Granulated metrial gland cells at implantation sites of the pregnant mouse uterus

Anat. Embryol. 160, 227 238 (1980) Anatomy and Embryology �9 by Springer-Verlag 1980

Granulated Metrial Gland Cells at Implantation Sites of the Pregnant Mouse Uterus

Ian Stewart and Sandra Peel

Human Morphology, Faculty of Medicine, University of Southampton, Southampton, UK

Summary. A study has been made of the distribution of, and synthesis of DNA by, granulated metrial gland cells at implantation sites in the pregnant mouse uterus. Granulated cells were found in small numbers randomly distributed throughout the endometrium on day 41/2 of pregnancy. Subse- quently cells of this type were lost from the antimesometrial and lateral decidua but increased dramatically in number in the developing decidua basalis. From day 71/2 granulated cells populated the mesometrial triangle to form the metrial gland. A high proportion of granulated cells was found to incorporate tritiated thymidine and the distribution of such cells is described. However, no granulated cells were found to incorporate tritiated thymidine at or after day 12 of pregnancy. In addition the loss of granulated metrial gland cells from the implantation site is described and is accounted for by degeneration in situ and also by migration via vascular channels. It is suggested that this latter route could be of functional significance.

Key words: Metrial gland cells (mouse) - Distribution - Differentiation - Degeneration - Light microscopy.

Introduction

Despite their name, granulated metrial gland cells in the pregnant mouse uterus are not restricted to the metrial gland itself, a structure which develops in the mesometrial triangle at each implantation site from about day 8 of pregnancy. A few granulated cells may be found throughout the endometrium in the pre- implantation uterus and following implantation their numbers increase dramatically in the developing decidua basalis (Stewart and Peel 1978). It has recently been suggested that the granulated cells in both mice (Stewart and Peel 1977) and rats (Peel and Bulmer 1977) differentiate in situ from a lymphocyte-like

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228 I. Stewart and S. Peel

precursor a l though previously it had been supposed that they arise f rom the f ibroblast- l ike s t romal cell popu la t ion (Selye and M c K e o w n 1935; Lark in and Cardell 1971). It is generally accepted, however, that following differentiat ion from their precursors, the granula ted cells pass th rough a proliferative period, and some may increase their ploidy, but this is followed by a final senescent per iod when they no longer synthesise D N A (Larkin and Schultz 1968; Bulmer and Peel 1974). In this present s tudy of the extent a nd life history of the granula ted metr ia l g land cell popu la t ion at imp lan ta t ion sites in the pregnant mouse uterus, the posi t ions of the granula ted cells have been mapped at stages f rom implan ta t ion unt i l after the fo rmat ion of the metr ial gland. In addi t ion an au torad iographic study has been made of D N A synthesis in individual cells after a recent inject ion of t r i t iated thymidine.

Metr ial g land development in the rat appears to reach its peak by abou t day 14 of pregnancy (Larkin and Schultz 1968) and few granula ted cells r emain at pa r tu r i t ion (Baker 1948). Cell lysis and degenerat ion dur ing the last week of pregnancy have been reported and it has been suggested that the release of the granule conten t into intercellular spaces may be of funct ional significance (Larkin 1972). The loss of the granula ted cells in the mouse and the degenerative changes which occur in them in the later stages of pregnancy, both in the decidua basalis and the metr ial gland, were therefore investigated.

Materials and Methods

Nulliparous albino mice were mated overnight, and the morning on which a vaginal plug was found was taken as day 0 of pregnancy. At least two animals were killed on each of days 41/2, 51/2, 61/2, 7, 71/2, 8, 8112, 9, 10, 11, 12, 13, 16 and 18 of pregnancy, at 9 a.m. or 9p.m. All animals, except those killed on day 18 of pregnancy, were given a single intraperitoneal injection of tritiated thymidine (3HTdR) (2 gCi/g body weight) 1 h before sacrifice. Implantation sites were prepared for glycol methacrylate embedding as described previously (Stewart and Peel 1977). Sections (1 g) were examined after being reacted with the periodic acid-Schiff (PAS) technique, with or without previous diastase digestion and counterstained with haematoxylin, or following staining with haematoxylin and eosin. Transverse sections through the centre of implantation sites were reacted with the PAS technique after diastase digestion and prepared for autoradiography using Ilford K5 emulsion. Slides were exposed for 50 days, developed in Kodak D19 developer, fixed, and counterstained with haematoxylin. A photographic montage of sections was prepared upon which the location of the granulated metrial gland cells was plotted. A granulated metrial gland cell was counted only when the profile contained both a nucleus and at least one of the typical glycoprotein granules. Cells which had at least five silver grains over the nucleus were counted as labelled. In addition each granulated cell was classified into one of the following categories :

1. Labelled within tissue 4. Unlabetled mitosis within tissue 2. Unlabelled within tissue 5. Labelled within maternal blood space 3. Labelled mitosis within tissue 6. Unlabelled within maternal blood space.

