J. Anat. (1976), 122, 3, pp. 485-498 With 13 figures Printed in Great Britain

485

An ultrastructural study of implantation in the golden hamster HI. Initial formation and differentiation of decidual cells

TERRY A. PARKENING* Department of Biology, University of Oregon, Eugene, Oregon 97403

(Accepted 27 September 1975) INTRODUCTION

During implantation, in many mammalian species, stromal cells located beneath the uterine epithelium and adjacent to the implanting blastocyst transform from fibroblasts into decidual cells. It is believed that these cells are involved in the nutrition of the embryo and in the protection of the uterus against the invading trophoblast (DeFeo, 1967; Finn, 1971). Ultrastructural studies of decidual cells during implantation have been reported in the rat (Enders & Schlafke, 1967; Tachi, Tachi & Lindner, 1969, 1970), mouse (Finn & Lawn, 1967; Potts, 1968), and hamster (McLennan, 1974). Hamster decidual cells have also been examined after implantation (Orsini, Wynn, Harris & Bulmash, 1970) and during pseudopregnancy (Brinsfield, Clark & Gerich, 1972; McLennan, 1974). In addition, the fine structure of artificially-induced deciduomata has been described in the rat (Jollie & Bencosme, 1965) and hamster (Orsini et al. 1970). The purpose of the present work was to study ultrastructurally the initial formation and subsequent maturation of decidual cells in golden hamsters pregnant for 3-51 days, and to relate the decidual events to other events involved in the implantation process. Previous papers by the author have dealt with the relationship of the blastocyst and the uterine epithelium at the ultrastructural level during these same stages of development (Parkening, 1976a,,b). MATERIALS AND METHODS

The female golden hamsters used for this study have been described in previous papers (Parkening, 1976a, b). A detailed account of the manner in which the material was prepared is given in the first paper (Parkening, 1976a). Twenty two female hamsters (3-5 months of age) pregnant for 3, 3i, 4, 41, 5 or 5j days postovulation were injected with Pontamine blue for the detection of implantation sites and then perfused with 2-5 % glutaraldehyde (phosphate buffered, pH 7 4) via the abdominal aorta. Areas of the uterus containing implantation sites made visible with the dye were dissected out or, if there was no reaction from the dye (as at 3 days), the entire uterus was removed and cut into smaller pieces. Specimens were fixed for an additional 1-2 hours in glutaraldehyde of similar concentration, post-fixed in 2% * Present address: Department of Anatomy, The University of Texas Medical Branch, Galveston, Texas 77550.

3I

A N A I122

486

TERRY A. PARKENING

.e

I

-s

*

.. K1

1-1,

I

.'

-_e

t'

X

"

*t1fL :'

rI..-

.41 -.-A,.

.q.

,

,

;It

. "t 4w

I A

1

2

'* 4EX;v

.

..% 'i"ki..

ttot: -

:

..

Ultrastructure of decidual cells in the hamster

487

osmium tetroxide, dehydrated, and then embedded in Epon-Araldite. Thick sections (1-3 ,tm), which were cut to locate blastocysts or implantationsites, were examined and photographed using phase contrast microscopy and were then re-embedded for electron microscopy (Schabtach & Parkening, 1974). Thin sections, prepared from a total of 89 embryos, were stained with lead citrate and uranyl acetate and examined in a Philips 300 electron microscope. The stromal cells examined were restricted to those in the immediate vicinity of the embryo. OBSERVATIONS

3 days Before attachment of the blastocyst to the uterine epithelium the fibroblasts of the endometrial stroma were mostly spindle-shaped, with sparsely scattered collagen fibres between the cells (Figs. 1, 2). The nucleus, which occupied most of the cell, had numerous pores and, in some cells, condensing chromatin (Fig. 1). In the relatively small amount of cytoplasm, Golgi cisternae, sometimes associated with secretory vesicles, were located near the nucleus. Some granular endoplasmic reticulum surrounded the nucleus and large numbers of polyribosomes were present throughout the cytoplasm. The mitochondrial profiles were round to cylindrical, with lamelliform cristae. In general there was little contact between cells, although a few junctions were present.

