0013-7227/78/1031-0086$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 1 Printed in U.S.A.
In Vitro Decidualization of Rat Endometrial Cells* NICOLE SANANES, SYLVAIN WEILLER, ETIENNE-EMILE BAULIEU, AND CLAUDE LE GOASCOGNE Unite de Recherches sur le Metabolisme Moleculaire et la Physiopathologie des Steroides de I'Institut National de la Sante et de la Recherche Medicale, Departement de Chimie Biologique, Faculte de Medecine de Bicetre, 78 avenue du General Leclerc, 94270 Bicetre, France be a prerequisite for such development and transformation of endometrial cells in vitro, since control experiments performed with nonhormonally injected castrated animals gave only isolated islets of epithelial cells and scattered fibroblast-like cells in culture, whereas the decidual transformation was still observed, although with some delay, with cells from progesterone (but not estradiol)-treated spayed rats. Such an in vitro system may provide a new opportunity to study the mechanism of decidualization and of its hormonal control. (Endocrinology 103: 86, 1978)
ABSTRACT. Rat endometrial cells were isolated from ovariectomized adults submitted to a progesterone-estradiol sequence proper to sensitize the uterus to respond to a deciduogenic stimulus, and then cultured in vitro in the presence of progesterone. They grew rapidly and became morphologically similar to the decidual cells occurring in vivo with regard to the binucleation and the accumulation of characteristic filamentous material in the cytoplasm. The life-span of the decidualized cells in vitro was comparable to the duration of the deciduoma in vivo. The progesterone treatment of the rat was found to
I
had a life-span limited to the duration of the deciduoma in vivo.
N THE RAT, at the time of blastocyst implantation, the endometrial cells undergo profound modifications and the stromal cells differentiate into decidual cells. These changes can be experimentally reproduced provided the animals are submitted to a precise hormonal sequence and to uterine stimulation. The process may be divided into three successive phases: sensitization, induction, and response (1-3). Although a proper hormonal sequence has been well described (4, 5), the mechanism of decidualization is not yet understood despite recent findings concerning the involvement of prostaglandin(s) (6). In order to study the hormonally controlled differentiation of decidual cells, a cell culture technique was applied. The preliminary results, reported here, show that the endometrial cells, hormonally sensitized in vivo, grew well in culture, acquired several of the characteristic features of the decidual cells, lacked contact inhibition, and
Materials and Methods Preparation of animals Five-month-old female rats of the Sprague-Dawley strain were ovariectomized under ether anesthesia 15 days before starting experiments. One group of animals received progesterone 5 mg daily for 4 consecutive days and estradiol 0.15 /xg on the 4th day, both hormones being given sc in 0.25 and 0.15 ml sesame oil, respectively. A second group of animals was treated only with progesterone and a third group (control) did not receive hormones. Animals were killed by bleeding under ether anesthesia 18 h after estradiol injection (first group), 18 h after the last progesterone injection (second group), or 20 days after ovariectomy (third group). Preparation of dispersed cells For each culture, uterine horns of three rats were dissected under sterile conditions, cleared of fat, and dipped into 10 ml phosphate-buffered saline, 0.2 M, pH 7.4, containing progesterone 0.5 JUM (PBS). They were rinsed three times in 10 ml PBS and subsequently incubated 5 min in trypsin (0.25 g trypsin in 100 ml PBS). After removing most of the liquid, the horns were thoroughly minced with iridectomy scissors. The fragments, suspended in 5
Received July 7, 1977. Requests for reprints should be addressed to: Nicole Sananes, Lab. Hormone, Universite Paris-Sud, 94-Bicetre, France. * Partial support for this study has been obtained from the Ford Foundation, WHO, DGRST, and the Faculte de Medecine Paris-Sud.