The distribution of granulated metrial gland cells was plotted from at least three sections at each stage of pregnancy from day 41/2 tO day 9. In animals from day 41/2-8 all granulated metrial gland cells found in each section were plotted. At day 81/2 and day 9 the granulated metrial gland ceils within the area bounded by the circular smooth muscle layer were plotted. In the metrial gland at day 81/2 and later, granulated cells were counted from photographs of three areas taken with a x25 objective lens so that the long axis on the 35 mm negative was directed towards the embryo. The areas chosen for photography covered both lateral and medial regions of the metrial gland. For the studies investigating DNA synthesis data were obtained

Granulated Metrial Gland Cells 229

from the plots, but additional sections were examined where necessary so that a minimum of 100 granulated metrial gland cells were counted at each stage. Granulated cells in the lumina of blood vessels were noted but were not included in the plots or in the data concerned with DNA synthesis.

Results

Distribution of Granulated Metrial Gland Cells

Although implantation had occurred at day 41/2 of pregnancy, decidualisation was only just apparent. At this stage a few thymidine labelled and unlabelled granulated metrial gland cells were observed, apparently randomly distributed throughout the endometrium (Fig. 1). By day 51/2 (Fig. 2) both labelled and unlabelled granulated cells were present in the developing decidua basalis in

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Fig. 2. The distribution of labelled ( • ) and unlabelled (.) granulated metrial gland cells one hour after an injection of tritiated thymidine, in one transverse section through the centre of an implantation site at day 51/2 of pregnancy. Mesometrial triangle (MT); Myometrium (M); Embryo (E)


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Fig. 3. The distribution of labelled ( x ) and unlabelled (.) granulated metrial gland cells one hour after an injection of tritiated thymidine, in one transverse section through the centre of an implantation site at day 71/2 of pregnancy. Metrial gland (MG); Myomet r ium (114); Developing embryo and placenta (E)

each section th rough an implan ta t ion site. Occasional granula ted metr ia l gland cells were present in the lateral regions of the implan ta t ion site but in cont ras t granula ted cells were only rarely seen in the developing an t imesomet r ia l decidua. F r o m d a y 61/2 of p regnancy no granula ted metr ia l gland cells were found in, the an t imesomet r ia l or lateral decidua but their numbers had increased in the decidua basalis where the dis tr ibut ion of labelled and unlabel led granula ted metr ia l gland cells was apparen t ly random. At day 7 and day 71/2 (Fig. 3), however , some areas of the decidua basalis adjacent to the developing embryo conta ined granula ted metr ia l gland cells but none of these was labelled; bo th labelled and unlabel led granula ted metr ia l gland cells were numerous in the


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Fig. 4. The distribution of labelled ( x ) and unlabelled (,) granulated metrial gland cells in the decidua basalis one hour after an injection of tritiated thymidine, in one transverse section through the centre of an implantat ion site at day 81/2 of pregnancy. No granulated cells were found in the antimesometrial part of the section. Metrial gland (MG); Myometr ium (M); Developing embryo and placenta (E)

Fig. 5. The percentage (means+ SE) of the granulated metrial gland cells which were in mitosis in the endometr ium of implantat ion sites at day 4 of pregnancy ([]) and in the decidua basalis (-.) and metrial gland (::) at later stages of pregnancy

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Fig. 6. Three granulated metrial gland cells in the lumen of a lateral sinusoid one hour after an injection of tritiated thymidine. One of the granulated cells (arrow) is labelled. Day 9 of pregnancy. Diastase, PAS and haematoxylin

Fig. 7. The percentage (means _+ SE) of the granulated metrial gland cells in the decidua basalis ( o - - - o ) and the metrial gland ( i - - n ) which were labelled one hour after an injection of tritiated thymidine

