FATE OF SPERMATOZOA IN THE FEMALE GENITAL TRACT

C. R. AUSTIN Division of Experimental Biology, Mattonai Institutefor Medical Research, London, N.W.j {Received 26th October 1959)

Summary. Spermatozoa were found to persist for 6 days in the uterine lumen of rabbits. They were also seen in the uterine glands in the greater horseshoe bat, the common pipistrelle bat, the guinea-pig, the stoat, the hedgehog and the mole. Sperm heads were identified in the uterine-gland mucosa in two guinea-pigs, and additional observations are reported on their presence in the Fallopian-tube mucosa in the greater horseshoe bat and the rat. In both regions, the spermatozoa were considered to lie within cells. There was evidence of dissolution of spermatozoa in the uterine glands of the horseshoe bat, and in the Fallopian-tube mucosa of the pipistrelle bat and the rat. The of one guinea-pig had localized regions of leucocytosis, probably owing to infection; in this specimen, many leucocytes contain¬ ing spermatozoa were found within some uterine glands and in the interstitial tissues at distances from the glands.

INTRODUCTION

tend to in the uterine in some animals have

Spermatozoa persist glands — they been described in this location in the dog (Marshall & Jolly, 1905), the shrew, the tarsier, the sooty mangabey, the gibbon, the Javanese fruit bat, the noctule bat (Kohlbrugge, 1910, 1913), the hedgehog, the stoat (Deanesly, 1934, 1935), the spiny anteater, the ewe and the antelope (E. C. Amoroso, personal com¬ munication, 1959), that is to say, in representatives of six Orders of mammals. Deanesly observed spermatozoa in the glands in almost all the pregnant hedge¬ hog uteri she examined, 'except very late in pregnancy, when little or no material between the foetuses was available for examination'. The presence of spermatozoa in uterine glands is not, however, a universal phenomenon: they have not been reported there in the rabbit, rat, mouse and man, in spite of the very numerous observations that have been made in these animals, and Hartman (1939) notes that he has never seen them in the opossum. Few useful data are available on the dissolution of spermatozoa within the female genital tract, although dissolution has often been invoked as one means of their disposal. In some animals, notably the mouse, extensive phagocytosis J51

Downloaded from Bioscientifica.com at 09/25/2021 03:03:07PM via free access 152 C. R. Austin of spermatozoa occurs in the uterus (see Austin, 1957), but there is no evidence that the products of digestion are released within the tract. On the other hand, there have been numerous reports describing the penetration of spermatozoa into tissues, both maternal and embryonic. Serious doubts on the validity of these findings have been raised by Vojtiskova (1956), who clearly showed that spermatozoa are very liable to become displaced from the lumen of the female tract during the manipulations involved in the pre¬ paration of material for histological study. More recently, however, new claims have been advanced in favour of the occurrence of tissue invasion by sperma¬ tozoa (Austin, 1959; Austin & Bishop, 1959), and reasons have been given for believing that their presence could not be ascribed to artefact. In the present paper, additional evidence is provided on the persistence and dissolution of spermatozoa within the female genital tract and on their entry into the tissues.

MATERIALS AND METHODS Investigations were made on the following animals : two greater horseshoe bats {Rhinolophusferrum-equinum), four common pipistrelle bats {Pipistrelluspipistrellus), ten rats {Rattus norvegicus), fourteen rabbits {Oryctolagus cuniculus), six guinea-pigs {Cavia porcellus), one hedgehog {Erinaceus europaeus), one mole {Talpa europaea) and two stoats {Mustela erminea). The rats, the guinea-pigs and nine of the rabbits were allowed natural mating; the remaining rabbits were inseminated artificially. Copulation plugs and uterine spermatozoa were found in the horseshoe bats. In the other animals, except in one pipistrelle bat, evidence that coitus had occurred was provided by the presence of spermatozoa in the uterus ; in the exceptional bat, no sperma¬ tozoa could be found in the uterus but there was an 8- to 1 o-cell embryo in one . Tissues intended for histological study were fixed in Bouin's fluid and embedded in paraffin; sections were cut at a thickness of 7 to 10 µ and stained with haematoxylin and eosin.

