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Effects of Sonication on Mature Rat Testes*†
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
Vol. 28, No.2, February 1977 Printed in U.S.A.
EFFECTS OF SONICATION ON MATURE RAT TESTES*t
ALAN DUMONTIER, B,S.t ALLAN BURDICK, PH,D,§ BERNARD EWIGMAN, B.S.:!: M. S. FAHIM, PH.D.~
Department of Obstetrics and Gynecology, School of Medicine, and Department of Biological Sciences, University of Missouri, Columbia, Missouri 65201
Exposure ofrat testes to high-frequency sound waves temporarily interrupted the spermatogenic process. Rats treated with 1 watt/sq cm for 10 minutes exhibited a degeneration ofadvanced germinal cells and were incapable ofimpregnating females for 150 days. Forty-eight hours after sonication, spermatocytes and spermatids developed irregular membranes and released their contents into the interstitium. Spermatogonia, Sertoli cells, and Leydig cells appeared normal, and no genetic anomalies could be detected in the progeny of treated animals. It was hypothesized that the reduction in sperm count was due to changes in membrane permeability which produced conditions unfavorable to maturation of testicular cells,
Scrotal testes of mammals are normally at a temperature several degrees (3" to 4° C) below that of the body core. In 1922, Crew 1 suggested that damage to the seminiferous epithelium of cryptorchid testes is the result of exposure of such testes to the higher intra-abdominal temperature. This suggestion has been supported by observations that intrascrotal testes exposed experimentally to elevated temperatures respond with rapid degeneration of the seminiferous epithelium. 2 - 4 However, not all of the cells degenerate during cryptorchidism. Sertoli cells, Leydig cells, and spermatogonia retain their morphology in cryptorchid testes. 5 Recently Fahim et al.,6 using various methods, exposed rat testes to above-normal temperatures
for short periods. The methods included hot-water baths, infrared spot-heating, microwave diathermy, and ultrasound. Results indicated that ultrasound was most effective in reducing sperm production at a lower temperature (3go C). Furthermore, the application of ultrasound was quick, painless, and easily administered. The use of ultrasound in medical diagnosis and therapy is accepted as an effective tool for many conditions. Ultrasound energy has a unique effect upon tissues because the sound waves selectively increase the temperature of areas difficult to heat by other means of diathermy. This thermal effect is greatest at interfaces between tissues of different acoustic impedance due to the scattering and conversion oflongitudinal sound waves into transverse components. 7, 8 Even relatively homogeneous material, such as testicular tissue, contains many interfaces which cause differential absorption and reflection of ultrasound energy. In addition to ultrasound's thermal characteristics, there are nonthermal effects which are thought to be both mechanical and chemical but are not clearly differentiated. 9 The study reported here was undertaken in order to examine the effects of ultrasound on the morphology and function of mature rat testicular cells.
Accepted August 18, 1976. *Supported in part by Whitewater Electronics. tPresented at the American Medical Student AssociationUniversity of Texas Medical Branch National Research Forum, April 1976, Galveston, Tex. :!:Medical student who presented the paper at the AMSAUTMB National Research Forum. §Professor of Genetics, Department of Biological Sciences. ~Professor and Chief, Section of Reproductive Biology. To whom reprint requests should be addressed at Department of Obstetrics and Gynecology, University Medical Center, Columbia, Mo. 62501.
195
196
DUMONTIER ET AL.
February 1977
FIG. 1. Apparatus designed for administering ultrasound to the testes of small animals. Sound waves emitted from the transducer at the base of the cup are mediated via distilled water before radiating the testes.
FIG. 2. Epon section of a seminiferous tubule from an untreated testis. Along the basement membrane are spermatogonia and Sertoli cells. Adjacent to these cells are the large nuclei of spermatocytes and, closer to the lumen, there are numerous spermatids. (x 1,540).