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IN VITRO DECIDUALIZATION ml trypsin, were transferred into an Erlenmeyer flask containing 25 ml trypsin and agitated by pipetting at room temperature. They were then incubated for 45 min with continuous magnetic stirring at 37 C under an atmosphere of 5% CO2 in air. The suspension was again pipetted, then left undisturbed until the largest fragments had sedimented. Twenty milliliters of the supernatant were removed and equally distributed into two plastic tubes containing each 0.15 ml fetal calf serum (Gibco) to stop the trypsin action. After 3 min at low speed centrifugation, the pellets were resuspended in 5 ml culture medium and kept at room temperature. Twenty milliliters of trypsin were added into the Erlenmeyer flask, the fragments were again pipetted, and the procedure described above was repeated twice more. Fragments still remaining in the flask were trypsinized once more by adding 2 ml of a 2.5% trypsin solution and incubation was carried out with magnetic stirring for 15 min. Thereafter, the suspension was centrifuged and the pellet was resuspended in 5 ml culture medium. All of the suspensions were pooled and filtered through three layers of sterile gauze into a 50-ml beaker. The volume was adjusted with culture medium in order to obtain approximately 5.105 cells/ml and 25 cm2 dishes (Falcon Plastics) were plated with 5 ml cell suspension. Culture medium The culture medium was Dulbecco modified Eagle medium (DME), supplemented with 20% heatinactivated calf serum (Gibco), plus antibiotics (penicillin, 100 U/ml; streptomycin, 100 jug/ml; nalidixic acid, 40 /ig/ml), and progesterone 0.5 /iM. The cultures were fed with fresh medium after 24 and 48 h, and then twice a week. Light and electron microscopy Living cells were observed and photographed with the phase contrast microscope. Cells, after different periods of culture, were rinsed in PBS and fixed in osmium tetroxide for 30 min. Some samples were stained with Giemsa and observed under light microscope while others were dehydrated through graded ethanol series and in situ embedded in Epon. Blocks were stained with toluidine blue in order to select areas of interest for preparing the ultrathin sections with Ultratome III from LKB. The sections were stained with uranyl acetate and lead citrate and observed in a Siemens Elmiskop 101. Several specimens were reembedded
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in Epon in order to cut sections perpendicularly to the cellular layers.
Results Culture of uterine cells from progesteroneestradioI- treated spayed rats Cells in culture. Within 1 h after seeding, most cells were attached to the substratum. At 24 h (Fig. la), the flasks were partially covered with an homogeneous population of well spread, fibroblast-like cells, frequently showing mitotic figures. After 48 h of culture (Fig. lb), the cells were larger, much more numerous, and very little intercellular space was left over; many cells were binucleated. There was no contact inhibition and several layers of cells were overlapping (Fig. le). At 72 h, piling and criss-crossing of the cells were extremely well marked (Fig. Id), and in the less densely plated zones, binucleated cells were abundant and mitotic figures were observed (Fig. lc). The general aspect of the culture observed at 72 h (Fig. 2a) remained identical until day 6 when some cells showed signs of degeneration. On day 13 (Fig. 2b), a large number of cells had died and they formed dense scattered clusters. Involution of culture was complete after 4 weeks (Fig. 2c). Ultrastructure. Electron microscopic analysis confirmed that binucleated cells observed in light microscopy (Fig. 3a) did possess two distinct nuclei (Fig. 3b). The nuclei were large, averaging 15 /xm in diameter. At 72 h, their size varied from one cell to another up to 30 jum. The chromatin was regularly dispersed. The nucleoli, which were densely stained with Giemsa (Fig. 3a), appeared as formed by the intermingled fibrillar and granular components, the latter prevailing (Figs. 3b and 5c). The number and size of nucleoli varied from cell to cell, but often were identical in the two nuclei of a same cell. One to eight nucleoli per nucleus were observed; the less numerous, the larger they were. The Golgi apparatus (Fig. 4a) and the rough endoplasmic reticulum (Fig. 4b) were well developed. Inside cisternae of the rough endoplasmic reticulum, a flocculent material accu-
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FIG. 1. Culture of endometrial cells from progesterone-estradiol-treated spayed rats and differentiation into decidual cells. Phase contrast micrographs of cells after 24 h (a), 48 h (b), and 72 h (c and d). Arrows point to binucleated cells (X115). At 72 h, the flask is irregularly covered with thick zones of several layers of cells (d) indicating lack of contact inhibition, and with zones of less density (c) where mitosis (m) and binucleated cells are easily identified, (e) Electron micrograph of cells cut perpendicularly to the surface attachment showing at 48 h the already superimposed cells: 1, 2, 3, 4 from bottom to top. N, nucleus (X8.700).
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FIG. 2. General aspect of endometrial cell cultures from progesterone-estradiol treated spayed rats at various times after seeding. Giemsa staining (x5). (a) 3-day culture showing the complete covering of the flask. Light areas correspond to Fig. lc and dark zones to Fig. Id. (b) 13-day culture: many dead cells are packed in dark clusters; living cells become less and less numerous and the areas devoid of cells are enlarging, (c) 28-day culture: regression is complete.
