SPERMATOZOA IN THE FEMALE GENITAL TRACT

Transcription

1 SPERMATOZOA IN THE FEMALE GENITAL TRACT D. G. EDGAR and S. A. ASDELL Department of Animal Husbandry, Cornell University, Ithaca, New York, U.S.A. (Received 18 June 1960) SUMMARY Ram spermatozoa were found at the ovarian end of the uterine (Fallopian) tubes of ewes at about 3 hr after mating. No radiopaque medium passed from the vagina to the uterine horns ofrabbits following vulval stimulation and copulation. Ram spermatozoa entered the tubes of ewes through a valve-like utero-tubal junction which prevented fluid from passing in the opposite direction. Almost all ram spermatozoa became immotile within 8 hr of their introduction into the tubes of ewes. The method of sperm transport in the female genital tract is discussed. INTRODUCTION Previous estimates of the time taken for spermatozoa to reach the ovarian end of the uterine (Fallopian) tubes of the ewe after mating have varied from 6 min to nearly 9hr (Table 1). Dauzier( 1958) was apparently convinced that at least 8 hr were necessary in the ewe and cow, and attributed the reports of more rapid ascent to faulty technique. Some observations are reported here which indicate that spermatozoa do in fact traverse the genital tract of the ewe in considerably less than 8 hr. Table 1. Previous reports of the time after mating when spermatozoa were found in ovarian end of uterine tubes of ewe Workers Quinlan, Mare & Roux (1932) Green & Winters (1935) Kelley (1937) Phillips & Andrews (1937) Lopyrin & Loginova (1939) Schott & Phillips (1941) Starke (1949) Dauzier (1958) Time after mating 5 In- 5 hr 5 hr 30 min 8 hr 40 min 20 min 6 min 8 hr When considering the relative importance of uterine and sperm motility for sperm transport, the question whether spermatozoa are accompanied by seminal fluid from the vagina into the uterus has significance. In some animals such as the pig and horse, whole semen apparently passes into the uterus (Mann, Polge & Rowson, 1956). The situation is less clear in the rabbit, ewe and cow. Krehbiel & Carstens (1939) found that stimulation of the vulva resulted in rapid transfer of radiopaque medium * Permanent address : Ruakura Animal Research Station, Hamilton, New Zealand. Appointment as Visiting Fellow at Cornell University supported by the International Cooperation Administration under the Visiting Research Scientists Program administered by the National Academy of Sciences of the U.S.A.

2 322 D. G. Edgar and S. A. Asdell from the vagina into the uteri of four of seven rabbits, but Noyes, Adams & Walton (1958) were unable to confirm these results. Some findings in rabbits are here described which agree with those of the latter workers. After the cervix the next important barrier to sperm progress appears to be the utero-tubal junction. Edgar & Asdell (1960) demonstrated the valve-like action of this structure in the ewe at, and shortly after, oestrus, when the uterine tube com menced to distend with fluid within 1 hr of the ligation of its ovarian end. This paper shows that in the ewe spermatozoa pass from the uterus through the utero-tubal junction into a tube that has been ligated at its ovarian end, although fluid does not pass in the opposite direction. Some observations are also reported on the survival of motility of ram spermatozoa introduced into the tubes of ewes and the method of sperm transport in the female genital tract is discussed. MATERIALS AND METHODS Western, Southdown, Shropshire, Hampshire and Corriedale ewes, all of which had lambed at least once, were used in the sheep experiments. Rate of sperm travel in reproductive tract of ewe After they had been detected in oestrus by an aproned ram, each of four ewes was allowed to mate and the time was noted. A true service was confirmed by the with drawal of some semen by pipette from the vagina. At laparotomy under local anaesthesia (2 % Procaine), the right uterine tube was removed and shortly after wards the ewe was killed and the left tube removed (Table 2). The ovarian third of each tube was flushed with warm saline within minutes of removal, and the flushings immediately examined for spermatozoa by phase-contrast microscopy at magnifications of 40 and 430. Table 2. Detection of spermatozoa in the tubes offour ewes Ovarian Presence No. of third of of ewe uterine tube Time after mating spermatozoa 15 R 55 min 0 L 2 hr 35 min + 16 R 1 hr 30 min 0 L 2 hr 30 min 0 18 R 2 hr 15 min 0 L 3 hr 15 min + 19 R 2 hr 15 min 0 L 3 hr 20 min + New glassware was used throughout and the study was made in a laboratory not previously used for work with semen so that the possibility of contamination with other sperm was reduced. Movement of radiopaque medium introduced into vagina of rabbit does From 2 to 5 ml. of radiopaque medium were introduced, by means of a glass inseminating tube, into the vagina of each of twelve New Zealand White rabbit does.

