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Observations of the cellular changes in cultured mouse metrial gland explants and of the maintenance of granulated metrial gland cells in vitro
TISSUE AND CELL, 1YY3 25 (6) 857-864 0 iYY3 Longman Group CJK Ltd.
D. D. Y. MUKHTAR
and I. J. STEWART
OBSERVATIONS OF THE CELLULAR CHANGES IN CULTURED MOUSE METRIAL GLAND EXPLANTS AND OF THE MAINTENANCE OF GRANULATED METRIAL GLAND CELLS IN VITRO Kcyword5.
Metrial
gland. explanr culture.
granulated
mctrial
gland cell. moux
ABSTRACT. Moue mctrial gland explants wrc cultured lot perwda ot up to 7 da!\ Granulated mctrial gland cells migrated from the explants onto the culture dish whbtratum with a peak in numhcrs of cm&rant cells usually found after l-3 days of culture. Histological examination of the cultured explants showed that the central cow of the explant\ rapidly dcgcneratcd although a viable supcrticial layer of tlssuc remained throughout the culture period. It is suggested that the degenerative changes and the emigration of many granulated mctrial gland cells from the explants was a roult of the large sire of the explants used and consequential nutrient deprivation. Granulated metrial gland cells wcrc found in the riahlc superficial layer of the explants and on their surface throughout the culture period. including day 7 of culture when few granulated mctrial gland cells remained on the culture dish \uhstratum. The retention of some granulated metrial gland cells on the surface of the explant\ when few rcmaincd on the culture dish substratum suggests that contact with \trt>mal cells 1s important for the maintcnancc of granulated mctrial gland cells.
Introduction The metrial gland is part of the maternal placenta which develops at the base of the mesometrium at each implantation site in mice from about day 8 of pregnancy and persists through pregnancy and into the postpartum period (Peel, 1989). In mice, as in other rodents, the metrial gland is characterised by the presence of granulated metrial gland (GMG) cells and fibroblast-like stroma1 cells (Selye and McKeown, 1935; Stewart and Peel, 1978). Granulated metrial gland cells are large rounded cells which contain numerous glycoprotein granules (Stewart and Peel, 1977). They are derived from bonemarrow precursors (Peel et al., 1983) which differentiate in the developing decidua basalis and metrial gland (Stewart and Peel. Human Morphology. Faculty of Medicine. University of Southampton. Bassett Crescent East, Southampton SOY 3TU Correspondence to Dr 1. J. Stewart. Received
26 May 1YY3
1977). It has been proposed that the differentiation and maintenance of GMG cells is influenced by decidual stromal cells or by the fibroblast-like stromal cells of the metrial gland (Stewart, 1983; 1987). Evidence in support of this proposal has not yet come forward. The function in pregnancy of GMG cells remains uncertain but in mice it has been shown that they migrate from the decidua basalis and metrial gland to the placenta where they are cytotoxic to some of the cytotrophoblast cells which line the maternal blood spaces of the labyrinthine placenta (Stewart, 1984; 1990). Granulated metrial gland cells will migrate from cultured explants of metrial gland tissue, maintained under standard tissue culture conditions, and become attached to the floor of the culture Aask (Mukhtar and Stenart, 1988). High numbers of GMG cells migrate from explants prepared from mice at day 10 of pregnancy but the numbers which are progressively lower from migrate explants obtained at progressively later
858
MUKHTAR
stages of pregnancy. Peak migration occurs during the first 2 days of culture and thereafter the numbers of GMG cells on the culture dish around the explants steadily declines and few remain after 7 days of culture (Mukhtar and Stewart, 1988). Small immature GMG cells which migrate from explants prepared from developing decidual tissue survive in culture for less than 24 hr although supplementing the culture medium with interleukin-2 may allow for long-term survival (Linnemeyer and Pollack, 1991). Although this explant culture system has been used as an effective source of a pure population of GMG cells the system also provides an opportunity for determining whether GMG cells which remain in contact with stromal cells, within or on the surface of the explants, will survive for longer than those which lose contact with the stromal cells by migrating onto the surface of the culture flask. We have therefore examined the changing morphology of metrial gland explants when cultured for periods of up to 7 days particularly with respect to the GMG cell population. Explants have been maintained in the presence or absence of progesterone, a hormone which has been suggested to influence directly (Sharma and Bulmer, 1983) or indirectly (Stewart, 1987) the differentiation of GMG cells and which is necessary for the maintenance of pregnancy. The surface of the cultured explants has been examined using scanning electron microscopy to more clearly assess the extent to which GMG cells come to lie at the interface between explant and culture flask. Further, the numbers of GMG cells which migrate from the explant onto the surface of the culture dish have been examined over the period of assessment of the changes occurring within the tissue explants. These studies have provided data which suggest why GMG cells migrate from cultured explants of metrial gland tissue but they also provide data which suggest that the maintenance of GMG cells is influenced by contact with stromal cells. Materials and Methods Animals
Random bred virgin female Porton mice aged 8-16 weeks from an in-house colony maintained under standard animal house con-
AND STEWART
ditions were used in this study. They were mated overnight with males of their own strain and the presence of a vaginal plug the following morning was taken as day 0 of pregnancy. Mice were killed by cervical dislocation under ether anaesthesia at days 10, 12 and 14 of pregnancy. At least six mice were killed at each stage of pregnancy. Some mice were killed at day 11 of pregnancy but these were only used for preparing explants for scanning electron microscopy. The uterus was removed from each animal under aseptic conditions and opened by a longitudinal incision along the antimesometrial wall. Fetuses were immediately killed by decapitation. Preparation of metrial gland explant cultures
Metrial glands were dissected from each implantation site and divided into 4 equal pieces with a sterile blade. One piece of metrial gland (explant) from each of 4 or 5 metrial glands from the same animal were attached to the base of 35 mm plastic culture dishes. Three ml of culture medium consisting of Eagles Minimal Essential Medium with Earle’s salts (Flow Laboratories, Irvine, UK) supplemented with 20% heat inactivated fetal calf serum (Seralab, Sussex, UK), 0.058% L-glutamine, 1OOIUml-’ penicillin and 100 pg ml-’ streptomycin were added to each culture dish. Cultures were further supplemented with either 225 ng progesterone in 25 ,ul ethanol or 25 ,ul ethanol alone (controls). Cultures were incubated at 37°C in a humid atmosphere of 5% CO2 in air for periods of up to 7 days. The day on which the explants of metrial gland tissue were prepared was taken as day 0 of culture. Medium was replaced on days 2, 4 and 6 of culture. Further details of the explant culture procedure are given in Mukhtar and Stewart (1988). Of the culture dishes prepared from each animal 2 dishes (1 with progesterone, 1 without progesterone) were used to determine the extent of migration of GMG cells from the culture explants and 2 dishes (1 with progesterone, 1 without progesterone) were used to assess the morphological changes occurring within the explant over the culture period. Additional cultures were established without any supplemented progesterone or ethanol to provide control material to assess
MOUSE
METRIAL
GLAND
R5Y
EXPLANTS
any possible effects of the small amounts of ethanol on the explant cultures.
