Exp. Geront. Vol. 14, pp. 193-199.
0531-5565/79/0801-0193502.00/0
~" Pergamon Press Ltd. 1979. Printed in Great Britain.
RELATIONSHIP BETWEEN SERUM CONCENTRATION OF GONADOTROPINS AND TESTOSTERONE IN HEATEXPOSED AGEING MALE RATS E. BEDRAK, Z. CHAP AND K. FRIED Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva84120, Israel (Received 7 January 1979)
INTRODUCTION AGEING, inter alia, goes hand in hand with decreased pituitary-testicular function and ultimately results in lower concentrations of circulating gonadotropins and testosterone (Ghanadian et al., 1976; Riegle and Meites, 1976; Chan et al., 1977; Gray, 1978). Exposure of intact sexually mature young males to elevated ambient temperature is also associated with lower testosterone production in the ram (Gomes et al., 1971), the bull (Rhynes and Ewing, 1973) and the rat (Bedrak et al., 1973). Recent findings (Bedrak and Chap, personal observations) indicated that the decrease in plasma testosterone concentration observed in heat-exposed rats was coupled with a transient decline in plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH). Moreover, heat-exposure inflicted histological damage to the testis and enhanced the activity of most testicular enzymes, except for 1713hydroxysteroid oxidoreductase (1713-HSD), synthesizing testosterone in the rat (Bedrak et al., 1971 ; 1973) and the mouse (Bedrak, 1972, 1974; Slonim and Bedrak, 1977). Cooling the testes eliminated the histological damage observed in the mouse, but the changes in the activity of testicular enzymes persisted (Chap et al., 1977). It was noted that the changes in the activity of the enzymes associated with androgen production by the testes of aged mice were similar to those observed in young, sexually mature heat-exposed mice (Bedrak, 1974; Slonim and Bedrak, 1977; Chap and Bedrak, 1977; Chap et aL, 1977). In view of this, it was suggested that prolonged exposure of the intact mammal to elevated ambient temperatures might interfere with normal testicular function by accelerating the disorganization of the enzyme synthesizing processes--a phenomenon which characterizes senescence. Since testicular function is controlled by the gonadotropins, it is important to establish the relationship between serum concentration of testosterone and gonadotropins in heat-exposed animals and correlate it with the activity of 1713-HSD. This is imperative in order to clarify whether heat-exposure also induces alteration in the pituitary function, comparable to those accompanying the process of ageing, or whether it is limited to the testes. MATERIALS AND M E T H O D S Animals
Three-month-old Wistar male rats from the colony of the Department of Hormone Research, Weizman Institute of Science, were used. They were kept either in a control (C) environment (20-22°C, 30-50~o relative humidity) or were exposed to heat (33--35°C,25-40~ relative humidity) for various durations (HA), as indicated in the individual experiments. The animals used for experimentation with hypothalamic disconnection, complete hypothalamic deafferentation (CHD), were 3.5-month-old rats obtained from the colony of the Hebrew University, Jerusalem. Deafferentation was performed by means of a stereotoxic instrument under anesthesia (Hal[tsz and Pupp, 1965). They were allowed 3 days for recovery before being used for experiments. Tap water and rat chow 193
194
E. B E D R A K , Z . C H A P A N D K . F R I E D
were available ad libitum throughout the experiments. Lighting regimen consisted of 14 h light and 10 h darkness. All animals were weighed weekly during the experiment as an indicative criterion for growth and development. Animals from both C and HA groups were killed at different ages as described for the individual experiments.
Blood and tissue collections All animals were bled between 10:00 and 12:00 by cardiac puncture under light ethyl ether anesthesia and killed for collection of tissues. The blood was allowed to clot for 2 h at room temperature and placed in a refrigerator at 4°C for 24 h. Serum was collected by centrifugation at 800 g for I0 rain at 2°C and stored at --20°C until hormone assays. Immediately after killing, the adenohypophysis, seminal vesicles and testes were excised and placed in ice-cold 0.25 M sucrose. The tissues were cleaned of debris, weighed and the testes were immediately stored at - 2 0 ° C .
Radioimmunoassay for the gonadotropins Both serum FSH and LH were measured in duplicate and in two dilutions by the double-antibody radioimmunoassay (Daane and Parlow, 1971) using kits supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD) and are expressed in terms of their respective N I A M D D rat ~'~P-1 reference preparations.
Radioimmunoassay for testosterone Plasma testosterone was determined by the radioimmunoassay without chromatographic purification according to the procedure routinely used in our laboratory and is briefly described here. Plasma aliquots of 0.25-0'5 ml were used for the extraction of testosterone. Prior to extraction, testosterone 1, 2, 6, 7-3H (2,000 dpm. sp. act 80-105 Ci/mmol, Amersham, England) was added to each sample to enable losses of testosterone which occurred during its extraction to be measured. Extraction was done by manual shaking of plasma aliquots with light petroleum ether (b.p. 40-60°C): benzene (4 : 1, V/V) at a ratio of 1 : 10 (V/V, plasma/organic solvent). The dried extract was dissolved in acetone and 20 ~ of it was removed to estimate the recovery of added tritiated testosterone. From the remainder, samples were assayed for testosterone in duplicate and at two dilutions. The buffer used in the RIA (RIA buffer) was 0'05 M Tris-base, 0'1 M NaCI, pH 8'0 containing 0'1 ~ gelatin and 0"1 ~ sodium azide. Each assay tube, containing unknown or standard, was incubated at 37°C for 1 h with 500 lal of a 1 : 9000 dilution of antiserum (anti-testosterone serum, batch 550/3, obtained from Dr. R. Etches, University of Reading, England) and 100 lal of RIA buffer containing approximately 12,000 dpm of tritiated testosterone. The assay tube was then incubated at 4°C for 16 h. Testosterone bound to the antibody was separated from the free steroid by the addition of 200 lal of a dextran-coated charcoal suspension (1 ~ Norit A and 0.1 ~ w/v dextran T-70 in RIA buffer). The solutions were mixed for 5 s, incubated at 4°C for 15 rain, centrifuged at 4°C and 600 g for 15 min and a portion of 500 !ul was taken to estimate the radioactivity bound to the antibody. The standard curve of percentage radioactivity bound was plotted against the logarithm of the concentrations of testosterone over the range of 20-1000 pg/tube. The testosterone content of extracts was determined by interpolation on the standard curve and the concentration in plasma calculated taking into account the losses during the extraction procedure. Generally, efficiency of extraction of tritiated testosterone tracer added to samples varied from 75 to 98 ~ . It was confirmed that the antiserum showed an appreciable cross-reaction (~,57 ~ ) w i t h 5ct-dihydrotestosterone (DHT) but not with other C19 androgens (Table 1). Consequently the assay of a sample containing both testosterone and DHT gives the total testosterone plus 5 7 ~ of the DHT concentration. However, since in the male blood the concentration of testosterone exceeds by far that of DHT, this assay gives the actual testosterone concentration, since the error due to interference by DHT is less than the inherent errors of the assay. "]'ABLE 1. CROSS-REACTION OF STEROIDS IN THE RADIOIMMUNOASSAY FOR TESTOSTERONE USING ANTI-TESTOSTERONE
-11u-BSA SERUM Steroid Testosterone 5u-dihydrotestosterone 5ct-androstane-3u, 1713 diol Androstenedione Androst-5-ene-313,17 fl diol Dehydroepiandrosterone * % cross reaction --
~ cross-reaction* 100.0 56-9 14"2 5.2 1.4 0.2
ng of testosterone causing a 50% drop in DPM bound - 100. ng of other steriods causing a 50 ~o drop in DPM
GONADOTROPINS AND TESTOSTERONE IN AGEING RAT
] 95
Activity of 17~-hydroxysteroid oxidoreductase in testicular tissue Preparation of testicular homogenate, incubation conditions, extraction, isolation and identification of metabolites was reported previously (Bedrak et al., 1971). The activity of the enzyme was estimated either from the conversion of androstenedione-4-~4C, fortified with an NADPH generating system, to testosterone. or by the transformation of testosterone-4-a~C, fortified with the oxidized cofactor, to androstenedione, The results are expressed as nmol product formed/rag protein/l h incubation. Protein determination was carried out according to Lowry et al. (1951).