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
MATERIALS AND METHODS
Eighty sexually mature male rats were anesthetized and their scrotums, containing both testes, were immersed in a 2-inch cylinder filled with water. The base of the cylinder consisted of an ultrasound transducer which emitted high frequency sound waves at 1,100,000 cps. Controls were treated in the same matter, but they were not exposed to ultrasound. The rats were divided into groups A and B. Group A received 1 wattlsq cm for 5 minutes. Group B received 1 watt/sq cm for 10 minutes. Six rats from each group were selected for ultrastructural examination. They were killed after 4 hours, 48 hours, 10 days, 20 days, 30 days, and 60 days, and their testes were prepared for electron microscopy. Fertility and progeny tests were performed on the remaining animals. Each fertility-tested male rat was placed with a proestrous female 24 hours after treatment. The females were replaced every 7 days until pregnancy occurred. The end-point for fertility was the length of time required for every surviving male in the treatment group to impregnate a female. Hematoxylin and eosinstained sections of the testes, seminal vesicles, and prostate were studied under a light microscope. The size and weight of the sex organs were
197
compared with those of controls. Animals were killed 2, 4, and 6 months after ultrasound treatment. The progeny of treated males was examined for indications of genetic damage. Inbreeding and outbreeding experiments involving three males and three females from each litter were carried out for two generations. To measure the temperature within the testis, the scrotum was cooled to 28" C by cold water application before ultrasound treatment was started. Because of possible damage to the testes resulting from thermocouple needle puncture, one testis was employed for temperature measurements, and the contralateral testis was used to study the histologic effects of heat treatment. In order to expose rat testes only, a Plexiglas restraining table with multiple adjustments for animal size and testicular position was built (Fig. 1). A lubricant was applied to the scrotum before ultrasound treatment. Rectal temperature and scrotal temperature were recorded by rectal telethermometer and digital thermometer. Animals were anesthetized with an intraperitoneal injection of Nembutal, 15 mg/kg, before ultrasound application.
FIG. 3. Paraplast section of a seminiferous tubule 48 hours after sonication. Intact spermatogonia and Sertoli cells can be seen along the basement membrane, while more advanced cells toward the center of this tubule have lost their integrity (x 578).
198
DUMONTIER ET AL.
February 1977
FIG. 4. Electron micrograph of a portion of a seminiferous tubule from an ~treated rat: Early spe~atid~ (St) occupy this entire section. Note that the lumen
Preparation of Testes for Light Microscopy. Rats were killed by cervical dislocation and their testes were immediately removed and immersed in Bouin's fixative for 48 hours. The testes were subsequently transferred to 70% ethanol and embedded in Paraplast. Sections (4 /Lm) were cut and stained with hemotoxylin and eosin. Preparation of Testes for Electron Microscopy. Testes were removed from rats killed by cervical dislocation, and the seminiferous tubules were chopped into 3-mm lengths. The tubules were fixed first with 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 hour. This was followed by a I-hour postfixation in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Tubules were dehydrated with ethanol and embedded in Epon 812, according to standard procedures. When the blocks had hardened, thick sections were cut and stained with toluidine blue for examination under the light microscope. Thin sections were stained with uranyl acetate for 15 minutes, followed by lead citrate for 5 minutes. These sections were studied and photographed in an RCA-3 electron microscope.
RESULTS
Fertility and Progeny Test. Testicular cells of rats treated with 1 wattJsq cm for 5 minutes (group A) exhibited no detectable structural anomalies and appeared similar to cells shown in Figure 2. However, such males did not impregnate females until after 40 days. In contrast, advanced germinal epithelial cells were absent 48 hours after treatment in approximately 35% of seminiferous tubules of group B rats treated with 1 wattJsq cm for 10 minutes (Fig. 3). These males did not impregnate the females until after 150 days. The teratology, sex ratio, and size oflitters were noted in the progeny tests. After three generations of matings, neither group A nor group B progeny showed any signs of genetic abnormalities. Characteristics of Group B Testes. There was no significant difference between treated and control animals in the size of testis, epididymis, prostate, or seminal vesicle. The intratesticular temperature after 10 minutes of ultrasound was 39 ± o.r C.
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
199
FIG.5. High magnification of a spermatid 48 hours after sonication. The shape ofthe nucleus W) is irregular, and arrows indicate regions of "flaking" on the cytoplasmic membrane. Note the debris within the lumen CLU) of the tubule (x 14,760).