6001
Quandtatiorz of GMG cell migration
400
Quantitation of GMG cell migration was performed as described in Mukhtar & Stewart (1988). On each of days 1, 2, 3, 4, and 7 of culture the number of GMG cells attached to the base of the culture dish around each explant was counted in an area defined by the field diameter (1.7 mm) of a X 10 objective lens around the edge of each explant. Cells only rarely migrated beyond this perimeter. On days when the culture medium was changed cell counts were carried out after the medium change. Data were analysed using a Student’s ttest.
300 200 100 0k 012
600 500 No. GMG
of
Scanning electron microscopy
Additional explants were prepared as described above and fixed in situ after 1, 2, 3. 4, 5 or 7 days of culture in 25% glutaraldehyde in supplement free Minimal Essential Medium (Eagles) for 3 hr at 4°C. They were then washed in phosphate buffered saline, dehydrated in ethanol and critical point dried. Pieces of culture dish with the explant attached were then mounted on stubs, sputter coated with platinum and examined in an Hitachi S800 scanning electron microscope. Results Quantitation
The numbers
of GMG cell migration
of GMG cells found around
3
4--
1
?
Day 12
400 300
Histological examination of metrial gland expfants
Explants were taken immediately after preparation (time 0), after attachment to the base of the culture dish (time 15 min) and after 1, 2, 3, 4, and 7 days of culture. The explants were fixed overnight in a mixture of 4% formaldehyde and 2% glutaraldehyde in 0.1 mol phosphate buffer 1-l pH 7.2-7.4 (modified from Karnovsky, 1965). Tissues were washed in buffer, dehydrated in ethanol and embedded in Araldite. Ten 1 ,um sections at different depths (-50 pm apart) were cut from each explant, stained with toluidine blue, and examined by bright field microscopy. At least three explants at each stage of culture were examined.
Day 10
500
0
1
2
3
4
7
600 Day 14
500 I 400. 300~ zooloo-
0 O-?
12
TV
4
7
Days of culture Fig. 1. Numbers (means + S.E.) of GMG cells which migrated from metrial gland explants cultured in the presence (75ngml-‘) and absence of progesterone. Each point on the graphs represents the number of GMG cells found around each e&ant. Metrial gland explants were obtained from at least h mice at each of days IO. 12 and 14 of pregnancy. Standard errors 13 are not shown. W Explants with progesterone. *-_* Explants with ethanol carrier.
the explants maintained in the presence of progesterone, and in the presence of the ethanol carrier only, are shown in Figure 1. These numbers are generally similar to those of a previous study where explants were cultured in the absence of both progesterone and the ethanol carrier (Mukhtar and Stewart, 1988). When the migration curves for the progesterone treated cultures were compared to the cultures supplemented with
MOUSE
METKIAL
GLAND
Shl
EXPLANTS
Generally, there was little or no difference between the morphology of comparable explants cultured in the presence, or in the absence, of progesterone. There appeared to be, however. more GMG cells in day 10 explants after 1 to 3 days in cultured in the presence of progesterone than in similar explants cultured in the absence of progesterone. In these progesterone stimulated explant cultures the stromal cells appeared to have fewer cytoplasmic inclusions than in control material. These differences were no longer discernible after 4 days of culture.
most of those found in explants from day 10 pregnant mice. Some GMG cells in day 10 explants were in mitosis (Fig. 2) but mitoses were rare in day 12 explants and absent from day 14 explants. There were some mitotic stromal cells in day 10 explants (Fig. 2), few in day 12 explants and only an occasional one in day 14 explants. Stromal cells in day 14 explants cytoplasmic contained many inclusions but these were not a feature of the stromal cells in day 10 explants and were only found to a limited extent in day 12 explants. An occasional degenerate GMG cell was found in the central part of day 14 explants. There was extensive damage at the periphery of each explant. presumably as a result of cutting the explants to size. Similar damaged areas were found in more central regions of some sections of explants fixed 15 mins after the explants were prepared but this damage was probably a result of the stabbing procedure used in fixing the explant\ to the base of the culture dish.
