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The role of the human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy*
Vol. 46, No.2, August 1986 Printed in U.SA.
FERTILITY AND STERILITY Copyright " 1986 The American Fertility Society
The role of the human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy*
Robert J. Schoysman, M.D.t J. Michael Bedford, Vet.M.B., Ph.D.:j:§ Free University of Brussels, Brussels, Belgium, and Cornell University Medical College, New York, New York
Epididymovasostomy has been used for examination of the role of particular regions of the epididymis in sperm maturation and storage, as reflected in fertility and in the motility, structural, and surface character, and also the number of the spermatozoa ejaculated. Human spermatozoa need be exposed only to the environment in the caput before passing into the vas deferens, in order to complete their maturation. However, the chance of pregnancy appears greater where the anastomosis is lower. Among the sperm characteristics examined, only motility was affected by the level of anastomosis, and there was a trend to higher numbers where the anastomosis was established beyond the upper caput. The storage function of the cauda is discussed in light of the finding that epididymovasostomy patients sometimes ejaculate sperm numbers seen in normal men. Fertil Steril46:293, 1986
The milieu in the epididymis provides specific support for the events of sperm maturation, and conditions in the cauda that are dependent on androgen and the low temperature of the scrotum permit a storage there of mature spermatozoa. In the species examined critically, significant numbers of mature spermatozoa first appear in the . midcorpus or lower corpus epididymidis with the pattern perhaps skewed somewhat more toward the cauda for hamster and rat. 1 In no mammal is
Received September 17,1985; revised and accepted March 19,1986. *Supported by the Special Program in Human Reproduction, World Health Organization, Geneva, Switzerland. tDepartment of Obstetrics and Gynecology, Free University of Brussels. :j:Departments of Obstetrics and Gynecology, Cell Biology, and Anatomy. §Reprint requests: J. Michael Bedford, Vet.M.B., Ph.D., Department of Obstetrics and Gynecology, Cornell University Medical College, 525 East 68th Street, New York, New York 10021.
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there evidence that spermatozoa can become fertile if retained in the testis, but it is not clear yet for any species which epididymal regions are essential to the maturation process. In trying to establish this, the relatively crude approach of ligation at defined points along the epididymis suggests that the caput may be the only critical region in the rabbit.2, 3 The hamster appears less flexible in this respect, in that ligation in the corpus epididymidis does not markedly skew the fertility profile toward the caput region. 4 By comparison with common animal models, relatively little has been gleaned yet about the human epididymis and the extent of its contribution to sperm maturation. It is known that maturing human spermatozoa undergo most of the subcellular changes seen in animals, i.e., modifications in their motility, metabolism, surface character, and structural quality.5 In terms of function, Moore and co-workers6 have provided the best available clue as to the fertility profile with the use of the hamster egg as a surrogate. This suggests that the first spermatozoa to com-
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plete their maturation in significant numbers do so while in the corpus epididymidis. One approach to analysis of the role of specific epididymal regions lies in the bypassing of them by means of epididymovasostomy. That technique has recently been used in studies of the rabbit,7 the results of which seem similar to the findings of the older ligation studies. However, epididymovasostomy has been employed for many years by clinicians as a means of bypassing epididymal blockage sites. 8 -1O This offers a fairly good chance of creating a bypass between patent epididymis and the vas deferens; and, long before maturation patterns were emphasized by the work of reproductive biologists, surgeons intuitively have attempted to include the greatest portion of epididymis. In the .present investigation, ejaculates were obtained from a relatively large group of patients in which epididymovasostomy had been performed successfully at defined levels of the epididymis. It seemed possible that examination of specific sperm parameters known to change during epididymal maturation5 might indicate the limits of the segment that is essential for maturation, and which of the several cellular facets of maturation depend specifically on epididymal factors for their completion. MATERIALS AND METHODS
Patients were recruited from a population of 723 individuals subject to epididymovasostomy for relief of epididymal blockage (or agenesis in one case) between 1 and 10 years earlier. The fertility rate, known only for 565 of these patients, reached 18%. To perform the investigation we had to know the exact level of the anastomosis on the epididymis. That was recorded at surgery as the distance between the proximal border of the caput and the proximal border of the anastomosis site. The distance was measured in millimeters because the different regions l l cannot always be accurately recognized from the surface anatomy in such cases. The 117 patients finally included in the fertility data of this report were those who had 20 x 106/ml or more spermatozoa in at least one previous ejaculate after either unilateral anastomosis or bilateral anastomosis at a similar site on both epididymides. The anastomosis between the epididymis and vas deferens was laterolateral. 12 The upper border was essentially coincident with the tubule that showed con294
tinuous sperm leakage after exploratory incision above the region of blockage. For sperm analysis, one ejaculate was obtained by masturbation from each of 35 of the 117 patients chosen on the basis of availability. These samples were subject to analysis within about 2 hours of collection. When delivered, the ejaculate was kept at 37°C, and the concentration and total number of spermatozoa were estimated with a hemocytometer. The other parameters studied were (1) the structural quality and free thiol status of the sperm nucleus and sperm tail; (2) the character of the sperm surface; and (3) swimming behavior (motility). STRUCTURAL QUALITY
The normal structural stability of spermatozoa during epididymal passage can be measured visually according to the swelling-disruption response to sodium dodecyl sulfate (SDS) with or without dithiothreitol (DTT), which cleaves disulfide bonds. 13 The latter determine the keratinQid stability typical of the sperm head and tail in all eutherian mammals, including man. To evaluate the structural quality of spermatozoa ejaculated by epididymovasostomy patients, an aliquot was assessed according to the head swelling response to either 1% SDS alone or to 1% SDS in borate buffer containing 2 x 10 - 3 M DTT p. 14 This reaction was stopped after a 15-minute exposure to SDS/DTT or after 60 minutes in SDS alone, by addition of an equal volume of 2.5% glutaraldehyde. The spermatozoa were assessed (> 200 sample) in successive fields in the phase-contrast microscope according to the degree of nuclear decondensation. The heads were classified as unchanged, moderately swollen, or grossly swollen. 14 Parallel observations were made on aliquots taken from ejaculates of fertile men. The assessments were subjective but always were made by one operator (J. M. B.). Because the changing stability of the sperm chromatin with epididymal passage appears to reflect the extent of - S - S - cross-linking of protein-bound free thiols,13. 15 the free thiol content of spermatozoa in aliquots from epididymovasostomy ejaculates was also examined by a direct chemical method. This depends on the specific uptake of 14C_iodoacetamide by the spermatozoa under given conditions. 15 This was done in two ways with samples from groups containing 9 and 13 patients, respe~ively. In the first, one aliquot
