
18
1'
19
20
En' 01pg )
Transfer of 5 days o!d blastocyst from fertilized adult
21
22
I
Autopsy
One-micron-thick sections, cut on an LKB ultramicrotome, were stained in Giemsa for tight microscopy. Ultrathin sections stained in uranyl acetate and lead citrate were observed with a Siemens Elmiscop 101 electron microscope.
Results I . Decidualisation in the Immature Rat
Deciduoma in the 3 week old rat, hormonally prepared according to the sequence shown in Fig. 1, can be induced either by oil instillation in the uterine lumen or transfer of blastocysts from the adult (Fig. 3).
Fig. 3. Procedure for induction of decidualisation by
blastocysts transfer
1. The Decidual Response
When sesame oil is instilled, the decidual response is massive with a regular swelling of the horn throughout its whole length. The diameter of the horn is 4-fold that of the control (Figs. 4a, b), with an average increased weight of 350%. Nevertheless, the magnitude of response varies according to individuals (Fig. 16). Nearly 50% give such a generalised reaction, 20% form 2-4 nodules (increased weight 100-200%) and the remaining 309h give no response. When blastocysts are transferred, the decidual reaction is always restricted to the implantation sites forming nodules.
Decidualisation in the Prepuberal Rat Uterus
135
Fig. 4. Histological comparison between the oil-instilled decidualised horn (b) and the opposite noninstilled control horn (a) AM, antimesometrial; M , mesometrial area. x 25 Fig. 5. An enlargement of part of Fig. 4b to display the histological differencesbetween the mesometrial (M) and antimesometrial (AM) regions. The epithelium (e),still preserved in the mesometrial area where stromal cells are loosely distributed, is flattening and degenerating in contact with the differentiated and compact decidual cells of the A M region. - L, uterine lumen. x 150
136
Figs. 6 and 7. (Legends see page 137)
N. Sananes and C. Le Goascogne:
Decidualisation in thc Prepuberal Rat Uterus
a) Light Microscopy. Two regions can be clearly distinguished on cross sections: the mesometrial and the antimesometrial zones (Fig. 5). Although the transition between these zones is progressive, they show different characteristic patterns in regard to the luminal epithelium and the stromal cells. In the antimesometrial area, epithelial cells show signs of degeneration: they are exremely flat, with little cytoplasm-containing lipids surrounding a clear nucleus. The subjacent stromal cells (Fig. 6 ) are large, tightly packed, with almost no intercellular space and abundant dense basophilic cytoplasm in which frequent lipid droplets are easily identified. They are binucleated and most of the nuclei, which have a reticulated aspect, contain 2-4 prominent nucleoli frequently observed in close contact to the nuclear membrane. These characteristic features progressively disappear towards periphery, and, near the myometrium, cells have the ordinary morphology of fibroblasts. They are small and spindle-shaped, separated by large intercellular space, with only one clear nucleus. Mitotic figures are frequently observed in this area. In the mesometrial zone, the luminal epithelium retains its palissadic aspect with relatively high cells, rich in lipid droplets accumulated beneath the nucleus. Stroma1 cells remain small, mononucleated with prominent intercellular space. Since alkaline phosphatase activity is considered as a good indicator of decidualisation [31, the distribution of the enzyme was histochemically investigated. An intense reaction for alkaline phosphatase activity is noted throughout the stroma of the oil-instilled horn, being more pronounced in the antimesometrial area. No activity at all is found in the luminal epithelium. On the contrary, in the control horn, alkaline phosphatase activity is restricted to the surface epithelium and is absent from the stroma. b) Electron Microscopy. The decidual cells are characterised by two nuclei, specialised junctions between cytoplasmic membranes of adjacent cells and accumulation of filaments within the cytoplasm.