Time 0, + 15 mins
After I-2 days of culture
Explants at time 0 and at + 15 mins generally appeared to be similar. All explants consisted of densely packed cells of which fibroblastlike stromal cells were the major cell type (Fig. 2). Granulated metrial gland cells were also numerous (Fig. 2). The GMG cells in explants from mice at day 12 and day 14 were larger, with more cytoplasmic granules than
Sections of explants cultured for l-2 days had a palely stained middle area which consisted mainly of degenerate cells with pyknotic nuclei and a more darkly stained periphery (Fig. 3). A few apparently normal stromal cells and smooth muscle cells were found in the degenerate middle areas. No healthv GMG cells were seen in the middle part o?’
the ethanol carrier there was only one significant difference: the peak value for the day 12 progesterone treated explants was significantly lower than with the carrier control treated explants (p < 0.05). Histology of metrial gland explants Explant cultures supplemented with progesterone
Fig. 7. Metrial gland explant at time 0. Densely packed fibroblast-like \trmnaI cell\ (small arrows), GMG cells (large arrows) and some smooth muscle cells (M) arc prcscnt. (‘ell\ in mltous can be seen (arrowheads). Explant from day 10 mouse. I pm Aralditc wctwn. ‘Ioluidmc hluc x4lJll. 1~1s.3. Metnal gland explant after I day in culture in the prescncc of progc~tcronc licalthy \tromal cells and healthy GMG cells (arrows) can be been in the pcrlphcral region 01 the explant. The central region of the explant (X) is dcgcncratc. Explant from day IO mowc I {rm Aralditc sectmn. Toluidine hluc. x400. Fig. 4. Metrial gland e&ant after 3 davs in culture in the presence of moccstcronc The pel-iphcral region contains’ healthy cells, i&ding GMG cell; (arrows). abd :as a Rattencd layer of cells at the surface. Some GMG cells (arrowheads) are on the wrface of the explant. The central region of the explant (X) is degenerate. Explant from day IO mouse I ,rm Aralditc wction Tolmdine hluc x400. Fir.._5. Mctrial eland exolant after7 davs in culture in the ~rescnce of ethanol carncr. Hcalthb cells arc present yn the peripheral region. GMG cells (arrows) can be seen on the wrtacc. ol the explant. Explant from day 10 mouse. 1 urn Aralditc section. Toluidinc hluc. xJIH). Fig. 6. Numerous GMG cells (G) on the surface of a metnal gland explant after I day m culture. Explant from day 10 mouse. Scanning electron microw)py 45’ tilt. x2175.
MUKHTAR
862
AND STEWART
Fig. 6. Numerous GMG cells (G) on the surface of a metrial gland explant after 1 day in culture. Explant from day 10 mouse. Scanning electron microscopy. 45” tilt. x2175.
the explants obtained from day 10 pregnant mice but a few healthy GMG cells were still present in the middle parts of the explants obtained from day 12 and day 14 mice. Healthy stromal cells and GMG cells were seen in the peripheral regions of each explant (Fig. 3). The stromal cells, including those obtained from day 10 pregnant mice and cultured for only 1 day contained cytoplasmic inclusions, many of which stained palely with toluidine blue. Mitoses were rare in 1 day cultured day 10 explants and were absent
from all other explants. By day 2 of culture all the healthy GMG cells seen in explants obtained from day 10 pregnant mice were large with numerous cytoplasmic granules although at 1 day of culture a few small GMG cells were still evident. At 1 and 2 days of culture, in explants from day 12 and day 14 mice all the healthy GMG cells were large. In all explants, not all of the GMG cells in the peripheral part of the explant were healthy; some had pyknotic nuclei and showed extensive cytoplasmic degeneration.
MOUSE METKIAL
GLAND
EXPLANTS
In addition. cellular debris including cytoplasmic granules was found in intercellular spaces and some of the inclusions in the stromal cells had a morphology and staining characteristics similar to GMG cell granules. Afivr 3. 4 and 7 days of culture After 3 days of culture most explants had developed an epithelial-like layer of cells around the edge of the explant (Fig. 4). All explants had a central region generally devoid of any cellular elements with a peripheral rim of healthy looking cells (Fig. 4). Most of these peripheral cells were stromal cells whose cytoplasm contained many inclusions. However, some healthy GMG cells were also found near to, or on, the surface of the explants (Fig. 4). Overall. there were fewer GMG cells than at earlier stages of culture and there were more in explants derived from day 10 pregnant mice than from mice killed at day 12 or day 14. All explants at day 7 of culture had at least one or two healthy GMG cells in them, or on their surface (Fig. 5) in each section examined. Scmning electron microscopy of cultured explants The surface of the explants after 1-2 days culture often appeared ragged but by day 4 of culture much of the surface of most explants appeared to be covered with epitheloid cells. Cells with a surface morphology typical of GMG cells, that is, rounded cells with numerous microvillous-like or leaf-like projections (Mukhtar and Stewart, 1993), were found on the surface of all explants (Fig. 6). Their numbers and distribution on the explants varied but generally they were most numerous on explants cultured for only 1 or 2 days. However, several were still found on explants cultured for 7 days. Occasionally, GMG cells were seen which appeared as if they were passing between surface cells to reach onto the surface of the explant. Discussion Migration of GMG cells from cultured explants of metrial gland tissue occurs in parallel with degenerative changes taking place within the central core of the explants. However, normal GMG cells were still found in the healthy peripheral rim of the explants