3 Spermatozoa in the female genital tract 323 Three radiopaque media were tested, Thorotrast (25% colloidal thorium dioxide; Testagar and Co., Detroit, Michigan, U.S.A.), Medopaque H (35% sodium orthoiodohippurate; Bell-Craig Inc., New York, New York, U.S.A.) and Iodochlorol (27% iodine and 7-5% chlorine in organic combination; Searle and Co., Chicago, Illinois, U.S.A.), each in four rabbits. The vulva of each female was stimulated with the finger for about 1 min, and the doe then allowed to mate with a buck. Just before, and at 2, 10 and 30 min after mating, a dorso-ventral radiograph was made of the posterior half of each doe. Passage of spermatozoa through utero-tubal junction of ewes Five ewes were detected in oestrus by an aproned ram, and one uterine tube of each ewe was then ligated at the ovarian end. The necessary laparotomy was per formed under 'Nembutal' anaesthesia in ewe no. 31, and under local anaesthesia (2 % Procaine) in ewes nos. 32, 33, 34 and 36. In the case of a sixth ewe, no. 38, the operation was carried out, under Nembutal anaesthesia, between oestrous periods. Table 3. Passage of spermatozoa through utero-tubal junction into the distended ligated, and non-ligated tubes of ewes Time of Time of examination mating after of tubes Presence No. of ewe ligation after mating (hr) of spermatozoa 31 4hr 6 L. 0 N.L hr 3 L. 0 NX hr 16 L. 0 NX hr 17 L. + N.L hr 15 L. 0 N.L days 5 L. + NX. -. = Ligated tube; N.L. = non-ligated tube. At various intervals (Table 3), long enough after the operation for the ligated tube to have commenced to distend, the ewes were allowed to mate. Both tubes were later removed, flushed immediately with warm saline, and the flushings examined by phase-contrast microscopy for the presence of spermatozoa. Survival of motility of ram spermatozoa in the tubes of ewes Semen samples were collected from rams by rectal electrical stimulation (Edgar, Inkster & MacDiarmid, 1956), and their density and motility subjectively assessed (Edgar, 1959) within 1 hr of collection. Approx ml. semen was introduced by microsyringe into a uterine tube via its abdominal opening, at laparotomy, in each of six oestrous ewes (Table 4). In a seventh instance, a semen sample was halved. One half was washed twice and then suspended in Ringer's solution containing fruc tose (Mann, 1954) and 500 u. penicillin and 500 µg dihydrostreptomycin/ml. The other half was centrifuged and resuspended in its seminal fluid. Approx ml. of the first suspension was introduced into one of the uterine tubes of ewe no. 39