Fig. 8. The percentage of the granulated metrial gland cells in the inner (o) and the outer (o) regions of the metrial gland which were labelled one hour after an injection of tritiated thymidine


compact zone. By this stage of pregnancy a few labelled and unlabelled granulated metrial gland cells were seen in the mesometrial triangle and the circular smooth muscle layer adjacent to it. At day 8 and day 81/2 (Fig. 4) large numbers of granulated metrial gland cells were usually present in the metrial gland but although granulated inertial gland cells were widely distributed in the decidua basalis, labelling of these cells was seen only in a limited area. Of the numerous granulated metrial gland cells in the decidua basalis at day 9 of pregnancy only a few were labelled and these were generally found in the central region below the metrial gland. Granulated metrial gland cells were found in the basal zone of the decidua basalis adjacent to the mesometrial triangle but the number of granulated cells in this area was not found to attain the levels reached in the rest of the decidua basalis or in the metrial gland. Many labelled granulated metrial gland cells were found in the metrial gland at days 9, 10 and 11 but no labelled granulated cells were found at day 12 of pregnancy or later.

Many of the larger granulated metrial gland cells examined appeared binu- cleate and in some of these both nuclei were labelled one hour after the injection of tritiated thymidine. Other granulated cells were found which contained mitotic figures and these were generally in areas which showed a high incidence of granulated cell labelling. The percentage of granulated cells which contained mitotic figures is shown in Figure 5 and a few of these mitotic figures were labelled.

Granulated metrial gland cells were found lying free within the lumina of blood vessels from day 7 of pregnancy in the decidua basalis. Some were seen in the lateral sinusoids of the decidua (Fig. 6) and others in the blood spaces of the labyrinth, junctional zone, secondary giant cell layer and in blood vessels of the metrial gland. The granulated metrial gland cells in the maternal blood channels generally appeared healthy and occasional ones had incorporated the tritiated thymidine (Fig. 6).

Proportion of Cells Incorporating Tritiated Thymidine

The percentage of granulated metrial gland cells in both the decidua basalis and metrial gland which had taken up 3HTdR one hour after injection at each stage of pregnancy is shown in Figure 7. In the decidua basalis a high percentage of granulated cells were labelled at day 51/2 and this reached a peak at day 7 71/2 after which the percentage of labelled granulated cells showed a steady fall to zero at day 10. In the metrial gland about half the granulated cells were labelled initially but after day 8 the percentage of labelling steadily decreased until at day 12 no labelled granulated cells were found. When the metrial gland was divided into inner and outer regions, and the proportion of labelled cells estimated for each region, a greater proportion was found to be labelled in the outer than in the inner region throughout the period between day 81/z and day 11 (Fig. 8).


Fig. 9. Granulated metrial gland cells in the metrial gland with well-defined nuclei and prominent nucleoli and containing many cytoplasmic glycoprotein granules. Day 13 of pregnancy. Diastase, PAS and haematoxylin


Loss of Granulated Metrial Gland Cells

In areas of the uterus which no longer contained cells actively incorporating thymidine the granulated metrial gland cells (Fig. 9) were usually large and relatively palely stained, and they contained numerous glycoprotein granules and much glycogen: the nuclei were rounded or kidney shaped, with only a thin rim of heterochromatin, and they contained one or two prominent nucleoli. In the last week of pregnancy changes were detected in the general organisation of the decidua basalis and the metrial gland and many granulated metrial gland cells appeared to be degenerating. At day 13 of pregnancy the decidua basalis cells in the area lying next to the trophoblastic giant cell layer were densely packed and many contained glycogen. However, in the rest of the decidua basalis the cells were less densely packed and contained little glycogen. Particu- larly in the more lateral region of the decidua basalis there were large intercellular spaces and although they were still present on day 15 of pregnancy they were reduced in number. At this stage the decidua basalis appeared reduced in area and most of the cells contained glycogen. By day 18 of pregnancy the decidua basalis was still present as a definable area but it showed extensive necrosis. Large intercellular spaces were also found in the metrial gland at day 13 (Fig. 10) and the area containing such spaces had increased by day 15. By day 18 the size of the metrial gland was reduced, but it still remained a prominent feature at the base of the mesometrium. The numbers of granulated metrial gland cells was noticeably reduced from day 13 in the decidua basalis and from about day 15 in the metrial gland. Certainly by day 18 only a few granulated metrial gland cells were found in sections of decidua basalis and metrial gland and most of these showed degenerative changes, Degenerative changes were often seen in granulated metrial gland cells from day 13 of pregnancy. They were initially most obvious in the decidua basalis in the area next to the trophoblastic giant cell layer but were subsequently found in the remainder of the decidua basalis and in the metrial gland. The nuclei of these degenerated metrial gland cells were often irregularly shaped and more densely stained than the nuclei of normal cells (Fig. 11). The cytoplasm sometimes appeared vacuolated, particularly in the area around the cytoplasmic granules. Some apparently dis- rupted cells were seen and diastase resistant PAS positive material was found which appeared to be lying in the intercellular spaces of both the decidua basalis and the metrial gland. Some of this glycoprotein material resembled, in form and staining characteristics, the cytoplasmic granules of granulated

