Phagocytosis of Spermatozoa by the Rabbit Vagina DAVID M.PHILLIPS AND SUSAN MAHLER The Population Council, The Rockefeller University, New York, New York 10021
ABSTRACT Twenty-four hours after mating in the rabbit, numerous spermatozoa are observed attached to the surface of the vaginal epithelium. Spermatozoa appear to be attached by their heads to microvilli of the simple columnar cells which compose the vaginal epithelium. Spermatozoa are taken up by the epithelial cells, and they are found within the cells in various stages of degeneration up to seven days after mating. Epithelial cells frequently appear to be filled with numerous vacuoles containing sperm components in various stages of degeneration. It is suggested that some property of the surface of sperm heads may render them particularly susceptible to phagocytosis. Phagocytosis of spermatozoa may not have long-term harmful effects for the epithelium since these epithelial cells presumably have a high rate of turnover. Spermatozoa are readily phagocytosed by a number of cell types of the reproductive tract. They have been reported to be ingested by leukocytes in the uterine lumen (Austin, '75, '60; Bedford, '65; Howe, '67; Howe and Black, '63; Marcus, '66; Mattner, '69a,b; Yanagimachi and Chang, '631, by uterine glands (Austin, '60; Zamboni, '71), uterine mucosa (SteinWerblowsky, '73), oviducal epithelium (Chakraborty and Nelson, '751, and cultured cervix cells (Reid, '64). In the epididymis, spermatozoa have been reported to invade the connective tissue (King, '55; Reid, '64) where they may be taken up by macrophages (Rouse1et al., '67). When the epididymis is obstructed, spermatozoa are absorbed by macrophages (Alexander, '72; Hoffer et al., '75) or by cells of the epididymal epithelium and epithelium of the vas deferens and vas deferens (Flickinger, '72; Hoffer et al., '75; Phadke, '64). When spermatozoa are added to established lines of fibroblasts in culture, the fibroblasts readily ingest spermatozoa and phagocytose them (Phillips et al., '76). In cases in which phagocytosis of spermatozoa has been described, it is not clear whether the live sperm may be ingested or whether only dead ones are subject to phagocytosis. This paper described the phagocytosis of large numbers of spermatozoa by the vaginal epithelium following normal mating in the rabbit. The extent of phagocytosis is such that several days after mating the vaginal epithelial cells are full of spermatozoa in various stages of degradation. ANAT. REC., 189: 61-72.
MATERIALS AND METHODS
Twenty female rabbits were sacrificed a t various times from 45 minutes to 7 days postcoitus. For transmission electron microscopy, tissues were fixed in 5% glutaraldehyde buffered with 0.2 M collidine, postfixed in 1% collidine-buffered OsO,, dehydrated in alcohol and embedded in EPON 812. For the SEM, tissues were fixed in collidine-buffered glutaraldehyde, dehydrated to 100%acetone, critical point dried, coated with gold-palladium and viewed in a ETEC autoscan. RESULTS
Sperm adhesion to vaginal epithelium Scanning electron micrographs of the vaginal surface three to twenty-four hours after coitus reveal the vaginal surface to be covered with spermatozoa (fig. 1).Frequently, spermatozoa are concentrated in crevices in the convoluted surface of the vagina (fig. 2). Sperm heads often appear to be intimately associated with microvilli of the simple columnar epithelium of the rabbit vagina (figs. 1,3). In scanning electron micrographs, microvilli appear to be fused or attached to sperm plasma membrane. The observation that spermatozoa are not removed in the fixation and dehydration procedure suggests that spermatozoa may be tightly bound to epithelial cells. In some scanning electron micrographs, spermatozoa are observed with their heads embedded in epithelium (fig. 4). In sectioned Received Aug. 4, '76. Accepted Feb. 14. '77.