Statistical analysis The data were statistically analyzed using the Student's t-test. The results were considered significantly different when the differences between the experimental groups differed at p --- 0-05. RESULTS
Effect of" continuous heat-exposure on weight of body testes, seminal vesicles and adenohypophysis C o n t i n u o u s exposure of rats to 35°C caused a significant decrease in body weight during the first week. Consequently, the body weight of H A rats was smaller than that of C t h r o u g h o u t the experiment. The increased d u r a t i o n of heat-exposure was associated with a high mortality rate, as was noted previously (Bedrak et at., 1971). The decrease in °',,,.organ weight of body weight due to heat-exposure was evident with the 4 - m o n t h - o l d rats and not with the ageing a n i m a l s (Table 2). It was noted that the color of the eyes of all the HA-10m o n t h - o l d rats t u r n from the n o r m a l shade of pink/red to white/grey a n d the animals appeared to be blind. This observation was not pursued any further. The relative organ weights of C rats decreased as the a n i m a l s ' ages increased (Table 2). TABI_.E 2. TREATMENT, AGE AND WEIGHT OE ANIMALS AT END OF EXPERIMENT (DEATH)
Treatment Control Acclimatized Control Acclimatized Control Acclimatized
(16) (15) (5) (4) (4) (4)
Age month) 4 4 6 6 10 10
Months in hot room 1 2 7
Body wt (g) 371 ± 18.2 269 H: 7.9 446 ± 22-6 322 ~- 6.5 489 5 32.2 354:2 9.6
~/oorgan of body wt Seminal AdenohyTestes vesicles pophysis* 0.91 -2- 0.019 0.46 ± 0.016 2.4 ± 0"08 0"70 ± 0.098 0-35± 0.019 2.5 ± 0.19 0.81 ~_ 0.021 0.32 L 0.022 2.1 ± 0-21 0'69 ± 0.058 0.38 I 0.019 1.9 ± 0.15 0.63 ~ 0.085 0.32± 0-002 1.9 ± 0.17 0.66 ~ 0.083 0.37 ~:: 0.013 1'7 ± 0.08
Figures in parenthesis represent number of observations. *Per cent organ weight of body weight x 10-2.
The effect of ageing and acclimatization on plasma concentration of gonadotropins and testosterone Neither prolonged exposure of male rats to a hot e n v i r o n m e n t n o r advancing in age, from 4 to 10 m o n t h s , caused a decrease in serum c o n c e n t r a t i o n of F S H and L H (Table 3). Similarly, advancing in age alone was n o t associated with lower serum testosterone concentration. O n the other hand, serum testosterone concentration declined during acclimatization to heat, irrespective of age or d u r a t i o n of exposure. The per cent decrease in a n d r o g e n c o n c e n t r a t i o n inflicted by exposure to heat was 25.9, 34.4 (p < 0.001) and 32.5 (p -" 0-001) for the group aged 4, 6 and 10 months, respectively.
The effect o f ageing and acclimatization on testicular act±v±O' of 1713-hydroa,t'steroid o xidoreduct ase (l 7-~ H S D ) A d v a n c i n g in age from 3 to l0 m o n t h s is accompanied by a decrease in the activity ol