4 324 D. G. Edgar and S. A. Asdell and the same amount of the second suspension was placed in the other tube. The numbers of spermatozoa introduced into each tube ranged approximately from 50 to 150 million. In all except ewe no. 23, the remainder of the untreated and treated semen was kept in vitro at 38 C and examined for motility at the same time as that recovered from the tubes. Table 4. Survival of motility of ram spermatozoa in the tubes of ewes Percentage Time in Percentage motile No. of ewe Material in tube motile tube (hr) in tube 26 Untreated semen Untreated semen Untreated semen Untreated semen 80 8 < 5 39 Resuspended spermatozoa 80 7J < 5 Washed spermatozoa + antibiotic 80 7i < 5 24 Untreated semen 40 4J 0* 23 Untreated semen * < 5 % motile in vitro. RESULTS AND DISCUSSION Rate of sperm travel in reproductive tract of ewe In three of the four experimental ewes, spermatozoa were found in the ovarian third of the second but not in the first uterine tube examined, the times after mating ranging from 2 hr 35 min to 3 hr 20 min. Sperm were found in neither tube of the fourth ewe, the examinations being made at 1 hr 30 min and 2 hr 30 min (Table 2). It appears therefore, that ram spermatozoa can reach the site of fertilization in the ewe in much less than the 8 hr considered minimal by Dauzier (1958). The rate of passage may vary considerably in different individuals or at different times in the same individual, and it seems that, at least in some cases, only minutes may be required (Table 1). Failure to detect the few sperm which actually reach the site may account for some of the discrepancies in the reported results (Table 1). Movement of radiopaque medium introduced into vagina of rabbit does In none of the twelve does tested did radiopaque material enter the uterine horns in response to vulval stimulation and/or copulation. These findings support those of Noyes et al. (1958) who were unable to repeat the results of Krehbiel & Carstens (1939) in rabbits. The latter workers spoke of vulval erection, but Noyes et al. and the present writers observed only momentary eversión of the vulva following digital stimulation, which suggests that there may be some variation in the endocrine state of the does. Ovulations following copulation were taken as evidence for a normal reproductive state in our rabbits. Walton (1930) obtained passage of neither dyes nor particles from the vagina to the uterine horns of rabbit does following copulation. Our radiographs confirm that the medium was apparently forced against each external os uteri by vaginal contractions and held in the vagina by a sphincter just cranial to the urethral orifice, as described by Krehbiel & Carstens and Noyes et al. This mechanism may normally assist in the passage of spermatozoa up the female reproductive tract.

5 Spermatozoa in the female genital tract 325 Passage of sperm through utero-tubal junction of ewes Although the valve-like utero-tubal junction would not permit fluid under pressure to pass from the uterine tubes into the uterus, spermatozoa were able, in two of the six ewes tested, to pass in the opposite direction. Spermatozoa were found neither in the ligated nor the non-ligated tubes of the remaining four ewes (Table 3). The mechanism whereby the sperm penetrated the utero-tubal junction is unknown, but Leonard & Perlman (1949), in rats, have shown the necessity for sperm motility for this purpose. Survival of motility of ram sperm in the tubes of ewes The semen samples contained varying proportions of motile sperm, but the motile spermatozoa in all samples were reasonably active when they were introduced into the uterine tubes of the experimental ewes. Many of the spermatozoa lost their motility after as little as 3^ hr, and nearly all were immotile after about 8 hr. Sperm motility had disappeared completely in three ewes examined after 16 or 24 hr (Table 4). Although none of the spermatozoa introduced into ewe no. 24 remained motile after 4J hr, a few from the same semen sample showed some motility after the same period in vitro at 38 C. AU semen samples incubated in vitro for longer periods showed no sperm motility, a result that was probably due to the accumulation of metabolites, as evidenced by a ph < 6. The removal of seminal fluid and the addition of antibiotics did not increase the proportion of spermatozoa which remained motile after 7\ hr in a uterine tube of ewe no. 39. Sperm motility in the female reproductive tract is probably adversely affected by some factor in addition to temperature, and other than lowered ph. Quinlan, Mare & Roux (1933) in sheep, and Chang (personal communication) in rabbits, also found that few spermatozoa remain motile after a few hours in the female genital tract. Many spermatozoa were observed to be disintegrating after only a few hours in the uterine tubes, some were adherent to mucosal strips flushed from the tubes, and some were surrounded by material which staining with periodic acid-schiff showed to be mucoid in nature. Hadek (1955) has shown that an acid mucopolysaccharide is present in the lumen of the tube of the ewe and that it is most abundant at the time of ovulation. This material probably assists in the breakdown of the spermatozoa. In one ewe in which a 20 in. length of polyethylene tubing had been ligated into the ovarian end of the tube, two spermatozoa were found in the lumen of the polyethylene tubing, indicating that a few spermatozoa probably escape into the abdominal cavity under normal conditions. Method of sperm transport in female genital tract Three kinds of evidence point to the conclusion that spermatozoa are carried pas sively up the female genital tract the movement of radiopaque fluid (Krehbiel & Carstens, 1939; Rowson, 1955), the rapidity with which spermatozoa traverse the tract compared with their slower rate of travel in vitro (Parker, 1931; Phillips & Andrews, 1937) although they may possibly propel themselves more rapidly in vivo, and the fact that non-motile spermatozoa can be transported up the tract (Van Demark & Moeller, 1951).

