Effect of Low Temperatures on Rat Spermatogenesis

Transcription

1 Effect of Low Temperatures on Rat Spermatogenesis Jean Macdonald, B.Sc., and R. G. Harrison, D.M. ALTHOUGH THERE HAVE BEEN several investigations of the effect of increased temperature on spermatogenesis in both the experimental animap2 and man,9 there are few reports of the changes produced in the testis by temperatures lower than that of the normal intratesticular temperature. Gronsky claimed that spraying the rat scrotum with ether produced an intrascrotal temperature of 3 C. and an exfoliation of all spermatogenic cells except some spermatogonia. Regeneration began within four weeks and was complete. On the other hand, Phillips and McKenzie found that applying running water at 6, 8, and 1 C. over the scrotum of rats caused no derangement of the germinal epithelium after ten days. In view of these discrepant results, it was decided to reinvestigate the problem, and to correlate the effects of low temperatures on spermatogenesis in the rat with the intratesticular temperature produced by the experimental procedure. METHOD Twenty-six mature male albino rats weighing 23-3 Gm. were used for. two types of experiments. In the first set of experiments, one testis was mobilized from the scrotum by severing the ligamentum inguinale (a procedure which in itself does not affect spermatogenesis), and removed through a midline abdominal incision, using ether anesthesia and full From the Department of Anatomy, University of Liverpool, Liverpool, England. This research has been aided by a grant from the Medical Research Council. We wish to thank Miss B. Birkett, Miss B. Constantine, L. G. Cooper, and C. FitzSimon for their technical assistance. 25

2 26 MACDONALD & HARRISON Fertility & Sterility aseptic precautions. The rat was placed prone on a wooden board, and the testis suspended through a hole in the board, which was so arranged that the testis was immersed in a bath of normal physiologic sodium chloride kept at temperatures varying between -8 and +2 C. by surrounding it with a container of ice-salt mixture (Fig. 1). The ligamentum inguinale of the contralateral testis was severed, and this testis, replaced in the scrotum, served as a control. The experimental testis was replaced TESTIS OF RAT _ SUBMERGED IN BEAKER CONTAINING SALINE AND POWDERED FROZEN SALINE. ICE AND SALT MIXTURE Fig. 1. The experimental method used to effect cooling of one testis of a rat by suspending it in saline at temperatures varying between -8 and +2 C. The contralateral testis, after removal from and replacement into the scrotum, served as a control. into the scrotum after exposure to low temperatures, and the abdominal incision closed. In the second set of experiments, the scrotum of the anesthetized rat was surrounded by ice-salt mixture at temperatures varying between -1 and _4 C. This was effected by placing the rat on its back, arranging rubber sheeting around its hind quarters, but leaving the scrotum exposed. In order to prevent retraction of one testis during the experiment, a ligature was placed through the cauda epididymidis on the experimental side and anchored to the skin of the scrotum. The terms used for the grades of degeneration of the seminiferous epithelium are those described by Oettle and Harrison: 1. Mild spermatogenic damage, in which the majority of the cells appear unaffected;

3 Vol. 5, No.3, 1954 TEMPERATURE AND SPERMATOGENESIS Moderate spermatogenic damage, intermediate between Grades 1 and 3; 3. Severe spermatogenic damage, where the majority of the spermatogenic cells are necrotic and only a few survive; 4. Spermatogenic destruction, in which only the surviving Sertoli cells can be seen; 5. Epithelial destruction, in which no living cells can be found in the tubule section. RESULTS Histologic Appearance of Germinal Epithelium Exposure of one testis. At 2 and c. for one hour, histologic examination at periods ot three, five, and fourteen days after the procedure showed no change whatever in the appearance of the testis as compared with the control. A temperature of _2" C. applied for one hour produced only mild spermatogenic damage in a few tubules at the periphery of the testis. kxposure of the testis to _4 C. for one hour, _5 C. for one hour, and _6 C. for fifteen minutes effected moderate spermatogenic damage throughout the testis when examined histologically three hours, and two, four, and fourteen days afterwards. This damage is temporary, since on examination of such specimens twenty-eight days after the application of low temperature, signs of regenerati()n were apparent. Only when temperatures of -6 and -8 a C. were applied for one hour was spermatogenic destruction found three hours, and two, four, and fourteen days later. After twenty-eight days, the seminiferous tubules were full of degenerating spermatogenic cells, with no surviving normal spermatogenic cells, and no attempt at regeneration. The control testes in all the above experiments showed normal spermatogenesis. A further experiment was performed in which one testis was immersed in physiologic sodium chloride at 32 a C. for one hour. When this testis was examined four days later, the histologic appearance was normal, and showed no difference from the control. This experiment demonstrates the innocuous nature of the experimental technic as such. Exposure of the scrotum. In these experiments, the anchored testis remained in situ, and the contralateral testis retracted into the abdomen in all experiments performed at temperatures above -4 C. Since the icesalt mixture was applied for periods of no longer than one hour, the retracted testis served as a control, and in all cases the histologic appearance of the germinal epithelium was normal. At _4 C. or lowr, however,