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DAVID M. PHILLIPS AND SUSAN MAHLER
material, heads of spermatozoa are observed in close association with microvilli of epithelial cells (fig. 5). Extra-cellular material (fuzz) on microvilli often appears in contact with the sperm plasmalemma overlying the acrosome (fig. 6). Spermatozoa are usually seen lying flattened on the surface of the epithelium (fig. l),however, they are sometimes seen a t an angle to the epithelium (figs. 3, 5 ) . We have not observed flanges of ectoplasma around sperm heads such as are generally observed during ingestion suggesting that spermatozoa remain adhering for sometime before ingestion. Numerous remnants of spermatozoa are observed in the epithelial cells of the vagina in animals sacrificed 24 to 72 hours after coitus (figs. 7, 8). The sperm nucleus is the most apparent structure. Numerous vacuoles, containing various types of material, are also observed in the cytoplasm of epithelial cells. Often, recognizable components of sperm tails in the process of being degraded are observed in the vacuoles, although the material within the vacuoles is generally nondescript (fig. 8). Vacuoles containing material which may be remnants of degraded spermatozoa are still observed in the vaginal epithelium seven days after mating. In the vaginal epithelium of unmated or hCG ovulated animals we only observe vacuoles with electron lucid contents. DISCUSSION
Spermatozoa appear to adhere to the surface of the vaginal epithelium by the plasma membrane of the sperm head. This could be a consequence of geometric factors such as the head being a large, broad area. It may however be a consequence of the surface of the head being qualitatively different from the surface of the rest of the cell. Studies on sperm surfaces structure employing freeze cleaving (Fawcett, '75; Friend and Fawcett, '74)or surface replicas (Phillips, '751,as well as studies on surface charge (Cooper and Bedford, '71; Yanagimachi and Teichman, '72) and distribution of carbohydrates (Nicolson and Yanagimachi, '72)' all suggest that the plasma membrane in the region of the sperm head is different from the plasma membrane on the rest of the cell. Spermatozoa are also known to stick to other spermatozoa and to substrates preferentially by their heads (Phillips, '72). Unfortunately, we do not know if the spermatozoa which we observe attached
to the vaginal epithelium are motile or viable or dead cells. Many appear morphologically normal-some have broken membranes, but this could occur during fixation. This complicates the interpretation of the preferential sticking by sperm heads. Degenerating sperm nuclei are much more obvious in the vaginal epithelium then other sperm components. This may be due to the fact that sperm nuclei are more resistant to degradation then components of the sperm tail. It is also possible that sperm tails are often severed and thus are not ingested by the epithelium. It is somewhat surprising that epithelial cells so readily phagocytose spermatozoa. I t may be due to some property of the cell surface of sperm heads since heads preferentially attach and spermatozoa are readily absorbed by other cell types, including epithelial cells (Alexander, '72;Chakraborty and Nelson, '75; Flickinger, '72;Hoffer et al., '75;Phadke, '64; Reid, '64; Stein-Werblowsky, '73). The vaginal epithelium is known to phagocytose living bacteria and carmine particles (Carleton, '31).I t is difficult to see the physiological significance of phagocytosis in the vaginal tract are not particularly harmful and there are numerous leukocytes involved in phagocytosis in the vagina (Phillips and Mahler, '77). It is possible that the surface of sperm heads is in some way similar to bacteria and that the epithelial cells therefore do not distinguish between bacteria and spermatozoa. In any event, phagocytosis of spermatozoa by the vaginal epithelium may not be particularly harmful to the organism, since the vaginal epithelial cells most likely are normally replaced a t a fairly rapid rate. LITERATURE CITED Alexander, N . J . 1972 Vasectomy: Long-term effects in the rhesus monkey. J. Reprod. Fert., 31: 399-406. 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. Rep. and Fert., I : 151-156. Bedford, J. M. 1965 Effect of environment on phagocytosis of rabbit spermatozoa. J. Reprod. Fert., 9; 249-256. Carleton, H.M. 1931 Studies on epithelial phagocytosis I. Proc. Roy. Soc. B., 108, I. Chakraborty, J. ,and L. Nelson 1975 Fate of surplus sperm in the fallopian tube of the white mouse. Biol. of Reprod., 12: 445-463. Cooper, G.W.,andJ. M. Bedord 1971 Acquisition of surface charge by the plasma membrane of mammalian spermatozoa during epididymal maturation. Anat. Rec., 269: 300-303.