196 TABLE 3.
E. BEDRAK, Z. CHAP AND K. FRIED
CONCENTRATION (MEAN ~z
S.E.M.) OF FSH, LH
AND TESTOSTERONE IN SERUM OF MATURE AND
AGED MALE RATS MAINTAINED AT A TEMPERATURE ENVIRONMENT (20-22°C, 30 50~/o RELATIVE HUMIDITY, CONTROL) AND AT A HOT ENVIRONMENT (33-3.SoC, 25-40~ RELATIVE HUMIDITY, ACCLIMATIZED) FOR VARIOUS DURATIONS
Age Months (month) in hot room --4 . . . . . 4 I 6 6 2 10 10 7
Treatment Control Acclimatized Control Acclimatized Control Acclimatized
FSH 1i04-~35i20) 1035 ~ 74(15) 980 F 43 (5) 1188 ! 12(4) 1120:!: 18(4) 1005 -~ 25 (4)
o/ ng hormone/ml serum /o LH Testosterone decrease* 8 8 ! 5--i20) 4'94 : 0'61(~5i..... ~-5~ 100~i 17(15) 3.66 : 0.78(5) 25.9 86 ~ 7(5) 4.45 :': 0"22 (5) 99.i 16(4) 2.92 ~ 0.44(41 34.4+ 85-~ 8(4) 4.58 ~ 0.32(4) 95 -~ 10(4) 3.09 ± 0.29(4) 32.55-
Figures in parenthesis represent number of observations per group. * °~o decrease in testosterone concentration observed in acclimatized rats as compared to controls of the same age-group. +p ,~ 0.001 : difference between control and acclimatized rats of the same age-group. 17[3-HSD in testicular h o m o g e n a t c . In general the decline in the e n z y m e a c t i v i t y was steady a n d r e a c h e d the m i n i m u m at 6 m o n t h s o f age. T h e per c e n t c h a n g e in e n z y m e a c t i v i t y o b s e r v e d in the v a r i o u s ages, as c o m p a r e d to the 3 - m o n t h - o l d C rats, w a s - - 3 6 . 4 , ~ 4 9 . 2 , - - 6 3 . 3 a n d - - 4 5 . 6 for rats aged 3.5, 4, 6 a n d 10 m o n t h s , respectively ( T a b l e 4). T h e increase in e n z y m e a c t i v i t y in testes o f 1 0 - m o n t h - o l d rats was o b s e r v e d also in 10m o n t h - o l d m i c e ( C h a p et al., 1977). T h e s u p p r e s s i o n o f the e n z y m e activity inflicted by p r o l o n g e d e x p o s u r e o f the intact rat to 35~'C is e v i d e n t in g r o u p s o f rats r a n g i n g f r o m 3.5 to 6 m o n t h s o f age. T h e increase in activity o f 1713-HSD o b s e r v e d in H A 1 0 - m o n t h - o l d rats r e s e m b l e d t h a t o b s e r v e d in H A 1 0 - m o n t h - o l d m i c e ( C h a p ctal., 1977). T h e increase in a c t i v i t y o b s e r v e d in H A y o u n g rats ( 3 - m o n t h - o l d ) is i n t r i g u i n g and r e q u i r e s a d d i t i o n a l w o r k . TABLE 4.
RELATIONSHIP BET"~'EEN AGE AND DURATION OF EXPOSURE TO HEAT ON ACTIVITY OF [ 7,~-HYDROXYSTEROID OXII)OREDUCTASE IN TESTICULAR TISSUE OF THE RAT
Treatment Control Acclimatized Control Acclimatized Control Acclimatized Control Acclimatized Control Acclimatized
(3) (3) (2) (2) (4) (4) (3) (5j (4) (4)
Age(month) 3.0 3.0 3.5 3"5 4.0 4.0 6.0 6"0 10.0 10'0
Months in hot rooms
2 7
Enzyme activity (a) 2"83 :i 0"132 3.60 .! 0-134 1.80 : 0"121 1.52 0.111 1.42 0'049 119 ! 0.333 1.04 :: 0.058 0.64 _~ 0.119 1.54 +- 0.089 1.90 ~L 0"010
°/o difference (b)
(c)
27-2* 36"4+ 15.6 49'2";" --16.2 63'3+ 38.5* 23'4";"
--45"6+
Figures in parenthesis represent number of experiments. (a) Nmol testosterone produced from androstenedione, in the presence of NADPH, per mg protein/h. (b) ?/,, change in enzyme activity observed in HA rats as compared to C of the same age-group. (c) o~ change in enzyme activity observed in C groups as compared to the 3-month-old C rats. *p - 0-05; + p - 0.01.
The relationship of contplete hypothalamic deafferentation (CHD) and acute heat exposure on testicular activity of l7~-hydroxysteroid oxidoreductase in 4-month-old rats T a b l e 5 d e m o n s t r a t e d t h a t C H D did not alter the activity o f the e n z y m e . A t e n d e n c y for a decline in a c t i v i t y o f t h e e n z y m e was o b s e r v e d in all a n i m a l s e x p o s e d to the a c u t e heatstress o f 4 11 for 5 c o n s e c u t i v e days. T h e c o m b i n e d effect o f a c u t e heat-stress a n d C H D was n o t g r e a t e r t h a n t h a t inflicted by the heat stress alone.
GONADOTROPINS AND TESTOSTERONE IN AGEING RAT
197
TABLE 5. THE EFFECT OF COMPLETE HYPOTHALAMIC DEAFFERENTATION (CHD) AND HEAT-EXPOSURE* ON THE ACTIVITY OF 17~-HYDROXYSTEROID OXIDOREDUCTASE IN TESTICULAR TISSUE OF a-MONTH-OLD RATS
Group and treatment Intact, control Intact, heat exposed CHD, control CHD, heat exposed
(3) (4) (3) (6)
Substrate and cofactor Androstenedione Testosterone and NADPH and NADP 1.35 ~ 0.144~" 0.76 :k_ 0"092 1"26 :k 0.212 0.72 ± 0.056 1.37 ~ 0'125 0.78 ± 0"086 1-20 ~ 0-105 0.74 ± 0"049
Figures in parenthesis represent number of experiments. *Rats exposed to a temperature of 36°C and 40% relative humidity for 4 h daily (09:00 - 13:00) for 5 consecutive days prior to killing. tNmol product formed/rag protein/h, mean and S.E.M. DISCUSSION The present study brings evidence that advancing in age, between 4 and 10 months, is not coupled with a decrease in serum concentration of FSH, LH and testosterone, an observation which agrees with the results of Gray (1978). Apparently the decrease in serum concentration of these hormones occurs in rats older than those used in the present investigation (Ghanadian et al., 1976; Riegle and Meites, 1976; Chan et al., 1977; Gray, 1978). Similarly, prolonged exposure of rats to 35°C did not inflict significant changes in serum concentration of FSH and LH. On the other hand, serum testosterone concentration declined during heat exposure, irrespective of the age of the animals. This decrease in testosterone concentration exists despite the increase in testicular blood flow known to exist in heat treated animals (Setchell et al., 1966) and is independent of changes in serum concentration of F S H and LH. It is difficult, without additional evidence, to offer a comprehensive explanation for the physiological processes leading to the lowering &testosterone concentration in peripheral blood of H A animals. One may speculate that acclimatization is associated with one or more of the following conditions that could lead to lower plasma concentration of the hormone: (a) increased catabolism and excretion rate of circulating testosterone, (b) damaged or smaller numbers of receptors for LH in the testis of HA rats, and (c) lower testosterone synthesizing capacity of the Leydig cell. Indeed, earlier work from this laboratory has demonstrated that the excretion