Testicular Innervation is Necessary for the Response of Plasma Testosterone Levels to Acute Stress

Transcription

1 BOLOGY OF RPRODUCTON 24, (1981) Testicular nnervation is Necessary for the Response of Plasma Testosterone Levels to Acute Stress ARTHUR. FRANKL2 and THL L. RYAN Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York 1391 ABSTRACT At 1, 2, 4, and 7 weeks or 15 months postsurgery, bilateral denervation of the testes of mature male rats had no effect on basal levels of plasma testosterone, testicular weight, spermatogenesis, or mating behavior. However, bilateral denervation blocked an acute stress-induced rise of plasma testosterone. This is the first report of a possible role of testicular innervation in the mature male rat. NTRODUCTON Although there have been many studies of hormonal regulation of testosterone secretion (Hall, 197), the neural regulation of testicular secretions has received little attention (Hodson, 197). arly investigations suggested the possibility of a neural contribution to testosterone regulation. Atrophy of the testes, degeneration of the seminiferous epithelium, and hypertrophy of interstitial secretory tissue were reported following disruption of neural pathways innervating the testes in guinea pigs (Coujard, 1952), rabbits (Hodson, 1965), and dogs (Kuntz, 1919; Marconi, 1923). Similar studies were not made on the mature rat, although Bacq (1931) reported impotence in three rats subjected to bilateral sympathectomy despite the maintenance of normal testicular weight. These studies suggest that denervation of the testis might have an effect upon the interstitial cells of Leydig, which are primarily responsible for testosterone secretion, and thus indirectly suppress reproductive function. However, most reports have failed to show neural innervation of interstitial cells, except possibly in man, rhesus monkey, bull, and swan (Hodson, 197). Alternatively, neural regulation of steroidal levels might occur indirectly by affecting blood flow (Damber and Janson, 1978). n either case, the question whether testicular innervation is involved in the regulation of plasma testosterone concentration appears important. n this study, we have attempted to determine chronic effects of testicular denervation on basal levels of testosterone in the male rat. Since the concentration of testosterone and blood flow in the spermatic vein are altered in rats subjected to laparotomy (Damber and Janson, 1978), we also subjected rats with denervated testes to systemic stress to determine whether loss of innervation to the testis would affect secretion of testosterone. Based on the observation that sympathetic-adrenal modulation of testicular blood flow is regulated by an immediate neurally mediated component and a delayed component that is influenced by adrenal medullary secretion (Cross and Silver, 1962), we also removed the adrenal medulla in this study. Throughout, we restricted denervation to testicular parenchyma, innervated only by adrenergic nerves in the rat according to Kuntz (1953), leaving pelvic ganglia and nerves intact, so as to preserve innervation to sexual accessory organs and to the testicular capsule. MATRALS AND MTHODS Animals Accepted November 19, 198. Received September 5, 198. Supported in part by NH grants AG-1155 and HD-481. Reprint requests. The research was carried out on male Sprague- Dawley rats, purchased at 8 days of age from Russell Miller Farms (Cazenovia, NY) or on animals derived from this stock. They were singly caged in a room maintained on a 12L: 12D cycle (lights on at 6 h) at a temperature of 2 ± 1#{176}C, and fed Purina rat chow 491 on 29 June 218

2 492 FRANKL AND RYAN ad libitum. The rats were days of age at the initiation of the experiments. Surgery Rats were denervated while anesthetized with sodium pentobarbital. With assistance of a Zeiss dissection microscope, right and left spermatic plexi were separated from the testicular artery and aorta, and excised along with portions of the nerves. Proximal ends of the nerves were sutured to underlying musculature to discourage reinnervation. Shamdenervated rats were treated similarly with the exception of separation and excision of the nerves. The adrenal medulla was expressed by compression through a nick made in the gland. Other Procedures Blood samples ( ml) were collected into heparinized syringes by cardiac puncture within 2 mm from the time the rats were anesthetized with ether. Plasma was separated by centrifugation, frozen, and stored at -2#{176} C for subsequent assay. Testosterone was measured by a radioimmunoassay developed in this laboratory (Frankel et al., 