PHAGOCYTOSIS 13 F SPERMATOZOA Fawcett, D. W. 1975 The mammalian spermatozoan. Developmental Biol., 44: 394436. Flickinger, C. J. 1972 Alterations in the fine structure of the rat epididymis after vasectomy. Anat. Rec., 173: 277-300. Friend, D.S.,and D. W. Fawcett 1974 Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol., 63: 641-664. Hoffer, A. P.,D. W. Hamilton and D. W. Fawcett 1975 Phagocytosis of spermatozoa by the epithelial cells of the ductuli efferentes after epididymal obstruction in the rat. J. Reprod. Fert., 44: 1-9. 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. King, E. S. J. 1955 Spermatozoa1 invasion of the epididymis. J. Path. Bact., 70: 459-467. 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 of 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. Nicolson, G. L., and R. Yanagimachi 1972 Terminal saccharides on sperm plasma membranes: Identification by specific agglutinins. Science, 177: 276-279. Phadke, A. M. 1964 Fate of spermatozoa in case of ob-
63
structive azoospermia after ligation of vas deferens in man. J. Reprod. Fert., 7: 1-12. Phillips, D.M. 1972 Comparative analysis of mammalian sperm motility. J. Cell Biol. 53: 561-573. 1975 Cell surfaces of rodent sperm heads. J. Exptl. Z O O ~ O 191: ~ Y , 1-8. Phillips, D. M.,and S. Mahler 1977 Leukocyte emigration and migration following mating in the rabbit. Anat. Rec., 189: 45-60. Phillips, S. G.,D. M. Phillips, V. G. Dev, D. A. Miller, 0. P. van Diggelen and 0. J. Miller 1976 Spontaneous cell hydridization involving somatic cells in sperm suspension. Exptl. Cell Res., 98: 429-443. Reid, B. L. 1964 The behavior of human sperm toward cultured fragments of human cervix, uteri. Lancet, 1: 2123. Rousel, J. D., 0. T. Stallcup and R. C. Austin 1967 Selective phagocytosis of spermatozoa in the epididymis of bulls, rabbits and monkeys. Fert. Steril., 18: 509-516. Stein-Werblowsky, R. 1973 Penetration of spermatozoa into the uterine mucosa: A possible cause of infertility in the human. Int. J. Fert., 18: 74-80. Yanagimachi, R., and M. C. Chang 1963 Infiltration of l e u kocytes into the uterine lumen of the golden hamster during the oestrous cycle and following mating. J. Reprod. Fert., 5: 389-396. Yanagimachi, R., and R. J. Teichman 1972 Cytochemical demonstration of acrosomal proteinase in mammalian and avian spermatozoa by a silver proteinate method. Biol. Reprod., 6: 87-97. Zamboni, L. 1971 Fine Morphology of Mammalian Fertilization and Implantation. K. S. Moghissi and E. S. E. Hafez, eds. Charles C. Thomas Publishers, Springfield Illinois, pp. 213-262.
PLATE I EXPLANATION OF FIGURES
1 Surface of t h e vagina 24 hours after natural mating. The vaginal surface is generally
covered with numerous spermatozoa. Groups of microvilli extend from the somewhat dome-shaped apical surfaces of the epithelial cells. X 3,000. 2 Scanning electron microscopy of the convoluted surface of the rabbit vagina. Spermatozoa frequently accumulate in crevices as seen here. Twenty-four hours after mating. X 1,500.
64
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
3 Sperm heads associated with microvilli of epithelial cells. In several places it appears that microvilli are attached to sperm heads. Twenty-four hours postcoitus. X 5,000. 4
66
The heads of these three spermatozoa are apparently inside the epithelial cells. The outline of the head can be seen in the spermatozoan on the left. Twenty-four hours postcoitus. X 3,600.
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
5 , 6 Sperm heads associated with the vaginal epithelium-24 hours postcoitus. Spermatozoa are frequently associated with microvilli of the epithelial cells by the plasma membrane overlying the acrosome. A thin region of extracellular fuzz that coats the microvilli appears to be associated with the sperm plasma membrane. Figure 5 X 6,000; figure 6 X 18,000.
68
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
7 Simple columnar cells of the vaginal epithelium two days postcoitus. Cells contain numerous recognizable sperm components. Sperm nuclei are particularly obvious. Numerous vacuoles, some containing recognizable sperm components, occur in the cytoplasm of the epithelial cells. X 3,000.
8 Region of a vaginal epithelial cell three hours after insemination. Numerous vacuoles, some containing recognizable sperm components (arrows), characterize the cytoplasm of these cells. X 12.000.