mulated. On their outer surface, numerous polysomes were attached and showed the particular configuration of cochleosomes (Fig. 4c). Large mitochondria, up to 2 jum in diameter, had cristae and a clear matrix. Scattered lipid droplets and lysosomes were also observed. In addition, large amounts of a filamentous material accumulated in the cytoplasm (Fig. 5a). The filaments were about 10 nm thick (Fig. 5b). Only a few polysomes were observed amidst the intermingled filaments. Culture of uterine cells from progesteronetreated spayed rats The same differentiation of the endometrial cells occurred with the above mentioned characteristics. However, the developmental process was slower and at 48 h of culture, the cells were less numerous when compared to those of the progesterone-estradiol-treated group and only a few of them were binucleated. On the 5th day after seeding, the development of the culture became similar to what was ob-
served after 72 h with cells from progesteroneestradiol-pretreated rats. Culture of uterine cells from untreated spayed rats Cells in culture. Contrary to endometrial cells from hormone-treated animals, uterine cells from untreated castrated rats were still floating in the culture medium 3 h after plating. Between 24 and 72 h (Fig. 6a), two types of cells were observed, epithelial cells in sparse islets and dispersed fibroblast-like elements, either of spindle or irregular stellate shape (Fig. 6b). The islets of epithelial cells were composed of 10-100 polygonal cells forming a continuous mosaic-like sheet (Fig. 6, c and d). Their nucleus contained one to four nucleoli, smaller when their number increased. These cultures were grown for 13 days and no decidual differentiation was observed. Ultrastructure. At 72 h, the cells of the islets looked like the uterine epithelial cells of
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FIG. 3. Main features of decidualized cells in the 72-h culture, (a) Light micrograph of cells fixed with osmium tetroxide and stained with Giemsa. Most of the cells are binucleated (X310). (b) Electron micrograph of a binucleated cell. N, nucleus; n, nucleolus (X5.800).
spayed rats (Fig. 7a). On their surface in contact with the culture medium, short and spaced microvilli were observed. A vacuolated Golgi apparatus was seen in the apical cytoplasm, just above the nucleus. Cisternae of the rough-surfaced endoplasmic reticulum were very few and polysomes mostly free. The cytoplasm contained a few lipid droplets, multivesicular bodies, autophagic vacuoles, and mitochondria. Adjacent cells were closely adherent and near to the apex, junctional complexes were observed (Fig. 7c) with, in addition, several desmosomes (Fig. 7d) with bundles of fibrils attached to the desmosomal plaques and penetrating deeply in the cytoplasm. Fibroblast-like cells (Fig. 7b) had no microvilli. Their Golgi apparatus was mostly vacu-
olar. A few cytoplasmic tonofilaments were observed in the cytoplasm. Furthermore, filamentous structures composed of 5-7 nm diameter microfilaments were frequently seen in the subsurface region (Fig. 7e), presumably in connection with the attachment of cells to the substratum. They differed from those accumulating in the decidual cells (Fig. 5b).
Discussion Decidualization in the rat is mainly characterized by the transformation of the stromal cells of the endometrium at the time of blastocyst implantation, and its occurrence depends on ovarian hormones. It can be experimentally reproduced in the castrated adult (7)
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FIG. 4. Cytoplasmic organelles of decidualized cells in the 72-h culture, (a) Extensive development of the Golgi apparatus (G); L, lipid droplet; N, nucleus (X14.000). (b) Well developed rough surfaced endoplasmic reticulum (ER) with attached polysomes (p) and containing a fuzzy material. M, large mitochondria; N, nucleus (x 14,000). (c) Detail of polysomes on the surface of ER, exhibiting the special figure of cochleosomes (x40,000).
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FIG. 5. Characteristic features of decidualized cells at 48-h culture, (a) Accumulation of filamentous material (f) in the cytoplasm. ER, rough-surfaced endoplasmic reticulum; Ly, lysosomes; M, mitochondria; p, polysomes (X 12,000). (b) Filaments at higher magnification (X40.000). (c) Large nucleolus with fibrillar (F) and granular (Gr) components loosely intermingled (X34.000).
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%•>. FIG. 6. Seventy-two-hour culture of endometrial cells from untreated spayed rats, (a) General aspect. Two distinct types of cells are recognized: islets of epithelial cells and dispersed fibroblast-like cells (X75). (b) Higher power view of the fibroblast-like cells showing spindle and irregular stellate shape cells. (X300). (c) Detail of an islet of epithelial cells (X300). (d) Same picture as (c) where the cells, difficult to individualize on (c), have been outlined with ink in order to show their mosaic arrangement (X300). a, b, c, d, Giemsa staining.
and in the immature (3) rat. Under these conditions, decidualization requires an hormonal sensitization with progesterone and estradiol (4). Results presented here showed that uterine cells from castrated adult animals, sensitized with the appropriate sequence of progesterone and estradiol, underwent a morphological transformation in vitro comparable to that observed in decidualization in vivo (3, 8). The typical cellular characteristics, such as binucleation, accumulation of filamentous mate-
rial, ballooning of mitochondria, increased number of lysosomes, development of the Golgi apparatus, and dilation of the rough surfaced endoplasmic reticulum, could be observed in vivo 2 days after induction and in vitro 2 days after seeding. Decidualization in vitro has also been obtained with endometrial cells from pseudopregnant rats (9). In vivo, decidualization occurs in hormonetreated animals only if an "inductive" stimulus is applied to the endometrium. Therefore, it is suggested that the mincing of the uterus
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Mv.