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Fertility and Sterility
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from each was frozen at - 80°C. Later, the sample was thawed and homogenized by sonication. After washing twice in isotonic saline, the sperm pellet was dissociated gently and the standard assay was carried out in a volume of 0.5 ml. 15 In view of initial results that showed no significant difference between epididymovasostomy and normal samples in this respect, the second group was included to control for the possibility that spontaneous oxidative crosslinking might occur in immature spermatozoa soon after ejaculation. Therefore, ejaculates collected were immediately counted, and an aliquot of some 30 x 106 spermatozoa was withdrawn and stored at 4°C. The aliquot was washed by double centrifugation in isotonic saline; and, after slight homogenization of the pellet with a stirring rod, 1ml of 14C_ iodoacetamide was mixed with it. The mixture was incubated at room temperature for 18 to 20 hours, then frozen in liquid nitrogen until assayed by counting of samples coming from patients with high or low anastomosis.1 5 SPERM SURFACE
Two techniques were used to evaluate the sperm surface. The first involved the binding pattern offluoresceinated concanavalin A (FITC-Con A) to spermatozoa. In these cases, sperm aliquots were first washed twice in Tyrode's solution and then fixed in 4% paraformaldehyde before staining and evaluation with epifluorescence optics. The other method for evaluation of the surface was chosen in light of obvious change in the binding pattern of positively charged colloidal ferric (Fe + + +) oxide particles, demonstrated during normal epididymal maturation of human spermaVol. 46, No.2, August 1986
Motility was evaluated subjectively in a phasecontrast microscope with the use of shallow well slides such that sperm movement was not restricted through frictional interaction with the glass. Motility was scored on the basis of the percentage of spermatozoa in at least ten fields moving vigorously and progressively. This assessment was made by J. M. B. within 2 hours of the collection of the semen samples.
RESULTS The present results suggest that the pattern of development of the ability to fertilize in the epididymal sperm population can be relatively flexible in man. Nonetheless, there is a greater chance for pregnancy where the anastomosis is made in the corpus, rather than at the level of the caput epididymidis.Figure 1 reveals occasional pregnancies in cases where an anastomosis had been made 8 mm or less from the proximal border of the caput, but there was a general absence of fertility in that group. Pregnancies were common, on the other hand, when the anastomosis site was > 10 mm from the proximal border in men having > 20 x 106 spermatozoa in a previous ejaculate. In examining ejaculated spermatozoa in terms of individual cellular parameters that change during passage of spermatozoa through the normal epididymis, only motility could be discerned to have a relationship to the level of anastomosis. Most men whose anastomosis was located in the proximal 8 mm ejaculated spermatozoa which were essentially immotile. By contrast, in all cases in which the anastomosis was > 10 mm from the caput border, some 20% to 90% of the spermatozoa were progressively motile (Fig. 2). Among the other sperm parameters studied, none appeared "immature," regardless of the level of the anastomosis. Spermatozoa taken from the caput of the normal human epididymis essentially fail to bind Fe + + + colloid at pH 1.8, in contrast to those of caudal spermatozoa. 5 However, there was no visible difference here in the pattern of binding of cationized Fe + + + colloid to
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the surface of spermatozoa from control and epididymovasostomy samples, regardless of the level of anastomosis. The Con-A binding patterns also showed no correlation between the level of anastomosis and a particular distribution of surface fluorescence on paraformaldehyde-fixed spermatozoa. Control Con-A-treated spermatozoa fluoresced moderately and uniformly, except for the region of the cytoplasmic droplet, which displayed a more intense fluorescence. Con-A-treated epididymovasostomy samples fluoresced with a pattern similar to that of the controls, irrespective of the level of anastomosis. It should be noted that the patterns observed were not similar to those localized by immunoperoxidase techniques. 16 According to the swelling response to SDS or SDS/DTT, there was no significant difference in the level of free thiol ( - SH) groups in ejaculated spermatozoa from normal controls and those from epididymovasostomy patients as a function of the level of anastomosis. The anastomosis patients as a group did not react differently from the controls, which themselves varied from individual to individual, as noted previously. The percentage of patients' spermatozoa that displayed detectable swelling in response to SDS alone varied from 4% to 22% (mean, 13.8%), but no pattern was seen according to the anastomosis level, and the mean did not differ significantly from that for control samples studied under the same conditions. Simi296
larly, spermatozoa ejaculated by patients with a high « 10 mm) anastomosis did not swell more readily in the face of SDSIDTT for 15 minutes than those from low anastomosis cases. The lightmicroscopic impression that control and all epididymovasostomy patients' spermatozoa had a similar - SH status was borne out by the experiments performed for evaluation of the free - SH content of spermatozoa as reflected in 14C-iodoacetamide incorporation. 15 As measured in 0.1ml aliquots of a sperm suspension, the samples from 22 epididymovasostomy patients studied displayed a mean specific activity of 2750 (range, 1471 to 5195) cpmll0 7 spermatozoa, compared with a mean of 3246 cpm/10 7 spermatozoa in parallel controls. Again, there was considerable variation among the ejaculates tested in both control and experimental groups; but, in the latter, this was in no way a reflection of the level of the anastomosis. In Figure 3, total numbers in the ejaculate are plotted for the unilateral and bilateral situation as a function of the level of the anastomosis. The lowest level is designated in the case of patients with bilateral anastomoses. Although it is difficult to draw firm conclusions, the numbers obtained suggest that fewer spermatozoa tend to be produced where high blockages are reversed. Attention is drawn to the fact that several patients, including some with a unilateral anastomosis, had numbers ranging from 160 to 535 x 106 in a single ejaculate. Because the cauda epididymidis is bypassed, this is of interest in light of the fact that these totals compare with the numbers produced in a first ejaculate by normal men with no malfunction of the reproductive tract.