137 The subepithelial cells are in close contact and form a compact area. Numerous and extensive junctions (Fig. 10) of different types are observed: maculae adherentes and tight junctions in which the outer lamellae of the cytoplasmic membranes are fused (Fig. 11). Towards the periphery, cellular junctions are less numerous and of reduced size, while intercellular spaces still persist with bundles of collagen fibrils. In the cytoplasm, most organelles are perinuclear and the accumulation of filamentous material is peripheral (Fig. 7). Mitochondria are numerous and large, with distinct cristae and relatively clear matrix. The cisternae of the rough-surfaced endoplasmic reticulum are loaded with tiny fibrils and free polysomes are abundant (Fig. 8). The Golgi apparatus does not look very active. Lipid droplets with irregular contour are scattered among these cytoplasmic organelles. The filamentous areas (Fig. 9) are almost entirely devoid of organelles except a few polysomes. They are composed of packed filaments 100 A thick and of indefinite length running in different plane sections. Two equally large and regularly shaped nuclei separated by a thin layer of cytoplasm occupy the centre of the cell (Fig. 12). They show very small clumps of condensed chromatin disposed along the lamina densa lining the inner nuclear membrane and interrupted at the level of the nuclear pores. Condensed chromatin is also associated with the nucleoli, while in the remaining nucleoplasm, uncondensed chromatin is evenly distributed. Prominent nucleoli, frequently attached to the nuclear membrane (Fig. 13), are characterised by densely packed nucleolonemas and intermingled fibrillar and granular components. A structure that might be compared to the coiled body 141 is frequently observed, so that it may be considered as a common nuclear organelle in these cells (Fig. 12). It has a thread-like appearance and stains as densely as the nucleolonemas in the nucleoli. Its average size is 0.5 p. The coiled thread is formed of minute fibrils and, in the clear zone that surrounds it, chromatin fibres are observed, establishing connections between this organelle and the chromatin network (Fig. 14).
Fig. 6. Antimesometrial zone. On this semithin Epon section stained in Giemsa, most of the cells are binucleated with prominent nucleoli. Several mitosis indicated by arrows are observed among them. x 1000
2. Implantation and Differentiation of Transferred Blastocysts
Fig. 7. Electron micrograph of the same region as Fig. 6 showing the ultrastructural characteristics of the decidual cells. Cytoplasmic organelles are located around the 2 nuclei (N). The light peripheral zone is filled with fdamentous material v). Cells are connected by cytoplasmic junctions (arrows). Cb, coiled body; n, nucleolus. x 4800
Free blastocysts, collected from the uterine fluid of adult rats on the 5th day after fertilisation and transferred into the uterine lumen of 18-day-old immature rats, hormonally prepared as indicated in Fig. 3, implant and differentiate typically.
Figs. 8-11. (Legends see page 140)
Figs. 12-14. (Legends see page 140)
140
Four days after blastocyst transfer, the recipient uterine horns show nodules at the implantation sites corresponding to those observed in the uterus of adults on day 9 of normal pregnancy. Serial histological cross sections reveal that each swelling is the result of decidual growth induced by an individual blastocyst (Fig. 15). The characteristics of this decidual reaction are in every respect comparable to those described above. Implantation occurs eccentrically in an antimesometrial crypt, the uterine lumen is closed and the development of the embryo is similar to that in the adult pregnant rat. Besides the ectoplacental cone and the trophoblastic cells, primary ectoderm, endoderm and mesoderm are differentiated and limit four cavities: ectoplacental, exocoelomic, amniotic and yolk sac. The two endodermal sheets lining the yolk sac cavity are themselves already distinct: large proximal endoderm cells and distal endoderm composed of small scattered cells along the Reichert membrane. Outside and along this membrane, giant trophoblastic cells migrate in contact with the decidual cells. Intense mitotic activity is seen in the ectodermic region surrounding the amniotic cavity where the embryo is to be formed.
N. Sananes and C. Le Goascogne:
II. Alteration of Uterine Responsiveness The frequency and magnitude (increased weight) of the decidual response induced by oil in the 3-week immature rat as described above, are modified by hormonal treatments performed between days 7 and 9. The response is completely abolished by progesterone pretreatment, while DES seems to increase it. As seen in Fig. 16, no response is obtained after pretreatment with progesterone. After DES, it seems that the response is amplified since the frequency of generalised deciduoma or nodule formation is increased as compared to the controls (not treated between days 7 and 9). The action of DES is also indicated by the increased size of the decidual cells in the subepithelial zone. Electron microscopy reveals that the cytoplasmic organelles are more numerous than in the control, especially the rough-surfaced endoplasmic reticulum (Fig. 19). In addition to abundant filamentous material, glycogen ( a and3! , forms) is also accumulated (Fig. 18). Cytoplasmic junctions are particularly well developed (Fig. 17).