FlG. 7. Ultrastructure of epithelial and fibroblast-like cells from untreated spayed rats in culture for 72 h. (a) Epithelial cell. G, Golgi apparatus; Mv, microvilli; N, nucleus; n, nucleolus (XlO.OOO). (b) Fibroblast-like cell. G, Golgi apparatus; N, nucleus; n, nucleolus (XlO.OOO). (c) Junctional complex between two epithelial cells near their free surface (x40,000). (d) Desmosomes distributed along the lateral membranes of two adjacent epithelial cells (x40,000). (e) Portion of the filamentous structure observed beneath the plasma membrane adherent to the substratum (X40.000).
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IN VITRO DECIDUALIZATION
and/or the plating of cells in the culture dish might be equivalent to the scratch performed in vivo, the effect of which appears to be mediated through prostaglandins (6,10). Thus it appears that stromal cells can be identically committed by hormones and trauma to become decidual cells, in vitro as well as in vivo. It may be observed that the average life-span of the decidualized cells in vitro is comparable to the development of the deciduoma in vivo. In the pseudopregnant female, decidual cells differentiate completely 5 days after induction and regression is almost achieved 12-13 days after the trauma (11). In culture, decidual cells also reached their maximal development around day 6 after seeding, and they progressively degenerated and most of them died after 2 weeks of culture. Control experiments indicated that uterine cells obtained from the endometrium of castrated rats, not hormonally treated, did not give more than a few islets of epithelial cells and rare isolated fibroblasts after 3 days of culture. These results evoke the necessary role of hormones in the sensitization of the uterine tissue. Furthermore, stromal cells differentiated into decidual cells, although less rapidly, when animals were treated only with progesterone without estradiol. Such an observation and the fact that decidualization can be induced in vivo by a traumatism of the endometrium in spayed rats treated by progesterone alone without estradiol, suggest an absolute need for progesterone and a facultative role for estradiol. The ability to grow in culture uterine stromal cells gives an opportunity to investigate conveniently decidualization, including its need in hormones, prostaglandins, and serum factors.
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Acknowledgments We thank Monique Gouezou and Francine Delahaye for skillful technical help, Christiane Barrier for illustrations, and Francoise Boussac for typing the manuscript.
References 1. Shelesnyak, M. C, G. J. Marcus, P. F. Kraicer, and B. Lobel, Experimental study of decidualization, Fifth World Congress on Fertility and Sterility, Excerpta Med Int Congr Ser 133: 363, 1966. 2. Glasser, S. R., and J. H. Clark, A determinant role for progesterone in the development of uterine sensitivity to decidualization and ovo-implantation, In Markert, C. L., and J. Papaconstantinou (eds), The Developmental Biology of Reproduction, Academic Press, New York, 1975, p. 311. 3. Sananes, N., and C. Le Goascogne, Decidualization in the prepuberal rat uterus, Differentiation 5:133,1976. 4. De Feo, V. J., Decidualization, In Wynn, R. M. (ed.), Cellular Biology of the Uterus, Appleton Century Crofts, New York, 1967, p. 191. 5. Psychoyos, A., Endocrine control of egg implantation, In Greep, R. 0., and E. B. Astwood (eds.), Handbook of Physiology, Endocrinology, vol. 2 Williams & Wilkins, Baltimore, 1973, p. 187. 6. Sananes, N., E. E. Baulieu, and C. Le Goascogne, Prostaglandin(s) as inductive factor of decidualization in the rat uterus, Mol Cell Endocrinol 6: 153, 1976. 7. Yochim, J. M., and V. J. De Feo, Hormonal control of the onset, magnitude and duration of uterine sensitivity in the rat by steroid hormones of the ovary, Endocrinology 72: 317, 1963. 8. Jollie, W. P., and S. A. Bencosme, Electron microscopic observations on primary decidua formation in the rat, Am J Anat 116: 217, 1965. 9. Vladimirsky, F., L. Chen, A. Amsterdam, U. Zor, and H. R. Lindner, Differentiation of decidual cells in cultures of rat endometrium, J Reprod Fertil 49: 61, 1977. 10. Castracane, V. D., S. K. Saksena, and A. A. Shaikh, Effect of IUDs, prostaglandins and indomethacin on decidual cell reaction in the rat, Prostaglandins 6: 397, 1974. 11. Velardo, J. T., A. B. Dawson, A. G. Olsen, and F. L. Hisaw, Sequence of histological changes in the uterus and vagina of the rat during prolongation of pseudopregnancy associated with the presence of deciduomata, Am J Anat 93: 273, 1953.
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Vol. 103, No. 1 Printed in U.S.A.