DISCUSSION
The results of this study support the general conclusion that in man, as in animals, the epididymis makes some critical contribution without which spermatozoa cannot develop the ability to fertilize. They also indicate that while much of the human epididymis can be dispensed with, the chance for pregnancy to occur is generally greater if the anastomosis is made low, in the corpus epididymidis, rather than high, in the caput region. The exact reason for this is not clear, because the percentage of the spermatozoa that are fully fertile in ejaculates produced after anastomosis at any level is not known. However, Figure 2 sug-
Schoysman and Bedford Epididymovasostomy and sperm function
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. gests that the percentage motility at least could be an important element. Precise interpretation of epididymovasostomy results is complicated to some degree by several factors. Where bilateral anastomoses are attempted, one cannot be sure whether both sides become patent and the number ejaculated represents the output of both testes. With side-to-side anastomosis, the level of the connecting cut epididymal tubule essentially reflects the upper boundary of the anastomosis site; but,in reality, some levels given in the tables here could err perhaps by 1 to 2 mm. Neither is a failure to establish· a pregnancy, seen variously in Figures 1 and 2, a sure indicator that the ejaculate studied does not contain fertile spermatozoa. On the other hand, despite these caveats, epididymovasostomy performed for purely clinical reasons clearly can provide valuable pointers as to the role of the human. epididymis in sperm maturation and storage. Although better overall fertility results are obtained with low anastomoses, Figure 1 and other studies 7, 17 show that some anastomoses situated within the first 8 mm of the duct can result in ejaculated spermatozoa able to establish a pregnancy. Thus, in evaluating the specific contribution of the epididymis, one can conclude that any essential dependence of spermatozoa on the milieu of the epididymis for maturation appears minimal beyond the caput region, specifically region 2.11 We presume that the majority of high anastomosis cases with poor motility and no fertility Vol. 46, No.2, August 1986
involved insufficient exposure of spermatozoa to the epididymal milieu before their passage into the vas deferens. However, in interpreting the consequences of epididymovasostomy, it should be appreciated that the human epididymis differs somewhat from that of many animal models. The vasa efferentia in man occupy the major part of what appears grossly as caput epididymidisl l ; and it is possible that a direct link between vasa efferentia and vas deferentia may have been established in some very high anastomosis patients here. One might anticipate an extremely poor quality of spermatozoa ejaculated in such cases. A relative flexibility of the functional relationship between spermatozoa and much of the human epididymis is implied in the fact that, compared with control spermatozoa, only motility was detectably different among the parameters tested as a function of the level of anastomosis. It is perhaps not surprising that the free - SH, and so structural status, of the epididymovasostomy samples appeared no different from that in controls, regardless of the level of anastomosis. Although the epididymal environment may have some modulating effect on this aspect of maturation,13 oxidation of - SH to - S - S - bonds proceeds readily even in vitro, in nonspecificconditions. Moreover, human sperm heads have a significantly lower cysteine content than those of other mammals. 18 With regard to the sperm surface, change in its membrane glycoprotein complement is a recognized special feature of epididymal maturation in a· number of mammals, and this aspect of the sperm surface character is believed to bear at least on the sperm's potential for capacitation and its ability to bind to the egg. Although comparable surface changes probably occur in human spermatozoa, as reflected in Fe + + + colloid bindin~ and the fluorescent expression of epididymal antigen,19 no consistent departure from controls was observed here in the FITC-Con A and Fe + + + binding patterns as a function of the level of anastomosis. However, these techniques will not detect all surface change, and the possibility that they are not specific enough is suggested by a case,20 reported after our observations were made, in which immotile spermatozoa and seminal plasma ejaculated by a high epididymovasostomy patient lacked a 38,000 molecular weight protein found on normal spermatozoa and in normal seminal plasma. Thus, the absence here of a correlation between the binding of such surface markers and
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the anastomosis level should perhaps be viewed with some reservation. Nonetheless, the fact that several pregnancies were established in this and another study17 when the lowest anastomosis was sited within 1 cm of the proximal border indicates that any essential contribution to the sperm surface can be acquired by at least some spermatozoa entirely in the caput epididymidis. With regard to motility, a considerable improvement has been reported during the first two postoperative years in a small series of high endto-end anastomosis patients,i 7 and this has raised some question as to the essential place of the human epididymis in maturation of the motility function. While the epididymis normally has a low rate of mitosis in the adult, the suggestion17 that a compensatory growth of the caput tubule could occur in such cases seems worthy of investigation. Here, samples from a few patients in the high anastomosis group also contained some spermatozoa with good progressive motility. However, in many more (and in the case described by Wong et al. 20) motility was persistently absent or very poor in spermatozoa ejaculated by the high « 10 mm) anastomosis group over a period of 2 years or longer (Fig. 2). In the rabbit, also, anastomosis high in the caput resulted in a persistent production of similarly poor quality spermatozoa. 7 The fact that a strong trend to better motility has been seen rather consistently where spermatozoa are able to pass through a longer section of epididymal duct reinforces5 the general view that the epididymis exerts some essential and specific control over the. functional maturation of the axoneme. As yet, the factors involved in motility maturation are still a matter for conjecture. 21 It must be appreciated that spermatozoa in the normal male do not yet possess the capacity for optimal motility if released from the very restricted proximal regions they can experience in the fertile high epididymovasostomy patients. Spermatozoa express this capacity in significant numbers only at a more distal point reached perhaps 48 to 72 hours or more after entering the epididymis. 5 A clear case in point is illustrated by micropuncture samples taken from the normal ram epididymis, the motility of which did not improve significantly until the proximal corpus region was reached. 22 Taken together with such results, the observations from epididymovasostomy cases suggest that while certain motility enhancement factors are provided in the proximal region of the 298
epididymis, more time than the short period spermatozoa stay in the caput is required for their effects to be expressed. In the case of "high" epididymovasostomy patients, this expression may well occur only after spermatozoa pass into the vas deferens. Finally, the present results raise a question that concerns sperm numbers. The data in Figure 3 show that a high anastomosis was more often followed by very low numbers in the ejaculate, though the reasons for this are not clear. On the other hand, in agreement with Silber,17 some men with high epididymal blockage in this series did produce a surprisingly high number even where only a unilateral patency was established. Moreover, "normal" numbers (160 to 535 x 106 ) were present in the ejaculates of several of our epididymovasostomy patients, as in other cases reported. 17 This raises a question as to the significance of the storage function of the cauda epididymidis, because the present results imply that the vas deferens alone can accommodate all the spermatozoa produced in a normal ejaculate. This appears to be true also for rabbits. 7 The issue of the role of the cauda epididymidis may be masked somewhat by the fact that the human caudal sperm population normally is relatively small. 23 Because body temperature reduces the storage capacity of the scrotal cauda,24 it is possible that its modest size in man partly reflects some suppressive effect of a higher scrotal temperature induced by clothing. However, regardless of species variation, in considering the fundamental significance of the cauda epididymidis, it should be realized that a sexually active male mammal may be required to deliver several sequential ejaculates within a short period in maximizing his fertility potential. This can involve the consecutive insemination of several females in polygynous species, or repeated insemination of the same female in the case of some rodents, felines, wolves, etc. 25 While the cauda epididymidis may not necessarily be of primary importance in the delivery of a first ejaculate, it is probably critical for the ability to deliver repeated ejaculates of high fertility within a limited time period.