FIG. 6. High magnification of a spermatid nucleus (St) 48 hours after sonication. The cytoplasm has diffused into the lumen of the seminiferous tubule, and the nuclear membrane is beginning to disintegrate (arrows) (x 17,306).
200
DUMONTIER ET AL.
February 1977
FIG. 7. Electron micrograph of a portion of a seminiferous tubule 20 days after sonication. Sertoli cell nuclei (Se) can be seen surrounded by an indeterminate amount of cytoplasm. These cells appear to engulf cell debris and digest it via the action of lysosomes (L) (x 5,387).
FIG. 8. Light micrograph of a seminiferous tubule 60 days after sonication. Every cell type is present at this time (1,540).
Vol. 28, No.2
EFFECTS OF SONH;ATION ON MATURE RAT TESTES
201
FIG. 9. Electron micrograph ofthe seminiferous tubule shown in Figure 8. Note the Sertoli cell nuclei (Se), abnormally maturing spermatid (Z), and bodies which resemble lysosomes eL). There is still a great amount of cell debris within the tubule, and cell boundaries are poorly delineated (x 5,387).
Examination with the electron microscope revealed no structural alterations of the seminiferous tubules 4 hours after sonication. However, between 4 and 48 hours after treatment, the membranes of advanced testicular cells (spermatids and spermatocytes) lost their integrity, and cells exhibited highly irregular shapes. Plasma membranes developed leaks, and cytoplasm escaped into the interstitial area. Discontinuous layers of membrane developed along the plasmalemma, giving it a "flaky" appearance. Endoplasmic reticulum swelled, as did mitochondria, which exhibited irregular cristae (Figs. 4 and 5). In some cases the cytoplasm was completely stripped away from the nucleus (Fig. 6). The preservation of stem cells and Sertoli cells appeared to be responsible for reversing the sterilizing effects of ultrasound. The majority of Sertoli cells remained intact and were predominant 20 days after treatment. This predominance may have been due to a proliferation of cells or to cell growth, which is manifested in an increased amount of cytoplasm. Twenty days after treatment, large amounts of cell debris could be seen enveloped by the cytoplasm of Sertoli cells. Also within the Sertoli cell cytoplasm at that time were
numerous large bodies resembling lysosomes (Fig. 7). Sixty days after sonication there was still a great deal of cell debris contained within the Sertoli cells (Figs. 8 and 9). Maturing spermatids were present along with the earlier cell types; however, the shape of the advanced spermatids did not correspond to their degree of nuclear condensation, giving these cells an atypical morphology. So, although all of the cell types seemed to be present after 60 days, the tubules were not capable for several more months of producing viable spermatozoa in enough quantities to impregnate females. It was previously determined in this laboratory that blood testosterone levels are not affected by sonication at either 1 watt/sq cm or 2 watts/sq cm for 10 minutes. 6 As might be expected, no significant decrease in the Leydig cell population could be detected in any of our samples, as represented by Figure 10. DISCUSSION
Setchell lO noted that substances passing from the blood into the seminiferous tubules must pass
202
DUMONTIER ET AL.
February 1977
FIG. 10. A, Light micrograph of a section from a control testis. Leydig cells can be seen between the cross-sections of seminiferous tubules. B, Light micrograph of a sonicated testis. Lumina of seminiferous tubules have been enlarged with the deterioration of advanced germinal epithelium, but the Leydig cell population appears to be unaffected (A and B, x 184).