In Vitro Decidualization of Rat Endometrial Cells* NICOLE SANANES, SYLVAIN WEILLER, ETIENNE-EMILE BAULIEU, AND CLAUDE LE GOASCOGNE Unite de Recherches sur le Metabolisme Moleculaire et la Physiopathologie des Steroides de I'Institut National de la Sante et de la Recherche Medicale, Departement de Chimie Biologique, Faculte de Medecine de Bicetre, 78 avenue du General Leclerc, 94270 Bicetre, France be a prerequisite for such development and transformation of endometrial cells in vitro, since control experiments performed with nonhormonally injected castrated animals gave only isolated islets of epithelial cells and scattered fibroblast-like cells in culture, whereas the decidual transformation was still observed, although with some delay, with cells from progesterone (but not estradiol)-treated spayed rats. Such an in vitro system may provide a new opportunity to study the mechanism of decidualization and of its hormonal control. (Endocrinology 103: 86, 1978)
ABSTRACT. Rat endometrial cells were isolated from ovariectomized adults submitted to a progesterone-estradiol sequence proper to sensitize the uterus to respond to a deciduogenic stimulus, and then cultured in vitro in the presence of progesterone. They grew rapidly and became morphologically similar to the decidual cells occurring in vivo with regard to the binucleation and the accumulation of characteristic filamentous material in the cytoplasm. The life-span of the decidualized cells in vitro was comparable to the duration of the deciduoma in vivo. The progesterone treatment of the rat was found to
I
had a life-span limited to the duration of the deciduoma in vivo.
N THE RAT, at the time of blastocyst implantation, the endometrial cells undergo profound modifications and the stromal cells differentiate into decidual cells. These changes can be experimentally reproduced provided the animals are submitted to a precise hormonal sequence and to uterine stimulation. The process may be divided into three successive phases: sensitization, induction, and response (1-3). Although a proper hormonal sequence has been well described (4, 5), the mechanism of decidualization is not yet understood despite recent findings concerning the involvement of prostaglandin(s) (6). In order to study the hormonally controlled differentiation of decidual cells, a cell culture technique was applied. The preliminary results, reported here, show that the endometrial cells, hormonally sensitized in vivo, grew well in culture, acquired several of the characteristic features of the decidual cells, lacked contact inhibition, and
Materials and Methods Preparation of animals Five-month-old female rats of the Sprague-Dawley strain were ovariectomized under ether anesthesia 15 days before starting experiments. One group of animals received progesterone 5 mg daily for 4 consecutive days and estradiol 0.15 /xg on the 4th day, both hormones being given sc in 0.25 and 0.15 ml sesame oil, respectively. A second group of animals was treated only with progesterone and a third group (control) did not receive hormones. Animals were killed by bleeding under ether anesthesia 18 h after estradiol injection (first group), 18 h after the last progesterone injection (second group), or 20 days after ovariectomy (third group). Preparation of dispersed cells For each culture, uterine horns of three rats were dissected under sterile conditions, cleared of fat, and dipped into 10 ml phosphate-buffered saline, 0.2 M, pH 7.4, containing progesterone 0.5 JUM (PBS). They were rinsed three times in 10 ml PBS and subsequently incubated 5 min in trypsin (0.25 g trypsin in 100 ml PBS). After removing most of the liquid, the horns were thoroughly minced with iridectomy scissors. The fragments, suspended in 5
Received July 7, 1977. Requests for reprints should be addressed to: Nicole Sananes, Lab. Hormone, Universite Paris-Sud, 94-Bicetre, France. * Partial support for this study has been obtained from the Ford Foundation, WHO, DGRST, and the Faculte de Medecine Paris-Sud.
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IN VITRO DECIDUALIZATION ml trypsin, were transferred into an Erlenmeyer flask containing 25 ml trypsin and agitated by pipetting at room temperature. They were then incubated for 45 min with continuous magnetic stirring at 37 C under an atmosphere of 5% CO2 in air. The suspension was again pipetted, then left undisturbed until the largest fragments had sedimented. Twenty milliliters of the supernatant were removed and equally distributed into two plastic tubes containing each 0.15 ml fetal calf serum (Gibco) to stop the trypsin action. After 3 min at low speed centrifugation, the pellets were resuspended in 5 ml culture medium and kept at room temperature. Twenty milliliters of trypsin were added into the Erlenmeyer flask, the fragments were again pipetted, and the procedure described above was repeated twice more. Fragments still remaining in the flask were trypsinized once more by adding 2 ml of a 2.5% trypsin solution and incubation was carried out with magnetic stirring for 15 min. Thereafter, the suspension was centrifuged and the pellet was resuspended in 5 ml culture medium. All of the suspensions were pooled and filtered through three layers of sterile gauze into a 50-ml beaker. The volume was adjusted with culture medium in order to obtain approximately 5.105 cells/ml and 25 cm2 dishes (Falcon Plastics) were plated with 5 ml cell suspension. Culture medium The culture medium was Dulbecco modified Eagle medium (DME), supplemented with 20% heatinactivated calf serum (Gibco), plus antibiotics (penicillin, 100 U/ml; streptomycin, 100 jug/ml; nalidixic acid, 40 /ig/ml), and progesterone 0.5 /iM. The cultures were fed with fresh medium after 24 and 48 h, and then twice a week. Light and electron microscopy Living cells were observed and photographed with the phase contrast microscope. Cells, after different periods of culture, were rinsed in PBS and fixed in osmium tetroxide for 30 min. Some samples were stained with Giemsa and observed under light microscope while others were dehydrated through graded ethanol series and in situ embedded in Epon. Blocks were stained with toluidine blue in order to select areas of interest for preparing the ultrathin sections with Ultratome III from LKB. The sections were stained with uranyl acetate and lead citrate and observed in a Siemens Elmiskop 101. Several specimens were reembedded
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in Epon in order to cut sections perpendicularly to the cellular layers.
