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Leukocyte emigration and migration in the vagina following mating in the rabbit.

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Leukocyte Emigration and Migration in the Vagina
Following Mating in the Rabbit
DAVID M. PHILLIPS AND SUSAN MAHLER
The Population Council, The Rockefeller University,New York, New York 10021
ABSTRACT
Within 45 minutes after mating in the rabbit, numerous
heterophil leukocytes adhere to the endothelium of venules in the vagina. Initial
association appears to occur via small protuberances from the leukocyte which
fit into small indentions in the endothelial cell. Following adherence, leukocytes
flatten and pass between endothelial cells. A regular intercellular space separates the leukocyte from the endothelial cells. Leukocytes subsequently migrate through the connective tissue to the epithelium. By three hours postcoitus,
the region beneath the basement lamina of the vaginal epithelium is crowded
with numerous juxtaposed leukocytes. Leukocytes subsequently migrate between epithelial cells into the vaginal lumen where they actively engulf spermatozoa. Spermatozoa appear to be ingested head first. Numerous small
filaments are observed in the leukocyte cytoplasm in the region adjacent to the
sperm head. Degranulation of azurophyl granules follows sperm uptake. The leukocyte response can be elicited either by spermatozoa (from the epididymis) or by
semen (from vasectomized bucks), but is not elicited by ovulation (with human
chorionic gonadotropin). It is suggested that the response may be initiated because the vagina does not distinguish between semen, spermatozoa and bacterial
infection.
Although only one or a few spermatozoa are
directly involved in fertilization, millions of
spermatozoa are transferred to the vagina or
uterus during insemination. The possible significance of such large numbers of spermatozoa has intrigued biologists for many years.
The fate of spermatozoa in the female tract,
particularly in the uterus, has been followed
in several species. From knowledge of fate of
spermatozoa in the female genital tract, one
might derive insight into the significance of
large numbers of spermatozoa or better
understanding of certain pathologies of the
female reproductive tract.
In all mammalian species which have been
studied, large numbers of leukocytes migrate
into the uterine lumen and actively phagocytose spermatozoa (Austin, '57, '60; Bedford,
'65; Howe, '67;Howe and Black, '63; Mattner,
'69a,b; Menge, '62; Yanagimachi and Chang,
'63). Leukocytes have also been reprted to be
involved in phagocytosis of spermatozoa in
the cervical canal (Mattner, '69a,b), vagina
(Allen, '22; Beaver, '60; Howe and Black, '63;
Long and Evans, '221, and oviducts (Howe,
ANAT. REC., 189: 45-60.
'67) of some species. The process of leukocyte
migration into the lumen of the uterus, vagina and oviducts has not been investigated,
although the morphology of emigration of leukocytes during the inflammatory response in
other tissues has been studied by several investigators (Florey and Grant, '61;Marchesi,
'61; Marchesi and Florey, '60; Williamson and
Grisham, '61;Yamamoto and %to, '66).This
report describes a morphological sequence of
events which occur in the rabbit vagina following coitus whereby large numbers of
heterophil leukocytes migrate from venules,
through the connective tissue, between epithelial cells and into the vaginal lumen where
they phagocytose spermatozoa.
MATERIALS AND METHODS
Twenty-four female rabbits were sacrificed
at intervals of 45 minutes to 7 days after
natural mating. Two rabbits were sacrificed
90 minutes after mating with vasectomized
bucks, and two 90 minutes after artificial insemination with spermatozoa from the cauda
Received Aug. 4, '76. Accepted Feb. 14, '77.
45
46
DAVID M. PHILLIPS AND SUSAN MAHLER
epididymis, and two after placing saline in the
vagina. Vaginas or lightly centrifuged vaginal washings were fixed in 5%glutaraldehyde
buffered with 0.2 M collidine, post-fixed in
% collidine-buffered OsO,, dehydrated in
alcohol, and embedded in Epon 812.
Animals which were induced to ovulate
were injected I.V. with 250 I.U. of human
chorionic gonadotropin. Ovaries were examined 24 hours after injection for haemorrhagic follicles.
RESULTS
Emigration of polymorphonuclear
leukocytes
Forty-five minutes after mating, large
numbers of polymorphonuclear leukocytes
were observed adhering to endothelial cells
and beneath the endothelial cells of venules in
the connective tissue underlying the vaginal
epithelium. (Vessels were judged to be
venules by morphological criteria.) Occasional leukocytes were observed passing between endothelial cells. Thus, polymorphonuclear leukocytes emigrated through the endothelium within 45 minutes of mating. That
very few leukocytes were seen actually passing between endothelial cells suggests that
this traversal is very rapid. The entire
emigration process is so rapid that micrographs from rabbits sacrificed 45 minutes or
90 minutes after mating show all stages of
emigration and can be arranged in what appears to be a logical series of events in leukocyte emigration (figs. 1-61.
