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Histochemistry of the cellular components of the metrial gland of the rat during prolonged pseudopregnancy.

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HISTOCHEMISTRY O F T H E
CELLULAR COMPONENTS O F THE METRIAL GLAND
O F THE RAT DURING PROLONGED
PSEUDOPR,EGNANCY
RICHARD A. ELLIS *
Biological Laboratories, Harvard University; Department of Anatomy, Harvard
Medical School and Arnold Biological Laboratory, Brown University
NlNE FIOUEES
INTRODUCTION
Velardo, et al. ( '53b) described the development of a metrial
gland near the mesometrial attachment of the traumatized
uteri of pseudopregnant rats. They associated the presence of
this structure with the maintenance of functional corpora
lutea and the inhibition of ovulation during prolonged pseudopregnancy. They noted the histological similarity between the
metrial gland associated with the secondary implantation site
of normal pregnancy and that of prolonged pseudopregnancy.
Some cytological characteristics of the cellular components
of the metrial gland of normal pregnancy have been described
in the literature (Seyle and McKeown, '35 ;Asplund, et al. '40 ;
Baker, '48 ;Bridgman, '48;and Wislocki, et al. '56). Attention
has been primarily focused on the large granular cells which
are found abundantly in the metrial gland, but Baker ('48)
described a second cell type which contained lipid. The lipid
gave histochemical reactions which he believed to be indicative
of steroids.
* Part of a thesis submitted to the Biology Department, Harvard University,
as partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Current address : Arnold Biological Laboratory, Brown University, Providence
12, Rhode Island.
39
40
RICHLLItD A. ELLIS
Although Seyle and McKeown ('35) have described some
cytological reactions of the metrial gland of normal pseudopregnancy, there has been no report on the cytochemistry of
the metrial gland developed during prolonged pseudopregnancy. With the physiological evidence of Velardo, et al.
('53b) at hand, it seemed that a thorough histochemical study
of cellular components of the metrial gland might aid in
establishing the functional significance of this structure during
prolonged pseudopregnancy.
MATERIALS AND METHODS
Pseudopregnancy was induced by electrical stimulation of
the uterine cervix of albino, estrous, virgin rats after the
method of Greep and Hisaw ( '38). Vaginal smears were
examined daily until the uteri were removed for histochemical
study. Four days after cervical stimulation the endometrium
of both cornua was traumatized by the method of Velardo,
et al. ('53b) to produce massive deciduomata and prolonged
pseudopregnancy (19 days 2.9, Velardo, et al., ( '53a). After
uterine traumatization, animals were killed at 24 hour intervals through to the twenty-first day of pseudognancy, and
uteri were removed for examination. The ages of the tissues
described are stated in terms of full days elapsed since cervical
stimulation, and the age of the metrial tissue is always four
days less than the indicated day of pseudopregnancy.
A variety of histochemical tests were applied to the sequence
of uteri. Sudan black B staining (Lillie, '54)' the Hayes ( '49)
modification of the Schiff plasma1 reaction, the Schultz test
for cholesterol, the Gomori ( '45) reaction for lipase-esterase
activity and the Pearse ('53) azo-dye method for alkaline
phosphatase were applied to frozen sections. Tissue slices
were also examined under polarized light for birefringence.
The following techniques were applied to 5 p paraffin sections after appropriate h a t i o n : Mallory 's methylene bluephloxin ; methyl green-pyronin (Brachet, '42) ; alkaline phos-
HISTOCHEMISTRY OF METRIAL GLAND
41
phatase (Gomori, ’52) ;acid phosphatase (Gomori, ’52) ; periodic acid Schiff (PAS) (Lillie, ’54) before and after salivary
digestion ; toluidin blue ; Mallory ’s triple connective tissue
stain; and Mitchell and Wislocki’s (’44)method for argyrophilic reticular fibers.
OBSERVATIONS
The “metrial gland” (Selye and McKeown, ’35) of prolonged pseudopregnancy is apparently derived from mesenchymal cells of the endometrium, myometrium and the
mesometrial uterine attachment (Velardo, et al., ’53b). The
focal point of development, however, lies within the so-called
lmesometrial triangle, an area bounded on two sides by the
mesometrial extensions of the external uterine muscle layer
and on the third side by the mesometrial quadrant of the
circular muscle coat (Young, ’51). A preponderant portion
of the metrial gland is thus equivalent to the “myometrial
gland” of Gerard (’25) and Asplund, et al. (’40).