Acknowledgments. We thank Harold I. Calvin, Ph.D., and Andrea Schoysman, M.D., for their help in assessment of iodoacetamide uptake by spermatozoa.
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REFERENCES 1. Bedford JM: Maturation transport and fate of spermatozoa in the epididymis. In American Handbook of Physiology, Sect 7, Vol 5, Edited by DW Hamilton, RO Greep. American Physiological Society, Washington, D.C., 1975, p 303 2. Bedford JM: Effect of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis. J Exp Zool 166:271, 1967 3. Orgebin-Crist MC: Studies on the function of the epididymis. BioI Reprod Suppl 1:155, 1969 4. Cummins JM: Effects of epididymal occlusion on sperm maturation in the hamster. J Exp ZooI197:187, 1976 5. Bedford JM, Calvin HI, Cooper GW: The maturation of spermatozoa in the human epididymis. J Reprod Fertil Suppl 18:199, 1973 6. Moore HDM, Hartman JD, Pryor JP: Fertilizing capacity of human spermatozoa. Int J Androl 6:310, 1983 7. Temple-Smith PD, Southwick G: Vasoepididymovasostomy in the rabbit: effects of level of anastomosis on semen quality and fertility. J Androl (Suppl) 6:30-P, 1985 8. Young DH: Surgical problems of the vas deferens. Proc Soc Study Fertil 3:40, 1951 9. Silber SJ: Microscopic vasoepididymovasostomy: specific microanastomosis to the epididymal tubule. Fertil Steril 30:565, 1978 10. Schoysman RJ: Microsurgery of the male genital tract. In Microsurgery in Infertility, Edited by E Cittadini, M Cognat, RJ Schoysman. Cofese, Palermo, 1981, p 83 11. Baumgarten HG, Holstein AF, Rosengren E: Arrangement ultrastructure and adrenergic innervation of smooth musculature of the ductuli efferentes, ductus epididymidis and ductus deferens in man. Z Zellforsch Mikrosk Anat 120:39, 1971 12. Schoysman RJ, Drouart JM: Progres recents dans la chirurgie de la sterilite masculine et feminine. Acta Clin Belg 71:261, 1972 13. Calvin HI, Bedford JM: Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil (Suppl) 13:65, 1971
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14. Bedford JM, Bent MJ, Calvin HI: Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J Reprod Fertil 33:19, 1973 15. Calvin HI, Yu CC, Bedford JM: Effects of epididymal maturation, zinc [II], copper [II] on the reactive sulfhydryl content of structural elements in rat spermatozoa. Exp Cell Res 81:333, 1973 16. Scacciati de Cerezo JM, Bueno MP, Skowrowski B, Cerezo AS: Immunohistochemical localization of conconavalin A and wheat germ receptors in normal human spermatozoa. Am J Reprod Immunol 2:246, 1982 17. Silber SJ: Vasoepididymovasostomy to the head of the epididymis: recovery of normal spermatozoal motility. Fertil Steril 34:149, 1980 18. Calvin HI: Comparative analysis of the nuclear basic proteins in rats, human, guinea pig, mouse, and rabbit spermatozoa. Biochem Biophys Acta 434:377,1976 19. Tezon JG, Ramella E, Cameo MS, Vazquez MH, Blaquier J A: Immunochemicallocalization of secretory antigens in the human epididymis and their association with spermatozoa. BioI Reprod 32:591, 1985 20. Wong PYD, Tsang AY, Wang C: Absence ofa sperm coating protein after epididymovasostomy. J Androl 3:205 1982 ' 21. Acott TS, Katz D, Hoskins DD: Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation. BioI Reprod 29:389, 1983 22. Pariset CC, Feinberg JMF, Dacheux JL, Weinman SJ: Changes in calmodulin levels and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa. J Reprod Fertil 74:105, 1985 23. Amann RP: A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl 2:31,1981 24. Foldesy RG, Bedford JM: Biology of the scrotum. I. Temperature and androgen as determinants of the sperm storage capacity of the cauda epididymidis. BioI Reprod 26:673, 1982 25. Parker GA: Sperm competition and the evolution of animal mating strategies. In Sperm Competition and the Evolution of Animal Mating Systems, Edited by RL Smith. New York, Academic Press, 1984, p 1
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FERTILITY AND STERILITY Copyright " 1986 The American Fertility Society
The role of the human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy*
Robert J. Schoysman, M.D.t J. Michael Bedford, Vet.M.B., Ph.D.:j:§ Free University of Brussels, Brussels, Belgium, and Cornell University Medical College, New York, New York
Epididymovasostomy has been used for examination of the role of particular regions of the epididymis in sperm maturation and storage, as reflected in fertility and in the motility, structural, and surface character, and also the number of the spermatozoa ejaculated. Human spermatozoa need be exposed only to the environment in the caput before passing into the vas deferens, in order to complete their maturation. However, the chance of pregnancy appears greater where the anastomosis is lower. Among the sperm characteristics examined, only motility was affected by the level of anastomosis, and there was a trend to higher numbers where the anastomosis was established beyond the upper caput. The storage function of the cauda is discussed in light of the finding that epididymovasostomy patients sometimes ejaculate sperm numbers seen in normal men. Fertil Steril46:293, 1986
The milieu in the epididymis provides specific support for the events of sperm maturation, and conditions in the cauda that are dependent on androgen and the low temperature of the scrotum permit a storage there of mature spermatozoa. In the species examined critically, significant numbers of mature spermatozoa first appear in the . midcorpus or lower corpus epididymidis with the pattern perhaps skewed somewhat more toward the cauda for hamster and rat. 1 In no mammal is
Received September 17,1985; revised and accepted March 19,1986. *Supported by the Special Program in Human Reproduction, World Health Organization, Geneva, Switzerland. tDepartment of Obstetrics and Gynecology, Free University of Brussels. :j:Departments of Obstetrics and Gynecology, Cell Biology, and Anatomy. §Reprint requests: J. Michael Bedford, Vet.M.B., Ph.D., Department of Obstetrics and Gynecology, Cornell University Medical College, 525 East 68th Street, New York, New York 10021.
Vol. 46, No.2, August 1986