Numerous leukocytes were observed adhering to the endothelial cells of venules. Most of
these cells were roughly spherical and appeared to be associated with endothelial cells
by small protuberances (fig. 1). The plasma
membrane of the leukocyte appeared slightly
more electron-dense a t the tips of these protuberances. In the areas where the protuberances were nearest to the endothelial
cell, the endothelial cells appeared as though
they were slightly indented to fit the contour
of the leukocytes protuberances. A minority
of leukocytes appeared to be flattened on the
surface juxtaposed to the endothelium with
small pseudopodia extending from the cell.
Presumably, spherical leukocytes first adhere
to the endotheluim and then flatten on the
surface of the endothelial cell.
Some heterophils were observed with a
pseudopodium protruding into the endothelium (fig. 2) or completely through the endo-
thelium (fig. 3). Presumably, heterophil leukocytes adhere to the endothelium by protuberances, flatten out, and subsequently
penetrate the endothelium by means of a
small pseudopodium. The pseudopodium characteristically appeared to pass between adjacent endothelial cells. A regular intercellular
space of about 200 A was seen between leukocyte and endothelial cell, suggesting that little or no plasma is lost from the venule during
this process. The cytoplasm in the pseudopod
was characteristically devoid of ribosomes,
granules, vacuoles and other organelles
(fig. 3).
After the pseudopod reached the outside of
the venule, the rest of the cell appeared to follow. Sometimes an hour-glass shaped leukocyte was seen compressed between a small
space between endothelial cells (fig. 4). In
other micrographs, the leukocyte appeared
more as though i t was slipping beneath endothelial cells (fig. 5). These observations suggest that there may be some variation in the
manner by which leukocytes pass through the
epithelium of venule. However, we always observed a close association between endothelial
cell and leukocyte.
Migration to the vaginal lumen
Once out of the venule lumen, leukocytes
apparently remain beneath the endothelium
for some time since large numbers of leukocytes were consistently observed there in vaginas of does sacrificed 90 minutes to 24 hours
postcoitus. Endothelial cells characteristically bulged into the vessel lumen because of the
underlying leukocytes (fig. 6). Leukocytes apparently subsequently progress through the
basement lamina and between pericytes
where large numbers of them were observed
in the fairly dense connective tissue below the
vaginal epithelium (fig. 7). Leukocytes in the
connective tissue appeared more elongated
than those associated with endothelium.
Irregularly shaped regions of cytoplasm, presumably pseudopodia, devoid of cytoplasmic
organelles were frequently observed in these
leukocytes (fig. 7).
Unlike most other mammalian species, the
rabbit vagina has a simple columnar epithelium. In tissues from animals sacrificed three
hours to three days after mating, large numbers of leukocytes were situated just beneath
the basement lamina of the vaginal epithelium (fig. 8). In vaginas fixed six to 24 hours
LEUKOCYTE EMIGRATION AND MIGRATION
postcoitus, it was typical to find the region
just under the vaginal epithelium completely
occupied by heterophil leukocytes. Leukocytes, therefore, apparently spend some time
under the epithelium before they migrate into
the vaginal lumen. Migrating leukocytes apparently pass between the columnar epithelial cells into the vaginal lumen, since they
were frequently observed between epithelial
cells (figs. 8, 9).Although spermatozoa were
not observed in leukocytes beneath the epithelium or in the epithelium, the leukocyte
cytoplasm frequently contained regions of
diffuse filamentous material suggesting that
there may have been some intercellular degranulation or degradation.
Vaginal lumen
Numerous leukocytes were observed in vaginal contents of rabbits 3 to 24 hours postcoitus. Many of these leukocytes contained remnants of spermatozoa, particularly sperm nuclei. Spermatozoa appeared to be taken up by
leukocytes head first. In cells which appeared
to be engulfing sperm heads, the leukocyte cytoplasm adjacent to the sperm head was characterized by a network of thin filaments (figs.
10, 11).Portions of spermatozoa within leukocytes were generally contained within
vacuoles. Leukocytes which contained phagocytic vacuoles with sperm components often
appeared to have undergone some degranulation, particularly of azurophil granules.
