J. Anat. (1980), 131, 2, pp. 255-262 255With 2 figuresPrinted in Great Britain

Migration of metrial gland cells in the mouse

A. D. DICKSONDivision of Morphological Science, University of Calgary,

Calgary, Alberta, Canada T2N 1N4

(Accepted 21 December 1979)

INTRODUCTION

The granulated cells typical of the rat metrial gland (Selye & McKeown, 1935)are of interest for several reasons. Similar cells have been described in the gravidhuman (Dallenbach-Hellweg & Nette, 1964) and monkey (Cardell, Hisaw & Dawson,1969). Functions ascribed to them include nutrition of the embryo (Selye &McKeown, 1935) and relaxin secretion (Dallenbach-Hellweg, Battista & Dallenbach,1965; Larkin, 1974). They contain hydrolytic enzymes (Bulmer, 1968; Carlsoo &Bloom, 1969) as well as glycogen (Bulmer & Dickson, 1960) and may be derivedfrom a cell of the lymphocyte series (Smith, 1966; Peel & Bulmer, 1977; Stewart &Peel, 1977), although this has been disputed (Larkin & Schultz, 1968; Larkin &Cardell, 1971).

Metrial gland cells are also found in the decidua basalis. Indeed they are presentearlier there than in the metrial gland in both the rat (Dickson & Bulmer, 1961)and the mouse (Stewart & Peel, 1978).Bridgman (1948 a, b) described these cells entering the lumina of maternal vessels

supplying the placenta and migrating along them on the ninth and tenth days in therat. Bulmer & Dickson (1960) and Dickson & Bulner (1961) found metrial glandcells very infrequently in these vessels and not at all in the placenta, though theydid find them in the ectoplacental cone. Stewart & Peel (1978) noted that, in themouse, the maternal vessels of the labyrinth commonly contain metrial gland cellson day 11 of gestation (which is equivalent to the twelfth day in this study).

Metrial gland cells are so much more frequent in the labyrinth of the mouse thanof the rat that it was decided to obtain a numerical estimate of them. Because of thepossibility raised by Bulmer & Peel (1977) that these cells may have a role in theimmunology of reproduction, comparison was made between the numerical esti-mates obtained in twa strains, one outbred (Swiss Webster/ALAS) and one inbred(C57BI/HPB), which would be expected to have different degrees of embryonicantigenicity of paternal origin. When it became apparent that fewer metrial glandcells were present in the C57B1/HPB than in the Swiss Webster/ALAS labyrinth,the lungs, where these cells had not previously been reported, were examined toshed light on the possibility that metrial gland cells of C57B1/HPB, but not ofSwiss Webster/ALAS, mice are able to reach them by passing through the maternallabyrinthine vessels.

MATERIALS AND METHODS

A complete uterine conceptual site was obtained each day from the eleventh tothe sixteenth day of gestation from each of 3 Swiss Webster/ALAS (SW) and of3 C57B1/HPB (C57B1) mice, the first day being the day of finding the vaginal plug

0021-8782/80/2828-7930 $02.00 © 1980 Anat. Soc. G.B. & I.17-2

at the 9 a.m. examination. They were fixed in a mixture of 2 % glutaraldehyde and4 % formaldehyde in 0 1 M phosphate buffer (modified from Karnovsky, 1965),embedded in paraffin wax, sectioned at 5 ,um perpendicular to the long axis of theuterus and stained by PAS with a haematoxylin counterstain, with and withoutprior diastase digestion.Both lungs from each mouse were processed similarly.Metrial gland cells were identified by observation of at least two of the following

characteristics: large size (Bridgman, 1948a), binuclearity, circumnuclear ring ofPAS-positive, diastase-fast granules (Wislocki, Weiss, Burgos & Ellis, 1957) and thepresence, adjacent to the nuclei and inside the ring of granules, of a structurelessarea that by comparison with electron micrographs (Larkin, 1971) appears to bethe Golgi region. The glycogen-containing peripheral cytoplasm was often notevident in the labyrinth or lung.