there evidence that spermatozoa can become fertile if retained in the testis, but it is not clear yet for any species which epididymal regions are essential to the maturation process. In trying to establish this, the relatively crude approach of ligation at defined points along the epididymis suggests that the caput may be the only critical region in the rabbit.2, 3 The hamster appears less flexible in this respect, in that ligation in the corpus epididymidis does not markedly skew the fertility profile toward the caput region. 4 By comparison with common animal models, relatively little has been gleaned yet about the human epididymis and the extent of its contribution to sperm maturation. It is known that maturing human spermatozoa undergo most of the subcellular changes seen in animals, i.e., modifications in their motility, metabolism, surface character, and structural quality.5 In terms of function, Moore and co-workers6 have provided the best available clue as to the fertility profile with the use of the hamster egg as a surrogate. This suggests that the first spermatozoa to com-
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plete their maturation in significant numbers do so while in the corpus epididymidis. One approach to analysis of the role of specific epididymal regions lies in the bypassing of them by means of epididymovasostomy. That technique has recently been used in studies of the rabbit,7 the results of which seem similar to the findings of the older ligation studies. However, epididymovasostomy has been employed for many years by clinicians as a means of bypassing epididymal blockage sites. 8 -1O This offers a fairly good chance of creating a bypass between patent epididymis and the vas deferens; and, long before maturation patterns were emphasized by the work of reproductive biologists, surgeons intuitively have attempted to include the greatest portion of epididymis. In the .present investigation, ejaculates were obtained from a relatively large group of patients in which epididymovasostomy had been performed successfully at defined levels of the epididymis. It seemed possible that examination of specific sperm parameters known to change during epididymal maturation5 might indicate the limits of the segment that is essential for maturation, and which of the several cellular facets of maturation depend specifically on epididymal factors for their completion. MATERIALS AND METHODS
Patients were recruited from a population of 723 individuals subject to epididymovasostomy for relief of epididymal blockage (or agenesis in one case) between 1 and 10 years earlier. The fertility rate, known only for 565 of these patients, reached 18%. To perform the investigation we had to know the exact level of the anastomosis on the epididymis. That was recorded at surgery as the distance between the proximal border of the caput and the proximal border of the anastomosis site. The distance was measured in millimeters because the different regions l l cannot always be accurately recognized from the surface anatomy in such cases. The 117 patients finally included in the fertility data of this report were those who had 20 x 106/ml or more spermatozoa in at least one previous ejaculate after either unilateral anastomosis or bilateral anastomosis at a similar site on both epididymides. The anastomosis between the epididymis and vas deferens was laterolateral. 12 The upper border was essentially coincident with the tubule that showed con294
tinuous sperm leakage after exploratory incision above the region of blockage. For sperm analysis, one ejaculate was obtained by masturbation from each of 35 of the 117 patients chosen on the basis of availability. These samples were subject to analysis within about 2 hours of collection. When delivered, the ejaculate was kept at 37°C, and the concentration and total number of spermatozoa were estimated with a hemocytometer. The other parameters studied were (1) the structural quality and free thiol status of the sperm nucleus and sperm tail; (2) the character of the sperm surface; and (3) swimming behavior (motility). STRUCTURAL QUALITY
The normal structural stability of spermatozoa during epididymal passage can be measured visually according to the swelling-disruption response to sodium dodecyl sulfate (SDS) with or without dithiothreitol (DTT), which cleaves disulfide bonds. 13 The latter determine the keratinQid stability typical of the sperm head and tail in all eutherian mammals, including man. To evaluate the structural quality of spermatozoa ejaculated by epididymovasostomy patients, an aliquot was assessed according to the head swelling response to either 1% SDS alone or to 1% SDS in borate buffer containing 2 x 10 - 3 M DTT p. 14 This reaction was stopped after a 15-minute exposure to SDS/DTT or after 60 minutes in SDS alone, by addition of an equal volume of 2.5% glutaraldehyde. The spermatozoa were assessed (> 200 sample) in successive fields in the phase-contrast microscope according to the degree of nuclear decondensation. The heads were classified as unchanged, moderately swollen, or grossly swollen. 14 Parallel observations were made on aliquots taken from ejaculates of fertile men. The assessments were subjective but always were made by one operator (J. M. B.). Because the changing stability of the sperm chromatin with epididymal passage appears to reflect the extent of - S - S - cross-linking of protein-bound free thiols,13. 15 the free thiol content of spermatozoa in aliquots from epididymovasostomy ejaculates was also examined by a direct chemical method. This depends on the specific uptake of 14C_iodoacetamide by the spermatozoa under given conditions. 15 This was done in two ways with samples from groups containing 9 and 13 patients, respe~ively. In the first, one aliquot
Schoysman and Bedford Epididymovasostomy and sperm function
Fertility and Sterility