3j days With the encompassment of the blastocyst by the uterine epithelium the organelles of the stromal cell cytoplasm remained similar to those described at 3 days, except that Golgi complexes were more extensive, exhibiting obvious secretory activity (Fig. 4). Changes also occurred within the nuclei of the cells as two types of chromatin differing in their electron densities became apparent. Dense chromatin occurred peripherally while less dense chromatin was associated with the nucleolus (Figs. 5, 7) which contained both granular and fibrillar components. Centrioles were seen in many cells and a few cells were in the process of dividing (Fig. 3) or had just completed division. Dividing cells generally had groups of microfilaments in the cytoplasm in addition to microtubules. Such microfilaments (Fig. 3) were only present in dividing or newly divided cells.

4 days At 4 days the shape of the stromal cells changed. While organelles such as mitochondria, Golgi bodies, polyribosomes and endoplasmic reticulum remained much the same, the amount of cytoplasm increased and the cells began to exhibit branched Fig. 1. A fibroblast located next to the uterine epithelium (UE) of a hamster pregnant for 3 days (post-ovulation). A blastocyst found free in the lumen was located just above the epithelial cells. Collagen fibres (C) are located around the elongated stromal cell, which consists primarily of a nucleus. Mitochondria, a Golgi complex and some granular endoplasmic reticulum are also visible within the cytoplasm. x 12 300. Fig. 2. Fibroblasts from a hamster at 3 days of pregnancy. The blastocyst, still free within the uterine lumen, was located a short distance from the cells. The cell on the left appears to have a reticulated nucleolus demonstrating both granular and fibrillar regions. x 10050. 31-2

488

TERRY A. PARKENING

A VI2 ---iP

.1.

Fig. 3. A dividing stromal cell located antimesometrially to a blastocyst newly enclosed by the uterine epithelium at 3i days of pregnancy. A network of fine filaments (arrow) is generally visible in the cytoplasm near the chromatin of dividing or newly divided cells. x 10600. Inset: An enlargement of the same area of microfilaments illustrated by the arrow. x 30300.

processes, which at times formed various types of junctions with adjoining cells. Most cellular contact was unspecialized, with a separation of 30 nm or more between the plasma membranes. At other times dense material formed along each side of the closely apposed surfaces and occasionally, in still other areas, tight junctions were formed (Fig. 6). The tight junctions were similar to those described in mouse decidual cells (Finn & Lawn, 1967) except that they were seldom cup-shaped. Instead, they were usually flattened, consisting of two opposed membranes which were sometimes separated by an electron-dense region which appeared to be periodically interrupted. The presence of this inner region indicates that these junctions were probably gap junctions. A few cells directly adjacent to the implanting blastocyst began to exhibit Fig. 4. A stromal cell immediately adjacent to a blastocyst newly enclosed by the uterine epithelium at 3j days of pregnancy. Fibroblasts undergoing their initial transformation to decidual cells exhibit more extensive Golgi complexes (G), with obvious secretory activity.

C, centriole. x 23800. Fig. 5. A newly forming decidual cell from a hamster at 4 days of pregnancy. The cell is located laterally to an implanting blastocyst. Fibrils (F) appear for the first time in a few cells at this stage of differentiation. Note the two types of chromatin within the nucleus. x 17400.

Ultrastructure of decidual cells in the hamster 9,

'4A,I

4

4

__

.3; ¶

- .:'AeP7. 9'"

;. 9s ':. ;^-S.ut >>r , *,.. F.*HE'

5-.,

.E;

j *

w5. wa.4x^x

^ *

i~F, *m. X e

490

TERRY A. PARKENING

'A

~

~

t'e'_-,~~~~~. I.7'-

Ultrastructure of decidual cells in the hamster

491

small quantities of glycogen and some cells for the first time contained a number of fine fibrils (about 6 nm width and of varying length) resembling tono-filaments. The fibrils, which had no specific orientation were in small patches except in a few cells where they occupied a major portion of the cytoplasm (Fig. 5). These fibrils were like the microfilaments confined earlier to dividing cells but were now present in all cells. As the blastocyst adhered to the uterine epithelium, polyribosomes within some of the developing decidual cells formed distinct, bead-like, coiled arrangements (Fig. 8). The number of ribosomes making up this pattern ranged from 18 to 26. These arrangements were not abundant: in some parts of the cytoplasm they existed singly; in other areas three or four arrangements existed as a group. Solitary cilia were also visible for the first time in a few of the differentiating stromal cells.

