GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

39, 336-342

(1979)

Levels of Testosterone in the Plasma and Testis of the Viviparous Lizard (Lacer& vivipara Jacquin) during the Annual cycle Y. COURTY Laboratoire

de Biologie

J. P. DUFAURE

AND

Cellulaire et Gk&tique, Complexe Scientifique Clermont-Ferrand II, B. P. 45, 63170 AubiPre. Accepted

June

des Ckzeaux, France

UniversitP

de

13, 1979

Testosterone concentration in the plasma and testis were measured by radioimmunoassay in 90 males of viviparous lizards. Determinations were performed at twice monthly intervals from March to October during two annual cycles. Plasma level of testosterone reached a peak of 445 rig/ml in the mating season, then fell abruptly to the value of 2 nglml in July, rose in September, then remained at a similar level of approximately 30-40 &ml before and after hibernation. Testosterone levels in the testis followed the plasma values except in June when the testicular levels were high although the plasma concentrations were low. This period (June) is characterized by atrophy of the epididymis (secondary sexual character) and of the seminiferous tubules and precedes the renewal of spermatogenesis (early testicular recrudescence). The plasma values of testosterone and their range of variations in this species are the highest presently known in vertebrates.

et al., 1976; Arslan et al., 1978). However

Seasonal changes in size and histology of the testis and of the accessory sexual organs (epididymis, sexual segment of the kidney) are well known in numerous species of reptiles (Saint Girons, 1963; Lofts, 1969) and correlated variations in androgen levels are documented but still poorly detailed. It has been shown by metabolic conversion of precursors that testosterone is the principal androgen in some reptiles as in mammals (Callard 1967; Tam et al., 1969; Lofts and Choy, 1971; Hews and Kime, 1978). In contrast, Bourne and Seamark (1978) have reported that epitestosterone is the major metabolite produced by the testes of an australian lizard Tiliqua rugosa incubated with various radioactive precursors, the rate of testosterone synthesis not exceeding 10%. Seasonal changes have also been found using in vitro studies (Tam et al., 1969; Boume and Seamark, 1978). Annual variations in the levels of circulating androgens measured by competitive protein binding technique or more recently by radioimmunoassay have been reported within this class (Boume and Seamark, 1975; Callard

very little information is available concerning the testicular concentration of androgens and their relation to the plasma levels (Arslan et al., 1978). Here we report the values of seasonal changes in plasma and testis testosterone concentrations observed on a temperate Lacertilian species, the viviparous lizard (Lacerta vivipara). The annual testicular cycle of Lacerta vivipara has been described by Mesure (1968) and Morat (1969) using morphological and histoenzymological criteria (variations of A5-3 P-hydroxysteroid dehydrogenase activity) and compared to variations of the accessory sexual organs (epididymis and sexual segment of the kidney). This cycle resembles that of closely related species (Licht et al., 1969). MATERIALS

Collection of’ animals. One hundred seventy adult males (weighing more than 2.0 g) of the viviparous lizard (Lacerta vi\,ipara Jacquin) were collected from March to October at twice monthly intervals (excepted for August) during two annual reproductive cycles (Table 2 reports data obtained during one cycle). They were captured in several populations localized in the 336

00166480/79/l

10336-07$01.00/o

Copyright @ 197’9 by Academic Press, Inc. AJI rights of reproduction in any form reserved.

AND METHODS

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--rLAJMA * ,.a, * AIY” ’ =I-

TESTIS

337

nation of recovery from testicular extracts and extracts of plasma. An additional aliquot was taken and 30,000 dpm of PHjestosterone was added. Testosterone standards, ranging from 0.02 to 1 ng. were pipetted in duplicate into identical tubes and 30,000 dpm of PHpestosterone was added to each tube. After evaporation, 300 ~1 of testosterone antiserum diluted to I = 45,000 was added to each sample and standard tube. After incubation overnight at 4”, antibody-bound and free hormone were separated using dextrancharcoal. The tubes were centrifuged and the supernatant, containing the bound steroid, was decanted into counting vials and counted for 10 min in PPOPOPOP-Triton X-lOO-toluene solution. Distilled water blanks were included in each assay and these blanks generally did not exceed 15 pg. Accuracy was determined by adding known amounts (50, 100, 500, 1000 pg) of testosterone to distilled water. The estimates (mean t SD) were 51 rt_ 10. 98 i 8, 513 i 34, 874 ? 25 pg, respectively. If known amounts (50 to 1000 pg) of testosterone were added to biological samples, initial values were found with mean variations of 6.0%. A highly significant correlation (r = 0.99) was found between assay values and the quantity of testis extracted. The intra- and interassay variations, determined by repeated measurements of the same biological samples, were 6.0 and 10.2’S, respectively. The lower limit of the assay was 50 pg. The antibody was prepared according to Veyssiere et ul.

Massif Central (France). Animals were housed in laboratory for a short period not exceeding 24 hr. Prrpurufion of‘ samyfrs. After weighing, animals were killed without anesthesia, between 9 and 11 AM. After decapitation, the blood was collected from carotid vessels and placed into heparinized tubes. Plasma samples obtained by centrifugation were frozen in liquid nitrogen and stored at - 18” until the testosterone analysis was performed. Testes and epididymides were weighed and a small sample of each was fixed in Bouin-Holland fluid for histological control. Testes were frozen and stored as described for plasma. Rudioimmunoassoy of testosterone. Testosterone was measured by the method of Mahoudeau and Bricaire (1972). All samples of plasma (10 to 40 ~1) and testis (7-60 mg) were extracted with 6 ml of isooctaneiethyl acetate (713, v/v) after adding 4000 dpm of PHjtestosterone (Amersham: S.A. 80 Ciimmol). The extracts were washed with distilled water and the organic layer was dried in an airstream and redissolved in 1 ml of isooctane for transfer to Celite columns. Each column. containing 0.8 g of Celite -0.8 ml of ethylenglycol, was washed and saturated with isooclane Dihydrotestosterone was eluted with 5 ml of isooctaneibenzene (75124. v/v) and the testosterone was then eluted with 5 ml of isooctane/ethyl acetate (75124, v/v). This eluate was dried and redissolved in 1 ml of ethanol. A 0.2.ml aliquot was taken for determi-

TABLE ANTISERUM

TESTOSTERONE

1

SPECIFICITY

Steroid

Percentage cross-reaction at BiBo = 50%

llp,17~,2l-Trihydroxy-4-pregnene-3.20-dione (cortisol) 3P-Hydroxy-5-pregnen-20-one (pregnenolone) 17a-Hydroxy-4-pregnene-3,20-dione (17-OH progesterone)

:0.30 co.30 io.30

C,,

17,9-Hydroxy-4-androstene-3-one (testosterone) 17@-Hydroxy-5a-androstan-3-one (dihydrotestosterone) 5a-Androstane-3o, l’lp-diol (3a diol) Sa-Androstane-3& l’lp-diol (3p diol) 4-Androstene-3, 17-dione (androstenedione) 5-Androstene-3& 17p-diol (androstenediol) 17a-Hydroxy-4-androstene-3-one (epitestosterone) 3a-Hydroxy-5cu-androstan-17-one (androsterone) 3a-Hydroxy-SP-androstan-17-one (ethiocholanolone) 3/3-Hydroxy-Sol-androstan-17-one (epiandrosterone) Sa-Androstane-3,17-dione (androstanedione) 3fi-Hydroxy-4-androstene-17-one (dehydroepiandrosterone)

100 74.30 3.40 5.30 1.80 1.03 0.32 co.30 co.30 co.30