Discussion Fig. 8. Cytoplasmic organelles in decidual cells. Amidst the numerous mitochondria (M) and the well-developed rough-surfaced endoplasmic reticulum (ER),free polysomes (p), several lysosomes (Ly) and a few lipid droplets (Lz]are observed. filamentous material. x 12,000 Fig. 9. The filamentous peripheral area is formed by accumulation of 100 A thick tangled filaments. N , nucleus; p , polysomes. x 40,000 Fig. 10. Cytodifferentiation of specialised junctions. Notice the alternation of the tight junctions (TJ) and maculae adherentes (MA). x 40.000 Fig. 11. Detail of a tight junction (TJ) in between two maculae adherentes (MA). The pentalaminar aspect results from the fusion of the external leaflets of the plasma membranes. x 120,000 Fig. 12. Typical nucleus of a decidual cell. The chromatin is evenly
dispersed in the nucleoplasm except at the periphery where it forms small clumps of condensed chromatin (H, heterochromatin) along the lamina densa (L)lining the inner nuclear membrane. The nucleolus (n) is large. A coiled body (Cb)is present. Arrows point to cytoplasmic junctions. Collagen fibrils (c) are seen in the intercellular spaces. x 12,000 Fig. 13. A large nucleolus attached to the nuclear membrane. Its fibrillar (F)and granular (0 components are intermingled. Notice persistance of the lamina densa (L),H , heterochromatin. x 40,000 Fig. 14. A coiled body. The dense coiled thread structure seems to be formed by tiny fibrils. This organelle is connected by chromatin fibers that are easily visible in the clear surrounding zone (arrow)
Decidualisation, normally occurring in the adult rat at the time of blastocyst implantation, requires a hormonal preparation of the uterus by a well-established progesterone-oestradiol sequence 151. The decidual response can also be experimentally induced in the castrated adult rat by oil instillation in animals given progesterone and oestradiol in such a way as to reproduce the physiological hormonal sequence. The fact that it was possible to provoke decidualisation on day 19 indicates that the uterus, at this stage of development, is not only equipped with the hormone receptors but that the complex interactions of oestradiol and progesterone may also take place, as well as in adults, at the level of epithelial-stroma1 interrelationship. Indeed, although the mechanism of decidualisation is far from being completely understood, it has been shown in the castrated adult rat that the mitogenic action of oestradiol on the epithelium is redirected towards the stroma after preparation with progesterone [6, 7, 81. Prenidatory release of oestrogen is supposed to sensitize the epithelium to blastocyst contact. In fact, oil or other substances I91 intraluminally instilled cannot induce decidualisation unless the progesterone-prepared castrated adult is given oestradiol. On the other hand, traumatism of the uterine horn, with mechanical alteration of both epithelium and stroma, does not require oestrogen but
Decidualisation in the Prepuberal Rat Uterus
14 1
Fig. 15. Implanted embryo in the uterus of a 3-week-old rat. AM, antimesometrial zone; dE, distal endoderm; EC, ectoplacental cav-
ity; EM, embryo; EP, ectoplacental cone; EX, exocoelomic cavity; L, uterine lumen; pE, proximal endoderm; Rm,Reichert membrane; YS, yolk-sac cavity. x 125
only progesterone to provoke deciduoma formation as obtained also in the prepuberal rat [lo]. Thus it can be concluded that the uterus of the 3week-old rat is competent to give a well-integrated expression of growth and differentiation, similar to the mature organ, long before sexual maturity which is achieved between days 35 and 31. Another aspect of the stromal-epithelium interrela-
tionship is well illustrated in the course of evolution of epithelial cells in contact with the differentiating mesenchymal cells. In the mesometrial region of the uterus, the luminal epithelium retains its morphology as well as the stromal cells. On the contrary, in the antimesometrial zone, epithelial cells in contact with decidualising ones degenerate. This is a good example of a short-range interaction between different cell types and it could serve
142
N. Sananes and C. Le Goascogne
n
E t$
75-
m
C 0 P
v)
a,
U
350 >.