4j days When the trophoblast cells penetrated and began phagocytosing the uterine epithelium, changes within the stromal cells were even more evident. Tight junctions became more abundant as the cytoplasm of the decidual cells expanded, enabling the cells to make more contact with adjoining cells (Fig. 7). Fibrils were present in small patches throughout most cells, as were small deposits of glycogen. Lipid droplets appeared in most cells, and some cisternae of the granular endoplasmic reticulum were moderately dilated.

5 days Decidual cells came closer together as blood lacunae increased around the implanting embryo. Cytoplasmic organelles remained similar to those described at 41 days. The nucleus of the cell changed slightly; the nucleoli were generally smaller, with most cells no longer exhibiting the less dense chromatin seen earlier.

5j days The fine structure of decidual cells after the trophoblast had phagocytosed the uterine epithelium was somewhat different from that previously described. The decidual cells formed close contacts with each other, leaving an even smaller volume of intercellular space. In some areas cells appeared to be binucleate (Fig. 12), while in other areas a membrane could be detected between nuclei. Junctions with dense cytoplasmic zones and tight junctions similar to those appearing earlier were present. The nuclei of many cells contained one round nucleolus located generally near the centre of the nucleus (Fig. 13). Deposits of glycogen were found in association with lipid droplets throughout many cells, with the greatest accumulation of glycogen in Fig. 6. Newly forming decidual cells located laterally to an implanting blastocyst at 4 days of pregnancy. Cells begin to extend processes, contacting one another and forming different types of junctions. Most junctions are unspecialized; however a few form dense cytoplasmic regions (upper arrow) or tight junctions (lower arrow). x 9500. Inset: A higher magnification of contact between two newly forming decidual cells at 4 days, illustrating the types of junctions described above. x 23 600. Fig. 7. A decidual cell from a hamster at 41 days of pregnancy. Glycogen (Gy), often in association with lipid droplets (L), appears regularly throughout most cells at this stage of pregnancy. Note the two types of chromatin within the nucleus. x 10900.

492

TERRY A. PARKENING

Ultrastructure of decidual cells in the hamster

493

Fig. 10. A decidual cell exhibiting a network of fibrils (F), from a hamster at 5+ days of pregnancy. There appears to be no pattern to the fibrils, which run in various directions. Crystalloid bodies (Cr) are often associated with the fibrils at this stage of pregnancy. x 20100.

cells located mesometrially to the embryo. Generally, the endoplasmic reticulum had widely dilated cisternae, and in places there were no ribosomes associated with the reticulum (Fig. 9). Two organelles, not detectable earlier, appeared in decidual cells. Crystalloid inclusions often associated with patches of fibrils were evident within the cytoplasm (Fig. 10). Such structures have not been described previously in association with decidual cells. Also found near the nucleus, and sometimes with fibrils, were electron-dense structures having a 'dumb-bell' appearance (Figs. 1 1,12). They generally consisted of an electron-dense matrix composed of two bulbs joined by a central shaft. A double membrane surrounded these structures, and the bulbs in cross section contained an indistinct number of electron-dense filaments. Such structures have not been reported by other researchers. Fig. 8. A newly forming decidual cell of a hamster at 4 days of pregnancy. The granular endoplasmic reticulum has become dilated in some parts of the cytoplasm, and ribosomes do not appear to be associated with some portions of the membrane. x 22600. Inset: After blastocyst attachment to the uterine epithelium ribosomes are occasionally found arranged in a characteristic coiled configuration (circle). x 73900. Fig. 9. A decidual cell from a hamster at 51 days of pregnancy. The granular endoplasmic reticulum (ER) has become more dilated, with fewer associated ribosomes than at previous stages (cf. Fig. 8). Gy, glycogen x 28 700.

