Spermatogenesis Following Experimental Testicular Ischemia

Transcription

1 Spermatogenesis Following Experimental Testicular Ischemia Frank Hinman, Jr, MD, and Gilbert I Smith, MD REGENERATION of the spermatogenic elements of the testis after depression by testosterone and by other agents-such as estrogens, febrile illnesses, and debilitating diseases-has in the last few years been clearly recognized, although the exact stimulating factors for such a rebound have yet to be exposed The present report concerns the effect of an entirely different noxious agent, temporary ischemia, on spermatogenesis, being a study of both damage and regeneration of the tubular epithelium The technic of producing temporary arterial obstruction in the testis of the dog has been described in a previous paper 2 In brief, after general anesthesia, a tourniquet is placed about the exposed spermatic cord (or in 9 later experiments, about the intact hemiscrotal skin), drawn just tight enough to stop bleeding from the site of the initial control biopsy, and left in place for periods of 1 to 14 hours Specimens were obtained 30 or 60 days after removal of the tourniquet, fixed in formalin or in Bouin's fluid, and stained with hematoxylin-eosin, and Masson's stain A total of 42 testes were so studied After 2 hours' ischemia, as described in the previous communications, spermatogenesis was depressed, with retention usually of the spermatogonia Leydig and Sertoli cells resisted damage, and the basement membrane remained intact (Table 1) After 4 hours, however, almost all sperma- From the Department of Surgery-Urology, University of California School of Medicine, San Francisco, California Supported by the Breon Fund for Medical Research Read before the Tenth Annual Meeting of the American Society for the Study of Sterility at San Francisco, California, June 18, 19, and 20,

2 444 HINMAN & SMITH Fertility & Sterility togenic elements were destroyed, and only a few viable Sertoli cells remained The Leydig cells formed a solid margin beneath the thick tunica albuginea (Fig 1) After periods of ischemia longer than 4 hours, all spermatogenic and Sertoli elements were destroyed, and if the ischemia lasted more than 10 hours, the Leydig cells perished also DESTRUCTION OF SPERMATOGENIC TISSUE To obtain more detailed data on the cytologic ehects of the shorter periods of ischemia on the spermatogenic cells, and on the possibility of regeneration during the period of repair, a study was made of a second, smaller group of 9 testes, obstructed by mass ligature for periods of 1 (2 testes), 2 (3 testes), 3 (2 testes), and 4 hours (2 testes), and removed 30 days or 60 days later and fixed in formalin and in Bouin's fluid Individual variation existed TABLE 1 Effect of Ischemia on Testicular Elements N-33 Spermatogenesis Sertoli cells Leydig cells Hours of ischemia required to Damage Destroy between the specimens from any given period, probably on the basis of vascular damage (? spasm) and animal age and nutrition In general, the specimens obtained in the second series, by obstruction of the entire scrotum, show greater damage than those reported earlier We have chosen representative sections for illustration The destructive changes will be traced in the illustrations: One-Hour Ischemia Although the basement membrane is still intact 60 days after 1 hour of ischemia (Fig 2), only a few spermatogonia remain among the disturbed Sertoli cells These few germinal cells have vacuolated cytoplasm, with occasional hyperchromatic, shrunken nuclei, or with granular chromatin In some tubules (Fig 3) epithelial disruption is more complete, although partial regeneration of the spermatic elements can be seen in certain areas in the 60-day specimens

3 Vol 6, No 5, 1955 SPERMATOGENESIS IN T'ESTICULAR ISCHEMIA 445 Fig 1 Leydig cell hyperplasia (from layer beneath tunica albuginea), 4-hour specimen, after 60 days (A~ X 80; B~ X 324) }i'ig 2 One hour's ischemia (Bouin's fluid; Masson stain) Few spermatogonia among distorted Sertoli cells (A~ X 80; B, X 324) Two-Hour Ischemia After two hours of ischemia, more extensive changes have taken place (Fig 4) The basement membrane is now greatly thickened All spermato-