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
through the walls of the testicular capillaries and then either through the tubular wall or into the rete testis and hence into the tubular lumen. Transport through the capillary wall is passive, but is comparatively unrestricted. In contrast, there is great variation in the rate at which substances enter the seminiferous tubules and, for some substances, specific transport systems may be involved. Water, ethanol, some steroids, and a-chlorohydrin enter readily; glucose and 3-0methylglucose are transported rapidly into the tubules by facilitated diffusion. Ions enter more slowly than water, but at appreciable rates, and the rate of entry of rubidium is reduced by ouabain. Cholesterol; carbohydrates such as sucrose, inositol, and inulin; amino acids; and proteins enter only very slowly. But follicle-stimulating hormone, luteinizing hormone, and growth hormones appear to enter more quickly than other proteins. Several studies have demonstrated an increased permeability of tissues in live animals and in isolated frog skin when subjected to ultrasound treatment.u- 13 Lehman and Krusen 14 reported that the increased permeability of biologic membranes was also facilitated by an additional stirring action elicited by ultrasonic energy.15 As a result, the diffusion potential increased to a point where there was no longer a selective movement of ions across membranes. It was further noted by Lehman and Biegler15 that an alteration in the charge of proteins as well as the isoelectric point of cell membranes occurred. These reactions persisted long after the application of ultrasound. When Lee 16 incubated testicular cell preparations at 37" C, he observed leakage of cytoplasmic constituents, primarily from spermatocytes and spermatids. A concurrent release of hydrolytic enzymes resulted from heating the lysosomal membrane and did not appear to initiate the cytoplasmic leakage. Lee hypothesized that the change in permeability of advanced cell membranes was the mechanism for cessation of spermatogenesis in cryptorchid testes. Our results were similar to Lee's in that membranes of advanced cells exposed to ultrasound were no longer capable of containing cytoplasmic constituents. Sertoli cells, Leydig cells, and spermatogonia retained their morphologic integrity. Differences in the composition of germinal epithelium might explain the specific effects produced by ultrasound in certain cell types. During maturation of the testes, when advanced cells begin to appear, there are marked alterations
203
of the fatty acid composition in the total lipid. 17. 18 It is known that more unsaturated fatty acids are present in testicular lipids than in other tissues of the rat.17 The evidence suggests that ultrasound does not immediately break down cell membranes. If this were the case, lesions would be detectable within 4 hours after treatment. Findings also indicate that changes elicited by ultrasound are irreversible over a long period. It is our hypothesis that ultrasound affects cell homeostasis within the seminiferous tubule by altering membrane permeability in certain cells, including those involved in the blood-testis barrier. Such an alteration in permeability would cause an ion imbalance which might produce the deterioration of the spermatogenic process seen 20 and 60 days after treatment. There were no indications in this study that ultrasound caused alterations in the genetics of germinal epithelial cells. This is in agreement with the findings of Lyon and Simpson,19 who subjected the gonads of male and female mice to pulsed and continuous ultrasound at 1.6 watts/sq cm for 15 minutes.
REFERENCES 1. Crew FAE: A suggestion as to the cause of the aspermatic
2. 3.
4.
5.
6.
7.
8. 9.
10.
condition of the imperfectly descended testis. J Anat 56: 98, 1922 Fukui N: Action of body temperature on the testicle. Jap Med World 3:160, 1923 Moore CR: Properties of the gonads as controllers of somatic and psychical characteristics. VIII. Heat application and testicular degeneration: the function of scrotum. Am J Anat 34:337,1924 Young WC: The influence of high temperature on the guinea-pig testes: histological changes and effects on reproduction. J Exp Zool 49:459, 1927 Vandemark ML, Free MJ: Temperature effects. In The Testis, Vol 3, Edited by AD Johnson, WR Gomes, ML Vandemark. New York, Academic Press, 1970, p 233 Fahim MS, Fahim Z, Der R, Hall DG, Harman J: Heat in male contraception (hot water 60' C, infrared, microwave, and ultrasound). Contraception 11:549, 1975 Horvath J: Experimentelle Untersuchungen uber die Verteilung der ultraschall Energie in monschlichen Gewebe. Aerztl Forsch 1:357, 1947 Fischer E: Basic biological effects of ultrasonic energy. Am J Phys Med 33:174,1954 Lehman JF: Ultrasound therapy. In Therapeutic Heat and Cold, Edited by S Licht, HL Kamenetz. Baltimore, Waverly Press, 1972, p 321 Setchell BP: The entry of substances into the seminiferous tubules. In Male Fertility and Sterility, Edited by RE Mancini, L Martini. New York, Academic Press, 1974, p 37
