Seasonal Changes in Plasma Testosterone and 5 α - Dihydrotestosterone Levels in the Adult Male

Seasonal changes in plasma levels of testosterone (T) and 5α-dihydrotestosterone (DTH) were examined in the adult male Japanese red-bellied newt. The concentration of plasma T increased significantly (p<0.05) from January to February and decreased drastically from April to July when it reached its minimum. After the summer decline, plasma T increase rapidly from July to October (p<0.05) and reached its maximum in October. Concerning the seasonal fluctuation of plasma DHT levels, three peaks were observed; one, the highest peak, appeared in the middle of May, and two lower peaks in early spring (Feb.-March) and autumn (Sept.-Oct.). In this report, the role of plasma T and DHT in the reproductive cycle is discussed. A number of investigations have been reported on seasonal changes in testicular function in amphibians, especially spermatogenic and steroidogenic activity (for review, see Lofts, 1974). With the development of a sensitive radioimmunoassay technique it has become possible to measure gonadal steroid levels in amphibians, such as Rana pipiens (Wada et al., 1976), Rana catesbeiana (Muller, 1977 a, b; Callard et al., 1978), Taricha granulosa (Moore and Muller, 1977; Moore et al., 1978) and Necturus maculosa (Bolaffi and Callard, 1979; Bolaffi and Callard, 1981). However, studies of plasma steroids during the annual reproductive cycle are limited to Rana esculenta (D'Istria et al., 1974; Rastogi et al., 1976), Taricha granulosa (Specker and Moore, 1980) and Bufo mauritanicus (Siboulet, 1981) only. In the Japanese redbellied newt, a detailed investigation of the relationship between testicular function and male sexual characteristics was carried out by histological and histochemical criteria (Tanaka and Iwasawa, 1979), but plasma steroid levels in the annual reproductive cycle were not studied. This study was conducted to investigate seasonal changes in plasma levels of testosterone (T) and 5α-dihydrotestosterone (DHT) in the adult male Japanese red-bellied newt. A summary of this work was presented at the 9th International Symposium on Comparative Endocrinology, Hong Kong, 1981. Materials and Methods Aduit male Japanese red-bellied newts, Cynops pyrrhogaster pyrrhogaster, were collected at an appointed station in Hayakawa, Niigata Prefecture, in the middle of every month throughout the year. Forty-five newts were divided randomly into three groups (fifteen newts/group). A blood sample collected from each group was used to determine the Received June 28, 1982. 2 TANAKA AND TAKIKAWA Endocrinol. Japon. February 1983 plasma testosterone (T) and 5α-dihydrotestosterone (DHT) concentrations. 1, 2, 6, 7-3H-testostosterone (3H-T) and 1, 2, 4, 5, 6, 7-3H-5α-dihydrotestosterone (3H-DHT) were obtained from New England Nuclear Co. Aliquots of the pooled plasma with 3H-T and 3H-DHT internal standard (c .a. 1,000dpm) added were extracted three times each with 10-20 vol. of diethyl ether, and the extracts were chromatographed on a Sephadex LH-20 (Pharmacia Fine Chemicals, Inc.) column using hexane: benzene: methanol, 90:5:5 (V:V:V) as a solvent system in order to separate T and DHT. Following column chromatography, T and DHT fractions were dried in a centrifugal evaporator and redissolved in 1ml of ethanol. An aliquot of each sample was taken for radioimmunoassay (RIA) and another was counted for recovery of the added internal standard. Recoveries for T and DHT ranged from 56.4-69.7% and 54.471.0%, respectively. Fractions were assayed for T and DHT, using an atiserum cospecific for T and DHT (Teikoku Hormone Mfg. Co., Ltd.). The RIA procedure was carried out according to the method of Shodono et al. (1975) with minor modification. Appropriate aliquots of the ethanol solution were added to new 10×75mm assay tubes, evaporated, and then redissolved in 200μl of 1% BSA (bovine serum albumin) in phosphate buffered saline (PBS: 0.01M sodium phosphate, 0.14M NaCl and 0.01% merthiolate, pH 7.5). The assay system consisted of 200μl of the standard or a sample dissolved in 1% BSA・PBS, 200μl of antiserum diluted in 1% normal rabbit serum-0.05M EDTA-PBS and 100μl of 3H-T or 3H-DHT (ca. 10,000dpm) in 1% BSAPBS. The first incubation was carried out overnight at 4°C. After incubation, 500μl of ice-cold dextran-charcoal suspension (0.25% Norit A and 0.025% Dextran T70 in PBS) was added to the above mixtures which had been cooled in ice water. The mixtures were vortexed thoroughly and allowed to stand at 0°C for 30min. They were then centrifuged at 3,000rpm for 15min, and 800μl aliquots of the supernatants were transfered to liquid scientillator vials. Ten ml of toluene scintillator containing 0.4% 2,5-diphenolozole (PPO) and 0.01% 1,4bis-2-(5-phenyloxazolyl)-benzene (POPOP) was added to each vial, vortexed vigorously and left to stand overnight. The radioactivity was then counted with a liquid scintillation spectrometer (Beckmann LS7500). The intra-assay coefficients of variation of 5 determinations with a single assay in extracts of pooled newt plasma were 8.3% for T and 9.6% for DHT, and the inter-assay coefficients of variation were 12.4% for T and 13.2% for DHT. All values were corrected for losses during extraction and chromatography and expressed as means±standard error of the three groups in each month. Statistical comparisons were made using the t-test. Results As shown in Fig. 1, two distinct peaks were observed in the seasonal fluctuation of plasma T levels; one in early spring (Feb.-April) and the other in autumn (Sept. -Nov .) The concentration of plasma T rose significantly (p<0.05) between January and February and reached its peak in February (21.33±2.77ng/ml). The values in March (17.82±2.80ng/ml) and April (17.87±1.90 ng/ml) were not significantly different from that of February. From April to July, the Plasma T level gradually dropped and finally reached its minimum (0.72±0.12ng/ml). After the summer decline, there was a rapid increase from a 4.80±0.24ng/ml plasma T level to 22.83±4.60ng/ml in September (P<0.05), and its highest peak was reached in October (26.03±8.83ng/ml). Subsequently, the plasma T remained at a high level until November and was not significantly different from that of October. From November to December, the value decreased significantly (P<0.05). Concerning the seasonal fluctuation in plasma DHT levels, three peaks were observed the highest peak appeared in the middle of May; and two lower peaks in early spring (Feb.-March) and autumn (Sept.-Oct.). The most interesting event was a rapid increase in plasm DHT in the middle of May (20.93±3.35ng/ml). This value was several times higher than those of the peaks in spring (6.21±1.52ng/ml) and autumn (9.29±1.29ng/ml). Furthermore, this DHT value was about 3times higher than the T value during this period. Vol.30, No.1 PLASMA ANDROGEN IN NEWT 3 Fig. 1. Seasonal changes in plasma testosterone and 5α-dihydrotestosterone in male newts. Points and curves represent mean values for three groups in each month, and vertical bars indicate standard errors.