Leukocyte response to seminal fluid
and spermatozoa
In order to determine whether the leukocyte response was caused by spermatozoa or
by seminal fluid, we mated two does to vasectomized bucks and inserted saline suspended
epidymial spermatozoa in the vagina of two
other does. Animals were sacrificed three
hours after insemination. In all animals the
leukocyte response was qualitatively similar
to the response in normally mated does. Large
numbers of leukocytes were found sticking to
endothelial cells and in the connective tissue,
suggesting that the leukocyte response can be
elicited either by seminal plasma or spermatozoa. Two other does were ovulated by I.V.
injection of human chorionic gonadotropin
and examined 24 hours later. Observation of
ovaries revealed haemorrhagic follicles, but
human chorionic gonadotropin induced ovulation did not result in leukocyte emigration.
Two control animals with saline deposited
47
into the vagina did not show a leukocyte response.
DISCUSSION
Electron micrographic observations on the
emigration of leukocytes from venules were
first described by Marchesi and Florey ('60)
and Marchesi ('61) in the mesentery of rats
following mild trauma. Since then there have
been a number of similar reports (Florey and
Grant, '61; Williamson and Grisham, '61;
Yamamoto and Sato, '66). Our observations
are similar to those reported by Marchesi and
Marchesi and Florey who found polymorphonuclear leukocytes adhering to the endothelium of small venules and emigrating
between endothelial cells. Therefore, morphologically, emigration of leukocytes as a response to mating in the rabbit appears to be
the same as the inflammatory response.
We have observed some minor differences
between our observations and previous observations by other workers. Some previous reports give the impression that sticking may
involve flattening of the leukocyte against
the endothelial surface (Florey and Grant,
'61; Marchesi, '61; Marchesi and Florey, '60).
Our observations suggest that polymorphonuclear leukocytes fist become associated with
endothelial cells a t the tips of small protuberances which project from the more or less
spherical leukocyte and that the plasma membrane of the leukocyte may be somewhat modified a t the protuberance since it is slightly
more dense in this region. The flattening
which is observed appears to occur subsequent
to the sticking since many cells are observed
to be stuck but not flattened. Williamson and
Grisham ('61), however, described endothelial
spikes which reportedly are "long finger-like
endothelial processes in which leukocytes become enmeshed" during acute inflammation
in the dog pancreas.
These endothelial spikes may be flattened
lamellar, rather than finger-like, processes of
endothelial cells under which leukocytes are
emigrating. Gwendolyn et al. ('74) have reported large gaps between endothelial cells in
the region of invading leukocytes. We have
not observed gaps and they are not reported
by earlier workers. Thus,large gaps in the endothelium probably represent a fixating artifact.
It is interesting that normal mating quickly elicit such a strong leukocyte response involving millions of heterophil leukocytes. The
response could be induced by sperm or by
48
DAVID
M. PHILLIPS AND SUSAN MAHLER
semen from vasectomized bucks. The biological significance of this response is unclear. It
is not obvious why the animal should have
need to phagocytose sperm in the vagina.
They are, in a sense, outside the animal and
would be expected to disappear shortly in any
case due to degradative activity of their own
hydrolases and normal outward flow of fluid
through the vagina. It is possible that there is
some harmful unknown effect of sperm if they
remain in the vagina, or it is possible that the
response we see is unrelated to the fact that
the invading cells are sperm, but may occur in
response to many foreign cell types. In other
words, the leukocyte response may be a form
of inflammatory reaction in a tissue which is
so sensitive that it initiates the process in response to any minor irritation. Possibly bacteria, spermatozoa, and a component of semen
bind to a common receptor on the vaginal epithelium which in some fashion triggers the
leukocyte response.
Whatever the basic cause of the leukocyte
reaction, i t provides a n excellent opportunity
to study leukocyte emigration in response to
well-defined, easily observable stimulus OCcurring a t a known point in time.
LITERATURE CITED
Allen, E. 1922 The estrous cycle in mouse. Am. J. Anat.,
30: 297-371.
Austin, C.R. 1957 Fate of spermatozoa in the uterus of the
mouse and rat. J. Endocrin., 14: 335-342.
1960 Fate of spermatozoa in the female genital
tract. J. Reprod. Fert., 1: 151-156.
Beaver, D. L. 1960 The hormonal induction of a vaginal
leukocyte exudate in germ-free mouse. Am. J. Path., 37:
-
769-773.
Bedford, J. M. 1965 Effect of environment on phagocytosis
of rabbit spermatozoa. J. Reprod. Fert., 9: 249-256.