rate of 17-ketosteroids is significantly higher in HA rats (Sod-Moriah and Bedrak, 1976). These steroidal metabolites include, inter alia, testosterone by-products and therefore support, in part, the thesis that heat-exposure is associated with enhanced metabolism and excretion rate of androgen. No data are available as yet as to the effect of heat-exposure on the number and function of receptors for L H in the Leydig cell. Regarding the testosteronesynthesizing capacity of the testes, it was demonstrated that heat-acclimatization of 4month-old rats coincided with an increased activity of the enzymes converting 5-ene C 21 steroid substrates, used for testosterone biosynthesis, to their 4-ene homologues (Bedrak et al., 1973). Moreover, the activity of all the enzymes, except for 1713-HSD, associated with testosterone metabolism via the 4-ene pathway was increased. The present findings indicate that the activity of 1713-HSD decreases with age (3-10 months) and also confirmed that the activity of this enzyme is reduced in 3.5-6 month-old HA rats. However, acclimatization of younger (3 months) or older animals (10 months) elicitated the opposite response. At the present time no data is available to elucidate the unique relationship existing between the activity of the enzyme and these two age-periods (3 and 10 months) of HA rats. It
198
E. BEDRAK,Z. CHAP AND K. FRill)
should be indicated that the increase in the activity of 17[~-HSD noted in 10-month-old HA rats was also noted in 10-month-old HA mice (Chap et al.. 1977) and in these two instances the animals appeared to be blind. Blindness, of course, affects the function of the hypothalamo-hypophyseal-gonadal axis, and tile role of this phenomenon in HA rats awaits further investigation. Is the decrease in the activity of 17[~-HSD responsible for the signiiicant drop in serum testosterone concentration observed in HA rats'? Evidence on hand does not entirely support this contention. Namely, while a single 4 h exposure of 3-4 month-old rats to 5 C reduced testosterone serum concentration by 43 i'~; (Bedrak and Chap, personal observation), a similar treatment to intact and C H D rats (Table 5) caused only a slight drop in the activity of 17[3-HSD. This decline in enzyme activity cannot be the sole cause for the significant drop in serum testosterone concentration observed in HA rats. This contention is supported also by the fact that, despite the decrease in activity of 17~-HSD noted in ageing C rats (Table 4), the concentration of serum testosterone remained practically the same (Table 3). Testicular function of young sexually mature HA rats is extremely sensitive to elevated ambient temperature. The hyperthermia which persists in the scrotum of HA rats (SodMoriah et al., 1974) is coupled on one hand with histological damage and lower fertility and on the other hand, with low testosterone concentration in peripheral blood. Such low levels of testosterone are characteristic of ageing rats (Chan et at., 1977; Gray, 1978). It is concluded that heat-exposure enhances senescence of the testis, independent of the pituitary function. SUMMARY Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone of control rats (20-22°C, 30-50 j°,o~relative humidity) did not vary at 4, 6 and 10 months of age. Prolonged maintenance of rats in a hot environment (33-35°C, 25-40 o;, relative humidity) was accompanied by a significant decrease in testosterone concentration, irrespective of the animal's age or duration of exposure to heat (1, 2 and 7 months), whereas the concentration of FSH and LH remained relatively unaffected. Activity of 17[3-hydroxysteroid oxidoreductase (17[~-HSD) in the testes of control rats was inversely related to the animals' age. In most cases prolonged exposure to heat augmented the decline in activity of 1715HSD associated with ageing. These data suggest that increasing in age is accompanied by a decrease in the activity of testicular 1713-HSD without affecting the concentration of FSH, LH and testosterone in blood serum. It is concluded that, within the age limits used in the present experiment, the significant drop in serum testosterone concentration characterizing rats exposed to heat is independent of serum gonadotropin concentration and testicular activity of 17[~-HSD, as well as the animal's age. Acknowledgements--Materials for radioimmunoassay of FSH and LH were kindly provided by the National Institute of Arthritis, Metabolic and Digestive Diseases through the Rat Pituitary Hormone Distribution Program. The authors are grateful to Dr. R. Etches, Department of Physiology and Biochemistry, University of Reading, England, for the supply of testosterone antiserum and to Dr. I ('bowers, Hadassa University Hospital, Jerusalem, for performing the hypothalal'rfic deafferentation.
REFERENCES BkDRAK, E., SAMOILOFF,V., SoD-MORIAH, U. A. and GOI.Dt~r.RCL S. (1971) I. 12"ttth~r. 51,489. B~:t)l~.aK, E. (1972) Gen comp. Endocr. 18, 524.
(;ONADOTROP1NS AND TESTOSTERONE IN A.GEING RAT
] 99
BEDRAK, E., SAMOILOFF, V. and SOD-MORIAE[, U. A. (1973) L Emtocr. 58, 207. BEDRAK~ E. (1974) Exp. Geront. 9, 235. CHAN, S. W. C., LEATHEM,J. H. and ESASHI, T. (1977) Endocrinology 101, 128. C H A P , Z. and BEDRAK, E. (1977) Exp. Geront. 12, 51. CHAP, Z., SLONIM, A. and BEDRAK, E. (1977) Exp. Geront. 12, 41. DAANE, T. A. and PARLOW, A. F. (1971) Endocrinology 88, 653. GHANADIAN, R., LEWIS, J. G. and CHISHOLM, G. D. (1976) Steroids 25, 753. GOMES, W. R., BUTLER, W. R. and JOrINSON, A. D. (1971) L ,4him, Sci. 33, 804. GRAY, G. D. (1978) I. Endocr, 76, 551. HALASZ, B. and PuPr,, L. (1965) Endocrinology 77, 553, LOWRY, O. H., ROSEBROUGH, N. H., FARR, A. L. and RANDALL, R. J. (1951) 1. biol. Chem. 193, 265. RHYNES, W. E. and EWING, L. L. (1973) Endocrinology 92, 509. RIEGLE, G. D. and ME|TES, J. (1976) Proc. Soc. exp. Biol. Med. 151, 507. SETCHELL, B. P., WAITES, G. M. H. and THORBURN, G. D. (1966) Circulation Res. 18, 755. SLOMM, A. and Br~DRAK, E. (1977) E.rp. Geront. 12, 35. SoD-MOI',I~,H, U. A., GOLDBERG, G. M. and BEDRAK, E. (1974) 1. Reprod. Fert. 37, 263. SoD-MORIAH, U. A. and BEDRAK, E. (1976) In: Progress in Animal Biometeorology (Edited by S.W. TROMP), Vol. 1, p. 292. Swets Zeitinger, Lisse.