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from each was frozen at - 80°C. Later, the sample was thawed and homogenized by sonication. After washing twice in isotonic saline, the sperm pellet was dissociated gently and the standard assay was carried out in a volume of 0.5 ml. 15 In view of initial results that showed no significant difference between epididymovasostomy and normal samples in this respect, the second group was included to control for the possibility that spontaneous oxidative crosslinking might occur in immature spermatozoa soon after ejaculation. Therefore, ejaculates collected were immediately counted, and an aliquot of some 30 x 106 spermatozoa was withdrawn and stored at 4°C. The aliquot was washed by double centrifugation in isotonic saline; and, after slight homogenization of the pellet with a stirring rod, 1ml of 14C_ iodoacetamide was mixed with it. The mixture was incubated at room temperature for 18 to 20 hours, then frozen in liquid nitrogen until assayed by counting of samples coming from patients with high or low anastomosis.1 5 SPERM SURFACE
Two techniques were used to evaluate the sperm surface. The first involved the binding pattern offluoresceinated concanavalin A (FITC-Con A) to spermatozoa. In these cases, sperm aliquots were first washed twice in Tyrode's solution and then fixed in 4% paraformaldehyde before staining and evaluation with epifluorescence optics. The other method for evaluation of the surface was chosen in light of obvious change in the binding pattern of positively charged colloidal ferric (Fe + + +) oxide particles, demonstrated during normal epididymal maturation of human spermaVol. 46, No.2, August 1986
Motility was evaluated subjectively in a phasecontrast microscope with the use of shallow well slides such that sperm movement was not restricted through frictional interaction with the glass. Motility was scored on the basis of the percentage of spermatozoa in at least ten fields moving vigorously and progressively. This assessment was made by J. M. B. within 2 hours of the collection of the semen samples.
RESULTS The present results suggest that the pattern of development of the ability to fertilize in the epididymal sperm population can be relatively flexible in man. Nonetheless, there is a greater chance for pregnancy where the anastomosis is made in the corpus, rather than at the level of the caput epididymidis.Figure 1 reveals occasional pregnancies in cases where an anastomosis had been made 8 mm or less from the proximal border of the caput, but there was a general absence of fertility in that group. Pregnancies were common, on the other hand, when the anastomosis site was > 10 mm from the proximal border in men having > 20 x 106 spermatozoa in a previous ejaculate. In examining ejaculated spermatozoa in terms of individual cellular parameters that change during passage of spermatozoa through the normal epididymis, only motility could be discerned to have a relationship to the level of anastomosis. Most men whose anastomosis was located in the proximal 8 mm ejaculated spermatozoa which were essentially immotile. By contrast, in all cases in which the anastomosis was > 10 mm from the caput border, some 20% to 90% of the spermatozoa were progressively motile (Fig. 2). Among the other sperm parameters studied, none appeared "immature," regardless of the level of the anastomosis. Spermatozoa taken from the caput of the normal human epididymis essentially fail to bind Fe + + + colloid at pH 1.8, in contrast to those of caudal spermatozoa. 5 However, there was no visible difference here in the pattern of binding of cationized Fe + + + colloid to
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the surface of spermatozoa from control and epididymovasostomy samples, regardless of the level of anastomosis. The Con-A binding patterns also showed no correlation between the level of anastomosis and a particular distribution of surface fluorescence on paraformaldehyde-fixed spermatozoa. Control Con-A-treated spermatozoa fluoresced moderately and uniformly, except for the region of the cytoplasmic droplet, which displayed a more intense fluorescence. Con-A-treated epididymovasostomy samples fluoresced with a pattern similar to that of the controls, irrespective of the level of anastomosis. It should be noted that the patterns observed were not similar to those localized by immunoperoxidase techniques. 16 According to the swelling response to SDS or SDS/DTT, there was no significant difference in the level of free thiol ( - SH) groups in ejaculated spermatozoa from normal controls and those from epididymovasostomy patients as a function of the level of anastomosis. The anastomosis patients as a group did not react differently from the controls, which themselves varied from individual to individual, as noted previously. The percentage of patients' spermatozoa that displayed detectable swelling in response to SDS alone varied from 4% to 22% (mean, 13.8%), but no pattern was seen according to the anastomosis level, and the mean did not differ significantly from that for control samples studied under the same conditions. Simi296
larly, spermatozoa ejaculated by patients with a high « 10 mm) anastomosis did not swell more readily in the face of SDSIDTT for 15 minutes than those from low anastomosis cases. The lightmicroscopic impression that control and all epididymovasostomy patients' spermatozoa had a similar - SH status was borne out by the experiments performed for evaluation of the free - SH content of spermatozoa as reflected in 14C-iodoacetamide incorporation. 15 As measured in 0.1ml aliquots of a sperm suspension, the samples from 22 epididymovasostomy patients studied displayed a mean specific activity of 2750 (range, 1471 to 5195) cpmll0 7 spermatozoa, compared with a mean of 3246 cpm/10 7 spermatozoa in parallel controls. Again, there was considerable variation among the ejaculates tested in both control and experimental groups; but, in the latter, this was in no way a reflection of the level of the anastomosis. In Figure 3, total numbers in the ejaculate are plotted for the unilateral and bilateral situation as a function of the level of the anastomosis. The lowest level is designated in the case of patients with bilateral anastomoses. Although it is difficult to draw firm conclusions, the numbers obtained suggest that fewer spermatozoa tend to be produced where high blockages are reversed. Attention is drawn to the fact that several patients, including some with a unilateral anastomosis, had numbers ranging from 160 to 535 x 106 in a single ejaculate. Because the cauda epididymidis is bypassed, this is of interest in light of the fact that these totals compare with the numbers produced in a first ejaculate by normal men with no malfunction of the reproductive tract.