494

TERRY A. PARKENING

Fig. 11. Decidual cells located laterally to an implanted embryo at 5j days of pregnancy. The cells are not directly adjacent to the embryo because blood lacunae have enlarged and these completely surround the embryo except for the region developing into the ectoplacental cone. 'Dumb-bell'-shaped structures (arrows) sometimes appear near the nucleus of the cell. Some junctions (crossed arrows), similar to thos3 at 4 days, exist between adjoining cells. Gy, glycogen. x 12950. DISCUSSION

Fibroblasts begin their transformation into decidual cells as soon as the blastocyst becomes enclosed by the uterine epithelium; the manner in which they are stimulated remains unknown. The blastocyst seemingly provides a specific stimulus, since plastic beads the size of blastocysts placed in a sensitized uterus fail to evoke a decidual reaction in mice (McLaren, 1968). The uterus, in order to respond to the stimulus, must be properly prepared by ovarian hormones; there are numerous Fig. 12. A decidual cell which appears to be binucleate, from a hamster at 5i days of pregnancy. Numerous fibrils (F) are present throughout the cytoplasm. An organelle resembling a 'dumbbell' (arrow), is actually a mitochondrion. x 18500. Inset: An enlargement of an organelle which appears quite commonly throughout decidual cells at this stage of pregnancy. These structures may be mitochondria or they may be similar to the 'dumb-bell' structures in Figure 11. x48200. Fig. 13. The nucleus of a decidual cell from a hamster at 5j days of pregnancy. A single rounded nucleolus is commonly found near the centre of the nucleus of decidual cells at this stage of pregnancy. This cell is located laterally to the region of the embryo where the ectoplacental cone is forming. Gy, glycogen. x 10050.

~~

495

Ultrastructure of decidual cells in the hamster

.~~~~~~~~~~~~. ~~~~~~~~ ~~~~~~~~~~~~~~

~

~

~'J

~

IA~~~~~~~~~~~~~~~~~~~~~~~~~~A 2 ~~~ *~~

t",~~~~~~~~~~~~~~~~~-~ 3

V

~8

v~~~~~~~~~~~~~~~~.

44~~~~~~~~~~ ~AI 4:

4t

13 -

t

V

'-~.~*~

496

TERRY A. PARKENING

theories as to the nature of this stimulus, the most controversial being the histamine theory (Shelesnyak, Marcus & Lindner, 1970; Finn, 1971; Glasser, 1972). The formation of decidual cells in the hamster is similar to that in the rat (Enders & Schlafke, 1967; Tachi et al. 1970) and mouse (Finn & Lawn, 1967; Potts, 1968). Cells begin to differentiate at the antimesometrial side of the uterus and quickly multiply to encompass the implanting blastocyst. As the cells increase in number they contact one another more frequently, forming various types of junctions. The formation of tight junctions has been described in an earlier study on the developing decidual cells of the mouse (Finn & Lawn, 1967). These junctions are now considered to be gap junctions (Finn & Porter, 1975). Gap junctions are specialized junctions which permit communication between the cells since these regions are associated with electrical conductivity and ionic permeability between cells (Loewenstein 1966). Since junctions are formed before trophoblastic penetration of the uterine epithelium, it has been suggested that the decidual cells may be forming a barrier against the trophoblastic invasion of the endometrium in rodents (Finn & Lawn, 1967). Cytoplasmic fibrils have been described in other studies of rodent decidual cells (Jollie & Bencosme, 1965; Enders & Schlafke, 1967; Tachi et al. 1970; Ljungkvist, 1973). The function of these fibrils is unknown, but they apparently exist in rat stromal cells before decidualization, and their formation may be dependent upon estrogen and progesterone, since they are absent in ovariectomized animals (Ljungkvist, 1973). Coil-shaped polyribosomes, first noted in rat stromal cells (Tachi et al. 1970), are identical to those in hamster stromal cells. In the rat the coiled polyribosomes are thought to be associated with the endoplasmic reticulum and whether or not they are visible depends on the plane of sectioning. In the hamster this arrangement of polyribosomes is present only during trophoblastic attachment and the penetration of the uterine epithelium and when the differentiating stromal cells begin forming mutual junctions (days 4 and 41 of pregnancy). Solitary cilia have been described in the rat uterus during early implantation, pseudopregnancy and the oestrous cycle (Tachi et al. 1969) and in the hamster during oestrus and pseudopregnancy (Clark & Brinsfield, 1973). While ciliogenesis in rat stromal cells has been considered to be dependent on the endocrine status of the uterus (Maraspin & Boccabella, 1971), a comparison of ciliary development in hamster stromal cells at oestrus and during pseudopregnancy did not reveal statistically significant differences (Clark & Brinsfield, 1973). Centrioles are visible in most stromal cells during implantation in the hamster (3J-4j days of pregnancy) and solitary cilia are capable of forming since some were noted, but cilia are not as abundant as those described at the time of implantation in the rat (Tachi et al. 1969). After the uterine epithelium has been eliminated (day 5O, centrioles are no longer evident in decidual cells adjacent to the hamster embryo. Crystalloid inclusions and 'dumb-bell' organelles have not been described previously in decidual cells. However, crystalloid bodies resembling those in hamster decidual cells have been found in mouse and rabbit blastocysts (Hadek & Swift, 1960; Enders & Schlafke, 1965). Since crystalloid accumulates during an increase in protein synthesis in the rabbit blastocyst, it has been suggested that these bodies