4 28 MACDONALD & HARRISON Fertility & Sterility the free testis did not retract. In such cases, therefore, no control testis was available, but since normal spermatogenesis was found in all control testes in other experiments, and since the animals used in the experiments came from an inbred stock, it was considered permissible to interpret the histologic appearances of both testes in these experiments as reflecting accurately the results of the experimental procedures used. At and above -4 C. for one hour, no change was observed in the germinal epithelium of the anchored testis when examined four days afterwards. This result also served to demonstrate the innocuous effect of the anchoring procedure on spermatogenesis. At _6 C. for one hour, mild spermatogenic damage was apparent in both testes after four days. A temperature of _8 C. applied for one hour produced moderate spermatogenic damage after four days, while after application of _1 C. for one hour, the histologic appearance of the seminiferous epithelium showed severe spermatogenic damage after four days, the tubules containing only a few spermatogonia and rare primary spermatocytes as well as Sertoli cells. From these experiments it is apparent that a much lower temperature must be applied to the scrotum to effect a given degree of degeneration of the seminiferous epithelium than in the case of direct application of low temperatures to the testis. These results suggested that there might be differences in the intra testicular temperature occasioned by the two procedures used, and experiments were therefore designed in order to examine this possibility. Intratesticular Temperature By means of fine (standard wire gauge 4) copper-constantan thermocouples, it is possible to measure the temperature within the testis while subjecting the testis or the scrotum to varying environmental temperatures. The thermocouple was made by soldering the copper and constantan wires end to end. It could then be threaded into the center of the testis, using a fine needle and taking care not to damage the testicular vessels. Readings were taken by means of a potentiometer, and the experimental procedures used were the same as before. The results obtained in 1 rats are shown in Figs. 2 and 3. From Fig. 2, which shows the effect of applying low temperatures directly to the testis, it may he noticed that intratesticular temperature varies between 6 and 8 C. above that of the surrounding physiologic sodium chloride bath. It may

5 Vol. 5, No.3, 1954 TEMPERATURE AND SPERMATOGENESIS 29 also be observed that spermatogenic destruction is produced at an intratesticular temperature of C. A temperature of 2 to 4 C. must be created inside the testis in order to effect spermatogenic damage. Thus, at an environmental temperature of -4 C., maintained for one hour, or of _6 C. for fifteen minutes, a temperature of 2 C. is found within the testis. A 2 B t wl4 a: ::;) I- 12 w.. UJ 1-1 a: «5 8!::! l- I/) UJ 6 a: I-!?:: 4 2 I -2 '= MINUTES Fig. 2. The intra testicular temperature during exposure of one testis to cooled saline at various temperatures by the method shown in Fig. 1. A indicates the point at which the testis was placed in the cooled saline, and B the point at which it was withdrawn. temperature of -6 or -8 C. for one hour ensures an intratesticular temperature of C. Figure 3 displays the intratesticular temperatures obtained by applying an ice-salt mixture direct to the scrotum, and it may be noticed here that the temperature within the testis is now approximately 1 to 12 C. above that of the freezing mixture applied to the scrotum. In other words, the presence of the scrotum ensures a temperature difference of 2 to 6 C.

6 21 MACDONALD & HARRISON Fertility & Sterility between its two surfaces, a degree of protective influence on the testis at low temperatures which cannot be ignored. This, furthermore, explains the necessity for application of lower temperatures to the testis within its scrotal sac in order to produce grades of degeneration comparable to those in the exposed testis. Thus, a temperature of 2 C. is reached in the testis only when an environmental temperature of _1 C. is applied to the 2 18 t B {- U 16 w 14 <{ a: 12 w..:1 «-' :::> -6 8 If) w!;( 6 : MINUTES Fig. 3. The intratesticular temperature during cooling of the scrotum by ice-saline mixture at various temperatures. A indicates the point at which cooling of the scrotum commenced, and B the point at which it ceased. scrotum for a period of approximately forty minutes, and histologically this testis shows severe spermatogenic damage. Since freezing a tissue induces pronounced vasoconstriction,2 and it has been established that ischemia effects marked degenerative changes in the seminiferous tubule,6, 7, 8, 13 it appeared probable that the degeneration seen after application of cold temperatures to the testis might be due to the resultant vasoconstriction. It was therefore decided to investigate the vascularization of the testis following its submission to a cold temperature.