at all stages of culture as well as on the surface of the explants. Granulated metrial gland cells are motile cells (Mukhtar and Stewart, 1988) and in oiuo migrate from the tissue of the decidua basalis and metrial gland into the lumina of blood vessels (Stewart and Peel, 1978; Stewart, 1991). Such migration in vivo occurs without evidence of degeneration of decidual/metrial gland tissue. It7 vitro, some of the migration of GMG celis onto the culture dish substratum may be a reflection of an inherent migratory capabilit) (Mukhtar and Stewart, 1988) but, from the appearance of the explant cores seen in the present study, it is more likely that migration occurs in the main to escape from the degenerate central core of the large explants to an area at the periphery, or surface of the explant, or free in the culture dish where there would be readily available nutrients. Changes in the stromal cells of the explant\ during the culture period were major. from their total degeneration in the central core region to the hypertrophy and the accumulation of inclusions in the stromal cells located at the periphery. Recently. it has been proposed that the inclusions in stromal cells of the metrial gland (Stewart and Peel. 1980) arise mainly as a result of endocytic activity associated with degenerative changes in the metrial gland and repair of the uterine wall following parturition and that monocyte derived macrophages do not play a major role in this activity (Stewart and Mitchell. 1992). The present study lends support to that proposal because of the changes observed in the stromal cells of the cultured explants where blood borne macrophases would not leave access. Generally, progesterone supplements had little effect on the morphology of the explant cultures or on the pattern of migration and maintenance of GMG cells outwith the explants. In a previous study of cultured rat metrial gland explants GMG cells were not maintained in the explants unless progesterone was present in the culture medium (Adam and Peel, 1983). The most obvious effect of progesterone in the present study was a small, but significant, reduction in the numbers of GMG cells which migrated from the cultured explants, from day 12 pregnant mice, at the peak migratory period. The changes to the GMG cell population in the explant cultures provides some insight
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MUKHTAR
into the mechanism by which GMG cell maintenance is supported. Progesterone supplements did not appear to influence GMG cell maintenance. Any soluble factors produced by the stromal cells would influence both the GMG cells in and on the explant and the cells on the culture dish substratum. However, GMG cells may need contact with the stromal cells. The possibility that the maintenance of GMG cells is regulated by a juxtacrine association with stromal cells requires further investigation. Immature GMG cells which migrate from metrial gland tissue explants obtained from mice killed between day 8 and day 10 of pregnancy do fail
AND STEWART
to mature into large GMG cells and generally die within 24 hr (Mukhtar and Stewart, 1993). It is perhaps at this time in the metrial gland when a juxtacrine mechanism is of greatest importance to the differentiation of GMG cells. Acknowledgements
We are grateful to C. Inman and his staff at the Electron Microscope Unit, Southampton General Hospital for their assistance with the scanning electron microscopy. The scanning electron microscopy was supported by the Wessex Medical School Trust.
References Adam, E. and Peel, S. 1983. The maintenance of rat metrial gland explants in uitro. in the presence and absence of progesterone. .I. Anal., 136, 321-328. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27, 137A. Linnemeyer, P. A. and Pollack, S. B. 1991. Murine granulated metrial gland cells at uterine implantation sites arc natural killer lincagc cells. J. fmmuno[. , 147, 253&2535. Mukhtar, D. D. Y. and Stewart, I. 1988. Migration of granulated metrial gland cells from cultured explants of mouse metrial gland tissue. Celf T&x. Res., 253, 413-417. Mukhtar, D. D. Y. and Stewart, I. J. 1993. The surface morphology of mouse metrial gland cells. J. Anor.. 182, 415424. Peel. S. 1989. Granulated metrial gland cells. Adv. Anat. Embryol. Cell Biol.. 115, 1-l 12. Peel, S., Stewart. I. J. and Bulmer. D. 1983. Experimental cvidence for the bone marrow origin of granulated mctrial gland cells of the mouse uterus. Cell Tin. Res., 233, 647-656. Selye. H. and McKeown. I. 1935. Studies on the physiology of the maternal placenta in the rat. Proc. Roy. Sot. B. 119, l-31. Sharma, R. and Bulmer, D. 1983. The effects of ovariectomy and subsequent progesterone rcplacemcnt on the uterus of the pregnant mouse. J. Amt.. 137, 695-703. Stewart, I. 1983. An investigation into the differentiation of granulated metrial gland cells in the early pregnant moue uterus. .I. Amt., 137, 85-93. Stewart, I. 1984. A morphological study of granulated metrial gland cells and trophoblast cells in the labyrinthmc placenta of the mouse. J. Anat., 139, 627-638. Stewart, I. 1987. Differentiation of granulated metrial gland cells in ovariectomized mice given ovarian hormones. J. Endow., 112, 23-26. Stewart, I. J. 1990. Granulated metrial gland cells in the mouse placenta. Placenta, 11, 263-275. Stewart, 1. J. 1991. Granulated metrial gland cells: pregnancy specific leukocytes? J. Leuk. Biol., 50, 19Pr207. Stewart, I. J. and Mitchell, B. S. 1992. Macrophages and other cndocytic cells in the mouse uterus during the second half of pregnancy and into the postpartum period. J. Arm., 181,11%126. Stewart, 1. and Peel, S. 1977. The structure and differentiation of granulated metrial gland cells of the prcgnam mouse uterus. Cell Tiss. Res., 184, 517-527. Stewart, I. and Peel, S. 1978. The differentiation of the decidua and the distribution of mctrial gland cells in the pregnant mouse uterus. Cell Tiss. Res., 187, 167-179. Stewart, I. and Peel, S. 1980. Granulated metrial gland cells at implantation sites of the pregnant mouse uterus. Amt. Embryol., 160, 227-238.