Two distinctly different cell types commonly differentiate
in the metrial gland during prolonged pseudopregnancy.
There are large, binucleate, granular metrial cells, and smaller,
hypertrophied fibroblasts which contain abundant lipid droplets. The two cell types develop independently of each other,
each reaching a maximum in number on different days of
pseudo pregnancy. They vary from being separately segregated in certain areas to almost equally intermingled in other
loci (figs. 3 and 4). A few mast cells are encountered in the
metrial gland; they are easily distinguished from the granular
metrial cells by their size and distinctly different granulation.
Fibroblasts are always present in the metrial gland, and
eosinophilic leucocytes invade the metrial gland as it becomes
at retic.
For the purposes of this paper we shall follow the development of the two major cell types characteristic of the metrial
gland separately.
42
RICHARD A. ELLIS
The grmular metrial cell
On the eighth day of pseudopregnancy some of the mesenchymal cells in the regions of the mesometrial triangle, myometrium and mesometrium show a change in form, The
cytoplasmic processes of these cells become rounded, their
cytoplasm becomes increasingly basophilic and the nuclear
chromatin is condensed (fig. 6). The basophilic material
assumes a perinuclear position and is densely aggregated near
the Golgi zone. Concurrently, glycogen accumulates in the
cytoplasm as finely stippled material demonstrable by the
PAS reaction.
By the tenth day of pseudopregnancy these enlarged cells
contain numerous granules of various size which are PAS
positive after salivary digestion and stain moderately with
acid dyes. They may also be weakly metachromatic after
staining with aqueous toluidine blue. Glycogen is more abundant in cells of this stage, and the basophilic material now
forms a ring surrounding the acidophilic granules (fig. 7).
All stages in the differentiation of the granular cells may be
observed on the twelfth day of pseudopregnancy. The largest
of these cells contain 30-60 granules of uniform size, which are
PAS positive after salivary digestion (fig. 2) intensely acidophilic (fig. 5), and weakly metachromatic. The cluster of
granules is surrounded by a basophilic recticulum (fig. 6), and
the peripheral cytoplasm is now occupied by large vacuoles of
glycogen (fig. 1).
Incomplete mitosia generally takes place in the differentiating granular metrial cell. This is apparently a stage in the
differentiation of these cells, for nearly all of the large granular metrial cells are binucleate. It is not uncommon to find
metrial cells with three or four nuclei. Since granules appear
early in the life span of the cells, they apparently do not
inhibit mitosis. Nevertheless, they interfere perhaps with
normal cell division and may thus be partly responsible for
the multinucleate nature of these cells. I n the binucleate cells
the granules are aggregated in the Golgi zone between the two
nuclei.
HISTOCHEMISTRY OF METRIAL GLAND
43
Until the fifteenth day of pseudopregnancy the granular
metrial cells may be identified by the histochemical reactions
previously described. In addition these cells give negative reactions for both alkaline and acid phosphatase, show a low
level of lipase-esterase activity, stain only faintly with Sudan
black B, and do not give a positive plasmal response or exhibit
birefringence. The faint sudanophilia is attributable to the
presence of small mitochondria as well as to a slight staining to
the periphery of the granules. The granules are unstained
with Baker’s acid hematein test f o r phospholipids. The
metachromatic staining of the granules with toluidine blue is
best demonstrated after Zenker-acetic and Carnoy fixation.
The metachromasia is not alcohol resistant but it is preserved
by acetone dehydration. The granules are not as uniformly or
as intensely metachromatic as the granules in the metrial cells
of the normal pregnant rat, and the metachromatic staining
is distinctly different from that seen in the granules of neighboring mast cells.
The population of granular metrial cells reaches a maximum
on about the fourteenth day of pseudopregnancy (Velardo, et
al., ’53b). At this time they are largely segregated to form
perivascular sheaths about the blood vessels of the metrial
gland. This perivascular localization persists through the
remainder of pseudopregnancy (fig. 4). The cells are usually
surrounded by a network of argyrophilic reticular fibers.