1975). Statistical analysis was performed on data which were transformed to natural longarithms. Analysis of variance for repeated measures was used, followed by comparison within and among groups by the Newman-Keuls test (Keppel, 1973). Serial sections of the testes were prepared according to the Guillery method (Luna, 1968) and examined for nerve fiber degeneration. Representative sections were stained with periodic acid-schiff stain and examined for spermatogenesis by enumeration of stage 7 spermatids per Sertoli cell (Clermont and Morgentaler, 1955). xperimental Design The effect of bilateral denervation on basal levels of plasma testosterone was studied in a set of three experiments in each of which there were three groups: intact control, sham bilaterally denervated, and bilaterally denetvated. n the first experiment, blood was collected between h 1 week before and 1, 2, and 4 weeks after surgery. This study was repeated with blood collected 1 week prior to surgery and 7 weeks after surgery. A third study was made of sexually experienced rats, from which blood was collected 1 week before, and at 1 week, 2 weeks, and 15 months after surgery. Procedures by which animals received sexual experience have been described (Ryan and Frankel, 1978). Once a month following surgery, the males were placed in a mating arena with an estrous female for 45 mm and allowed to mate to ejaculation. n a second set of studies, the effect of laparotomy and repeated etherization was tested as a possible acute seressor upon plasma testosterone levels by studying five groups of rats which were subjected to 1) less than 2 mm anesthetization with ether, 2) 5 mm of anesthetization with ether, 3) laparotomy and 5 min of anesthetization with ether, 4) 3 mm anesthetization with ether, and 5) laparotomy and 3 mm of anesthetization with ether. Anesthetization was maintained by manipulation of an etherized nose cone; the rat never regained consciousness. Blood was collected by cardiac puncture between 1 and 113 h in all animals immediately after the test procedure. The 3 mm regimen of both stressors proved to provide the greatest effect upon plasma testosterone concentration and was utilized in a study of four groups: intact, sham-operated, bilaterally denervated, and bilaterally adrenodemedullated. Seven weeks after surgery, a basal blood sample was collected by cardiac puncture. Seven days later, the animals were acutely stressed, and a blood sample was taken immediately. RSULTS ffect of Bilateral Denervation on Basal Plasma Testosterone Concentration There was no significant change in plasma testosterone concentration during the first and second week after surgery in any group in the first study (Table 1). By the 4th week after surgery (Table 1), plasma testosterone levels were significantly lower in both sham-denervated and denervated rats (P<.1). Testicular weights of sham-denervated and denervated rats were similar (3..69 ±.7 g and 3.95 ±.11 g, respectively). Pyrid inc-silver strain of testicular sections (cut at 15,um) revealed degenerating nerve fibers in bilaterally denervated rats. Repetitive blood sampling was singled out as possibly depressing hormone levels during the first 7 week study. Consequently, in the second study only two blood collections were used. Plasma testosterone concentration was significantly (P<.5) lower 7 weeks after surgery in all three groups (Fig. 1); however, there was no effect of treatment. n the third study, initial levels of testosterone before treatment in intact, sham- and bilaterally denervated rats were 2.96 ±.84, 3.88 ± 1.6, and 3.49 ±.91 ng/ml, respectively. Plasma testosterone levels varied from each other during the first month but not significantly. By 15 months, they were significantly lower in all three groups (.99 ±.26,.84 ±.16, and.83 ±.15 ng/ml, respectively) (P<.1), but not significantly different from each other. Animals (n = 4) in all three groups mated to ejaculation within 3 mm up to 15 months after surgery. At that time, they were over 18 months old. Spermatogenesis was normal in both sham- and bilateeally denervated rats in which histological sections were prepared and examined. ffect of Bilateral Denervation on the Plasma Testosterone Response to Acute Stress Among the acutely stressed groups, both ether and combined ether plus laparotomy for a on 29 June 218