D. D. Y. MUKHTAR
and I. J. STEWART
OBSERVATIONS OF THE CELLULAR CHANGES IN CULTURED MOUSE METRIAL GLAND EXPLANTS AND OF THE MAINTENANCE OF GRANULATED METRIAL GLAND CELLS IN VITRO Kcyword5.
Metrial
gland. explanr culture.
granulated
mctrial
gland cell. moux
ABSTRACT. Moue mctrial gland explants wrc cultured lot perwda ot up to 7 da!\ Granulated mctrial gland cells migrated from the explants onto the culture dish whbtratum with a peak in numhcrs of cm&rant cells usually found after l-3 days of culture. Histological examination of the cultured explants showed that the central cow of the explant\ rapidly dcgcneratcd although a viable supcrticial layer of tlssuc remained throughout the culture period. It is suggested that the degenerative changes and the emigration of many granulated mctrial gland cells from the explants was a roult of the large sire of the explants used and consequential nutrient deprivation. Granulated metrial gland cells wcrc found in the riahlc superficial layer of the explants and on their surface throughout the culture period. including day 7 of culture when few granulated mctrial gland cells remained on the culture dish \uhstratum. The retention of some granulated metrial gland cells on the surface of the explant\ when few rcmaincd on the culture dish substratum suggests that contact with \trt>mal cells 1s important for the maintcnancc of granulated mctrial gland cells.
Introduction The metrial gland is part of the maternal placenta which develops at the base of the mesometrium at each implantation site in mice from about day 8 of pregnancy and persists through pregnancy and into the postpartum period (Peel, 1989). In mice, as in other rodents, the metrial gland is characterised by the presence of granulated metrial gland (GMG) cells and fibroblast-like stroma1 cells (Selye and McKeown, 1935; Stewart and Peel, 1978). Granulated metrial gland cells are large rounded cells which contain numerous glycoprotein granules (Stewart and Peel, 1977). They are derived from bonemarrow precursors (Peel et al., 1983) which differentiate in the developing decidua basalis and metrial gland (Stewart and Peel. Human Morphology. Faculty of Medicine. University of Southampton. Bassett Crescent East, Southampton SOY 3TU Correspondence to Dr 1. J. Stewart. Received
26 May 1YY3
1977). It has been proposed that the differentiation and maintenance of GMG cells is influenced by decidual stromal cells or by the fibroblast-like stromal cells of the metrial gland (Stewart, 1983; 1987). Evidence in support of this proposal has not yet come forward. The function in pregnancy of GMG cells remains uncertain but in mice it has been shown that they migrate from the decidua basalis and metrial gland to the placenta where they are cytotoxic to some of the cytotrophoblast cells which line the maternal blood spaces of the labyrinthine placenta (Stewart, 1984; 1990). Granulated metrial gland cells will migrate from cultured explants of metrial gland tissue, maintained under standard tissue culture conditions, and become attached to the floor of the culture Aask (Mukhtar and Stenart, 1988). High numbers of GMG cells migrate from explants prepared from mice at day 10 of pregnancy but the numbers which are progressively lower from migrate explants obtained at progressively later
858
MUKHTAR
stages of pregnancy. Peak migration occurs during the first 2 days of culture and thereafter the numbers of GMG cells on the culture dish around the explants steadily declines and few remain after 7 days of culture (Mukhtar and Stewart, 1988). Small immature GMG cells which migrate from explants prepared from developing decidual tissue survive in culture for less than 24 hr although supplementing the culture medium with interleukin-2 may allow for long-term survival (Linnemeyer and Pollack, 1991). Although this explant culture system has been used as an effective source of a pure population of GMG cells the system also provides an opportunity for determining whether GMG cells which remain in contact with stromal cells, within or on the surface of the explants, will survive for longer than those which lose contact with the stromal cells by migrating onto the surface of the culture flask. We have therefore examined the changing morphology of metrial gland explants when cultured for periods of up to 7 days particularly with respect to the GMG cell population. Explants have been maintained in the presence or absence of progesterone, a hormone which has been suggested to influence directly (Sharma and Bulmer, 1983) or indirectly (Stewart, 1987) the differentiation of GMG cells and which is necessary for the maintenance of pregnancy. The surface of the cultured explants has been examined using scanning electron microscopy to more clearly assess the extent to which GMG cells come to lie at the interface between explant and culture flask. Further, the numbers of GMG cells which migrate from the explant onto the surface of the culture dish have been examined over the period of assessment of the changes occurring within the tissue explants. These studies have provided data which suggest why GMG cells migrate from cultured explants of metrial gland tissue but they also provide data which suggest that the maintenance of GMG cells is influenced by contact with stromal cells. Materials and Methods Animals
Random bred virgin female Porton mice aged 8-16 weeks from an in-house colony maintained under standard animal house con-
AND STEWART
ditions were used in this study. They were mated overnight with males of their own strain and the presence of a vaginal plug the following morning was taken as day 0 of pregnancy. Mice were killed by cervical dislocation under ether anaesthesia at days 10, 12 and 14 of pregnancy. At least six mice were killed at each stage of pregnancy. Some mice were killed at day 11 of pregnancy but these were only used for preparing explants for scanning electron microscopy. The uterus was removed from each animal under aseptic conditions and opened by a longitudinal incision along the antimesometrial wall. Fetuses were immediately killed by decapitation. Preparation of metrial gland explant cultures
Metrial glands were dissected from each implantation site and divided into 4 equal pieces with a sterile blade. One piece of metrial gland (explant) from each of 4 or 5 metrial glands from the same animal were attached to the base of 35 mm plastic culture dishes. Three ml of culture medium consisting of Eagles Minimal Essential Medium with Earle’s salts (Flow Laboratories, Irvine, UK) supplemented with 20% heat inactivated fetal calf serum (Seralab, Sussex, UK), 0.058% L-glutamine, 1OOIUml-’ penicillin and 100 pg ml-’ streptomycin were added to each culture dish. Cultures were further supplemented with either 225 ng progesterone in 25 ,ul ethanol or 25 ,ul ethanol alone (controls). Cultures were incubated at 37°C in a humid atmosphere of 5% CO2 in air for periods of up to 7 days. The day on which the explants of metrial gland tissue were prepared was taken as day 0 of culture. Medium was replaced on days 2, 4 and 6 of culture. Further details of the explant culture procedure are given in Mukhtar and Stewart (1988). Of the culture dishes prepared from each animal 2 dishes (1 with progesterone, 1 without progesterone) were used to determine the extent of migration of GMG cells from the culture explants and 2 dishes (1 with progesterone, 1 without progesterone) were used to assess the morphological changes occurring within the explant over the culture period. Additional cultures were established without any supplemented progesterone or ethanol to provide control material to assess
MOUSE
METRIAL
GLAND
R5Y
EXPLANTS
any possible effects of the small amounts of ethanol on the explant cultures.