7 Vol. 5, No.3, 1954 TEMPERATURE AND SPERMATOGENESIS 211 Testis Vascularization Experiments were performed in which one testis was removed from the scrotum and suspended in cold physiologic sodium chloride as in the first set of experiments. Temperatures of -2, -4 and _8 C. were used. Following subjection of the testis to these temperatures for a period of one hour, the rats were injected with Micropaque* through the thoracic aorta, at a pressure of 19 mm. Hg, prior to radiography. This substance, because of its particle size, will not pass through the capillary bed, and therefore Fig. 4. The radiographic appearance of the experimental (right) and ontrol (left) testes of a rat following injection of Micropaque into the thoracic aorta immediately after suspending the experimental testis in saline at a temperature of _2 C. for one hour. While the arterial system in the control testis is well injected, the finer intratesticular branches of the experimental testis are not shown, although the epididymal vessels are clearly visible. demonstrates only the arterial pattern. Injection was made immediately after withdrawing the experimental testis from its cold physiologic sodium chloride bath. A pressure of 19 mm. Hg was used in order to ensure as complete an injection as possible of the testicular artery and its branches. Since injection was made through the thoracic aorta, both experimental and control testes would be injected under similar conditions. In the experiment performed at _2 C., both experimental and control testes were well injected apart from the finer terminal intratesticular branches of the testicular artery in the experimental testis (Fig. 4). Fol* Damancy & Co. Ltd.

8 212 MACDONALD & HARRISON Fertility & Sterility lowing application of a temperature of _4 C., the testicular artery of the experimental testis is clearly not injected beyond the point in its intratesticular course at which it normally begins to give off its branches (Fig. 5). Injection of the thoracic aorta after application of a temperature of -8 C. displays, on subsequent radiography, that the radiopaque medium in the experimental testis has not progressed beyond the point at which the testicular artery curves posteriorly into the parenchyma of the testis (Fig. 6). In a further experiment in which the testis was exposed to _8 C. Fig. 5. The arterial system of experimental (left) and control (right) testes of a rat as shown by radiography following injection of Micropaque into the thoracic aorta immediately after suspending the experimental testis in saline at _4 C. for one hour. The arterial supply of the control testis is well injected, as are the vessels to the caput epididymidis on the experimental side. The testicular artery of the experimental testis is, however, not injected beyond the point in its intratesticular course at which it normally begins to give off branches. for one hour, injection of the thoracic aorta seven days later failed to fill the testicular artery in the experimental testis, although the control testis was easily injected. It can only be concluded, therefore, that the absence of filling of the testicular artery beyond the points mentioned in these cases, which may be due to vasoconstriction, must result iri ischemia of the testis. Whatever the etiology of this arterial block, the untoward results of application of cold temperatures to the testis could accrue from minor degrees of ischemia, since ischemia of only one hour's duration is sufficient to produce profound damage within the seminiferous tubule. III

9 Vol. 5, No.3, TEMPERATURE AND SPERMATOGENESIS DISCUSSION It has been clearly demonstrated that mature spermatozoa are able to survive submission to very low temperaturesy' 16, 17 This has been confirmed in our experiments since, even when the damage produced in the seminiferous tubules after application of cold temperatures had progressed to spermatogenic destruction, normal-staining mature spermatozoa could still be found in the epididymal tubules and, in some cases, within the lumina of the seminiferous tubules themselves. Fig. 6. The radiographic appearance of experimental (left) and control (right) testes of a rat after injection of Micropaque into the thoracic aorta immediately after suspending the experimental testis in saline at a temperature of _8 C. for one hour. The radiopaque medium in the experimental testis has not progressed beyond the point at which the testicular artery curves posteriorly into the testicular parenchyma. The control testis is very well injected, however. The process of spermatogenesis in the rat is clearly very susceptible to a low environmental temperature, and the experiments described in this communication provide an explanation for the discrepancies to be found in the literature. Thus, an intrascrotal temperature of 3 C. produced by spraying the rat's scrotum with 12 cc. ether for ten minutes daily over a period of two weeks,3 together with the fat-solvent action of the ether, might ehect mild spermatogenic damage which would progress to regeneration after two to four weeks. Preliminary experiments, in which 1 cc. ethyl chloride were sprayed on the rat scrotum over a period of six minutes, demonstrate that the intra