DISCUSSION
The results of this study support the general conclusion that in man, as in animals, the epididymis makes some critical contribution without which spermatozoa cannot develop the ability to fertilize. They also indicate that while much of the human epididymis can be dispensed with, the chance for pregnancy to occur is generally greater if the anastomosis is made low, in the corpus epididymidis, rather than high, in the caput region. The exact reason for this is not clear, because the percentage of the spermatozoa that are fully fertile in ejaculates produced after anastomosis at any level is not known. However, Figure 2 sug-
Schoysman and Bedford Epididymovasostomy and sperm function
Fertility and Sterility
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. gests that the percentage motility at least could be an important element. Precise interpretation of epididymovasostomy results is complicated to some degree by several factors. Where bilateral anastomoses are attempted, one cannot be sure whether both sides become patent and the number ejaculated represents the output of both testes. With side-to-side anastomosis, the level of the connecting cut epididymal tubule essentially reflects the upper boundary of the anastomosis site; but,in reality, some levels given in the tables here could err perhaps by 1 to 2 mm. Neither is a failure to establish· a pregnancy, seen variously in Figures 1 and 2, a sure indicator that the ejaculate studied does not contain fertile spermatozoa. On the other hand, despite these caveats, epididymovasostomy performed for purely clinical reasons clearly can provide valuable pointers as to the role of the human. epididymis in sperm maturation and storage. Although better overall fertility results are obtained with low anastomoses, Figure 1 and other studies 7, 17 show that some anastomoses situated within the first 8 mm of the duct can result in ejaculated spermatozoa able to establish a pregnancy. Thus, in evaluating the specific contribution of the epididymis, one can conclude that any essential dependence of spermatozoa on the milieu of the epididymis for maturation appears minimal beyond the caput region, specifically region 2.11 We presume that the majority of high anastomosis cases with poor motility and no fertility Vol. 46, No.2, August 1986
involved insufficient exposure of spermatozoa to the epididymal milieu before their passage into the vas deferens. However, in interpreting the consequences of epididymovasostomy, it should be appreciated that the human epididymis differs somewhat from that of many animal models. The vasa efferentia in man occupy the major part of what appears grossly as caput epididymidisl l ; and it is possible that a direct link between vasa efferentia and vas deferentia may have been established in some very high anastomosis patients here. One might anticipate an extremely poor quality of spermatozoa ejaculated in such cases. A relative flexibility of the functional relationship between spermatozoa and much of the human epididymis is implied in the fact that, compared with control spermatozoa, only motility was detectably different among the parameters tested as a function of the level of anastomosis. It is perhaps not surprising that the free - SH, and so structural status, of the epididymovasostomy samples appeared no different from that in controls, regardless of the level of anastomosis. Although the epididymal environment may have some modulating effect on this aspect of maturation,13 oxidation of - SH to - S - S - bonds proceeds readily even in vitro, in nonspecificconditions. Moreover, human sperm heads have a significantly lower cysteine content than those of other mammals. 18 With regard to the sperm surface, change in its membrane glycoprotein complement is a recognized special feature of epididymal maturation in a· number of mammals, and this aspect of the sperm surface character is believed to bear at least on the sperm's potential for capacitation and its ability to bind to the egg. Although comparable surface changes probably occur in human spermatozoa, as reflected in Fe + + + colloid bindin~ and the fluorescent expression of epididymal antigen,19 no consistent departure from controls was observed here in the FITC-Con A and Fe + + + binding patterns as a function of the level of anastomosis. However, these techniques will not detect all surface change, and the possibility that they are not specific enough is suggested by a case,20 reported after our observations were made, in which immotile spermatozoa and seminal plasma ejaculated by a high epididymovasostomy patient lacked a 38,000 molecular weight protein found on normal spermatozoa and in normal seminal plasma. Thus, the absence here of a correlation between the binding of such surface markers and
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the anastomosis level should perhaps be viewed with some reservation. Nonetheless, the fact that several pregnancies were established in this and another study17 when the lowest anastomosis was sited within 1 cm of the proximal border indicates that any essential contribution to the sperm surface can be acquired by at least some spermatozoa entirely in the caput epididymidis. With regard to motility, a considerable improvement has been reported during the first two postoperative years in a small series of high endto-end anastomosis patients,i 7 and this has raised some question as to the essential place of the human epididymis in maturation of the motility function. While the epididymis normally has a low rate of mitosis in the adult, the suggestion17 that a compensatory growth of the caput tubule could occur in such cases seems worthy of investigation. Here, samples from a few patients in the high anastomosis group also contained some spermatozoa with good progressive motility. However, in many more (and in the case described by Wong et al. 20) motility was persistently absent or very poor in spermatozoa ejaculated by the high « 10 mm) anastomosis group over a period of 2 years or longer (Fig. 2). In the rabbit, also, anastomosis high in the caput resulted in a persistent production of similarly poor quality spermatozoa. 7 The fact that a strong trend to better motility has been seen rather consistently where spermatozoa are able to pass through a longer section of epididymal duct reinforces5 the general view that the epididymis exerts some essential and specific control over the. functional maturation of the axoneme. As yet, the factors involved in motility maturation are still a matter for conjecture. 21 It must be appreciated that spermatozoa in the normal male do not yet possess the capacity for optimal motility if released from the very restricted proximal regions they can experience in the fertile high epididymovasostomy patients. Spermatozoa express this capacity in significant numbers only at a more distal point reached perhaps 48 to 72 hours or more after entering the epididymis. 5 A clear case in point is illustrated by micropuncture samples taken from the normal ram epididymis, the motility of which did not improve significantly until the proximal corpus region was reached. 22 Taken together with such results, the observations from epididymovasostomy cases suggest that while certain motility enhancement factors are provided in the proximal region of the 298
epididymis, more time than the short period spermatozoa stay in the caput is required for their effects to be expressed. In the case of "high" epididymovasostomy patients, this expression may well occur only after spermatozoa pass into the vas deferens. Finally, the present results raise a question that concerns sperm numbers. The data in Figure 3 show that a high anastomosis was more often followed by very low numbers in the ejaculate, though the reasons for this are not clear. On the other hand, in agreement with Silber,17 some men with high epididymal blockage in this series did produce a surprisingly high number even where only a unilateral patency was established. Moreover, "normal" numbers (160 to 535 x 106 ) were present in the ejaculates of several of our epididymovasostomy patients, as in other cases reported. 17 This raises a question as to the significance of the storage function of the cauda epididymidis, because the present results imply that the vas deferens alone can accommodate all the spermatozoa produced in a normal ejaculate. This appears to be true also for rabbits. 7 The issue of the role of the cauda epididymidis may be masked somewhat by the fact that the human caudal sperm population normally is relatively small. 23 Because body temperature reduces the storage capacity of the scrotal cauda,24 it is possible that its modest size in man partly reflects some suppressive effect of a higher scrotal temperature induced by clothing. However, regardless of species variation, in considering the fundamental significance of the cauda epididymidis, it should be realized that a sexually active male mammal may be required to deliver several sequential ejaculates within a short period in maximizing his fertility potential. This can involve the consecutive insemination of several females in polygynous species, or repeated insemination of the same female in the case of some rodents, felines, wolves, etc. 25 While the cauda epididymidis may not necessarily be of primary importance in the delivery of a first ejaculate, it is probably critical for the ability to deliver repeated ejaculates of high fertility within a limited time period.