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PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Swan Mahler
PLATE 4
ABSTRACT Twenty-four hours after mating in the rabbit, numerous spermatozoa are observed attached to the surface of the vaginal epithelium. Spermatozoa appear to be attached by their heads to microvilli of the simple columnar cells which compose the vaginal epithelium. Spermatozoa are taken up by the epithelial cells, and they are found within the cells in various stages of degeneration up to seven days after mating. Epithelial cells frequently appear to be filled with numerous vacuoles containing sperm components in various stages of degeneration. It is suggested that some property of the surface of sperm heads may render them particularly susceptible to phagocytosis. Phagocytosis of spermatozoa may not have long-term harmful effects for the epithelium since these epithelial cells presumably have a high rate of turnover. Spermatozoa are readily phagocytosed by a number of cell types of the reproductive tract. They have been reported to be ingested by leukocytes in the uterine lumen (Austin, '75, '60; Bedford, '65; Howe, '67; Howe and Black, '63; Marcus, '66; Mattner, '69a,b; Yanagimachi and Chang, '631, by uterine glands (Austin, '60; Zamboni, '71), uterine mucosa (SteinWerblowsky, '73), oviducal epithelium (Chakraborty and Nelson, '751, and cultured cervix cells (Reid, '64). In the epididymis, spermatozoa have been reported to invade the connective tissue (King, '55; Reid, '64) where they may be taken up by macrophages (Rouse1et al., '67). When the epididymis is obstructed, spermatozoa are absorbed by macrophages (Alexander, '72; Hoffer et al., '75) or by cells of the epididymal epithelium and epithelium of the vas deferens and vas deferens (Flickinger, '72; Hoffer et al., '75; Phadke, '64). When spermatozoa are added to established lines of fibroblasts in culture, the fibroblasts readily ingest spermatozoa and phagocytose them (Phillips et al., '76). In cases in which phagocytosis of spermatozoa has been described, it is not clear whether the live sperm may be ingested or whether only dead ones are subject to phagocytosis. This paper described the phagocytosis of large numbers of spermatozoa by the vaginal epithelium following normal mating in the rabbit. The extent of phagocytosis is such that several days after mating the vaginal epithelial cells are full of spermatozoa in various stages of degradation. ANAT. REC., 189: 61-72.
MATERIALS AND METHODS
Twenty female rabbits were sacrificed a t various times from 45 minutes to 7 days postcoitus. For transmission electron microscopy, tissues were fixed in 5% glutaraldehyde buffered with 0.2 M collidine, postfixed in 1% collidine-buffered OsO,, dehydrated in alcohol and embedded in EPON 812. For the SEM, tissues were fixed in collidine-buffered glutaraldehyde, dehydrated to 100%acetone, critical point dried, coated with gold-palladium and viewed in a ETEC autoscan. RESULTS
Sperm adhesion to vaginal epithelium Scanning electron micrographs of the vaginal surface three to twenty-four hours after coitus reveal the vaginal surface to be covered with spermatozoa (fig. 1).Frequently, spermatozoa are concentrated in crevices in the convoluted surface of the vagina (fig. 2). Sperm heads often appear to be intimately associated with microvilli of the simple columnar epithelium of the rabbit vagina (figs. 1,3). In scanning electron micrographs, microvilli appear to be fused or attached to sperm plasma membrane. The observation that spermatozoa are not removed in the fixation and dehydration procedure suggests that spermatozoa may be tightly bound to epithelial cells. In some scanning electron micrographs, spermatozoa are observed with their heads embedded in epithelium (fig. 4). In sectioned Received Aug. 4, '76. Accepted Feb. 14. '77.