0 C a, 3 U
a,
25
0
--
Generalized
2-4 nodules ._
Magnitude of Responses Fig. 16. Comparison of the effects of progesterone (P)or diethylstilboestrol (DES) pretreatments on the decidual response. C , control not pretreated
as a model for the study of the importance of epithelialmesenchymal interactions that intervene in a variety of morphogenetic processes. A characeristic aspect of the decidualisation is the progressive spreading of stromal cell differentiation from the subepithelial zone towards periphery. This transformation comprises the cytodifferentiation of cellular junctions including tight junctions, where transfer of substances from cell to cell is supposed to take place, and junctions of the desmosome type (maculae adherentes), ensuring mechanical adhesion between cells. These contacts suggest some kind of close communication in the cell-to-cell interactions of which the functional aspect in this tissue is still a matter of speculation 1111. Differentiation of decidua involves the formation of binucleated and polyploid cells. It has been shown in the mouse that binucleated cells do not result from cell fusion 1123 and that in the rat, formation of binucleated cells precedes polyploidy [ 13 1. Binucleated cells are transformed into polyploid ones during mitosis by fusion at metaphase of chromosomes from the two simultaneously dividing nuclei. The significance of such an
evolution towards polyploidisation, which also occurs in the regenerating liver, remains obscure. Some hormonal-dependent ultrastructural modifications of the uterine epithelial cell nuclei occur in the prepuberal rat between days 8 and 10. The number of nuclear bodies [141 that appear at that time is increased by DES and reduced by progesterone (Le Goascogne, C. and Baulieu, E. E., unpubhshed). Correlation of these observations with several other hormonally related changes occurring at the same time, lead to the suggestion that the 8-10 day period might be critical in the maturation process of the uterus (Le Goascogne, C . and Baulieu, E. E., unpublished). If this were true, disturbance of this maturation could be responsible for altered responses of the organ to physiological solicitations at a later period of development. Results show that the decidual response under present experimental conditions is completely inhibited after progesterone injection on days 7, 8 and 9. Furthermore, it seems that DES treatment during the same period can induce a slight amplification of the decidual reaction. These results corroborate the assumption that the 8- 10 day period may be of particular significance in the maturation of the rat uterus. Implantation and development of the transferred blastocyst indicate that the decidual reaction obtained in the prepuberal rat, is morphologically comparable to that experimentally induced in the adult [151 and even functionally similar. In the present experiments, further development of the embryo and of the foetus has not been studied. Transfer of blastocysts to the immature offers new possibilities to investigate the mechanism of nidation. Acknowledgements: The authors are thankful to E.E. Baulieu and A. Psychoyos for continuous interest and critical reading of the manuscript. They are also grateful to Francine Delahaye for expert technical assistance, to Christiane Barrier for preparing the illustrations and to Daniele Prod’homme for the typing of the manuscript.
Figs. 17-19. Effects of DES injected on days 8 and 9, on the ultrastructure of decidual cells. Fig. 17. A large decidual cell with numerous cytoplasmic organelles and extremely well developed cellular junctions. x 4800 Fig. 18. Glycogen storage. The bulk of the glycogen is in the form of aggregates ( a rosettes) of the individual p particles. x 38,000 Fig. 19. Detail of the abundant rough endoplasmic reticulum (ER) and of the almost continuous cytoplasmic junctions. Li,lipid droplets; N , nucleus. x 16,000
Figs. 17-19. (Legends see page 142)
144
References 1. De Feo, V. J.: In: Cellular Biology of the Uterus, Wynn, R. M. (ed.), p. 191. Amsterdam: North Holland Publishing Co. 1967 2. Gomori, G.: In: Microscopic Histochernistry. Univ. Chicago Press, 1952 3. Finn, C. A., Hinchliffe, J. R.: J. Reprod. Fertil. 8, 331, 1964 4. Monneron, A., Bernhard, W.: J. Ultrastruct. Res. 21, 266, 1969 5. Psychoyos, A.: In: Vitamins and Hormones. Hams, R. S., Munson, P. L., Diczfalusy, E., Glover, J. (eds.), Vol. XXXI, p. 201. New York: Academic Press 1973 6.Martin, L., Finn, C. A.: J. Endocr. 41, 363, 1968 7.Clark, B. F.: J. Endocr. 50, 527, 1971
N. Sananes and C. Le Goascogne
8. Tachi, C., Tachi, S., Lindner, €1. R.: J. Reprod. Fertil. 31, 59, 1972 9. Psychoyos, A.: In: Adv. Reprod. Physiol., McLaren, A. (ed.), Vol. 11, p. 257, London: Logos Press 1967 10. Madjerek, Z. S.: Acta Morphol. Neerl. Scand. 10, 259, 1972 1 1 . Finn, C. A., Lawn, A. M.: J. Ultrastruct. Res. 20, 321, 1967 12. Ansell, J. D., Barlow, P. W., McLaren, A.: J. Ernbryol. exp. Morph. 31, 223, 1974 13. Dupont, H., Duluc, A. J., Mayer, G.: C. R . Acad. Sci. (Paris) 212, 2360, 1971 14. Dupuy-Coin, A. M., Kalifat, S. R., Bouteille, M.: J. Ultrastruct. Res. 38, 174, 1972 15. Jollie, P. W., Bencosme, S . A,: Am. J. Anat. 116, 217, 1965