Acknowledgments. We thank Harold I. Calvin, Ph.D., and Andrea Schoysman, M.D., for their help in assessment of iodoacetamide uptake by spermatozoa.
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REFERENCES 1. Bedford JM: Maturation transport and fate of spermatozoa in the epididymis. In American Handbook of Physiology, Sect 7, Vol 5, Edited by DW Hamilton, RO Greep. American Physiological Society, Washington, D.C., 1975, p 303 2. Bedford JM: Effect of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis. J Exp Zool 166:271, 1967 3. Orgebin-Crist MC: Studies on the function of the epididymis. BioI Reprod Suppl 1:155, 1969 4. Cummins JM: Effects of epididymal occlusion on sperm maturation in the hamster. J Exp ZooI197:187, 1976 5. Bedford JM, Calvin HI, Cooper GW: The maturation of spermatozoa in the human epididymis. J Reprod Fertil Suppl 18:199, 1973 6. Moore HDM, Hartman JD, Pryor JP: Fertilizing capacity of human spermatozoa. Int J Androl 6:310, 1983 7. Temple-Smith PD, Southwick G: Vasoepididymovasostomy in the rabbit: effects of level of anastomosis on semen quality and fertility. J Androl (Suppl) 6:30-P, 1985 8. Young DH: Surgical problems of the vas deferens. Proc Soc Study Fertil 3:40, 1951 9. Silber SJ: Microscopic vasoepididymovasostomy: specific microanastomosis to the epididymal tubule. Fertil Steril 30:565, 1978 10. Schoysman RJ: Microsurgery of the male genital tract. In Microsurgery in Infertility, Edited by E Cittadini, M Cognat, RJ Schoysman. Cofese, Palermo, 1981, p 83 11. Baumgarten HG, Holstein AF, Rosengren E: Arrangement ultrastructure and adrenergic innervation of smooth musculature of the ductuli efferentes, ductus epididymidis and ductus deferens in man. Z Zellforsch Mikrosk Anat 120:39, 1971 12. Schoysman RJ, Drouart JM: Progres recents dans la chirurgie de la sterilite masculine et feminine. Acta Clin Belg 71:261, 1972 13. Calvin HI, Bedford JM: Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil (Suppl) 13:65, 1971
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14. Bedford JM, Bent MJ, Calvin HI: Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J Reprod Fertil 33:19, 1973 15. Calvin HI, Yu CC, Bedford JM: Effects of epididymal maturation, zinc [II], copper [II] on the reactive sulfhydryl content of structural elements in rat spermatozoa. Exp Cell Res 81:333, 1973 16. Scacciati de Cerezo JM, Bueno MP, Skowrowski B, Cerezo AS: Immunohistochemical localization of conconavalin A and wheat germ receptors in normal human spermatozoa. Am J Reprod Immunol 2:246, 1982 17. Silber SJ: Vasoepididymovasostomy to the head of the epididymis: recovery of normal spermatozoal motility. Fertil Steril 34:149, 1980 18. Calvin HI: Comparative analysis of the nuclear basic proteins in rats, human, guinea pig, mouse, and rabbit spermatozoa. Biochem Biophys Acta 434:377,1976 19. Tezon JG, Ramella E, Cameo MS, Vazquez MH, Blaquier J A: Immunochemicallocalization of secretory antigens in the human epididymis and their association with spermatozoa. BioI Reprod 32:591, 1985 20. Wong PYD, Tsang AY, Wang C: Absence ofa sperm coating protein after epididymovasostomy. J Androl 3:205 1982 ' 21. Acott TS, Katz D, Hoskins DD: Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation. BioI Reprod 29:389, 1983 22. Pariset CC, Feinberg JMF, Dacheux JL, Weinman SJ: Changes in calmodulin levels and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa. J Reprod Fertil 74:105, 1985 23. Amann RP: A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl 2:31,1981 24. Foldesy RG, Bedford JM: Biology of the scrotum. I. Temperature and androgen as determinants of the sperm storage capacity of the cauda epididymidis. BioI Reprod 26:673, 1982 25. Parker GA: Sperm competition and the evolution of animal mating strategies. In Sperm Competition and the Evolution of Animal Mating Systems, Edited by RL Smith. New York, Academic Press, 1984, p 1
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