Fig. 10. Large intercellular spaces (S) in the lateral area of the metrial gland. Day 13 of pregnancy. PAS and haematoxylin

Fig. 11. Granulated metrial gland cells with irreguiarly shaped, densely stained nuclei (arrows) in the decidua basalis. Day 13 of pregnancy. Diastase, PAS and haematoxylin

Fig. 12. Large inclusions (arrows) containing glycoprotein material in association with stromal cells of the metrial gland. Other smaller, paler staining inclusions (arrowheads) are numerous in stromal cells. Day 16 of pregnancy. Diastase, PAS and haematoxylin


metrial gland cells. Large accumulations of diastase resistant PAS positive material often appeared as inclusions within the cytoplasm of the decidual cells in the decidua basalis and in the stromal cells of the metrial gland (Fig. 12). At this time the stromal cells of the metrial gland contained other small inclusions, many of which gave a pale reaction with the diastase PAS technique (Fig. 12). Many stromal cells enlarged and in late pregnancy could no longer be described as fibroblast-like.

Discussion

In the two days following implantation, granulated metrial gland cells were lost from the antimesometrial and lateral regions of the decidualising endometrium, but they increased in numbers in the developing decidua basalis. On day 71/2 of pregnancy (Fig. 3), when granulated cells were numerous in the decidua basalis, a few were seen in the mesometrial triangle forming the begin- nings of the metrial gland. Such a progression, their appearance in the decidua basalis being followed by their appearance in the mesometrial triangle, has also been reported for analogous cells in the rat (Dickson and Bulmer 1961; Dallenbach-Hellweg et al. 1965) and the hamster (Pijnenborg et al. 1974).

When the granulated metrial gland cells in the decidua basalis and metrial gland were examined following an injection of tritiated thymidine, the distribution of labelled and unlabelled ceils in each area appeared, initially, to be random (Figs. 2, 8). However, areas containing only unlabelled cells were found in the decidua basalis from day 71/2 (Fig. 3). At later stages of pregnancy such areas increased in size so that by day 9 in the decidua basalis labelled cells were found only in the central area and in the area adjacent to the metrial gland. At this stage of pregnancy both labelled and unlabelled granulated metrial gland cells were distributed throughout the metrial gland but the percentage of labelled cells was higher in its outer region. From day 12, no labelled granulated cells were seen in the metrial gland. The pattern produced as granulated cells cease to incorporate thymidine in the different regions of the implantation site parallels the changes in the distribution of precursor cells (Stewart and Peel 1978) and probably represents an ordered sequence in the cessation of proliferation in the various regions. Whether the loss of granulated cells from the antimesometrial and lateral decidua as well as the loss of granulated cells actively incorporating thymidine from the different areas of the implantation site is due to these tissues providing an increasingly inhospitable environment, either actively or passively, is uncertain.

A high percentage of labelled granulated metrial gland cells was found in both the decidua basalis and the metrial gland one hour after an injection of tritiated thymidine (Fig. 7). The difference in the peak of the percentage labelling of granulated cells in the decidua basalis and metrial gland may be explained by a variation in the proportion of granulated cells actively in cycle and in the resting Go period (Stewart 1980). In addition, many granulated cells were in mitosis and some of these had also incorporated the thymidine. It would seem likely that the granulated metrial gland cells undergo a high


rate of proliferation with a short G 2 phase in the cell cycle. These observations also suggest that, whatever the original stem cell, there is a rapid multiplication of the granulated cells themselves, and the stem cell pool may well be relatively small. However, proliferation of granulated metrial gland cells appears to cease by day 12 of pregnancy (Fig. 7).