OBSERVATIONS

PERSISTENCE AND DISSOLUTION OF SPERMATOZOA IN THE LUMINA OF UTERUS AND UTERINE GLANDS

Twelve rabbits were killed on different days after mating or artificial insemina¬ tion. Spermatozoa were found in the washings from the uterus in animals killed on Days 1, 3, 4, 5 and 6 (Table 1). They showed no obvious morphological change other than separation of heads and tails, which was common, and they were not found in the washings from animals killed on Days 7, 8 and 9. In studies of histological sections, aggregations of spermatozoa in uterine glands were seen in the horseshoe bat (PL 1, Fig. 1), the pipistrelle bat, the guinea-pig, the stoat, the hedgehog and the mole. One of the guinea-pigs, in which these spermatozoa were found, had been killed on the 5th day of pregnancy, the others earlier.

Downloaded from Bioscientifica.com at 09/25/2021 03:03:07PM via free access Fate of Spermatozoa 153 Evidence of dissolution of spermatozoa was seen in some of the uterine glands of the horseshoe bats: these glands contained numerous objects that resembled sperm heads, but were distinctly smaller, ranging down in size to minute rodlets (PI. 1, Fig. 2). The appearance presented conformed with the descriptions of Metschnikoff (1899) and Hoehner & Behne (1914) of sperm heads undergoing dissolution within leucocytes.

Table i the number of spermatozoa found in o.05-ml aliquots of i -ml washings from the uteri of rabbits killed after mating or artificial insemination on day o

Mated (M) or Day No. rabbits No. spermatozoa artificially inseminated (AI) 185 AI 8 M 31 AI 86 & o both M o & 23 M & AI o & o both M o M o & o both M

ENTRY OF SPERMATOZOA INTO TISSUES Uterus Careful examination of sections of twenty-four uteri (from two horseshoe bats, four pipistrelles, six rats, two rabbits, six guinea-pigs, one hedgehog, one mole and two stoats) failed to reveal any evidence of tissue invasion by spermatozoa, except in specimens taken from two of the guinea-pigs. These two animals had been killed on the day after the finding of the copulation plug, and they provided five instances of a sperm head lying in the mucous membrane of a uterine gland (PI. 1, Fig. 5). The location appeared to be intracellular, and in one section there were no fewer than four heads evidently embedded in one cell. In one guinea-pig, many sperm heads were found within leucocytes migrating through the submucous tissues. In this animal, the whole section of the uterus presented an unusual picture, for large numbers of leucocytes were evident in some of the glands and in the immediately surrounding tissue (PI. i, Fig. 3). In addition, discreet regions of intense leucocytosis were found unassociated with glands. The general appearance was most distinctive and quite unlike that of the other uteri examined in this investigation. Leucocytes bearing sperm heads were abundant both in the gland lumina and in the interstitial regions of leucocytosis (PL 1, Fig. 4). Approximate counts were made over thirty con¬ secutive sections and it was estimated that, in the 200- to 300-µ length of uterus represented, there were about a thousand spermatozoa being carried through the tissue in this way. Fallopian Tube The results described here are complementary to those that have already been reported (Austin, 1959; Austin & Bishop, 1959). Additional cytological features

Downloaded from Bioscientifica.com at 09/25/2021 03:03:07PM via free access 154 C. R. Austin are presented in PL i, Fig. 6, which depicts sections passing horizontally through the mucosa, at the level of the nuclei, in a Fallopian tube of a horseshoe bat. Several sperm heads can be seen, mostly in association with mucosal-cell nuclei, and it is evident that the presence of the sperm head often appears to have some effect on the nearby nucleus. Some of the heads seemed to lie within small vesicles in the cytoplasm. Vesicles associated with sperm heads were also detected in rat and rabbit mucosa. These local effects seem likely to be because the sperm head occupied a position within the mucosal cell rather than between cells, and this is certainly consistent with the impression gained from the close study of many such heads. Sperm heads were far more commonly seen in the mucosa lining the crypts of the Fallopian tube, and the great majority of the cells that were presumed to contain the heads were relatively undifferentiated in form, lacking cilia or visible signs of mucus secretion. The frequency with which spermatozoa were found in the mucosa was never very high; actual counts were made in the horseshoe bat and the rat, and 138 and 51 examples, respectively, were found in a length of tube of about 0.25 mm. In the same regions, the number of mucosal cells was estimated to be of the order of 8000 to 10,000. The frequency was even lower in the other animals. It did not differ appreciably in uteri placed in fixative at different intervals of time after the death of the animal (30 sec, 5 min and 1 hr), and so the possibility that spermatozoon invasion was a post-mortem phenomenon can reasonably be excluded. Observations on the rats gave some information on the persistence in the mucosa of invading spermatozoa, for these animals had been killed at 6 hr (two rats), 18 hr (five rats), 42 hr (one rat), 68 hr (one rat) and 92 hr (one rat) after coitus. Instances were seen at about the same frequency at 6 hr and 18 hr, but were noticeably fewer at 42 hr, and none was seen at 68 hr and 92 hr. Disappearance may have been owing to dissolution of spermatozoa or to the normal loss of the enveloping cells. The occurrence of dissolution is favoured by the fact that some of the sperm heads seen in rat Fallopian tubes had clearly suffered whilst still recognizable, were altered in or change — they shape reduced in size, or had diminished staining affinity. In addition, in the pipistrelle bat that had an 8- to -cell embryo in the Fallopian tube, the mucosa pre¬ sented small objects that could have been sperm heads in various stages of dissolution. In no species was there any evidence that invading sperm heads become resolved into their constituent chromosomes or unite with mucosal-cell nuclei. DISCUSSION The data recorded here, and the reports of previous investigators already cited, show that, at least in some species, spermatozoa may persist in the uterine lumen until the time of implantation of the embryo or even later. Observations on the horseshoe bat indicate that dissolution of spermatozoa can occur in the uterine glands. In addition, it is well known that spermatozoa are likely to accompany eggs on their passage into the uterus: in the , attached to the surface of the , or, in the rabbit, embedded in the mucin layer. The cells of the embryonic are believed to be capable