Ultrastructure of decidual cells in the hamster

497

may be associated with this synthesis (Enders, 1971). The present author is unaware of any ultrastructural studies describing organelles resembling the 'dumb-bell'shaped structures found in the hamster decidual cells. These structures, which vary somewhat in electron density, are relatively common throughout decidual cells at 5j days of pregnancy. Some of the structures resemble mitochondria, and it is possible that even the more electron-dense bodies are in fact modified mitochondria. It is apparent that a considerable number of ultrastructural changes occur as fibroblasts transform into decidual cells. For an account of the ultrastructural characteristics of decidual cells in hamsters from 5 days, 19 hours to 7 days, 12 hours of pregnancy, consult Orsini et al. (1970). SUMMARY

The transformation of fibroblasts into decidual cells was studied ultrastructurally in golden hamsters pregnant for 3, 3j, 4, 41, 5, or 54 days (post-ovulation). Cells within the endometrial stroma were generally separated from one another and spindle-shaped before blastocyst contact with the uterine epithelium. Once the epithelium enclosed the blastocyst (3- days), stromal cells adjacent to the blastocyst antimesometrially began to exhibit more extensive Golgi complexes with increased secretory activity. As differentiation proceeded the amount of cellular contact increased and various types of junctions formed between the cells. Throughout the period examined, intercellular space progressively decreased, while the cisternal width of the granular endoplasmic reticulum continually increased. Special organelles, whose functions remain unknown, first appeared in differentiating cells at 4 days (fibrils) and 5j days (crystalloid and 'dumb-bell' structures). I wish to express my appreciation to Dr A. L. Soderwall for his interest and support during this investigation. This study was supported in part by USPHS Grant No. HD 04234-03 and by NIH Physiology Training Grant No. 5 TOI GM00336. REFERENCES

BRINSFIELD, T. H., CLARK, M. V. & GERICH, V. (1972). Ultrastructural changes of the hamster endometrial stromal cell during the last half of pseudopregnancy. Journal of Reproduction and Fertility 30, 185-190. CLARK, M. V. & BRINSFIELD, T. H. (1973). Ciliated stromal cells in the hamster uterus. Biology of Reproduction 8, 100-102. DEFEO, V. J. (1967). Decidualization. In Cellular Biology of the Uterus (ed. R. M. Wynn), pp. 191-290. New York: Appleton-Century-Crofts. ENDERS, A. C. (1971). The fine structure of the blastocyst. In The Biology of the Blastocyst (ed. R. J. Blandau), pp. 71-94. Chicago: University of Chicago. ENDERS, A. C. & SCHLAFKE, S. (1965). The fine structure of the blastocyst: some comparative studies. In Preimplantation Stages of Pregnancy (ed. G. E. W. Wolstenholme & M. O'Connor), pp. 29-54. London: Churchill. ENDERS, A. C. & SCHLAFKE, S. (1967). A morphological analysis of the early implantation stages in the rat. American Journal of Anatomy 120, 185-226. FINN, C. A. (1971). The biology of decidual cells. Advances in Reproductive Physiology 5, 1-26. FINN, C. A. & LAWN, A. M. (1967). Specialised junctions between decidual cells in the uterus of the pregnant mouse. Journal of Ultrastructure Research 20, 321-327. FINN, C. A. & PORTER, D. G. (1975) The decidual cell reaction. In The Uterus, Reproductive Biology Handbooks, Vol. 1, pp. 74-85. Acton, Massachusetts: Publishing Sciences Group, Inc. GLASSER, S. R. (1972). The uterine environment in implantation and decidualization. In Reproductive Biology (ed. H. Balin & S. Glasser), pp. 776-833. Amsterdam: Excerpta Medica.