0531-5565/79/0801-0193502.00/0
~" Pergamon Press Ltd. 1979. Printed in Great Britain.
RELATIONSHIP BETWEEN SERUM CONCENTRATION OF GONADOTROPINS AND TESTOSTERONE IN HEATEXPOSED AGEING MALE RATS E. BEDRAK, Z. CHAP AND K. FRIED Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva84120, Israel (Received 7 January 1979)
INTRODUCTION AGEING, inter alia, goes hand in hand with decreased pituitary-testicular function and ultimately results in lower concentrations of circulating gonadotropins and testosterone (Ghanadian et al., 1976; Riegle and Meites, 1976; Chan et al., 1977; Gray, 1978). Exposure of intact sexually mature young males to elevated ambient temperature is also associated with lower testosterone production in the ram (Gomes et al., 1971), the bull (Rhynes and Ewing, 1973) and the rat (Bedrak et al., 1973). Recent findings (Bedrak and Chap, personal observations) indicated that the decrease in plasma testosterone concentration observed in heat-exposed rats was coupled with a transient decline in plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH). Moreover, heat-exposure inflicted histological damage to the testis and enhanced the activity of most testicular enzymes, except for 1713hydroxysteroid oxidoreductase (1713-HSD), synthesizing testosterone in the rat (Bedrak et al., 1971 ; 1973) and the mouse (Bedrak, 1972, 1974; Slonim and Bedrak, 1977). Cooling the testes eliminated the histological damage observed in the mouse, but the changes in the activity of testicular enzymes persisted (Chap et al., 1977). It was noted that the changes in the activity of the enzymes associated with androgen production by the testes of aged mice were similar to those observed in young, sexually mature heat-exposed mice (Bedrak, 1974; Slonim and Bedrak, 1977; Chap and Bedrak, 1977; Chap et aL, 1977). In view of this, it was suggested that prolonged exposure of the intact mammal to elevated ambient temperatures might interfere with normal testicular function by accelerating the disorganization of the enzyme synthesizing processes--a phenomenon which characterizes senescence. Since testicular function is controlled by the gonadotropins, it is important to establish the relationship between serum concentration of testosterone and gonadotropins in heat-exposed animals and correlate it with the activity of 1713-HSD. This is imperative in order to clarify whether heat-exposure also induces alteration in the pituitary function, comparable to those accompanying the process of ageing, or whether it is limited to the testes. MATERIALS AND M E T H O D S Animals
Three-month-old Wistar male rats from the colony of the Department of Hormone Research, Weizman Institute of Science, were used. They were kept either in a control (C) environment (20-22°C, 30-50~o relative humidity) or were exposed to heat (33--35°C,25-40~ relative humidity) for various durations (HA), as indicated in the individual experiments. The animals used for experimentation with hypothalamic disconnection, complete hypothalamic deafferentation (CHD), were 3.5-month-old rats obtained from the colony of the Hebrew University, Jerusalem. Deafferentation was performed by means of a stereotoxic instrument under anesthesia (Hal[tsz and Pupp, 1965). They were allowed 3 days for recovery before being used for experiments. Tap water and rat chow 193
194
E. B E D R A K , Z . C H A P A N D K . F R I E D
were available ad libitum throughout the experiments. Lighting regimen consisted of 14 h light and 10 h darkness. All animals were weighed weekly during the experiment as an indicative criterion for growth and development. Animals from both C and HA groups were killed at different ages as described for the individual experiments.
Blood and tissue collections All animals were bled between 10:00 and 12:00 by cardiac puncture under light ethyl ether anesthesia and killed for collection of tissues. The blood was allowed to clot for 2 h at room temperature and placed in a refrigerator at 4°C for 24 h. Serum was collected by centrifugation at 800 g for I0 rain at 2°C and stored at --20°C until hormone assays. Immediately after killing, the adenohypophysis, seminal vesicles and testes were excised and placed in ice-cold 0.25 M sucrose. The tissues were cleaned of debris, weighed and the testes were immediately stored at - 2 0 ° C .
Radioimmunoassay for the gonadotropins Both serum FSH and LH were measured in duplicate and in two dilutions by the double-antibody radioimmunoassay (Daane and Parlow, 1971) using kits supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD) and are expressed in terms of their respective N I A M D D rat ~'~P-1 reference preparations.
Radioimmunoassay for testosterone Plasma testosterone was determined by the radioimmunoassay without chromatographic purification according to the procedure routinely used in our laboratory and is briefly described here. Plasma aliquots of 0.25-0'5 ml were used for the extraction of testosterone. Prior to extraction, testosterone 1, 2, 6, 7-3H (2,000 dpm. sp. act 80-105 Ci/mmol, Amersham, England) was added to each sample to enable losses of testosterone which occurred during its extraction to be measured. Extraction was done by manual shaking of plasma aliquots with light petroleum ether (b.p. 40-60°C): benzene (4 : 1, V/V) at a ratio of 1 : 10 (V/V, plasma/organic solvent). The dried extract was dissolved in acetone and 20 ~ of it was removed to estimate the recovery of added tritiated testosterone. From the remainder, samples were assayed for testosterone in duplicate and at two dilutions. The buffer used in the RIA (RIA buffer) was 0'05 M Tris-base, 0'1 M NaCI, pH 8'0 containing 0'1 ~ gelatin and 0"1 ~ sodium azide. Each