4 446 HINMAN & SMITH Fertility & Sterility genic elements have vanished All that remains within the tubules are Sertoli cells with disintegrated cytoplasm and pyknotic, shrunken nuclei Leydig cells are, however, relatively undamaged at this stage 3 Fig 3 One hour's ischemia (Bouin s fluid; Masson, X 324) :More complete epithelial disruption No evidence of regeneration Fig 4 Two hours' ischemia (Bouin' s fluid; Masson, X 324) Spermatogenic elements destroyed Sertolicells disintegrating Basement membrane greatly thickened Fig 5 Three hours' ischemia (Bouin' s fluid; Masson, X 324) Very thick basement membrane surrounds a few unidentifiable cells Edematous, fibrous connective tissue 4 Three-Hour Ischemia Ischemia of 3 hours' duration (Fig 5) produces extren1e damage to the spermatogenic elements The very thick basement membrane surrounds a few cells which cannot be identified The interstitial tissue is edematous and fibrous and the Leydig cells (not shown in these sections) are clumped 5

5 Vol 6, No5, 1955 SPERMATOGENESIS IN TESTICULAR ISCHEMIA 447 Four-Hour Ischemia At four hours, there is almost complete loss of tubular identity, and the Leydig cells form masses at the periphery (Fig 1) Fig Fig 6 6 Attempt at regeneration, regeneration, two-hour two-hour specimen specimen (A, (A, X X 80; 80; B, X 324 Attempt at B, X 324 Spec Spec 160L, hematoxylin-eosin) hematoxylin-eosin) ## 160L, Fig Fig 7 7 Same, specimen (A, (A, X X 80; 80; B, B, X X 324 Same, two-hour two-hour specimen 324 Spec Spec # IlR, llr, hematoxylinhematoxylineosin) eosin) :

6 HINMAN & SMITH 448 Fertility & Sterility REGENERATION OF SPERMATOGENIC TISSUE Regeneration of the genninal epithelium occurred only to an insignificant degree by the end of the experiment (60 days) Only a few specimens showed any regeneration at all Three testes out of a total of 6 examined 60 days after two hours or less of ischemia (Figs 6, 7, 8) showed significant regeneration of spermatogonia Testes examined at 30 days and those sub- Fig 8 Same, two-hour specimen (Spec # 2 R, hematoxylin-eosin) jected to 3 or more hours of ischemia failed to show regeneration In the specimens illustrated, nuclei containing clumped chromatin and granular, somewhat vacuolated cytoplasm can be seen Occasional spermatids are seen among the rather unifonn masses of earlier cells, and complete maturation is hard to find In other words, a rebound phenomenon in a clinical sense did not occur under the conditions of these experiments IMPLICATIONS That vascular obstruction can produce testicular damage is readily demonstrated Our experiments show the effects of extreme degrees of testicular anoxia, greater than would be expected even in complete torsion of the testis One might speculate that less complete but recurrent ischemia could occur (? subclinical torsion), causing lesser degrees of spennatogenic damage The changes noted after shorter periods of complete ischemia resemble

7 Vol 6, No5, 1955 SPERMATOGENESIS IN TESTICULAR ISCHEMIA 449 those seen in certain patients with infertility (loss of mature forms, thickening of the basement membrane, etc) It would appear that complete vascular obstruction of more than 1 or 2 hours causes irreversible changes in the dog testis The interval in the human being might be slightly longer because of the known greater resistance of human tissue to ischemia In contrast, we have found a report on testicular ischemia in the rat l in which even shorter periods of obstruction causes irreversible damage The chance for subsequent fertility from the human testis after complete torsion would be expected to be small should the obstruction continue for more than 2 hours SUMMARY 1 Complete ischemia was produced in 42 dog testes for periods varying from 1 to 14 hours Specimens were examined SO or 60 days later 2 One or 2 hours' ischemia produces irreversible damage, although some incomplete regeneration occurs No effective rebound phenomenon was seen under the conditions of the experiments S Although the ischemia in clinical torsion of the testis is seldom as great as in experimental animals, these studies show the most extreme effects which could be expected on the spermatogenic elements REFERENCES 1 OETTLE, A C, and HARRISON, R C The histological changes produced in the rat testis by temporary and permanent occlusion of the testicular artery J Path Bact 64:273, SMITH, C I Cellular changes from graded testicular ischemia ] Urol, in press