61
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DAVID M. PHILLIPS AND SUSAN MAHLER
material, heads of spermatozoa are observed in close association with microvilli of epithelial cells (fig. 5). Extra-cellular material (fuzz) on microvilli often appears in contact with the sperm plasmalemma overlying the acrosome (fig. 6). Spermatozoa are usually seen lying flattened on the surface of the epithelium (fig. l),however, they are sometimes seen a t an angle to the epithelium (figs. 3, 5 ) . We have not observed flanges of ectoplasma around sperm heads such as are generally observed during ingestion suggesting that spermatozoa remain adhering for sometime before ingestion. Numerous remnants of spermatozoa are observed in the epithelial cells of the vagina in animals sacrificed 24 to 72 hours after coitus (figs. 7, 8). The sperm nucleus is the most apparent structure. Numerous vacuoles, containing various types of material, are also observed in the cytoplasm of epithelial cells. Often, recognizable components of sperm tails in the process of being degraded are observed in the vacuoles, although the material within the vacuoles is generally nondescript (fig. 8). Vacuoles containing material which may be remnants of degraded spermatozoa are still observed in the vaginal epithelium seven days after mating. In the vaginal epithelium of unmated or hCG ovulated animals we only observe vacuoles with electron lucid contents. DISCUSSION
Spermatozoa appear to adhere to the surface of the vaginal epithelium by the plasma membrane of the sperm head. This could be a consequence of geometric factors such as the head being a large, broad area. It may however be a consequence of the surface of the head being qualitatively different from the surface of the rest of the cell. Studies on sperm surfaces structure employing freeze cleaving (Fawcett, '75; Friend and Fawcett, '74)or surface replicas (Phillips, '751,as well as studies on surface charge (Cooper and Bedford, '71; Yanagimachi and Teichman, '72) and distribution of carbohydrates (Nicolson and Yanagimachi, '72)' all suggest that the plasma membrane in the region of the sperm head is different from the plasma membrane on the rest of the cell. Spermatozoa are also known to stick to other spermatozoa and to substrates preferentially by their heads (Phillips, '72). Unfortunately, we do not know if the spermatozoa which we observe attached
to the vaginal epithelium are motile or viable or dead cells. Many appear morphologically normal-some have broken membranes, but this could occur during fixation. This complicates the interpretation of the preferential sticking by sperm heads. Degenerating sperm nuclei are much more obvious in the vaginal epithelium then other sperm components. This may be due to the fact that sperm nuclei are more resistant to degradation then components of the sperm tail. It is also possible that sperm tails are often severed and thus are not ingested by the epithelium. It is somewhat surprising that epithelial cells so readily phagocytose spermatozoa. I t may be due to some property of the cell surface of sperm heads since heads preferentially attach and spermatozoa are readily absorbed by other cell types, including epithelial cells (Alexander, '72;Chakraborty and Nelson, '75; Flickinger, '72;Hoffer et al., '75;Phadke, '64; Reid, '64; Stein-Werblowsky, '73). The vaginal epithelium is known to phagocytose living bacteria and carmine particles (Carleton, '31).I t is difficult to see the physiological significance of phagocytosis in the vaginal tract are not particularly harmful and there are numerous leukocytes involved in phagocytosis in the vagina (Phillips and Mahler, '77). It is possible that the surface of sperm heads is in some way similar to bacteria and that the epithelial cells therefore do not distinguish between bacteria and spermatozoa. In any event, phagocytosis of spermatozoa by the vaginal epithelium may not be particularly harmful to the organism, since the vaginal epithelial cells most likely are normally replaced a t a fairly rapid rate. LITERATURE CITED Alexander, N . J . 1972 Vasectomy: Long-term effects in the rhesus monkey. J. Reprod. Fert., 31: 399-406. 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. Rep. and Fert., I : 151-156. Bedford, J. M. 1965 Effect of environment on phagocytosis of rabbit spermatozoa. J. Reprod. Fert., 9; 249-256. Carleton, H.M. 1931 Studies on epithelial phagocytosis I. Proc. Roy. Soc. B., 108, I. Chakraborty, J. ,and L. Nelson 1975 Fate of surplus sperm in the fallopian tube of the white mouse. Biol. of Reprod., 12: 445-463. Cooper, G.W.,andJ. M. Bedord 1971 Acquisition of surface charge by the plasma membrane of mammalian spermatozoa during epididymal maturation. Anat. Rec., 269: 300-303.