assay tube, containing unknown or standard, was incubated at 37°C for 1 h with 500 lal of a 1 : 9000 dilution of antiserum (anti-testosterone serum, batch 550/3, obtained from Dr. R. Etches, University of Reading, England) and 100 lal of RIA buffer containing approximately 12,000 dpm of tritiated testosterone. The assay tube was then incubated at 4°C for 16 h. Testosterone bound to the antibody was separated from the free steroid by the addition of 200 lal of a dextran-coated charcoal suspension (1 ~ Norit A and 0.1 ~ w/v dextran T-70 in RIA buffer). The solutions were mixed for 5 s, incubated at 4°C for 15 rain, centrifuged at 4°C and 600 g for 15 min and a portion of 500 !ul was taken to estimate the radioactivity bound to the antibody. The standard curve of percentage radioactivity bound was plotted against the logarithm of the concentrations of testosterone over the range of 20-1000 pg/tube. The testosterone content of extracts was determined by interpolation on the standard curve and the concentration in plasma calculated taking into account the losses during the extraction procedure. Generally, efficiency of extraction of tritiated testosterone tracer added to samples varied from 75 to 98 ~ . It was confirmed that the antiserum showed an appreciable cross-reaction (~,57 ~ ) w i t h 5ct-dihydrotestosterone (DHT) but not with other C19 androgens (Table 1). Consequently the assay of a sample containing both testosterone and DHT gives the total testosterone plus 5 7 ~ of the DHT concentration. However, since in the male blood the concentration of testosterone exceeds by far that of DHT, this assay gives the actual testosterone concentration, since the error due to interference by DHT is less than the inherent errors of the assay. "]'ABLE 1. CROSS-REACTION OF STEROIDS IN THE RADIOIMMUNOASSAY FOR TESTOSTERONE USING ANTI-TESTOSTERONE
-11u-BSA SERUM Steroid Testosterone 5u-dihydrotestosterone 5ct-androstane-3u, 1713 diol Androstenedione Androst-5-ene-313,17 fl diol Dehydroepiandrosterone * % cross reaction --
~ cross-reaction* 100.0 56-9 14"2 5.2 1.4 0.2
ng of testosterone causing a 50% drop in DPM bound - 100. ng of other steriods causing a 50 ~o drop in DPM
GONADOTROPINS AND TESTOSTERONE IN AGEING RAT
] 95
Activity of 17~-hydroxysteroid oxidoreductase in testicular tissue Preparation of testicular homogenate, incubation conditions, extraction, isolation and identification of metabolites was reported previously (Bedrak et al., 1971). The activity of the enzyme was estimated either from the conversion of androstenedione-4-~4C, fortified with an NADPH generating system, to testosterone. or by the transformation of testosterone-4-a~C, fortified with the oxidized cofactor, to androstenedione, The results are expressed as nmol product formed/rag protein/l h incubation. Protein determination was carried out according to Lowry et al. (1951).
Statistical analysis The data were statistically analyzed using the Student's t-test. The results were considered significantly different when the differences between the experimental groups differed at p --- 0-05. RESULTS
Effect of" continuous heat-exposure on weight of body testes, seminal vesicles and adenohypophysis C o n t i n u o u s exposure of rats to 35°C caused a significant decrease in body weight during the first week. Consequently, the body weight of H A rats was smaller than that of C t h r o u g h o u t the experiment. The increased d u r a t i o n of heat-exposure was associated with a high mortality rate, as was noted previously (Bedrak et at., 1971). The decrease in °',,,.organ weight of body weight due to heat-exposure was evident with the 4 - m o n t h - o l d rats and not with the ageing a n i m a l s (Table 2). It was noted that the color of the eyes of all the HA-10m o n t h - o l d rats t u r n from the n o r m a l shade of pink/red to white/grey a n d the animals appeared to be blind. This observation was not pursued any further. The relative organ weights of C rats decreased as the a n i m a l s ' ages increased (Table 2). TABI_.E 2. TREATMENT, AGE AND WEIGHT OE ANIMALS AT END OF EXPERIMENT (DEATH)
Treatment Control Acclimatized Control Acclimatized Control Acclimatized
(16) (15) (5) (4) (4) (4)
Age month) 4 4 6 6 10 10
Months in hot room 1 2 7
Body wt (g) 371 ± 18.2 269 H: 7.9 446 ± 22-6 322 ~- 6.5 489 5 32.2 354:2 9.6
~/oorgan of body wt Seminal AdenohyTestes vesicles pophysis* 0.91 -2- 0.019 0.46 ± 0.016 2.4 ± 0"08 0"70 ± 0.098 0-35± 0.019 2.5 ± 0.19 0.81 ~_ 0.021 0.32 L 0.022 2.1 ± 0-21 0'69 ± 0.058 0.38 I 0.019 1.9 ± 0.15 0.63 ~ 0.085 0.32± 0-002 1.9 ± 0.17 0.66 ~ 0.083 0.37 ~:: 0.013 1'7 ± 0.08
Figures in parenthesis represent number of observations. *Per cent organ weight of body weight x 10-2.
The effect of ageing and acclimatization on plasma concentration of gonadotropins and testosterone Neither prolonged exposure of male rats to a hot e n v i r o n m e n t n o r advancing in age, from 4 to 10 m o n t h s , caused a decrease in serum c o n c e n t r a t i o n of F S H and L H (Table 3). Similarly, advancing in age alone was n o t associated with lower serum testosterone concentration. O n the other hand, serum testosterone concentration declined during acclimatization to heat, irrespective of age or d u r a t i o n of exposure. The per cent decrease in a n d r o g e n c o n c e n t r a t i o n inflicted by exposure to heat was 25.9, 34.4 (p < 0.001) and 32.5 (p -" 0-001) for the group aged 4, 6 and 10 months, respectively.
The effect o f ageing and acclimatization on testicular act±v±O' of 1713-hydroa,t'steroid o xidoreduct ase (l 7-~ H S D ) A d v a n c i n g in age from 3 to l0 m o n t h s is accompanied by a decrease in the activity ol