Florey, H. W.,and L. H. Grant 1961 Leukocyte migration
from small blood vessels stimulated with ultraviolet
light: An electron microscope study. J. Path. Bact., 82:
13-17.
Gwendolyn, J. S., W. G. M. Ritchie and P. R. Lynch 1974
Venous endothelial damage produced by massive sticking and emigration of leukocytes. Am. J. Path., 74: 507-
522.
Howe, G. R. 1967 Leukocyte response to spermatozoa in
ligated segments of the rabbit vagina, uterus and
oviduct. J. Reprod. Fert., 13: 563-566.
Howe, G. R.,and D. L. Black 1963 Spermatozoa transport
and leukocyte responses in the reproductive tract of
calves. J. Reprod. Fert., 6: 305-311.
Long, J. A., and H. M. Evans 1922 The estrous cycle in the
rat and its associated phenomena. Mem. Univ. Calif., 5:
5-124.
Marchesi, V. T. 1961 The site of leukocyte emigration during inflammation. Quart. J. Exp. Physiol., 46: 115-118.
Marchesi, V. T., and H. W. Florey 1960 Electron micrographic observations on t h e emigration of leukocytes.
Quart. J. Exp. Physiol., 45: 343-348.
Marcus, S. L. 1966 Influence of ovarian hormones on leukocytic response to spermatozoa in the uterus of the
golden hamster. Fert. Steril., 17: 212-220.
Mattner, P. E. 1969a Differential leukocyte response to
spermatozoa in the cervix and the uterus in ewes. J.
Reprod. Fert., 18: 297-303.
1969b Phagocytosis of spermatozoa by leukocytes
in bovine cervical mucus in uitro. J. Reprod. Fert., 20:
133-134.
Menge, A. C., W. J. Tyler and L. E. Casida 1962 Factors affecting the removal of spermatozoa from the rabbit
uterus. J. Reprod. Fert., 3: 396-404.
Williamson, J. R.,and J. W. Grisham 1961 Electron microscopy of leukocytic margination and emigration in
acute infiammation in dog pancreas. Am. J. Path., 39:
239-256.
Yamamoto, T. Y., and S. Sat0 1966 Electron microscope
studies on the vessel wall and the leukocyte emigration.
Archivum Histogicum Japonicum, 27: 297-310.
Yanagimachi, R., and M. C. Chang 1963 Infiltration of leukocytes into the uterine lumen of the golden hamster
during the oestrous cycle and following mating. J.
Reprod. Fert., 5: 389-396.
PLATE 1
EXPLANATION OF FIGURE
1 Leukocyte associated with endothelial cell of venule. Neutrophils appear to adhere to
the endothelium by small protuberances. X 42,000.
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan MahIer
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
2 A small pseudopodium appears to be protruding from the leukocyte between cells of
the venous endothelium. Forty-five minutes postcoitus.
X
9,000.
3 Leukocyte pseudopodium extending into connective tissue beneath the endothelium
of the venule. The cytoplasm of the pseudopodium is characterized by small filaments
and is devoid of other structures. Endothelial cells are closely associated with plasma
membrane of the pseudopodium. Forty-five minutes postcoitus. X 30,000.
50
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan Mahler
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
4.5
52
Constricted neutrophils emigrating between endothelial cells of a venule. Ninety
minutes postcoitus. X 7,000.
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan Mahler
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
6 Leukocytes just beneath the endothelium cause the endothelium to bulge into the
vessel lumen. Ninety minutes postcoitus. X 30,000.
7 Four neutrophils in the areolar connective tissue to the vagina. Ninety minutes
postcoitus. X 3,000.
54
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan Mahler
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
8
Simple columnar epithelium of the rabbit vagina (Top). Numerous polymorphonuclear leukocytes are situated just below the basement lamina of the epithelium-one
leukocyte is situated between epithelial cells. Six hours after mating. X 2,000.
9 Section through t h e vaginal epithelium parallel to t h e vaginal surface. One day after
mating. Leukocytes are observed between epithelial cells. Some sperm remnants are
observed in epithelial cells, but not in leukocytes. X 2,600.
56
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan Mahler
PLATE 5
PLATE 6
EXPLANATION OF FIGURES
10 Sperm heads apparently being engulfed by leukocytes in the vaginal lumen. Six
hours postcoitus. X 6,000.
11 Fibrous material within heterophil cytoplasm adjacent to a sperm head which is apparently being ingested. Six hours after mating. X 48,000.
58
LEUKOCYTE EMIGRATION AND MIGRATION
David M. Phillips and Susan Mahler
PLATE 6
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