3 FFCT OF DNRVATD TSTS ON PLASMA TSTOSTRON 493 TABL 1. Chronic effect of bilateral denervation on basal levels of plasma testosterone.a Weeks before (-) or after (+) surgery Plasma testosterone (ng/ml ± SM) Control (9) Sham-Denervated (9) Denervated (1) 2.52 ± ± ± ± ± ± ± ± ± ± ± ±.29* adenervation or sham-denervation occurred on Day. Blood was collected by cardiac puncture from rats anesthetized with ether. Number within parentheses is number of animals within group. P<.1 in comparison with previous sample in same group. 3 mm period stimulated a significant rise of plasma testosterone concentration (Fig. 2). Since the combined stressors caused a more significant rise (P<.1), this treatment was chosen to challenge both the bilaterally denervated and bilaterally adrenodemedullated rats. There was a significant rise of plasma testosterone following application of the stressors to intact and sham-surgery groups (P<.5, Fig. 3). There was no rise in either the bilaterally denervated or bilaterally adrenodemedullated groups. DSCUSSON We conclude that bilateral denervation of the rat testis has no acute or chronic effect upon testicular weight, spermatogenesis, sexual behavior, or basal plasma testosterone levels. This is in marked contrast to data for guinea pigs, rabbits, and dogs. The lack of response in O BASAL, WK PRSURGRY n BASAL. 7WKSPOSTSURGRY z -, w C w z - C,) DNRVATD DNRVATD FG. 1. Plasma testosterone levels (ng/ml, mean ± SM) following bilateral denervation of the testes of mature male rats. Blood was collected by cardiac puncture within 2 mm of ether anesthesia, 1 week before, and 7 weeks after surgery. Number of animals within each group is indicated. Plasma testosterone levels fell significantly (P<.1) within each group at 7 weeks postsurgery, but there was no significant effect of treatment. -Y BASAL THR THR THR THR 5MN AND 3OMN AND FG. 2. Plasma testosterone levels (ng/ml, mean ± SM) following ether or combined effect of ether plus laparotomy for 5 or 3 min in intact male rats. Testosterone increased significantly (*P<.5, **P<.ol) 3 mm after stress compared with basal levels. Blood was collected by cardiac puncture within 2 mm of ether anesthesia. Number of animals within each group is indicated. LAP LAP on 29 June 218

4 494 FRANKL AND RYAN, C O BASAL THR AND LAPAROTOMY FOR 3 MN NTACT SHAM DN Au,N- SURGRY 6 #{149}1 6 DMD FG. 3. Plasma testosterone levels (ng/ml, mean ± SM) following 3 mm ether plus laparotomy 7 weeks after bilateral denervation or bilateral adrenal demedullation. Testosterone increased significantly (P<.5) compared with basal levels in intact and sham-surgery groups only. Blood was collected by cardiac puncture within 2 mm of ether anesthesia; basal levels were measured week prior to collection after stress. Number of animals within each group is indicated. the rat was observed in both young and old animals. Possibly, testicular damage in other species was due to back pressure, as a result of inhibition of sperm transit, following denervation of the testicular capsule and epididymides (Hodson, 1965). n our study, we did not disturb capsular innervation, which is thought to regulate contractions involved in propulsion of sperm following sperm iation (Davis et al., 197; Bell and McLean, 1973). t now is apparent that innervation plays a role in the testicular response to stress, and its absence blocks a sharp elevation of plasma testosterone concentrations following ether stress and laparotomy. This early elevation in response to ether is the beginning of a complex reaction to stress which is followed by a decrease in plasma testosterone levels in the rat within 4 h, and a restoration of normal levels within 8 h (Frankel and Wright, unpublished data). The response to ether previously has been noted as a single depression of testosterone levels in the rat testis within 5 mm (Fariss et al., 1969) or in the rat testicular vein blood within 1 or 2 h (Bardin and Peterson, 1967; Free et al., 198). However, the initial rise in peripheral testosterone levels within 1 mm in response to handling has been noted consistently in our laboratory (Kamel and Frankel, 1978), and it is associated with an earlier rise in plasma LH, as noted by Krulich et al. (1974), Turpen et al. (1976) and uker et al., (1975). The inability of the rat with bilaterally denervated testes to respond initially to ether and laparotomy is matched by the bilaterally adrenodemedullated rat. This suggests a possible role of catecholamines in the normal response. However, since catecholamines are,known to depress plasma testosterone concentration, and decrease testicular blood flow (Free, 1977), their involvement in what appears to be an LH-mediated event is unresolved. This is the first report to examine the #{149}- possible role of innervation on testicular function in the male rat. Despite a seemingly passive role in the regulation of basal plasma testosterone levels, and in