PHAGOCYTOSIS 13 F SPERMATOZOA Fawcett, D. W. 1975 The mammalian spermatozoan. Developmental Biol., 44: 394436. Flickinger, C. J. 1972 Alterations in the fine structure of the rat epididymis after vasectomy. Anat. Rec., 173: 277-300. Friend, D.S.,and D. W. Fawcett 1974 Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol., 63: 641-664. Hoffer, A. P.,D. W. Hamilton and D. W. Fawcett 1975 Phagocytosis of spermatozoa by the epithelial cells of the ductuli efferentes after epididymal obstruction in the rat. J. Reprod. Fert., 44: 1-9. 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. King, E. S. J. 1955 Spermatozoa1 invasion of the epididymis. J. Path. Bact., 70: 459-467. 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 of 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. Nicolson, G. L., and R. Yanagimachi 1972 Terminal saccharides on sperm plasma membranes: Identification by specific agglutinins. Science, 177: 276-279. Phadke, A. M. 1964 Fate of spermatozoa in case of ob-
63
structive azoospermia after ligation of vas deferens in man. J. Reprod. Fert., 7: 1-12. Phillips, D.M. 1972 Comparative analysis of mammalian sperm motility. J. Cell Biol. 53: 561-573. 1975 Cell surfaces of rodent sperm heads. J. Exptl. Z O O ~ O 191: ~ Y , 1-8. Phillips, D. M.,and S. Mahler 1977 Leukocyte emigration and migration following mating in the rabbit. Anat. Rec., 189: 45-60. Phillips, S. G.,D. M. Phillips, V. G. Dev, D. A. Miller, 0. P. van Diggelen and 0. J. Miller 1976 Spontaneous cell hydridization involving somatic cells in sperm suspension. Exptl. Cell Res., 98: 429-443. Reid, B. L. 1964 The behavior of human sperm toward cultured fragments of human cervix, uteri. Lancet, 1: 2123. Rousel, J. D., 0. T. Stallcup and R. C. Austin 1967 Selective phagocytosis of spermatozoa in the epididymis of bulls, rabbits and monkeys. Fert. Steril., 18: 509-516. Stein-Werblowsky, R. 1973 Penetration of spermatozoa into the uterine mucosa: A possible cause of infertility in the human. Int. J. Fert., 18: 74-80. Yanagimachi, R., and M. C. Chang 1963 Infiltration of l e u kocytes into the uterine lumen of the golden hamster during the oestrous cycle and following mating. J. Reprod. Fert., 5: 389-396. Yanagimachi, R., and R. J. Teichman 1972 Cytochemical demonstration of acrosomal proteinase in mammalian and avian spermatozoa by a silver proteinate method. Biol. Reprod., 6: 87-97. Zamboni, L. 1971 Fine Morphology of Mammalian Fertilization and Implantation. K. S. Moghissi and E. S. E. Hafez, eds. Charles C. Thomas Publishers, Springfield Illinois, pp. 213-262.
PLATE I EXPLANATION OF FIGURES
1 Surface of t h e vagina 24 hours after natural mating. The vaginal surface is generally
covered with numerous spermatozoa. Groups of microvilli extend from the somewhat dome-shaped apical surfaces of the epithelial cells. X 3,000. 2 Scanning electron microscopy of the convoluted surface of the rabbit vagina. Spermatozoa frequently accumulate in crevices as seen here. Twenty-four hours after mating. X 1,500.
64
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
3 Sperm heads associated with microvilli of epithelial cells. In several places it appears that microvilli are attached to sperm heads. Twenty-four hours postcoitus. X 5,000. 4
66
The heads of these three spermatozoa are apparently inside the epithelial cells. The outline of the head can be seen in the spermatozoan on the left. Twenty-four hours postcoitus. X 3,600.
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
5 , 6 Sperm heads associated with the vaginal epithelium-24 hours postcoitus. Spermatozoa are frequently associated with microvilli of the epithelial cells by the plasma membrane overlying the acrosome. A thin region of extracellular fuzz that coats the microvilli appears to be associated with the sperm plasma membrane. Figure 5 X 6,000; figure 6 X 18,000.
68
PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Susan Mahler
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
7 Simple columnar cells of the vaginal epithelium two days postcoitus. Cells contain numerous recognizable sperm components. Sperm nuclei are particularly obvious. Numerous vacuoles, some containing recognizable sperm components, occur in the cytoplasm of the epithelial cells. X 3,000.
8 Region of a vaginal epithelial cell three hours after insemination. Numerous vacuoles, some containing recognizable sperm components (arrows), characterize the cytoplasm of these cells. X 12.000.
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PHAGOCYTOSIS OF SPERMATOZOA David M. Phillips and Swan Mahler
PLATE 4