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11. Lehman J, Becker G: Uber die permeabilitatssteigernde Wirkung von Ultraschallwellen auf biologische Membranen. Strahlentherapie 79:553, 1949 12. Lehman J, Becker G, Jaenicke W: Uber die Wirkung von Ultraschall-Wellen auf den Ionendurchtritt durch biologische Membranen als Beitrag zur Theorie des therapeuticschen Wirkungsmechanismus. Strahlentherapie 83:311, 1950 13. Lehman J, Becker G: Uber histologische Veranderungen nach Ultraschall und anderen Physikalischen Einwirkungen bei Permeabilitatsuntersuchungen an der Froschhaut. Strahlentherapie 84:306, 1951 14. Lehman JF, Krusen FH: Effect of pulsed and continuous application of ultrasound on transport of ions through biologic membranes. Arch Phys Med Rehab 35:20, 1954
February 1977 15. Lehman JF, Biegler R: Changes of potentials and temperature gradients in membranes caused by ultrasound. Arch Phys Med Rehab 35:287,1954 16. Lee LPK: Temperature effect on the permeability ofplasrna membranes of advanced germinal cells of the rat testis. Can J Biochem 52:586, 1973 17. Davis JT, Bridges RB, Coniglio JG: Changes in lipid composition of the maturing rat testis. Biochem J 98:342, 1966 18. Oshima M, Carpenter MP: The lipid composition of the prepubertal and adult rat testis. Biochim Biophys Acta 152:479, 1968 19. Lyon MF, Simpson GM: An investigation into the possible genetic hazards of ultrasound. Br J Radiol 47:712, 1974
Vol. 28, No.2, February 1977 Printed in U.S.A.
EFFECTS OF SONICATION ON MATURE RAT TESTES*t
ALAN DUMONTIER, B,S.t ALLAN BURDICK, PH,D,§ BERNARD EWIGMAN, B.S.:!: M. S. FAHIM, PH.D.~
Department of Obstetrics and Gynecology, School of Medicine, and Department of Biological Sciences, University of Missouri, Columbia, Missouri 65201
Exposure ofrat testes to high-frequency sound waves temporarily interrupted the spermatogenic process. Rats treated with 1 watt/sq cm for 10 minutes exhibited a degeneration ofadvanced germinal cells and were incapable ofimpregnating females for 150 days. Forty-eight hours after sonication, spermatocytes and spermatids developed irregular membranes and released their contents into the interstitium. Spermatogonia, Sertoli cells, and Leydig cells appeared normal, and no genetic anomalies could be detected in the progeny of treated animals. It was hypothesized that the reduction in sperm count was due to changes in membrane permeability which produced conditions unfavorable to maturation of testicular cells,
Scrotal testes of mammals are normally at a temperature several degrees (3" to 4° C) below that of the body core. In 1922, Crew 1 suggested that damage to the seminiferous epithelium of cryptorchid testes is the result of exposure of such testes to the higher intra-abdominal temperature. This suggestion has been supported by observations that intrascrotal testes exposed experimentally to elevated temperatures respond with rapid degeneration of the seminiferous epithelium. 2 - 4 However, not all of the cells degenerate during cryptorchidism. Sertoli cells, Leydig cells, and spermatogonia retain their morphology in cryptorchid testes. 5 Recently Fahim et al.,6 using various methods, exposed rat testes to above-normal temperatures
for short periods. The methods included hot-water baths, infrared spot-heating, microwave diathermy, and ultrasound. Results indicated that ultrasound was most effective in reducing sperm production at a lower temperature (3go C). Furthermore, the application of ultrasound was quick, painless, and easily administered. The use of ultrasound in medical diagnosis and therapy is accepted as an effective tool for many conditions. Ultrasound energy has a unique effect upon tissues because the sound waves selectively increase the temperature of areas difficult to heat by other means of diathermy. This thermal effect is greatest at interfaces between tissues of different acoustic impedance due to the scattering and conversion oflongitudinal sound waves into transverse components. 7, 8 Even relatively homogeneous material, such as testicular tissue, contains many interfaces which cause differential absorption and reflection of ultrasound energy. In addition to ultrasound's thermal characteristics, there are nonthermal effects which are thought to be both mechanical and chemical but are not clearly differentiated. 9 The study reported here was undertaken in order to examine the effects of ultrasound on the morphology and function of mature rat testicular cells.