maintenance of spermatogenesis, testicular weight, and sexual behavior, innervation is essential to at least one testicular function, i.e., the sudden rise of plasma testosterone levels in response to acute stress. nnervation may play a similar role in other acute responses of plasma testosterone to handling (Wright and Frankel, 1973), to sexual behavior (Kamel and Frankel, 1978), etc. We conclude that there are important neural elements in the regulation of the testis in the male laboratory rat. ACKNOWLDGMNTS We thank Dr. B. V. Cadwell, Yale University School of Medicine, for a generous donation of antibody to testosterone, and Mrs. M. R. Frankel for typing the manuscript. We are indebted to Dr. R. T. Hill, when at NH, for first suggesting to us that innervation might play some role in testicular regulation in the male rat. RFRNCS Bacq, Z. M. (1931). mpotence of the male rodent after sympathetic denervation of the genital organs. Am. J. Physiol. 96, Bardin, C. W. and Peterson, R.. (1967). Studies of androgen production by the rat: Testosterone and androstenedione content of blood. ndocrinology 8, Bell, C. and McLean, J. R. (1973). The autonomic innervation of the rat testicular capsule. J. Reprod. Fertil. 32, Clermont, Y. and Morgentaler, H. (1955). Quantitative study of spermatogenesis in the hypophysectomized rat. ndocrinology 57, Coujard, R. (1952). ffets sur c testicule et l epididyme de a phenolisation de fibres nerveuses. C. R. Soc. Biol. 146, Cross, B. A. and Silver,. A. (1962). Neurovascular control of oxygen tension in the testis and epididymis. J. Reprod. Fertil. 3, on 29 June 218

5 FFCT OF DNRVATD TSTS ON PLASMA TSTOSTRON 495 Damber, J.-. and Janson, P.. (1978). Testicular blood flow and testosterone concentrations in spermatic venous blood of anaesthetmzed rats. J. Reprod. Fertil. 52, Davis, J. R., Langford, C. A. and Kirby, P. J. (197). The testicular capsule. n: The Testis. Vol. 1. (A. D. Johnson, W. R. Gomes and N. L. Vandemark, eds.). Academic Press, New York. pp uker, J. S., Meites, J. and Riegle, G. D. (1975). ffects of acute stress on serum LH and prolactin in intact, castrate and dexamethasone-treated male rats. ndocrinology 96, Fariss, B. L., Hurley, T. J., Hane, S. and Forsham, P. H. (1969). Reduction of testicular testosterone in rats by ether anesthesia. ndocrinology 84, Frankel, A.., Mock,. J., Wright, W. W. and Kamel, F. (1975). Characterization and physiological validation of a radioimmunoassay for plasma testosterone in the male rat. Steroids 25, Free, M. J. (1977). Blood supply to the testis and its role in local exchange and transport of hormones. n: The Testis. VoL 4. (A. D. Johnson and W. R. Comes, eds.). Academic Press, New York. pp Free, M. J., Jaffe, R. A. and Cheng, H-C. (198). ffect of anesthetics on testosterone production in rats. J. Androl. 1, Hall, P. F. (197). ndocrinology of the testis. n: The Testis. Vol. 2. (A. D. Johnson, W. t. Gomes and N. L. Vandemark, eds.). Academic Press, New York. PP Hodson, N. (1965). Sympathetic nerves and reproductive organs in the male rabbit. J. Reprod. Fertil. 1, Hodson, N. (197). The nerves of the testis, epididymis, and scrotum. n: The Testis. Vol. 1. (A. D. Johnson, W. R. Comes and N. L. Vandemark, eds.). Academic Press, New York. pp Kamel, F. and Frankel, A.. (1978). Hormone release during mating in the male rat: Time course, relation to sexual behavior, and interaction with handling procedures. ndocrinology 13, Keppel, C. (1973). Design and Analysis. Prentice-Hall, nglewood Cliffs, NJ. Krulich, L., Hefco,., lner, P. and Read, C. B. (1974). The effects of acute stress on the secretion of LH, FSH, prolactin and GH in the normal male rat, with comments on their statistical evaluation. Neuroendocrmnology 16, Kuntz, A. (1919). xperimental degeneration in the testis of the dog. Anat. Rec. 17, Kuntz, A. (1963). The Autonomic Nervous System. 4th d. Lea and Febiger, Philadelphia. Luna, L. C. (1968), Manual of Histologic Staining Methods of the Armed Forces nstitute of Pathology. 3rd d. McGraw Hill, New York. p. 19. Marconi, P. (1923). Atrophic testiculaire par lesions nerveuses. C. R. Soc. Biol. 88, Ryan,. L. and Frankel, A. 1. (1978). Studies on the role of the medial preoptic area in sexual behavior and hormonal response to sexual behavior in the mature male laboratory rat. Biol. Reprod. 19, Turpen, C., Johnson, D. C. and Dunn, J. D. (1976). Stress-induced gonadotropmn and prolactin secretory patterns. Neuroendocrinology 2, Wright, W. W. and Frankel, A. 1. (1973). The effects of prehandling and repeated blood collection on plasma testosterone in the adult male rat. Am. Zool. 13, 1287 Abstr. on 29 June 218