Metrial gland cells in the maternal labyrinthine vessels of a placenta were countedin 9 PAS/diastase sections spaced 100 ,um apart, the middle section transecting theattachment of the umbilical vessels. Three placentae were used from each strain oneach day. A metrial gland cell was judged to be in a maternal labyrinthine vesselwhen maternal erythrocytes accompanied it. Cells in the main maternal vesselssupplying the labyrinth were not included. The count was therefore of metrial glandcells in maternal vessels in a position in which interchange of gases, and othersubstances, with blood in embryonic vessels could take place.By measuring its area in an image projected at known magnification, the area of

the placental labyrinth (excluding major maternal and embryonic vessels and largetongues of junctional zone trophoblast projecting into it) was determined in thecentral section and in each of the extreme sections used for counting metrial glandcells. The areas of these sections were added together and multiplied by 3 to give anestimate, for each mouse, of the total area of labyrinth in which metrial glandcells were counted.

Metrial gland cells were counted in one section of each lung. Three mice of eachstrain were used on the eleventh day and one of each strain from the twelfth to thesixteenth day. Random orientation of the plane of section (Underwood, 1970) wasensured by random orientation of the lung in the paraffin wax at the time ofembedding. Sections of lung of approximately equal area were used. The area ofeach section (for eleventh day mice only) was measured as above.

In case there might be a difference between the sections of SW and C57BI mouselungs in the ratio of lung tissue to air space, which could be normal or result fromdifferential collapse or other artifact but which might tend to influence the numberof metrial gland cells found in a given section area, the ratios were compared in theeleventh day mice. A Zeiss eyepiece integrating disc was used to determine thepercentage of intersection points falling on lung substance in sample areas. Thesame sections employed for counting metrial gland cells and for measurements ofarea were utilized for this purpose also. The hilar region, full of large blood vesselsand bronchi, was avoided but otherwise selection of areas was random, a pre-selected pattern of movement of the mechanical stage being used to arrive atsuccessive sample areas. The percentage was determined in 10 areas for each section.There being two sections per mouse, one from each lung, and three eleventh daymice per strain, 60 determinations were made for each strain.

256 A. D. DICKSON

Mouse metrial gland cells 257

~ /

'1 '"2' ,o < t_MGC MGC

Fig. 1. A mletrial gland cell (MGC) in a maternal vessel of the placental labyrinth of a twelfth daySwiss Webster/ALAS mouse embryo. x 500.Fig. 2. A metrial gland cell (MGC) in the lung of a C57BI/HPB mouse on the eleventh day ofgestation. x 500.

RESULTS

A metrial gland cell in a maternal channel in the labyrinth is shown in Figure 1.The number of metrial gland cells counted in maternal vessels in nine spaced sectionsthrough its placental labyrinth is indicated for each mouse in Table 1. When thefigures in the SW column are compared with those in the C57B1 column using a rankcorrelation method (Hettmansperger, 1975), there is an overall difference (P< 0 0001) between the columns. It might be concluded that more metrial gland cellsare present in the maternal labyrinthine vessels from the eleventh to the sixteenthday in SW than in C57Bl mice.However, if metrial gland cells were uniformly distributed in the same density in

the labyrinth of mice of the two strains, the number of metrial gland cells countedin a fixed number of consistently placed and equally spaced sections through alabyrinth would depend on its size. Table 2 gives the sum of the areas of the labyrinthmeasured in the central and two extreme sections (each 400 ,um from the centralsection) of the nine sections per labyrinth in which metrial gland cells were counted.If the figures in the SW column are compared with those in the C57Bl by the rankcorrelation test used above, there is an overall difference (P < 0 002). The figuresfor the SW mice are again larger.Table 3 shows the number of metrial gland cells per labyrinth taking the area

measured into account in accordance with the formula

N-=(u Number of metrial gland cells x 105(Smof areas of central and extreme sections) x 3

The multiplier 105 is included to make the quotients whole numbers and thefactor 3 to recognise that only one third of the sections per labyrinth in whichmetrial gland cells were counted had their areas measured. Their inclusion makes

258 A. D. DICKSON

Table 1. Number of metrial gland cells in maternal blood vessels of the placentallabyrinth from the eleventh to the sixteenth day ofgestation. Each entry refers to onemouse

Mice

Day SW C57B1

2 111 1 0

2 016 6

12 31 710 329 5

13 7 523 11107 18

14 44 4382 10185 20

15 93 23102 5434 4

16 36 1337 16

Table 2. Sum of areas (sq mm) of centre and extreme sections of the placental laby-rinth in which metrial gland cells were counted. Each entry refers to the mouse in thecorresponding position in Table 1