6001
Quandtatiorz of GMG cell migration
400
Quantitation of GMG cell migration was performed as described in Mukhtar & Stewart (1988). On each of days 1, 2, 3, 4, and 7 of culture the number of GMG cells attached to the base of the culture dish around each explant was counted in an area defined by the field diameter (1.7 mm) of a X 10 objective lens around the edge of each explant. Cells only rarely migrated beyond this perimeter. On days when the culture medium was changed cell counts were carried out after the medium change. Data were analysed using a Student’s ttest.
300 200 100 0k 012
600 500 No. GMG
of
Scanning electron microscopy
Additional explants were prepared as described above and fixed in situ after 1, 2, 3. 4, 5 or 7 days of culture in 25% glutaraldehyde in supplement free Minimal Essential Medium (Eagles) for 3 hr at 4°C. They were then washed in phosphate buffered saline, dehydrated in ethanol and critical point dried. Pieces of culture dish with the explant attached were then mounted on stubs, sputter coated with platinum and examined in an Hitachi S800 scanning electron microscope. Results Quantitation
The numbers
of GMG cell migration
of GMG cells found around
3
4--
1
?
Day 12
400 300
Histological examination of metrial gland expfants
Explants were taken immediately after preparation (time 0), after attachment to the base of the culture dish (time 15 min) and after 1, 2, 3, 4, and 7 days of culture. The explants were fixed overnight in a mixture of 4% formaldehyde and 2% glutaraldehyde in 0.1 mol phosphate buffer 1-l pH 7.2-7.4 (modified from Karnovsky, 1965). Tissues were washed in buffer, dehydrated in ethanol and embedded in Araldite. Ten 1 ,um sections at different depths (-50 pm apart) were cut from each explant, stained with toluidine blue, and examined by bright field microscopy. At least three explants at each stage of culture were examined.
Day 10
500
0
1
2
3
4
7
600 Day 14
500 I 400. 300~ zooloo-
0 O-?
12
TV
4
7
Days of culture Fig. 1. Numbers (means + S.E.) of GMG cells which migrated from metrial gland explants cultured in the presence (75ngml-‘) and absence of progesterone. Each point on the graphs represents the number of GMG cells found around each e&ant. Metrial gland explants were obtained from at least h mice at each of days IO. 12 and 14 of pregnancy. Standard errors 13 are not shown. W Explants with progesterone. *-_* Explants with ethanol carrier.
the explants maintained in the presence of progesterone, and in the presence of the ethanol carrier only, are shown in Figure 1. These numbers are generally similar to those of a previous study where explants were cultured in the absence of both progesterone and the ethanol carrier (Mukhtar and Stewart, 1988). When the migration curves for the progesterone treated cultures were compared to the cultures supplemented with
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Generally, there was little or no difference between the morphology of comparable explants cultured in the presence, or in the absence, of progesterone. There appeared to be, however. more GMG cells in day 10 explants after 1 to 3 days in cultured in the presence of progesterone than in similar explants cultured in the absence of progesterone. In these progesterone stimulated explant cultures the stromal cells appeared to have fewer cytoplasmic inclusions than in control material. These differences were no longer discernible after 4 days of culture.
most of those found in explants from day 10 pregnant mice. Some GMG cells in day 10 explants were in mitosis (Fig. 2) but mitoses were rare in day 12 explants and absent from day 14 explants. There were some mitotic stromal cells in day 10 explants (Fig. 2), few in day 12 explants and only an occasional one in day 14 explants. Stromal cells in day 14 explants cytoplasmic contained many inclusions but these were not a feature of the stromal cells in day 10 explants and were only found to a limited extent in day 12 explants. An occasional degenerate GMG cell was found in the central part of day 14 explants. There was extensive damage at the periphery of each explant. presumably as a result of cutting the explants to size. Similar damaged areas were found in more central regions of some sections of explants fixed 15 mins after the explants were prepared but this damage was probably a result of the stabbing procedure used in fixing the explant\ to the base of the culture dish.