Accepted August 18, 1976. *Supported in part by Whitewater Electronics. tPresented at the American Medical Student AssociationUniversity of Texas Medical Branch National Research Forum, April 1976, Galveston, Tex. :!:Medical student who presented the paper at the AMSAUTMB National Research Forum. §Professor of Genetics, Department of Biological Sciences. ~Professor and Chief, Section of Reproductive Biology. To whom reprint requests should be addressed at Department of Obstetrics and Gynecology, University Medical Center, Columbia, Mo. 62501.
195
196
DUMONTIER ET AL.
February 1977
FIG. 1. Apparatus designed for administering ultrasound to the testes of small animals. Sound waves emitted from the transducer at the base of the cup are mediated via distilled water before radiating the testes.
FIG. 2. Epon section of a seminiferous tubule from an untreated testis. Along the basement membrane are spermatogonia and Sertoli cells. Adjacent to these cells are the large nuclei of spermatocytes and, closer to the lumen, there are numerous spermatids. (x 1,540).
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
MATERIALS AND METHODS
Eighty sexually mature male rats were anesthetized and their scrotums, containing both testes, were immersed in a 2-inch cylinder filled with water. The base of the cylinder consisted of an ultrasound transducer which emitted high frequency sound waves at 1,100,000 cps. Controls were treated in the same matter, but they were not exposed to ultrasound. The rats were divided into groups A and B. Group A received 1 wattlsq cm for 5 minutes. Group B received 1 watt/sq cm for 10 minutes. Six rats from each group were selected for ultrastructural examination. They were killed after 4 hours, 48 hours, 10 days, 20 days, 30 days, and 60 days, and their testes were prepared for electron microscopy. Fertility and progeny tests were performed on the remaining animals. Each fertility-tested male rat was placed with a proestrous female 24 hours after treatment. The females were replaced every 7 days until pregnancy occurred. The end-point for fertility was the length of time required for every surviving male in the treatment group to impregnate a female. Hematoxylin and eosinstained sections of the testes, seminal vesicles, and prostate were studied under a light microscope. The size and weight of the sex organs were
197
compared with those of controls. Animals were killed 2, 4, and 6 months after ultrasound treatment. The progeny of treated males was examined for indications of genetic damage. Inbreeding and outbreeding experiments involving three males and three females from each litter were carried out for two generations. To measure the temperature within the testis, the scrotum was cooled to 28" C by cold water application before ultrasound treatment was started. Because of possible damage to the testes resulting from thermocouple needle puncture, one testis was employed for temperature measurements, and the contralateral testis was used to study the histologic effects of heat treatment. In order to expose rat testes only, a Plexiglas restraining table with multiple adjustments for animal size and testicular position was built (Fig. 1). A lubricant was applied to the scrotum before ultrasound treatment. Rectal temperature and scrotal temperature were recorded by rectal telethermometer and digital thermometer. Animals were anesthetized with an intraperitoneal injection of Nembutal, 15 mg/kg, before ultrasound application.
FIG. 3. Paraplast section of a seminiferous tubule 48 hours after sonication. Intact spermatogonia and Sertoli cells can be seen along the basement membrane, while more advanced cells toward the center of this tubule have lost their integrity (x 578).
198
DUMONTIER ET AL.
February 1977
FIG. 4. Electron micrograph of a portion of a seminiferous tubule from an ~treated rat: Early spe~atid~ (St) occupy this entire section. Note that the lumen
Preparation of Testes for Light Microscopy. Rats were killed by cervical dislocation and their testes were immediately removed and immersed in Bouin's fixative for 48 hours. The testes were subsequently transferred to 70% ethanol and embedded in Paraplast. Sections (4 /Lm) were cut and stained with hemotoxylin and eosin. Preparation of Testes for Electron Microscopy. Testes were removed from rats killed by cervical dislocation, and the seminiferous tubules were chopped into 3-mm lengths. The tubules were fixed first with 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 hour. This was followed by a I-hour postfixation in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Tubules were dehydrated with ethanol and embedded in Epon 812, according to standard procedures. When the blocks had hardened, thick sections were cut and stained with toluidine blue for examination under the light microscope. Thin sections were stained with uranyl acetate for 15 minutes, followed by lead citrate for 5 minutes. These sections were studied and photographed in an RCA-3 electron microscope.