Downloaded from Bioscientifica.com at 09/25/2021 03:03:07PM via free access Fate of Spermatozoa 155 of active phagocytosis (Huggett & Hammond, 1952), and there must therefore be good possibilities that entire spermatozoa, or their breakdown products, are normally taken up by the embryo, but the results of such absorption are at present entirely conjectural. The probable passage of sperm heads into the cytoplasm of the mucous- membrane cells, and the fact that the heads often appear to lie within small vesicles, strongly suggests that a form of phagocytosis is involved. Mucosal cells do not seem normally to be phagocytic, though they can be entered by lymphocytes (Trowell, 1958; Nilsson, 1958). Within the mucosal cells, the heads apparently undergo dissolution and there was no evidence of any form of union or syngamy between sperm nucleus and cell nucleus. This is consistent with the failure of Edwards & Sirlin (1959) to find any labelled mucosal-cell nuclei in the Fallopian tubes of mice inseminated at coitus with spermatozoa in which the deoxyribonucleic acid contained 14C. The incidence of sperm entry is very low, but if the cells entered are stem cells, which seems quite likely in view of their relatively undifferentiated form and their location in crypts, then appreciable areas of mucosa might eventually receive whatever influence spermatozoa can transmit under these circumstances. entry into the submucous uterine tissues is clearly rare it was Sperm — only seen with leucocytes acting as vectors and appeared to be associated with a pathological state of the organ. Nevertheless, in the instance encountered, there was no doubt that quite large numbers were implicated and this fact, together with certain other considerations now to be made, suggests that such an occurrence could have major significance. The submucous tissues of the human epididymis have been reported to have been invaded by spermatozoa in a number of cases (King, 1955), and it is believed that this is generally attributable to the effects of local infection upon the integrity of the epithelium. The exceptional guinea-pig uterus of the present series may well have been an example of the consequences of infection. The migration of sperm-laden leuco¬ cytes in this specimen is also strongly reminiscent of the retreat into the maternal tissues of leucocytes carrying fragments of dead foetuses, as described by Henderson (1954). Henderson traced the laden leucocytes to the subserous lymphoid tissue and it is probable that particulate antigen was thus brought into relation with the sites of antibody formation. It is reasonable to suppose that spermatozoa, too, can be carried to these sites. Infection, or simply inflam¬ mation, is known to augment the response to antigens (Boyd, 1956). Con¬ sequently, the existence of a pathological state in the genital tract might not only permit spermatozoa to enter submucous tissues and so greatly improve their chances of exerting antigenic influence, but would also increase the intensity of the resulting antibody response. In this way, a single coitus could conceivably affect subsequent fertility, if at the time of mating the female tract is in an infected or damaged state.