Time 0, + 15 mins
After I-2 days of culture
Explants at time 0 and at + 15 mins generally appeared to be similar. All explants consisted of densely packed cells of which fibroblastlike stromal cells were the major cell type (Fig. 2). Granulated metrial gland cells were also numerous (Fig. 2). The GMG cells in explants from mice at day 12 and day 14 were larger, with more cytoplasmic granules than
Sections of explants cultured for l-2 days had a palely stained middle area which consisted mainly of degenerate cells with pyknotic nuclei and a more darkly stained periphery (Fig. 3). A few apparently normal stromal cells and smooth muscle cells were found in the degenerate middle areas. No healthv GMG cells were seen in the middle part o?’
the ethanol carrier there was only one significant difference: the peak value for the day 12 progesterone treated explants was significantly lower than with the carrier control treated explants (p < 0.05). Histology of metrial gland explants Explant cultures supplemented with progesterone
Fig. 7. Metrial gland explant at time 0. Densely packed fibroblast-like \trmnaI cell\ (small arrows), GMG cells (large arrows) and some smooth muscle cells (M) arc prcscnt. (‘ell\ in mltous can be seen (arrowheads). Explant from day 10 mouse. I pm Aralditc wctwn. ‘Ioluidmc hluc x4lJll. 1~1s.3. Metnal gland explant after I day in culture in the prescncc of progc~tcronc licalthy \tromal cells and healthy GMG cells (arrows) can be been in the pcrlphcral region 01 the explant. The central region of the explant (X) is dcgcncratc. Explant from day IO mowc I {rm Aralditc sectmn. Toluidine hluc. x400. Fig. 4. Metrial gland e&ant after 3 davs in culture in the presence of moccstcronc The pel-iphcral region contains’ healthy cells, i&ding GMG cell; (arrows). abd :as a Rattencd layer of cells at the surface. Some GMG cells (arrowheads) are on the wrface of the explant. The central region of the explant (X) is degenerate. Explant from day IO mouse I ,rm Aralditc wction Tolmdine hluc x400. Fir.._5. Mctrial eland exolant after7 davs in culture in the ~rescnce of ethanol carncr. Hcalthb cells arc present yn the peripheral region. GMG cells (arrows) can be seen on the wrtacc. ol the explant. Explant from day 10 mouse. 1 urn Aralditc section. Toluidinc hluc. xJIH). Fig. 6. Numerous GMG cells (G) on the surface of a metnal gland explant after I day m culture. Explant from day 10 mouse. Scanning electron microw)py 45’ tilt. x2175.
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Fig. 6. Numerous GMG cells (G) on the surface of a metrial gland explant after 1 day in culture. Explant from day 10 mouse. Scanning electron microscopy. 45” tilt. x2175.
the explants obtained from day 10 pregnant mice but a few healthy GMG cells were still present in the middle parts of the explants obtained from day 12 and day 14 mice. Healthy stromal cells and GMG cells were seen in the peripheral regions of each explant (Fig. 3). The stromal cells, including those obtained from day 10 pregnant mice and cultured for only 1 day contained cytoplasmic inclusions, many of which stained palely with toluidine blue. Mitoses were rare in 1 day cultured day 10 explants and were absent
from all other explants. By day 2 of culture all the healthy GMG cells seen in explants obtained from day 10 pregnant mice were large with numerous cytoplasmic granules although at 1 day of culture a few small GMG cells were still evident. At 1 and 2 days of culture, in explants from day 12 and day 14 mice all the healthy GMG cells were large. In all explants, not all of the GMG cells in the peripheral part of the explant were healthy; some had pyknotic nuclei and showed extensive cytoplasmic degeneration.
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In addition. cellular debris including cytoplasmic granules was found in intercellular spaces and some of the inclusions in the stromal cells had a morphology and staining characteristics similar to GMG cell granules. Afivr 3. 4 and 7 days of culture After 3 days of culture most explants had developed an epithelial-like layer of cells around the edge of the explant (Fig. 4). All explants had a central region generally devoid of any cellular elements with a peripheral rim of healthy looking cells (Fig. 4). Most of these peripheral cells were stromal cells whose cytoplasm contained many inclusions. However, some healthy GMG cells were also found near to, or on, the surface of the explants (Fig. 4). Overall. there were fewer GMG cells than at earlier stages of culture and there were more in explants derived from day 10 pregnant mice than from mice killed at day 12 or day 14. All explants at day 7 of culture had at least one or two healthy GMG cells in them, or on their surface (Fig. 5) in each section examined. Scmning electron microscopy of cultured explants The surface of the explants after 1-2 days culture often appeared ragged but by day 4 of culture much of the surface of most explants appeared to be covered with epitheloid cells. Cells with a surface morphology typical of GMG cells, that is, rounded cells with numerous microvillous-like or leaf-like projections (Mukhtar and Stewart, 1993), were found on the surface of all explants (Fig. 6). Their numbers and distribution on the explants varied but generally they were most numerous on explants cultured for only 1 or 2 days. However, several were still found on explants cultured for 7 days. Occasionally, GMG cells were seen which appeared as if they were passing between surface cells to reach onto the surface of the explant. Discussion Migration of GMG cells from cultured explants of metrial gland tissue occurs in parallel with degenerative changes taking place within the central core of the explants. However, normal GMG cells were still found in the healthy peripheral rim of the explants