After the sixteenth day, the granules and cytoplasm of the
metrial cells stain more heavily with Sudan black B, and a
moderate plasmal reaction is also observed. At this time the
granules in some cells may stain lightly with Baker’s acid
hematein. This is probably indicative of an increase in the
amount of phospholipid in these cells. Many of the cells are beginning t o atrophy by the eighteenth day of pseudopregnancy
and by the nineteenth or twentieth day degeneration has
become widespread. The nuclei become pycnotic, the granules
disappear and the large vacuoles of glycogen vanish. No active
secretion of the granular metrial cells or their contents into
44
RICHARD A. ELLIS
the capillaries of the metrial gland was observed. Typical
granular metrial cells may persist in the metrial gland as late
as the twenty-first day of pseudopregnancy.
The lipid-bearing cells
Throughout pseudopregnancy fibroblasts are always present in the mesometrial triangle and in the modified circular
muscle layer adjacent to that region. Some of these cells
apparently give rise to the granular metrial cells, while many
remain unmodified. During the seventeenth and eighteenth
days of pseudopregnancy great numbers of these fibroblastic
elements undergo cytomorphosis. The nuclei become enlarged
and acquire smooth ovoid outlines. The nuclear chromatin
may be clumped or finely dispersed (fig. 8). With Sudan black
B staining cytoplasmic lipid droplets are visible. These are
frequently very large and numerous, often nearly filling the
cytoplasm (fig. 9). A major portion of the lipid in these cells
is Schultz positive for cholesterol, gives a positive plasma1
reaction and is birefringent under polarized light.
The lipid-bearing cells are most numerous on the nineteenth and twentieth days of pseudopregnancy. At this time
these hypertrophied fibroblasts fill the portions of the metrial
gland not occupied by the granular metrial cells and pervade
the circular muscle layer (figs. 3 and 4). They show moderate
to heavy lipase-esterase activity but do not give positive acid
or alkaline phosphatase reactions. Their cytoplasm may be
slightly basophilic and occasionally a few particles of glycogen
may be discerned near the nuclei. Collagenous fibers closely
invest these cells, and there is little or no transformation of
this associated connective tissue into argyrophilic reticular
fibers. By the end of the twentieth day of prolonged pseudopregnancy, the entire metrial gland is usually atrophic. The
lipid decreases in amount in the enlarged fibroblasts, and at
the termination of prolonged pseudopregnancy little, if any;
lipid is still detectable.
HISTOCHEMISTRY OF METRIAL GLAND
45
DISCUSSION
The histochemical reactions of the cellular components of
the metrial glands which develop during prolonged pseudopregnancy and during normal gestation are nearly identical.
The lipid-bearing cells are similar to those described by Baker
( '48) in thc mctrial gland of the gravid uterus of the rat. They
differentiate at the same time, in the same sites, from the same
cell types, and the lipid component of the cells is similar
cytochemically. They differ only in their life spans. I n normal
pregnancy they may persist through the twenty-second day of
lactation, but in prolonged pseudopregnancy they disappear
with the onset of estrus. The granular metrial cells give the
same histochemical reactions as those described by TVislocki,
et al. ( '56) in the metrial gland formed in the rat uterus during
normal pregnancy. The only exception is the weaker metachromasia of the specific granules during pseudopregnancy.
This would indicate that fcwer acidic prosthetic groups are
associated with these granules than with those of the metrial
cells of pregnant rats.
The glycogen, the basophilic substance, and the specific
granules contained within the granular metrial cells are the
products of intracellular metabolism and are not accumulated
by phagocytosis as suggested by Asplund, et al. ('40). The
differentiation of the granular metrial cells as observed by
the various histochemical tests leads to this conclusion. The
basophilic material which accumulates around the Golgi zone
at an early stage in the differentiation of these cells is most
certainly intracellular in origin. The subsequent appearance
of the specific granules in the Golgi zone surrounded by the
basophilic ring indicates that the elaboration of the specific
granules is intimately associated with thc basophilic material.