During the last week of pregnancy the numbers of granulated metrial gland cells appeared to decrease in both the decidua basalis and the metrial gland and although some were still present on day 18 of pregnancy many of these showed signs of degeneration. The loss of the granulated cells may be due to their degeneration in situ although they have been seen apparently passing between endothelial cells (Stewart and Peel 1978). The large intercellular spaces seen in the decidua basalis and metrial gland (Fig. 10) may be a result of the migration of granulated cells into the lumina of blood vessels. However, the lysis of granulated metrial gland cells in situ has been reported by previous workers and it has been suggested that this may be of functional significance (Cardell et al. 1969; Larkin 1972). Peel and Stewart (1979) found extracellular PAS positive material and granules but no evidence to suggest that dissolution of granules occurred in the intercellular space of the rat. It was suggested that the stromal cells which have been reported to endocytose marker proteins (Sharma and Peel 1978) were involved in the endocytosis of the products of granulated metrial gland cell degeneration. It could be, therefore, that the granulated cells which pass into the blood vessels are of functional significance and that those which remain and lyse in situ in the decidua basalis and metriaI gland are in excess of requirements. Granulated cells in areas associated with the maternal-foetal barrier may be of importance, particularly in relation to recent findings of cytoplasmic IgG in these cells (Bulmer and Peel 1977). The presence of granulated cells in vessels which apparently drain the implantation site may represent a surplus to requirements but it cannot be excluded that they have a function outwith the uterus.

Acknowledgements. We are grateful to Professor D. Bulmer for his advice and constructive criticism. This work was supported by the Wellcome Trust

References

Baker BL (1948) Histochemical variations in the metrial gland of the rat during pregnancy and lactation. Proc Soc Exp Biol Med 68:492-496

Bulmer D, Peel S (1974) An autoradiographic study of cellular proliferation in the uterus and placenta of the pregnant rat. J Anat (Lond) 117:433441

Buhner D, Peel S (1977) The demonstration of immunoglobulin in the metrial gland cells of the rat placenta. J Reprod Fertil 49:143-145

Cardell RR, Hisaw FL, Dawson AB (1969) The fine structure of granular cells in the uterine endometrium of the Rhesus monkey (Macaca mulatta) with a discussion of the possible function of these cells in relaxin secretion. Am J Anat 124:30%340

Dallenbach-Hellweg 0, Battista JV, Dallenbach FD (1965) Immunohistological and histochemical localization of relaxin in the metrial gland of the pregnant rat. Am J Anat 117:433-450

Dickson AD, Bulmer D (I961) Observations on the origin of metriaI gland cells in the rat placenta. J Anat (Lond) 95:262-273


Larkin LH (1972) Electron microscopy of granule release in metrial gland cells of the pregnant rat. Anat Rec 172:109-126

Larkin LH, Cardell RR (1971) Differentiation of granulated metrial gland cells in the uterus of the pregnant rat: an electron microscope study. Am J Anat 132:241-258

Larkin LH, Schultz RL (1968) Histochemical and autoradiographic studies of the formation of the metrial gland in the pregnant rat. Am J Anat 122:607-619

Peel S, Bnlmer D (1977) The fine structure of the rat metrial gland in relation to the origin of the granulated cells. J Anat (Lond) 123:687-696

Peel S, Stewart I (1979) Ultrastructural changes in the rat metrial gland in the latter half of pregnaricy. Anat Embryol 155: 209-219

Pijnenborg R, Robertson WB, Brosens I (1974) The arterial migration of trophoblast in the uterus of the golden hamster, Mesocricetus auratus. J Reprod Fertil 40 : 269-280

Selye H, McKeown I (1935) Studies on the physiology of the maternal placenta in the rat. Proc R Soc, B 119:1 31

Sharma RP, Peel S (1978) The uptake of proteins by the uterus and placenta of the pregnant rat. J Anat (Lond) 126:410411

Stewart IJ (1980) PhD Thesis, University of Southampton, UK Stewart I, Peel S (1977) The structure and differentiation of granulated metrial gland cells of

the pregnant mouse uterus. Cell Tissue Res 184:517-527 Stewart I, Peel S (1978) The differentiation of the decidua and the distribution of metrial gland

cells in the pregnant mouse uterus. Cell Tissue Res 187:167-179

Accepted May 10, 1980

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Granulated metrial gland cells at implantation sites of the pregnant mouse uterus