at all stages of culture as well as on the surface of the explants. Granulated metrial gland cells are motile cells (Mukhtar and Stewart, 1988) and in oiuo migrate from the tissue of the decidua basalis and metrial gland into the lumina of blood vessels (Stewart and Peel, 1978; Stewart, 1991). Such migration in vivo occurs without evidence of degeneration of decidual/metrial gland tissue. It7 vitro, some of the migration of GMG celis onto the culture dish substratum may be a reflection of an inherent migratory capabilit) (Mukhtar and Stewart, 1988) but, from the appearance of the explant cores seen in the present study, it is more likely that migration occurs in the main to escape from the degenerate central core of the large explants to an area at the periphery, or surface of the explant, or free in the culture dish where there would be readily available nutrients. Changes in the stromal cells of the explant\ during the culture period were major. from their total degeneration in the central core region to the hypertrophy and the accumulation of inclusions in the stromal cells located at the periphery. Recently. it has been proposed that the inclusions in stromal cells of the metrial gland (Stewart and Peel. 1980) arise mainly as a result of endocytic activity associated with degenerative changes in the metrial gland and repair of the uterine wall following parturition and that monocyte derived macrophages do not play a major role in this activity (Stewart and Mitchell. 1992). The present study lends support to that proposal because of the changes observed in the stromal cells of the cultured explants where blood borne macrophases would not leave access. Generally, progesterone supplements had little effect on the morphology of the explant cultures or on the pattern of migration and maintenance of GMG cells outwith the explants. In a previous study of cultured rat metrial gland explants GMG cells were not maintained in the explants unless progesterone was present in the culture medium (Adam and Peel, 1983). The most obvious effect of progesterone in the present study was a small, but significant, reduction in the numbers of GMG cells which migrated from the cultured explants, from day 12 pregnant mice, at the peak migratory period. The changes to the GMG cell population in the explant cultures provides some insight
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into the mechanism by which GMG cell maintenance is supported. Progesterone supplements did not appear to influence GMG cell maintenance. Any soluble factors produced by the stromal cells would influence both the GMG cells in and on the explant and the cells on the culture dish substratum. However, GMG cells may need contact with the stromal cells. The possibility that the maintenance of GMG cells is regulated by a juxtacrine association with stromal cells requires further investigation. Immature GMG cells which migrate from metrial gland tissue explants obtained from mice killed between day 8 and day 10 of pregnancy do fail
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to mature into large GMG cells and generally die within 24 hr (Mukhtar and Stewart, 1993). It is perhaps at this time in the metrial gland when a juxtacrine mechanism is of greatest importance to the differentiation of GMG cells. Acknowledgements
We are grateful to C. Inman and his staff at the Electron Microscope Unit, Southampton General Hospital for their assistance with the scanning electron microscopy. The scanning electron microscopy was supported by the Wessex Medical School Trust.
References Adam, E. and Peel, S. 1983. The maintenance of rat metrial gland explants in uitro. in the presence and absence of progesterone. .I. Anal., 136, 321-328. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27, 137A. Linnemeyer, P. A. and Pollack, S. B. 1991. Murine granulated metrial gland cells at uterine implantation sites arc natural killer lincagc cells. J. fmmuno[. , 147, 253&2535. Mukhtar, D. D. Y. and Stewart, I. 1988. Migration of granulated metrial gland cells from cultured explants of mouse metrial gland tissue. Celf T&x. Res., 253, 413-417. Mukhtar, D. D. Y. and Stewart, I. J. 1993. The surface morphology of mouse metrial gland cells. J. Anor.. 182, 415424. Peel. S. 1989. Granulated metrial gland cells. Adv. Anat. Embryol. Cell Biol.. 115, 1-l 12. Peel, S., Stewart. I. J. and Bulmer. D. 1983. Experimental cvidence for the bone marrow origin of granulated mctrial gland cells of the mouse uterus. Cell Tin. Res., 233, 647-656. Selye. H. and McKeown. I. 1935. Studies on the physiology of the maternal placenta in the rat. Proc. Roy. Sot. B. 119, l-31. Sharma, R. and Bulmer, D. 1983. The effects of ovariectomy and subsequent progesterone rcplacemcnt on the uterus of the pregnant mouse. J. Amt.. 137, 695-703. Stewart, I. 1983. An investigation into the differentiation of granulated metrial gland cells in the early pregnant moue uterus. .I. Amt., 137, 85-93. Stewart, I. 1984. A morphological study of granulated metrial gland cells and trophoblast cells in the labyrinthmc placenta of the mouse. J. Anat., 139, 627-638. Stewart, I. 1987. Differentiation of granulated metrial gland cells in ovariectomized mice given ovarian hormones. J. Endow., 112, 23-26. Stewart, I. J. 1990. Granulated metrial gland cells in the mouse placenta. Placenta, 11, 263-275. Stewart, 1. J. 1991. Granulated metrial gland cells: pregnancy specific leukocytes? J. Leuk. Biol., 50, 19Pr207. Stewart, I. J. and Mitchell, B. S. 1992. Macrophages and other cndocytic cells in the mouse uterus during the second half of pregnancy and into the postpartum period. J. Arm., 181,11%126. Stewart, 1. and Peel, S. 1977. The structure and differentiation of granulated metrial gland cells of the prcgnam mouse uterus. Cell Tiss. Res., 184, 517-527. Stewart, I. and Peel, S. 1978. The differentiation of the decidua and the distribution of mctrial gland cells in the pregnant mouse uterus. Cell Tiss. Res., 187, 167-179. Stewart, I. and Peel, S. 1980. Granulated metrial gland cells at implantation sites of the pregnant mouse uterus. Amt. Embryol., 160, 227-238.