ACKNOWLEDGMENTS I should like to acknowledge my gratitude to Dr Ruth Deanesly for making available to me her collection of histological preparations from specimens of the common pipistrelle bat, the hedgehog, the mole and the stoat; the uterus

Downloaded from Bioscientifica.com at 09/25/2021 03:03:07PM via free access 156 C. R. Austin of the guinea-pig killed on the 5th day of pregnancy was also kindly provided by her. I am indebted to Mr Michael Blackmore for collecting the greater horseshoe bats, and to Mr M. R. Young for preparing the photographic enlargements.

REFERENCES Austin, C. R. (ig57) Fate of spermatozoa in the uterus of the mouse and rat. J. Endocrin. 14, 335. Austin, C. R. (igsg) Entry of spermatozoa into the Fallopian-tube mucosa. Nature, Lond. 183, go8. Austin, C. R. & Bishop, M. W. . (ig5g) Presence of spermatozoa in the uterine-tube mucosa of bats. J. Endocrin. 18, viii. Boyd, W. C. (1956) Fundamentals of immunology, 3rd edn. Interscience Publishers, New York. Deanesly, R. (ig34) The reproductive processes of certain mammals. Part VI. The reproductive cycle of the female hedgehog. Phil. Trans., B, 223, 23g. Deanesly, R. (ig35) The reproductive processes of certain mammals. Part IX. Growth and reproduc¬ tion in the stoat (Mustela erminea). Phil. Trans., B, 225, 45g. Edwards, R. G. & Sirlin, J. (195g) Fate of spermatozoa penetrating into the tissues of the Fallopian tube. Nature, Lond. 183, 1744. Hartman, C. G. (1939) Ovulation, fertilization and the transport and viability of eggs and spermatozoa. Sex and Internal Secretions, 2nd edn. Ed. E. Allen. Baillierè, Tindall & Cox, London. Henderson, M. (ig54) Foetal regression in rabbits; experimental study of histolysis and phagocytosis. Proc. roy Soc, B, 142, 88. Hoehner, O. & Behne, K. (1914) Über die Lebensdauer homologer und heterologer Spermatozoen im weiblichen Genitalapparat und in der Bauchhöhle. Zbl. Gynäk. 38, 5. Huggett, A. St. G. & Hammond, J. (1952) Physiology of the placenta. Marshall's Physiology of Reproduc¬ tion, 2nd edn, vol. 2, p. 238. Ed. A. S. Parkes. Longmans, Green & Co., London. King, E. S. J. (ig55) Spermatozoal invasion of the epididymis. j1. Path. Bad. 70, 45g. Kohlbrugge, J. H. F. (igio) Der Einfluss der Spermatozoïden auf den Uterus, . Morph. Anthr. ia, 359· Kohlbrugge, J. H. F. (igi3) Die Verbreitung der Spermatozoïden im weiblichen Körper und im befruchteten Ei. Arch. EntwMech. Org. 35, 165. Marshall, F. H. A. & Jolly, W. A. (1905) Contributions to the physiology of mammalian reproduc¬ tion. I. The oestrus cycle in the dog. Phil. Trans., B, 198, gg. Metschnikoff, E. (i8gg) Étude sur la résorption des cellules. Ann. Inst. Pasteur, 13, 737. Nilsson, O. (ig58) Electronmicroscopy of the Fallopian tube epithelium of rabbits in oestrus. Exp. Cell Res. 14, 341. Trowell, O. A. (ig58) The lymphocyte. Int. Rev. Cytol. 7, 235. Vojtiskova, M. ( 1956) The question of the participation of non-fertilizing sperms in the sexual process. Folia biol. 2, 239.

/ DESCRIPTION OF PLATE 1

Figs, i and 2. Cross-sectional views of uterine glands in the greater horseshoe bat. In Fig. 1, most of the sperm heads are unaltered in size, whereas in Fig. 2 there are numerous small objects that could be sperm heads at different degrees of reduction in size. X 1000. Figs. 3 and 4. Sections of the uterus of a guinea-pig. In Fig. 3, one of the glands is filled with leucocytes, which also exist in large numbers in the surrounding tissue. X 270. In Fig. 4, a small part of the interglandular tissue is shown, in which there are many leucocytes; sperm heads can be seen in three cells. X 1300. Fig. 5. Cross-sectional view of part of a uterine gland in a guinea-pig, showing a sperm head within the mucosa, 1300. Fig. 6. Views of tangential sections at nuclear level through the mucous-membrane lining of crypts in the Fallopian tube of a greater horseshoe bat. Several sperm heads can be seen, mostly in transverse section. X 1300.

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(Facing p. 156)

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