196 TABLE 3.
E. BEDRAK, Z. CHAP AND K. FRIED
CONCENTRATION (MEAN ~z
S.E.M.) OF FSH, LH
AND TESTOSTERONE IN SERUM OF MATURE AND
AGED MALE RATS MAINTAINED AT A TEMPERATURE ENVIRONMENT (20-22°C, 30 50~/o RELATIVE HUMIDITY, CONTROL) AND AT A HOT ENVIRONMENT (33-3.SoC, 25-40~ RELATIVE HUMIDITY, ACCLIMATIZED) FOR VARIOUS DURATIONS
Age Months (month) in hot room --4 . . . . . 4 I 6 6 2 10 10 7
Treatment Control Acclimatized Control Acclimatized Control Acclimatized
FSH 1i04-~35i20) 1035 ~ 74(15) 980 F 43 (5) 1188 ! 12(4) 1120:!: 18(4) 1005 -~ 25 (4)
o/ ng hormone/ml serum /o LH Testosterone decrease* 8 8 ! 5--i20) 4'94 : 0'61(~5i..... ~-5~ 100~i 17(15) 3.66 : 0.78(5) 25.9 86 ~ 7(5) 4.45 :': 0"22 (5) 99.i 16(4) 2.92 ~ 0.44(41 34.4+ 85-~ 8(4) 4.58 ~ 0.32(4) 95 -~ 10(4) 3.09 ± 0.29(4) 32.55-
Figures in parenthesis represent number of observations per group. * °~o decrease in testosterone concentration observed in acclimatized rats as compared to controls of the same age-group. +p ,~ 0.001 : difference between control and acclimatized rats of the same age-group. 17[3-HSD in testicular h o m o g e n a t c . In general the decline in the e n z y m e a c t i v i t y was steady a n d r e a c h e d the m i n i m u m at 6 m o n t h s o f age. T h e per c e n t c h a n g e in e n z y m e a c t i v i t y o b s e r v e d in the v a r i o u s ages, as c o m p a r e d to the 3 - m o n t h - o l d C rats, w a s - - 3 6 . 4 , ~ 4 9 . 2 , - - 6 3 . 3 a n d - - 4 5 . 6 for rats aged 3.5, 4, 6 a n d 10 m o n t h s , respectively ( T a b l e 4). T h e increase in e n z y m e a c t i v i t y in testes o f 1 0 - m o n t h - o l d rats was o b s e r v e d also in 10m o n t h - o l d m i c e ( C h a p et al., 1977). T h e s u p p r e s s i o n o f the e n z y m e activity inflicted by p r o l o n g e d e x p o s u r e o f the intact rat to 35~'C is e v i d e n t in g r o u p s o f rats r a n g i n g f r o m 3.5 to 6 m o n t h s o f age. T h e increase in activity o f 1713-HSD o b s e r v e d in H A 1 0 - m o n t h - o l d rats r e s e m b l e d t h a t o b s e r v e d in H A 1 0 - m o n t h - o l d m i c e ( C h a p ctal., 1977). T h e increase in a c t i v i t y o b s e r v e d in H A y o u n g rats ( 3 - m o n t h - o l d ) is i n t r i g u i n g and r e q u i r e s a d d i t i o n a l w o r k . TABLE 4.
RELATIONSHIP BET"~'EEN AGE AND DURATION OF EXPOSURE TO HEAT ON ACTIVITY OF [ 7,~-HYDROXYSTEROID OXII)OREDUCTASE IN TESTICULAR TISSUE OF THE RAT
Treatment Control Acclimatized Control Acclimatized Control Acclimatized Control Acclimatized Control Acclimatized
(3) (3) (2) (2) (4) (4) (3) (5j (4) (4)
Age(month) 3.0 3.0 3.5 3"5 4.0 4.0 6.0 6"0 10.0 10'0
Months in hot rooms
2 7
Enzyme activity (a) 2"83 :i 0"132 3.60 .! 0-134 1.80 : 0"121 1.52 0.111 1.42 0'049 119 ! 0.333 1.04 :: 0.058 0.64 _~ 0.119 1.54 +- 0.089 1.90 ~L 0"010
°/o difference (b)
(c)
27-2* 36"4+ 15.6 49'2";" --16.2 63'3+ 38.5* 23'4";"
--45"6+
Figures in parenthesis represent number of experiments. (a) Nmol testosterone produced from androstenedione, in the presence of NADPH, per mg protein/h. (b) ?/,, change in enzyme activity observed in HA rats as compared to C of the same age-group. (c) o~ change in enzyme activity observed in C groups as compared to the 3-month-old C rats. *p - 0-05; + p - 0.01.
The relationship of contplete hypothalamic deafferentation (CHD) and acute heat exposure on testicular activity of l7~-hydroxysteroid oxidoreductase in 4-month-old rats T a b l e 5 d e m o n s t r a t e d t h a t C H D did not alter the activity o f the e n z y m e . A t e n d e n c y for a decline in a c t i v i t y o f t h e e n z y m e was o b s e r v e d in all a n i m a l s e x p o s e d to the a c u t e heatstress o f 4 11 for 5 c o n s e c u t i v e days. T h e c o m b i n e d effect o f a c u t e heat-stress a n d C H D was n o t g r e a t e r t h a n t h a t inflicted by the heat stress alone.
GONADOTROPINS AND TESTOSTERONE IN AGEING RAT
197
TABLE 5. THE EFFECT OF COMPLETE HYPOTHALAMIC DEAFFERENTATION (CHD) AND HEAT-EXPOSURE* ON THE ACTIVITY OF 17~-HYDROXYSTEROID OXIDOREDUCTASE IN TESTICULAR TISSUE OF a-MONTH-OLD RATS
Group and treatment Intact, control Intact, heat exposed CHD, control CHD, heat exposed
(3) (4) (3) (6)
Substrate and cofactor Androstenedione Testosterone and NADPH and NADP 1.35 ~ 0.144~" 0.76 :k_ 0"092 1"26 :k 0.212 0.72 ± 0.056 1.37 ~ 0'125 0.78 ± 0"086 1-20 ~ 0-105 0.74 ± 0"049
Figures in parenthesis represent number of experiments. *Rats exposed to a temperature of 36°C and 40% relative humidity for 4 h daily (09:00 - 13:00) for 5 consecutive days prior to killing. tNmol product formed/rag protein/h, mean and S.E.M. DISCUSSION The present study brings evidence that advancing in age, between 4 and 10 months, is not coupled with a decrease in serum concentration of FSH, LH and testosterone, an observation which agrees with the results of Gray (1978). Apparently the decrease in serum concentration of these hormones occurs in rats older than those used in the present investigation (Ghanadian et al., 1976; Riegle and Meites, 1976; Chan et al., 1977; Gray, 1978). Similarly, prolonged exposure of rats to 35°C did not inflict significant changes in serum concentration of FSH and LH. On the other hand, serum testosterone concentration declined during heat exposure, irrespective of the age of the animals. This decrease in testosterone concentration exists despite the increase in testicular blood flow known to exist in heat treated animals (Setchell et al., 1966) and is independent of changes in serum concentration of F S H and LH. It is difficult, without additional evidence, to offer a comprehensive explanation for the physiological processes leading to the lowering &testosterone concentration in peripheral blood of H A animals. One may speculate that acclimatization is associated with one or more of the following conditions that could lead to lower plasma concentration of the hormone: (a) increased catabolism and excretion rate of circulating testosterone, (b) damaged or smaller numbers of receptors for LH in the testis of HA rats, and (c) lower testosterone synthesizing capacity of the Leydig cell. Indeed, earlier work from