Results Culture of uterine cells from progesteroneestradioI- treated spayed rats Cells in culture. Within 1 h after seeding, most cells were attached to the substratum. At 24 h (Fig. la), the flasks were partially covered with an homogeneous population of well spread, fibroblast-like cells, frequently showing mitotic figures. After 48 h of culture (Fig. lb), the cells were larger, much more numerous, and very little intercellular space was left over; many cells were binucleated. There was no contact inhibition and several layers of cells were overlapping (Fig. le). At 72 h, piling and criss-crossing of the cells were extremely well marked (Fig. Id), and in the less densely plated zones, binucleated cells were abundant and mitotic figures were observed (Fig. lc). The general aspect of the culture observed at 72 h (Fig. 2a) remained identical until day 6 when some cells showed signs of degeneration. On day 13 (Fig. 2b), a large number of cells had died and they formed dense scattered clusters. Involution of culture was complete after 4 weeks (Fig. 2c). Ultrastructure. Electron microscopic analysis confirmed that binucleated cells observed in light microscopy (Fig. 3a) did possess two distinct nuclei (Fig. 3b). The nuclei were large, averaging 15 /xm in diameter. At 72 h, their size varied from one cell to another up to 30 jum. The chromatin was regularly dispersed. The nucleoli, which were densely stained with Giemsa (Fig. 3a), appeared as formed by the intermingled fibrillar and granular components, the latter prevailing (Figs. 3b and 5c). The number and size of nucleoli varied from cell to cell, but often were identical in the two nuclei of a same cell. One to eight nucleoli per nucleus were observed; the less numerous, the larger they were. The Golgi apparatus (Fig. 4a) and the rough endoplasmic reticulum (Fig. 4b) were well developed. Inside cisternae of the rough endoplasmic reticulum, a flocculent material accu-
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FIG. 1. Culture of endometrial cells from progesterone-estradiol-treated spayed rats and differentiation into decidual cells. Phase contrast micrographs of cells after 24 h (a), 48 h (b), and 72 h (c and d). Arrows point to binucleated cells (X115). At 72 h, the flask is irregularly covered with thick zones of several layers of cells (d) indicating lack of contact inhibition, and with zones of less density (c) where mitosis (m) and binucleated cells are easily identified, (e) Electron micrograph of cells cut perpendicularly to the surface attachment showing at 48 h the already superimposed cells: 1, 2, 3, 4 from bottom to top. N, nucleus (X8.700).
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FIG. 2. General aspect of endometrial cell cultures from progesterone-estradiol treated spayed rats at various times after seeding. Giemsa staining (x5). (a) 3-day culture showing the complete covering of the flask. Light areas correspond to Fig. lc and dark zones to Fig. Id. (b) 13-day culture: many dead cells are packed in dark clusters; living cells become less and less numerous and the areas devoid of cells are enlarging, (c) 28-day culture: regression is complete.
