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.

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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

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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

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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:

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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.

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LEUKOCYTE EMIGRATION AND MIGRATION David M. Phillips and Susan Mahler

PLATE 2

PLATE 3 EXPLANATION OF FIGURES 4.5

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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.

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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.

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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.

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LEUKOCYTE EMIGRATION AND MIGRATION David M. Phillips and Susan Mahler

PLATE 6

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

Leukocyte Emigration and Migration in the Vagina Following Mating in the Rabbit DAVID M. PHILLIPS AND SUSAN MAHLER The Population Council, The Rockefe...
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