498

TERRY A. PARKENING

HADEK, R. & SwIFr, H. (1960). A crystalloid inclusion in the rabbit blastocyst. Journal of Biophysical and Biochemical Cytology 8, 836-841. JOLLIE, W. P. & BENCOSME, S. A. (1965). Electron microscopic observations on primary decidua formation in the rat. American Journal of Anatomy 116, 217-236. LJUNGKVIST, I. (1973). Uterine stromal morphology of the spayed, virgin rat when prepared with progesterone and oestrogen for implantation. Zeitschrift fur Anatomie und Entwicklungsgeschichte 141, 161-169. LOEWENSTEIN, W. R (1966). Permeability of membrane junctions. Annals of the New York Academy of Sciences 137, 441-472. MARASPIN, L. E. & BOCCABELLA, A. V. (1971). Solitary cilia in endometrial fibroblasts. Journal of Reproduction and Fertility 25, 343-347. MCLAREN, A. (1968). Can beads stimulate a decidual response in the mouse uterus? Journal ofReproduction and Fertility 15, 313-315. McLENNAN, J. G. (1974). Ultrastructural studies of early nidation in pregnancy and pseudopregnancy. American Journal of Obstetrics and Gynecology 120, 319-334. ORSINI, M. W., WYNN, R. M., HARRIS, J. A. & BULMASH, J. M. (1970). Comparative ultrastructure of the decidua in pregnancy and pseudopregnancy. American Journal of Obstetrics and Gynecology 106, 14-25. PARKENING, T. A. (1976a). An ultrastructural study of implantation in the golden hamster. I. Loss of the zona pellucida and initial attachment to the uterine epithelium. Journal of Anatomy 121, 161-184. PARKENING, T. A. (1976b). An ultrastructural study of implantation in the golden hamster. II. Trophoblastic invasion and removal of the uterine epithelium. Journal of Anatomy 122, 211-230. PoTTs, D. M. (1968). The ultrastructure of implantation in the mouse. Journal of Anatomy 103, 77-90. SCHABTACH, E. & PARKENING, T. A. (1974). A method for sequential high resolution light and electronmicroscopy of selected areas of the same material. Journal of Cell Biology 61, 261-264. SHELESNYAK, M. C., MARCUS, G. J. & LINDNER, H. R. (1970). Determinants of the decidual reaction. In Ovo-Implantation. Human Gonadotropins and Prolactin (ed. P.O. Hubinont, F. Leroy, C. Robyn & P. Leleux), pp. 118-129. Basel: S. Karger. TACHI, S. TACHI, C. & LINDNER, H. R. (1969). Cilia-bearing stromal cells in the rat uterus. Journal of Anatomy 104, 295-308. TACHI, S., TACHI, C. & LINDNER, H. R. (1970). Ultrastructural features of blastocyst attachment and trophoblastic invasion in the rat. Journal of Reproduction and Fertility 21, 37-56.

An ultrastructural study of implantation in the golden hamster. III. Initial formation and differentiation of decidual cells.

The transformation of fibroblasts into decidual cells was studied ultrastructurally in golden hamsters pregnant for 3, 3 1/2, 4, 4 1/2, 5, or 5 1/2 da...
8MB Sizes 0 Downloads 0 Views