this laboratory has demonstrated that the excretion rate of 17-ketosteroids is significantly higher in HA rats (Sod-Moriah and Bedrak, 1976). These steroidal metabolites include, inter alia, testosterone by-products and therefore support, in part, the thesis that heat-exposure is associated with enhanced metabolism and excretion rate of androgen. No data are available as yet as to the effect of heat-exposure on the number and function of receptors for L H in the Leydig cell. Regarding the testosteronesynthesizing capacity of the testes, it was demonstrated that heat-acclimatization of 4month-old rats coincided with an increased activity of the enzymes converting 5-ene C 21 steroid substrates, used for testosterone biosynthesis, to their 4-ene homologues (Bedrak et al., 1973). Moreover, the activity of all the enzymes, except for 1713-HSD, associated with testosterone metabolism via the 4-ene pathway was increased. The present findings indicate that the activity of 1713-HSD decreases with age (3-10 months) and also confirmed that the activity of this enzyme is reduced in 3.5-6 month-old HA rats. However, acclimatization of younger (3 months) or older animals (10 months) elicitated the opposite response. At the present time no data is available to elucidate the unique relationship existing between the activity of the enzyme and these two age-periods (3 and 10 months) of HA rats. It
198
E. BEDRAK,Z. CHAP AND K. FRill)
should be indicated that the increase in the activity of 17[~-HSD noted in 10-month-old HA rats was also noted in 10-month-old HA mice (Chap et al.. 1977) and in these two instances the animals appeared to be blind. Blindness, of course, affects the function of the hypothalamo-hypophyseal-gonadal axis, and tile role of this phenomenon in HA rats awaits further investigation. Is the decrease in the activity of 17[~-HSD responsible for the signiiicant drop in serum testosterone concentration observed in HA rats'? Evidence on hand does not entirely support this contention. Namely, while a single 4 h exposure of 3-4 month-old rats to 5 C reduced testosterone serum concentration by 43 i'~; (Bedrak and Chap, personal observation), a similar treatment to intact and C H D rats (Table 5) caused only a slight drop in the activity of 17[3-HSD. This decline in enzyme activity cannot be the sole cause for the significant drop in serum testosterone concentration observed in HA rats. This contention is supported also by the fact that, despite the decrease in activity of 17~-HSD noted in ageing C rats (Table 4), the concentration of serum testosterone remained practically the same (Table 3). Testicular function of young sexually mature HA rats is extremely sensitive to elevated ambient temperature. The hyperthermia which persists in the scrotum of HA rats (SodMoriah et al., 1974) is coupled on one hand with histological damage and lower fertility and on the other hand, with low testosterone concentration in peripheral blood. Such low levels of testosterone are characteristic of ageing rats (Chan et at., 1977; Gray, 1978). It is concluded that heat-exposure enhances senescence of the testis, independent of the pituitary function. SUMMARY Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone of control rats (20-22°C, 30-50 j°,o~relative humidity) did not vary at 4, 6 and 10 months of age. Prolonged maintenance of rats in a hot environment (33-35°C, 25-40 o;, relative humidity) was accompanied by a significant decrease in testosterone concentration, irrespective of the animal's age or duration of exposure to heat (1, 2 and 7 months), whereas the concentration of FSH and LH remained relatively unaffected. Activity of 17[3-hydroxysteroid oxidoreductase (17[~-HSD) in the testes of control rats was inversely related to the animals' age. In most cases prolonged exposure to heat augmented the decline in activity of 1715HSD associated with ageing. These data suggest that increasing in age is accompanied by a decrease in the activity of testicular 1713-HSD without affecting the concentration of FSH, LH and testosterone in blood serum. It is concluded that, within the age limits used in the present experiment, the significant drop in serum testosterone concentration characterizing rats exposed to heat is independent of serum gonadotropin concentration and testicular activity of 17[~-HSD, as well as the animal's age. Acknowledgements--Materials for radioimmunoassay of FSH and LH were kindly provided by the National Institute of Arthritis, Metabolic and Digestive Diseases through the Rat Pituitary Hormone Distribution Program. The authors are grateful to Dr. R. Etches, Department of Physiology and Biochemistry, University of Reading, England, for the supply of testosterone antiserum and to Dr. I ('bowers, Hadassa University Hospital, Jerusalem, for performing the hypothalal'rfic deafferentation.
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(;ONADOTROP1NS AND TESTOSTERONE IN A.GEING RAT
] 99
BEDRAK, E., SAMOILOFF, V. and SOD-MORIAE[, U. A. (1973) L Emtocr. 58, 207. BEDRAK~ E. (1974) Exp. Geront. 9, 235. CHAN, S. W. C., LEATHEM,J. H. and ESASHI, T. (1977) Endocrinology 101, 128. C H A P , Z. and BEDRAK, E. (1977) Exp. Geront. 12, 51. CHAP, Z., SLONIM, A. and BEDRAK, E. (1977) Exp. Geront. 12, 41. DAANE, T. A. and PARLOW, A. F. (1971) Endocrinology 88, 653. GHANADIAN, R., LEWIS, J. G. and CHISHOLM, G. D. (1976) Steroids 25, 753. GOMES, W. R., BUTLER, W. R. and JOrINSON, A. D. (1971) L ,4him, Sci. 33, 804. GRAY, G. D. (1978) I. Endocr, 76, 551. HALASZ, B. and PuPr,, L. (1965) Endocrinology 77, 553, LOWRY, O. H., ROSEBROUGH, N. H., FARR, A. L. and RANDALL, R. J. (1951) 1. biol. Chem. 193, 265. RHYNES, W. E. and EWING, L. L. (1973) Endocrinology 92, 509. RIEGLE, G. D. and ME|TES, J. (1976) Proc. Soc. exp. Biol. Med. 151, 507. SETCHELL, B. P., WAITES, G. M. H. and THORBURN, G. D. (1966) Circulation Res. 18, 755. SLOMM, A. and Br~DRAK, E. (1977) E.rp. Geront. 12, 35. SoD-MOI',I~,H, U. A., GOLDBERG, G. M. and BEDRAK, E. (1974) 1. Reprod. Fert. 37, 263. SoD-MORIAH, U. A. and BEDRAK, E. (1976) In: Progress in Animal Biometeorology (Edited by S.W. TROMP), Vol. 1, p. 292. Swets Zeitinger, Lisse.