10 214 MACDONALD & HARRISON Fertility & Sterility testicular temperature falls to almost C. at the end of the experiment (Fig. 7). It should be noted, however,. that it is difficult to avoid anesthetizing the experimental animal to the point of death during this procedure using the quantities of volatile anesthetic recommended by Gronsky. It is also to be expected that ethyl chloride would have a greater effect in lowering intratesticular temperature than ether, since the latent heat of vaporization at 2 C. of the former is 91.9, and of the latter The lowest environmental temperature to which the rat scrotum was subjected by Phillips and McKenzie was 6 C., and this would be unlikely to produce Fig ,---<r , 14 1:5 12 =>" ';{ ::1 9 ::-: J B6 j:: 5 (/) ::-:4 :5 I- 2 1 OL---L2-34--L- MINUTES The intratesticular temperature during spraying of 1 cc. ethyl chloride on the rat scrotum over a period of six minutes. any damage, since the application of a temperature of -4 C. to the scrotum only effects an intratesticular temperature of 6 C. (Fig. 3), and this produced no damage whatever in the testis when applied for one hour. Moore 1o has shown that the application of heat to the scrotum of the guinea pig is effective in producing complete degeneration of the germinal epithelium at temperatures of 46 to 47 C. for ten minutes. This temperature is some 1 to 12 above that of the testis in the normal animaf Our experiments have shown that a temperature of _1 C. applied to the scrotum for one hour is necessary to produce a similar degree of damage. It is suggested, therefore, that the intrascrotal mammalian testis is able to withstand relatively large variations in environmental temperature. The scrotum

11 Vol. 5, Nc.. 3, 1954 TEMPERATURE AND SPERMATOGENESIS 215 itself acts as an insulator in this function, because of the temperature difference between its two surfaces. Also, a less degree of thermal variation when applied directly to the testis is necessary to cause spermatogenic degeneration. Thus, artificial cryptorchidism,l1 which would produce an increase in environmental temperature of only 8 to go c., and application of a temperature of only _6 C. for one hour, are capable of causing spermatogenic destruction in the rat. There is a suggestion in these experiments that some part of the structure of the testis has a thermoregulatory capacity. Thus, as may be seen from Fig. 2, the intratesticular temperature at an environmental temperature of _6 C. varies between and.5 C. after thirty minutes' application. Yet the intratesticular temperature following application of an environmental temperature of _8 C. does not fall lower than -.1 C. This cannot be due to the thermoregulatory function of the scrotum, since the variations in environmental temperature were applied direct to the testis. An arrangement which could effect this temperature regulation has already been shown to exist in the vessels supplying the testis, 8 the testicular artery and veins constituting a preheating and precooling mechanism. The intrinsic heat production of the testis itself cannot be ignored, however, and must playa large part in these phenomena. The clinical applications of this research are of some importance. Scrotal temperature in man is only 2.2 c., on an average below that of the abdomen.! It is possible, therefore, that less cooling of the human testis than in the rat may be effective in producing sterility. SUMMARY 1. Experiments have been performed in the rat in which low temperatures have been applied directly to the testis and to the scrotum. 2. Temperatures of -6 and _8 c., when applied directly to the testis for one hour, caused spermatogenic destruction, with no evidence or possibility of regeneration. S. A temperature of _1 C. applied to the scrotum for one hour is necessary to produce severe spermatogenic damage, from which some regeneration might occur. 4. The differences in these results can be accounted for by the protective influence of the scrotum and the effect of the two procedures on intratesticular temperature.

12 216 MACDONALD & HARRISON Fertility & Sterility 5. The untoward results of application of low temperatures on spermatogenesis are probably caused by ischemia, which in turn may be caused by vasoconstriction produced by the low temperature. REFERENCES 1. BADENOCH, A. W. Brit. M.I. 2:61, BELLMAN, S. Actaradiol.12:73, CRONSKY, N. Anat. Anz. 69:'228, li.arrison, R. G. ]. Anat. 83:267, HARRISON, R. G. Fertil. & Steril. 3:366, HARRISON, R. G., and OETTLE, A. C. Proc. Soc. Study of Fertil. 2:6, HARRISON, R. G., and WEINER, J. S. ]. Physiol. 17 :48, HARRISON, R. G., and WEINER, J. S. ]. Exper. Biol. 26:34, MACLEOD, J., and HOTCHKISS, R. S. Endocrinology 28:78, MOORE, C. R. Am. I. Anat. 34:337, MOORE, C. R. Am.]. Anat. 34:269, MOORE, C. R. In Allen, E. Sex and Internal Secretions. London, England, Bailliere, Tindall & Cox, OETTLE, A. C., and HARRISON, R. G. ]. Path. & Bact. 64:273, PARKES, A. S. Brit. M. I. 2:212, PmLLIPs, R. W., and MCKENZIE, F. F. Bull. Mo. Agric. Exper. Station. 217, POLGE, C. Nature, London 167:949, POLGE, C., SMITH, A. U., and PARKES, A. S. Nature, London 164:666, 1949.