Mice

Day SW C57BI

85.3 61-811 149-3 131-7

162-3 54-7198-2 170-3

12 315-2 166-4279-8 214 6237-3 253-6

13 277-3 205-4273-7 276-3400-0 320-0

14 491-7 362-7405 4 380-8411 7 327-3

15 413 6 353 6341-8 420-0517-2 419-0

16 478-2 479-1460 9 459.0

Mouse metrial gland cells

Table 3. Number of metrial gland cells in the placental labyrinth standardized forarea. Each entry refers to the mouse in the corresponding position in Tables 1 and 2

Mice

Day SW C57Bl

391 011 447 253

205 02691 1174

12 1058 14023693 4664074 657

13 841 8112801 13278917 1875

14 2983 39526742 87514975 2037

15 7495 21689947 42862191 318

16 2509 9042676 1162

no difference to the rank correlation test. By this test there is overall a greaternumber of metrial gland cells per unit area measured in the SW labyrinth than inthe C57BI from the eleventh to the sixteenth day of gestation (P < 00001).The number of metrial gland cells per unit area of labyrinth measured (Table 3)

appears to increase from the eleventh to the fifteenth day and to decrease to thesixteenth day. By the Mann-Whitney test (Mann & Whitney, 1947), the peaknumberof metrial gland cells per unit area of labyrinth on the fifteenth day is higher(P = 005) in the SW than in the C57B1 mice.When metrial gland cells were sought in the lungs, they were almost invariably

found in interalveolar septa (Fig. 2) and only very rarely in pulmonary arterybranches. They were usually elongated in one dimension, which was presumablyparallel to the long axis of the vessel in which obturation had occurred. It wasfrequently impossible to verify by the microscopical methods used that such a cellwas in fact contained within a blood vessel. No indication was noted of preferentialdistribution of metrial gland cells to either lung or part thereof.

It was unusual to find metrial gland cells in the lungs after the eleventh day,only one being found in SW lungs on the thirteenth day and 3 and 5 on the twelfthand thirteenth days, respectively, in C57Bl lungs. This was in the same number ofsections per mouse as on the eleventh day. Table 4 shows the total number of metrialgland cells found in one section of each lung of each of 3 mice of each strain on theeleventh day of gestation. It also shows the sum of the areas of these sections. Thenumbers of metrial gland cells standardized for area are shown in the right column.The number of metrial gland cells per unit area is higher in C57B1 than in SW mouselung on the eleventh day (Mann-Whitney test, P= 005).The mean percentage of eyepiece integrating disc intersections with lung substance

rather than air spaces was 43-7 (S.D. 100) in the eleventh day SW mice and 31-0(S.D. 6 8) in C57B1 mice. By Student's t test, there is a difference between the means

259

A. D. DICKSON

Table 4. Metrial gland cells in the lungs on the eleventh day of gestation. Each entryin the left column gives the total number of metrial gland cells in two randomly placedsections of the lungs of one mouse. The corresponding entry in the middle columngives the combined area of these two sections

Number of Metrial glandmetrial Area cells x 103

gland cells (sq mm) Area

2 55-0 36SW 1 47-8 21

5 57-3 8725 61 8 404

C57BI 37 70-8 52331 70-3 441

(P < 0-0001). This indicates that the SW lung has, for whatever reason, a higherratio of substance to air space than the C57BI. Metrial gland cells might, since theylie in lung substance, be expected to occur more frequently in a given area of SWlung. However, as indicated above, metrial gland cells are more numerous ineleventh day C57BI than SW lungs. The relative frequency in C57B1 lungs would,of course, be increased if a correction for this factor were applied.

DISCUSSION

The results reported above can be summarized by saying that (1) there is a greateraccumulation of metrial gland cells in the maternal labyrinthine vessels in SW thanin C57BI mice, (2) the accumulation in these vessels rises to a higher peak on thefifteenth day in the SW strain and (3) the accumulation in the lungs, greater in theC57BI than the SW strain, appears to occur too early to account for the relativepaucity of metrial gland cells noted in the C57BI labyrinth.