mulated. On their outer surface, numerous polysomes were attached and showed the particular configuration of cochleosomes (Fig. 4c). Large mitochondria, up to 2 jum in diameter, had cristae and a clear matrix. Scattered lipid droplets and lysosomes were also observed. In addition, large amounts of a filamentous material accumulated in the cytoplasm (Fig. 5a). The filaments were about 10 nm thick (Fig. 5b). Only a few polysomes were observed amidst the intermingled filaments. Culture of uterine cells from progesteronetreated spayed rats The same differentiation of the endometrial cells occurred with the above mentioned characteristics. However, the developmental process was slower and at 48 h of culture, the cells were less numerous when compared to those of the progesterone-estradiol-treated group and only a few of them were binucleated. On the 5th day after seeding, the development of the culture became similar to what was ob-
served after 72 h with cells from progesteroneestradiol-pretreated rats. Culture of uterine cells from untreated spayed rats Cells in culture. Contrary to endometrial cells from hormone-treated animals, uterine cells from untreated castrated rats were still floating in the culture medium 3 h after plating. Between 24 and 72 h (Fig. 6a), two types of cells were observed, epithelial cells in sparse islets and dispersed fibroblast-like elements, either of spindle or irregular stellate shape (Fig. 6b). The islets of epithelial cells were composed of 10-100 polygonal cells forming a continuous mosaic-like sheet (Fig. 6, c and d). Their nucleus contained one to four nucleoli, smaller when their number increased. These cultures were grown for 13 days and no decidual differentiation was observed. Ultrastructure. At 72 h, the cells of the islets looked like the uterine epithelial cells of
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Endo • 1978 Vol 103 • No 1
FIG. 3. Main features of decidualized cells in the 72-h culture, (a) Light micrograph of cells fixed with osmium tetroxide and stained with Giemsa. Most of the cells are binucleated (X310). (b) Electron micrograph of a binucleated cell. N, nucleus; n, nucleolus (X5.800).
spayed rats (Fig. 7a). On their surface in contact with the culture medium, short and spaced microvilli were observed. A vacuolated Golgi apparatus was seen in the apical cytoplasm, just above the nucleus. Cisternae of the rough-surfaced endoplasmic reticulum were very few and polysomes mostly free. The cytoplasm contained a few lipid droplets, multivesicular bodies, autophagic vacuoles, and mitochondria. Adjacent cells were closely adherent and near to the apex, junctional complexes were observed (Fig. 7c) with, in addition, several desmosomes (Fig. 7d) with bundles of fibrils attached to the desmosomal plaques and penetrating deeply in the cytoplasm. Fibroblast-like cells (Fig. 7b) had no microvilli. Their Golgi apparatus was mostly vacu-
olar. A few cytoplasmic tonofilaments were observed in the cytoplasm. Furthermore, filamentous structures composed of 5-7 nm diameter microfilaments were frequently seen in the subsurface region (Fig. 7e), presumably in connection with the attachment of cells to the substratum. They differed from those accumulating in the decidual cells (Fig. 5b).
Discussion Decidualization in the rat is mainly characterized by the transformation of the stromal cells of the endometrium at the time of blastocyst implantation, and its occurrence depends on ovarian hormones. It can be experimentally reproduced in the castrated adult (7)
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FIG. 4. Cytoplasmic organelles of decidualized cells in the 72-h culture, (a) Extensive development of the Golgi apparatus (G); L, lipid droplet; N, nucleus (X14.000). (b) Well developed rough surfaced endoplasmic reticulum (ER) with attached polysomes (p) and containing a fuzzy material. M, large mitochondria; N, nucleus (x 14,000). (c) Detail of polysomes on the surface of ER, exhibiting the special figure of cochleosomes (x40,000).
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SANANES ET AL.
Kndo • 1978 Vol 103 • No 1
FIG. 5. Characteristic features of decidualized cells at 48-h culture, (a) Accumulation of filamentous material (f) in the cytoplasm. ER, rough-surfaced endoplasmic reticulum; Ly, lysosomes; M, mitochondria; p, polysomes (X 12,000). (b) Filaments at higher magnification (X40.000). (c) Large nucleolus with fibrillar (F) and granular (Gr) components loosely intermingled (X34.000).
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IN VITRO DECIDUALIZATION
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:\
a
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V < w
• •
%•>. FIG. 6. Seventy-two-hour culture of endometrial cells from untreated spayed rats, (a) General aspect. Two distinct types of cells are recognized: islets of epithelial cells and dispersed fibroblast-like cells (X75). (b) Higher power view of the fibroblast-like cells showing spindle and irregular stellate shape cells. (X300). (c) Detail of an islet of epithelial cells (X300). (d) Same picture as (c) where the cells, difficult to individualize on (c), have been outlined with ink in order to show their mosaic arrangement (X300). a, b, c, d, Giemsa staining.
and in the immature (3) rat. Under these conditions, decidualization requires an hormonal sensitization with progesterone and estradiol (4). Results presented here showed that uterine cells from castrated adult animals, sensitized with the appropriate sequence of progesterone and estradiol, underwent a morphological transformation in vitro comparable to that observed in decidualization in vivo (3, 8). The typical cellular characteristics, such as binucleation, accumulation of filamentous mate-
rial, ballooning of mitochondria, increased number of lysosomes, development of the Golgi apparatus, and dilation of the rough surfaced endoplasmic reticulum, could be observed in vivo 2 days after induction and in vitro 2 days after seeding. Decidualization in vitro has also been obtained with endometrial cells from pseudopregnant rats (9). In vivo, decidualization occurs in hormonetreated animals only if an "inductive" stimulus is applied to the endometrium. Therefore, it is suggested that the mincing of the uterus
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Mv.