Observations with the electron microscope (Wislocki et al.,
'56) provide further evidence on this point. The glycogen
stores characteristic of the large granular metrial cells probably reflect the altered metabolism of these cells, and are not
accumulated by phagocytosis.
46
RICHARD A. ELLIS
An analysis of the histochemistry of the specific granules in
the metrial cells of pregnant rats as well as a theory as to
their possible function is given by Wislocki et al. ('56). Since
these cells seem to be alamost identical with those formed during prolonged pseudopregnancy there is little need to repeat
their discussion here.
Pseudopregnancy normally persists in rats for only thirteen
days (Velardo, et al., '53a). Since the lipid-bearing cells of
the metrial gland do not develop until after the fourteenth
day of pseudopregnancy, they are peculiarly associated with
massive deciduomata and prolonged pseudopregnancy. The
differentiation of these cells is apparently a recapitulation of
the progressive differentiation of similar cells in the metrial
gland of normal pregnancy and probably has no other significance. The lipid contained in these cells stained with Sudan
black B, is Schultz positive for cholesterol, gives a positive
plasma1 reaction, and is birefringent under polarized light.
Baker ( '48) finds that the lipid-bearing cells developed in the
metrial gland during pregnancy and lactation give similar
reactions and in addition stain rose with Nile blue sulphate.
He considers tests indicative of steriods. Since Velardo, et
al., ('53a) have associated the metrial gland with the maintenance of prolong pseudopregnancy, it seems possible that
these lipid-bearing cells may contribute in some way to the
steroid metabolism of the rat.
SUbdMARY
1. Metrial glands that had developed in the mesometrial
wall of the traumatized uteri of pseudopregnant rats were
examined histochemically.
2. The differentiation of two distinct cell types, the granular metrial cells and lipid-bearing fibroblasts, is observed in
the metrial gland.
3. The granular cells are characterized by an early cytoplasmic basophilia ; an abundance of glycogen in their
cytoplasm ; and specific granules which are weakly meta-
HISTOCHEMISTRY OF METRIAL GLAND
47
chromatic, stain with acid dyes, and are PAS possitive after
salivary digestion.
4. The lipid-bearing cells appear near the termination of
prolonged pseudopregnancy. They contain large amounts of
sudanophilic lipid some of which is Schultz positive and birefringent under polarized light.
5. Both cell types are atretic at the termination of prolonged pseudopregnancy.
ACKNOWLEDGMENT
I wish to thank Professor A. B. Dawson for his constant
encouragement and valuable criticism of this work, and Professors J. Walter Wilson and William Montagna for reading
the manuscript.
LITERATURE CITED
ASPLUND,J., V. BORELLAND H. HoLMGREN 1940 In der Uteruswand wahrend
Graviditiit auftretende metachromatische granuleirete Zellverbande und
ihre Stellung zur ‘ ‘Glandula myometralis. ” Z. mikr.-anat. Forach.,
48: 478-528.
BAKER,B. L. 1948 Histochemical variations in the metrial gland of the rat
during pregnancy and lactation. Proc. Soc. Exp. Biol. and Med., 68:
492496.
BRACHET,
J. 1942 L a localisation des acides pentosenuclhique dans les tissus
animaux e t les oeufs d’amphibiens en voie de developpement. Arch.
Biol. Paris, 53: 207-257.
BEIDGMAN,
J. 1948 A morphological study of the development of the placenta
of the rat. J. Morph., 83: 61-86.
GERARD,
P. 1925 Bur un gland myometnale de la souris e t du rat. C. R. Soe.
de Biol., 98: 457-459.
GoYoRI, G. 1945 The microtechnical demonstration of sites of lipase activity.
Proc. Boc. Exp. Biol. and Med., 68: 362-364.
1952 Microscopic Histochemistry. Chicago: Univ. of Chicago Prom
273 pp.
GREEP, R. O., AND F. L. HISAW 1938 Pseudopregnancies from electrical stimulation of the cervix in the diestrum. Proc. Soc. Exp. Biol. and Med.,
S9: 359-360.
HAYES,E. R. 1949 The plasma1 reaction. Stain Tech., $4: 19-23.
LILLIE, R. D. 1954 Histopathological Technique and Practical Histochemistry.