RESULTS
Fertility and Progeny Test. Testicular cells of rats treated with 1 wattJsq cm for 5 minutes (group A) exhibited no detectable structural anomalies and appeared similar to cells shown in Figure 2. However, such males did not impregnate females until after 40 days. In contrast, advanced germinal epithelial cells were absent 48 hours after treatment in approximately 35% of seminiferous tubules of group B rats treated with 1 wattJsq cm for 10 minutes (Fig. 3). These males did not impregnate the females until after 150 days. The teratology, sex ratio, and size oflitters were noted in the progeny tests. After three generations of matings, neither group A nor group B progeny showed any signs of genetic abnormalities. Characteristics of Group B Testes. There was no significant difference between treated and control animals in the size of testis, epididymis, prostate, or seminal vesicle. The intratesticular temperature after 10 minutes of ultrasound was 39 ± o.r C.
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
199
FIG.5. High magnification of a spermatid 48 hours after sonication. The shape ofthe nucleus W) is irregular, and arrows indicate regions of "flaking" on the cytoplasmic membrane. Note the debris within the lumen CLU) of the tubule (x 14,760).
FIG. 6. High magnification of a spermatid nucleus (St) 48 hours after sonication. The cytoplasm has diffused into the lumen of the seminiferous tubule, and the nuclear membrane is beginning to disintegrate (arrows) (x 17,306).
200
DUMONTIER ET AL.
February 1977
FIG. 7. Electron micrograph of a portion of a seminiferous tubule 20 days after sonication. Sertoli cell nuclei (Se) can be seen surrounded by an indeterminate amount of cytoplasm. These cells appear to engulf cell debris and digest it via the action of lysosomes (L) (x 5,387).
FIG. 8. Light micrograph of a seminiferous tubule 60 days after sonication. Every cell type is present at this time (1,540).
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EFFECTS OF SONH;ATION ON MATURE RAT TESTES
201
FIG. 9. Electron micrograph ofthe seminiferous tubule shown in Figure 8. Note the Sertoli cell nuclei (Se), abnormally maturing spermatid (Z), and bodies which resemble lysosomes eL). There is still a great amount of cell debris within the tubule, and cell boundaries are poorly delineated (x 5,387).
Examination with the electron microscope revealed no structural alterations of the seminiferous tubules 4 hours after sonication. However, between 4 and 48 hours after treatment, the membranes of advanced testicular cells (spermatids and spermatocytes) lost their integrity, and cells exhibited highly irregular shapes. Plasma membranes developed leaks, and cytoplasm escaped into the interstitial area. Discontinuous layers of membrane developed along the plasmalemma, giving it a "flaky" appearance. Endoplasmic reticulum swelled, as did mitochondria, which exhibited irregular cristae (Figs. 4 and 5). In some cases the cytoplasm was completely stripped away from the nucleus (Fig. 6). The preservation of stem cells and Sertoli cells appeared to be responsible for reversing the sterilizing effects of ultrasound. The majority of Sertoli cells remained intact and were predominant 20 days after treatment. This predominance may have been due to a proliferation of cells or to cell growth, which is manifested in an increased amount of cytoplasm. Twenty days after treatment, large amounts of cell debris could be seen enveloped by the cytoplasm of Sertoli cells. Also within the Sertoli cell cytoplasm at that time were
numerous large bodies resembling lysosomes (Fig. 7). Sixty days after sonication there was still a great deal of cell debris contained within the Sertoli cells (Figs. 8 and 9). Maturing spermatids were present along with the earlier cell types; however, the shape of the advanced spermatids did not correspond to their degree of nuclear condensation, giving these cells an atypical morphology. So, although all of the cell types seemed to be present after 60 days, the tubules were not capable for several more months of producing viable spermatozoa in enough quantities to impregnate females. It was previously determined in this laboratory that blood testosterone levels are not affected by sonication at either 1 watt/sq cm or 2 watts/sq cm for 10 minutes. 6 As might be expected, no significant decrease in the Leydig cell population could be detected in any of our samples, as represented by Figure 10. DISCUSSION
Setchell lO noted that substances passing from the blood into the seminiferous tubules must pass
202
DUMONTIER ET AL.