FlG. 7. Ultrastructure of epithelial and fibroblast-like cells from untreated spayed rats in culture for 72 h. (a) Epithelial cell. G, Golgi apparatus; Mv, microvilli; N, nucleus; n, nucleolus (XlO.OOO). (b) Fibroblast-like cell. G, Golgi apparatus; N, nucleus; n, nucleolus (XlO.OOO). (c) Junctional complex between two epithelial cells near their free surface (x40,000). (d) Desmosomes distributed along the lateral membranes of two adjacent epithelial cells (x40,000). (e) Portion of the filamentous structure observed beneath the plasma membrane adherent to the substratum (X40.000).
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IN VITRO DECIDUALIZATION
and/or the plating of cells in the culture dish might be equivalent to the scratch performed in vivo, the effect of which appears to be mediated through prostaglandins (6,10). Thus it appears that stromal cells can be identically committed by hormones and trauma to become decidual cells, in vitro as well as in vivo. It may be observed that the average life-span of the decidualized cells in vitro is comparable to the development of the deciduoma in vivo. In the pseudopregnant female, decidual cells differentiate completely 5 days after induction and regression is almost achieved 12-13 days after the trauma (11). In culture, decidual cells also reached their maximal development around day 6 after seeding, and they progressively degenerated and most of them died after 2 weeks of culture. Control experiments indicated that uterine cells obtained from the endometrium of castrated rats, not hormonally treated, did not give more than a few islets of epithelial cells and rare isolated fibroblasts after 3 days of culture. These results evoke the necessary role of hormones in the sensitization of the uterine tissue. Furthermore, stromal cells differentiated into decidual cells, although less rapidly, when animals were treated only with progesterone without estradiol. Such an observation and the fact that decidualization can be induced in vivo by a traumatism of the endometrium in spayed rats treated by progesterone alone without estradiol, suggest an absolute need for progesterone and a facultative role for estradiol. The ability to grow in culture uterine stromal cells gives an opportunity to investigate conveniently decidualization, including its need in hormones, prostaglandins, and serum factors.
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Acknowledgments We thank Monique Gouezou and Francine Delahaye for skillful technical help, Christiane Barrier for illustrations, and Francoise Boussac for typing the manuscript.
References 1. Shelesnyak, M. C, G. J. Marcus, P. F. Kraicer, and B. Lobel, Experimental study of decidualization, Fifth World Congress on Fertility and Sterility, Excerpta Med Int Congr Ser 133: 363, 1966. 2. Glasser, S. R., and J. H. Clark, A determinant role for progesterone in the development of uterine sensitivity to decidualization and ovo-implantation, In Markert, C. L., and J. Papaconstantinou (eds), The Developmental Biology of Reproduction, Academic Press, New York, 1975, p. 311. 3. Sananes, N., and C. Le Goascogne, Decidualization in the prepuberal rat uterus, Differentiation 5:133,1976. 4. De Feo, V. J., Decidualization, In Wynn, R. M. (ed.), Cellular Biology of the Uterus, Appleton Century Crofts, New York, 1967, p. 191. 5. Psychoyos, A., Endocrine control of egg implantation, In Greep, R. 0., and E. B. Astwood (eds.), Handbook of Physiology, Endocrinology, vol. 2 Williams & Wilkins, Baltimore, 1973, p. 187. 6. Sananes, N., E. E. Baulieu, and C. Le Goascogne, Prostaglandin(s) as inductive factor of decidualization in the rat uterus, Mol Cell Endocrinol 6: 153, 1976. 7. Yochim, J. M., and V. J. De Feo, Hormonal control of the onset, magnitude and duration of uterine sensitivity in the rat by steroid hormones of the ovary, Endocrinology 72: 317, 1963. 8. Jollie, W. P., and S. A. Bencosme, Electron microscopic observations on primary decidua formation in the rat, Am J Anat 116: 217, 1965. 9. Vladimirsky, F., L. Chen, A. Amsterdam, U. Zor, and H. R. Lindner, Differentiation of decidual cells in cultures of rat endometrium, J Reprod Fertil 49: 61, 1977. 10. Castracane, V. D., S. K. Saksena, and A. A. Shaikh, Effect of IUDs, prostaglandins and indomethacin on decidual cell reaction in the rat, Prostaglandins 6: 397, 1974. 11. Velardo, J. T., A. B. Dawson, A. G. Olsen, and F. L. Hisaw, Sequence of histological changes in the uterus and vagina of the rat during prolongation of pseudopregnancy associated with the presence of deciduomata, Am J Anat 93: 273, 1953.
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