New Pork: The Blakiston Co., Inc. 501 pp.
48
RICHARD A. ELLIS
MITCHELL,A. J.,
AND G. B. WISLOCKI 1944 Selective staining of glycogen by
ammoniacal silver nitrate: A new method. Anat. Rec., 90: 261-266.
PEARSE,
A. C. E. 1953 Histochemistry, Theoretical and Applied. Boston:
Little, Brown, and Go. 530 pp.
SELYE,
H.,A N D T. MCKEOWN 1935 Studies on the physiology of the maternal
pIacenta in the rat. Proc. Roy. SOC.B., 119: 1-31.
VELARDO, J. T., A. G. OLSEN, F. L. HISAWAND A. B. DAWSON 1953a The
influence of decidual tissue upon pseudopregnancy. Endoerin., 63:
216-220.
VELARDO,
J. T., A. B. DAWSON,A. G. OLSEN AND F. L. HISAW 1953b Sequcnre of histological changes in the utcrus and vagina of the r a t
during prolongation of pseudopregnancy associated with the presence
of deciduomata. Am. J. Anat., 95: 273-305.
WISLOCKI,
G. B., L. P. WEISS, M. H. BURGOS
AND R. A. ELLIS1957 The
cytology, histoehemistry and electron microscopy of the granular cells
of the metrial glarid of the gravid rat. J. Anat., 91: 131-140.
YOUNG, A. 1951 Vascular architecture of the rat uterus. Proc. Roy. SOC.
Edinburgh, 64B: 292-311.
PLATE 1
EXPLANATION OF FIQURES
1 Portion of the metrial gland on the 14th day of prolonged pseudopregnancy.
The granular metrial cells contain large vacuoles filled with glycogen.
Rossman's fixation, stained with P A S ( x 400).
2
Similar section after salivary digestion (30 min. at 37" C). PAS positive
granules remain in the ceUs while the glycogen deposits have vanished.
Rossman 's fixation, stained with PAS and light hematoxylin, counter-stain
( x 400).
3 Traumatized uterus on the 20th day of prolonged pseudopregnancy. The
lipid-bearing cells delimit, the metrial gland, and the perivascular cuffs of
granular metrial cells remain almost unstained. Formalin fixation, Sudan
black B, 10 p frozen section ( X 20).
4 Higher magnification of a region of the metrial gIand in figure 3. The
distribution of the two cell types is shown. There is some confluence of the
granular and the lipid-bearing cells ( X 120).
HTSTOCHEMTSTRY O F METKIAL CILANI)
K l C H A R D A. ELLIS
PLATE 2
EXPLANATION OF FIGURES
5
Acidophilic granules in a fully differentiated metrial cell. The peripheral
vacuolation is apparently the result of the dissolution of glycogen from the
cell. Only onc of the two nuclei is visible in the photograph. Zenker-acetic
fixation, Nallory 's methylene blue-eosin stain ( X 1000).
6
A fully differentiated granular metrial cell showing the basophilic ring
investing a cluster of granules (unstained) in the Golgi zone. A young
metrial cell is seen a t lower left possessing a highly basophilic cytoplasm with
8 reticular pattern.
Cilrnoy 's fixation, methyl green-pyronin staining
( X 1000).
7
A partially differentiatd granular metrial cell.
A ring of basophilir
material surrounds the Golgi zone. Note the indentation of the nucleus.
Carnoy 's fixation, methyl green-pyronin staining ( x 1000).
8 Kuclei of lipid-bearing fibroblasts on the 18th day of prolonged pseudopregnancy. Although the nuclear chromatin sliows considerable variation,
the chromatin pattern seen in a positive Hooker-Forbcs assay for progesterone is frequently observed (arrow). Zenker-acetic fixation, Mallory 's
methylene blue-main ( X 750).
9
Hypertrophied fibroblasts containing many glohulw of lipid on the 20th day of
prolonged pseudopregnancy. Much of thi, f a t is Schultz positive for cholesterol
and birefringent with po1:rrized light. Formalin fixation, Sudan black B
( X 1000).
50
HISTOCHEMISTRY OF METRIAL GLAND
RICHARD A . ELLIS
PLATE 2
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