February 1977
FIG. 10. A, Light micrograph of a section from a control testis. Leydig cells can be seen between the cross-sections of seminiferous tubules. B, Light micrograph of a sonicated testis. Lumina of seminiferous tubules have been enlarged with the deterioration of advanced germinal epithelium, but the Leydig cell population appears to be unaffected (A and B, x 184).
Vol. 28, No.2
EFFECTS OF SONICATION ON MATURE RAT TESTES
through the walls of the testicular capillaries and then either through the tubular wall or into the rete testis and hence into the tubular lumen. Transport through the capillary wall is passive, but is comparatively unrestricted. In contrast, there is great variation in the rate at which substances enter the seminiferous tubules and, for some substances, specific transport systems may be involved. Water, ethanol, some steroids, and a-chlorohydrin enter readily; glucose and 3-0methylglucose are transported rapidly into the tubules by facilitated diffusion. Ions enter more slowly than water, but at appreciable rates, and the rate of entry of rubidium is reduced by ouabain. Cholesterol; carbohydrates such as sucrose, inositol, and inulin; amino acids; and proteins enter only very slowly. But follicle-stimulating hormone, luteinizing hormone, and growth hormones appear to enter more quickly than other proteins. Several studies have demonstrated an increased permeability of tissues in live animals and in isolated frog skin when subjected to ultrasound treatment.u- 13 Lehman and Krusen 14 reported that the increased permeability of biologic membranes was also facilitated by an additional stirring action elicited by ultrasonic energy.15 As a result, the diffusion potential increased to a point where there was no longer a selective movement of ions across membranes. It was further noted by Lehman and Biegler15 that an alteration in the charge of proteins as well as the isoelectric point of cell membranes occurred. These reactions persisted long after the application of ultrasound. When Lee 16 incubated testicular cell preparations at 37" C, he observed leakage of cytoplasmic constituents, primarily from spermatocytes and spermatids. A concurrent release of hydrolytic enzymes resulted from heating the lysosomal membrane and did not appear to initiate the cytoplasmic leakage. Lee hypothesized that the change in permeability of advanced cell membranes was the mechanism for cessation of spermatogenesis in cryptorchid testes. Our results were similar to Lee's in that membranes of advanced cells exposed to ultrasound were no longer capable of containing cytoplasmic constituents. Sertoli cells, Leydig cells, and spermatogonia retained their morphologic integrity. Differences in the composition of germinal epithelium might explain the specific effects produced by ultrasound in certain cell types. During maturation of the testes, when advanced cells begin to appear, there are marked alterations
203
of the fatty acid composition in the total lipid. 17. 18 It is known that more unsaturated fatty acids are present in testicular lipids than in other tissues of the rat.17 The evidence suggests that ultrasound does not immediately break down cell membranes. If this were the case, lesions would be detectable within 4 hours after treatment. Findings also indicate that changes elicited by ultrasound are irreversible over a long period. It is our hypothesis that ultrasound affects cell homeostasis within the seminiferous tubule by altering membrane permeability in certain cells, including those involved in the blood-testis barrier. Such an alteration in permeability would cause an ion imbalance which might produce the deterioration of the spermatogenic process seen 20 and 60 days after treatment. There were no indications in this study that ultrasound caused alterations in the genetics of germinal epithelial cells. This is in agreement with the findings of Lyon and Simpson,19 who subjected the gonads of male and female mice to pulsed and continuous ultrasound at 1.6 watts/sq cm for 15 minutes.
REFERENCES 1. Crew FAE: A suggestion as to the cause of the aspermatic
2. 3.
4.
5.
6.
7.
8. 9.
10.
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