It is impossible to state categorically that the cells found in the labyrinthinematernal vessels are metrial gland cells. However, in favour of this are their simi-larity, in morphology and in response to the PAS reaction, to those cells situatedin the metrial gland and decidua basalis and the existence of an apparent migrationpathway from the decidua basalis and/or metrial gland to the labyrinth in thisspecies (Stewart & Peel, 1978; personal observation).Assuming, until there is conclusive evidence to the contrary, that they are metrial

gland cells, an overall difference was noted in their numbers in the maternal laby-rinthine vessels of the two strains investigated, the SW having more than the C57B1.Since the difference was present after standardizing for the size of the labyrinth, itis important that the method indicative of labyrinth size (i.e. volume) be appropriate.The placing of sections, the number of sections per labyrinth and the number ofmice per strain were selected to reflect the size of the disc-shaped labyrinth and totake into account such factors as deviations from perfection of the disc shape andnon-parallelism of disc axis and plane of section.

There is no evidence to indicate the underlying basis for the accumulation ofmetrial gland cells in the labyrinth. It may be that the cells are too large and/ortoo inflexible to pass through or that they become, and remain, attached to the

260

Mouse metrial gland cells 261trophoblast lining the labyrinthine maternal vessels. On the other hand, if thenumber found post mortem simply mirrors the number in transit at death, theapparent accumulation (Table 3) may be due to an increase in migration with theadvance of gestation.Turning now to the apparent metrial gland cells found in the lungs, the question

of whether they really are metrial gland cells arises once more. The morphologicaland histochemical evidence is as strong as ever but in this case there is no obser-vational evidence for passage between the labyrinth and the lung. However, anobvious route for migration is evident through the uterine veins, posterior venacava and heart to the pulmonary arteries and their branches. A major questionconcerning the accumulation in the lungs is whether it could account for thedifference found between the strains in numbers of these cells found in the labyrinth.At first glance it would appear that it could not, for the greatest accumulation inthe labyrinth appears to be towards the end of the period studied while in the lungit seems to be at the beginning. However, this could be influenced by the relativelengths of time, presently unknown, that the cells persist once they have lodged inthe vessels in the lung and labyrinth.The fact that there has been no mention above of metrial gland cells n other

locations does not imply that they are not to be found elsewhere. They have beennoted in the junctional zone trophoblast and, apparently more frequently, in thegiant cell circulation, where they have also been noted by Stewart & Peel (1978).The numbers in these and other locations will be the subject of further study.The function subserved by migration of metrial gland cells to the labyrinth

remains puzzling. Bridgman (1948 b) suggested that the migrating cells carry glycogento the trophoblast. A more attractive proposal was made by Bulmer & Peel (1977).who demonstrated that these cells, in both the metrial gland and the decidua basalis,showed a strong reaction with fluorescein-labelled IgG. They postulated that thecells are involved in transmitting a blocking antibody to the trophoblast. The greaternumber of metrial gland cells in the labyrinth of the random-bred SW strain,compared with the inbred C57B1 strain, could be construed as consistent with thispostulate.

If this hypothesis were correct, it would be difficult to account for the migrationof metrial gland cells to the lungs. It might be a mere side-effect, some cells beingoutside the size spectrum of those that lodge in the labyrinth. The diminishingdifferential migration to the lungs in the two strains could, for example, be due todifferential reduction in calibre of the maternal labyrinthine vessels as the placentadevelops.

SUMMARY

Using Swiss Webster/ALAS and C57BI/HPB mice from the eleventh to thesixteenth day of gestation, comparative estimates have been made of the numbersof metrial gland cells in (a) the maternal vessels of the chorio-allantoic placenta and(b) the maternal lungs. Overall, more metrial gland cells were found in the maternallabyrinthine vessels in SW than in C57Bl mice.There appears to be a peak in numbers on the fifteenth day in both strains, the

level attained being higher in the SW strain. More metrial gland cells were found inthe lungs of C57B1 than in those of SW mice on the eleventh day of gestation. Afterthat day, they were uncommon in the lungs of both strains.

Financial support from the Medical Research Council of Canada, co-operationwith the statistics by Dr A. Rademaker and technical assistance by Mrs Lesley Bartonand Mary Collins are gratefully acknowledged.

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