6 326 D. G. Edgar and S. A. Asdell However, Noyes et al. (1958) showed that motility was necessary for spermatozoa to pass the cervical barrier in rabbits and Leonard & Perlman (1949) demonstrated that only motile spermatozoa passed through the utero-tubal junction in rats. These findings and those of Braden (1953) in rabbits, and Quinlan et al. (1933), Warbritton, McKenzie, Berliner & Andrews (1937) and Dauzier (1958) in sheep, that the cervix and utero-tubal junction are passed only by a small proportion of the spermatozoa which reach them, that very few arrive at the ovarian end ofthe uterine tube, and that sperm progression continues over a period of several hours, all suggest that sperm motility usually plays an essential role in sperm transport in the female genital tract. The distension of the uterine tube after ligation of its ovarian end, at and shortly after oestrus (Edgar & Asdell, 1960), means that tubai fluids normally flow into the abdominal cavity during that time. This may aid the passage of spermatozoa after they have passed the utero-tubal junction. Under some conditions, as, for instance, when uterine muscular activity is stimulated by mating (Van Demark & Hays, 1952), the muscular activity of the female genital tract may be such as to carry a few live and/or dead sperm rapidly to the site of fertilization, and probably most sperm owe much of their slower progress along the tract to its subsequently reduced muscular activity. However, to penetrate ova sperm must apparently possess motility (Blandau & Odor, 1952), and it seems probable that most of them would not have passed the cervical and/or utero-tubal barriers without this capacity. Gratitude is expressed to Drs D. L. Black, R. W. Dougherty, R. H. Foote, D. E. Hogue, K. McEntee and K. L. Turk for helpful discussion and facilities. REFERENCES Blandau, R. J. & Odor, D. L. (1952). Fértil, db Steril. 3, 13. Braden, A. W. H. (1953). Aust. J. biol. Sci. 6, 693. Dauzier, L. (1958). Published Doctoral Thesis, University of Paris. Edgar, D. G. (1959). N.Z. Vet. J. 7, 61. Edgar, D. G. & Asdell, S. A. (1960). J. Endocrin. 21, 315. Edgar, D. G., Inkster, I. J. & MacDiarmid, H. J. (1956). N.Z. Vet. J. 4, 20. Green, W. W. & Winters, L. M. (1935). Anat. Ree. 61, 457. Hadek, R. (1955). Anat. Ree. 121, 187. Kelley, R. B. (1937). Bull. Coun. Sci. Indust. Res. Austr. no Krehbiel, R. H. & Carstens, S. C. P. (1939). Amer. J. Physiol. 125, 571. Leonard, S. L. & Perlman, P. L. (1949). Anat. Ree. 104, 89. Lopyrin, A. I. & Loginova, N. V. (1939). Sovetsk. Zoo-tech. 3, 144 (cited by Dauzier, 1958). Mann, T. (1954). The biochemistry of semen. London: Methuen. Mann, T., Polge, C. & Rowson, L. E. A. (1956). J. Endocrin. 13, 133. Noyes, R. H., Adams, C. E. & Walton, A. (1958). Fértil, éc Steril. 9, 288. Parker, G. H. (1931). Phil. Trans., 219, 381. Phillips, R. W. & Andrews, F.. (1937). Anat. Ree. 68, 127. Quinlan, J., Mare, G. S. & Roux, L. L. (1932). XVIlIth Rep. Dir. Vet. Ser. Anim. Industr. Union of South Africa, p Quinlan, J., Mare, G. S. & Roux, L. L. (1933). Onderstepoort J. vet. Sci. 1, 136. Rowson, L. E. A. (1955). Brit. Vet. J. 111, 334. Schott, R. G. & Phillips, R. W. (1941). Anat. Ree. 79, 531. Starke, N. C. (1949). Onderstepoort J. vet. Sci. 22, 415. Van Demark, N. L. & Hays, R. L. (1952). Amer. J. Physiol. 170, 518. Van Demark, N. L. & Moeller, A. N. (1951). Amer. J. Physiol. 165, 674. Walton, A. (1930). J. Obstet. Qynaec. Brit. Emp. 37, 92. Warbritton, V., McKenzie, F. F., Berliner, V. & Andrews, F.. (1937). Proc. Amer. Soc. Anim. Prod. 30, 142.