Influence of adrenal cortex on testicular activity in the toad during the breeding season

Life Sciences, Vol. 66, No. 13, pp. 1253-1260,2OCG Copyright 0 2000 Elsevier Science Inc. Printedin the USA. All rights reserved 0024-3205/OO/s_See front matter

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ELSEVIER

INFLUENCE OF ADRENAL CORTEX ON TESTICULAR ACTIVITY IN THE TOAD DURING THE BREEDING SEASON * N.M.Biswas, Gargi Ray Chaudhurt, MSarkar and RBengupta Reproductive Physiology Unit, Department of Physiology, University College of Science and Technology, 92, A.P.C. Road, Calcutta - 700009. India (Received in final form November 1, 1999)

Summary The aim of the present study was to determine the role of adrenals in gonadal activity in the male toad during the breeding season. Exogenous administration of corticosterone or metapyrone for 6 days inhibited adrenal A5-3 P(delta 5-3 beta) hydroxysteroid ( As-3P_ HSD) and testicular 17p (17 beta) hydroxysteroid dehydrogenase (17p -HSD) activities, decreased the serum levels of testosterone and inhibited spermatogenesis. When toads were treated with corticosterone a significant rise of serum corticosterone was noted while metapyrone treatment appeared to decrease serum corticosterone levels. It is concluded that adrenocortical hormone plays an indirect role in testicular activity in toads during the breeding season. Key Words:

adrenal, testis, metapyrone, toad, breeding season

Since Selye (1) reported his initial finding on an increased ACTH secretion from the anterior pituitary at the expense of gonadotropin, several investigators have tried to find out the relationship between adrenal and gonadal activities in different species of animals including man. It is well known that hypo and hyperfunction of the adrenal cortex can result in the depression of sexual function in man. Our previous study shows the adrenocortical hyperactivity and results in the inhibition of testicular steroid hormone synthesis in arsenite-treated rats (2,3). Adrenalectomy reduces testicular A 5-3p (delta 5-3 beta) hydroxysteroid dehydrogenase and spermatogenesis in rat (4). Chemical adrenalectomy also results in testicular atrophy in birds@. On the otherhand in vivo and in vitro studies shows that glucocorticoids have direct inhibitory effects on Leydig cell steroidogenesis(6). Similar inhibitory effect of glucocorticoids on Leydig cell steroidogenesis due to repression of cholesterol side chain cleavage cytochrome P 450 have been reported (7). Corticosterone administration also induces testicular regression in lizard during the breeding phase (8). But our recent observation shows that corticosterone increases testicular endocrine activity in toads during the hibernation when gonads remains at a quiescent state (9) The purpose of the present investigation was to determine whether corticosterone has an influence on testicular activity in toads during the breeding season. We also used metapyrone an inhibitor of adrenal steroidogenesis to study the effect of corticosterone deficiency in the testes in toads. Methods Chemicals: Corticosterone, BSA, EDTA, Testosterone, NADP were purchased from Sigma l

Reprint request to Dr. N.M.Biswas

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Chemical, USA. Corticosterone was dissolved in 10% alcohol and metapyrone was dissolved in 20% propylene glycol. Metapyrone was a gift from Ciba-Geigy, Basel, Switzerland. Animals : Experiments were carried out during the months of June and July (Breeding season). Sixty four male toads (Bufo melanosrictus) of average body weight 5Og were used for the present investigations. They were maintained in natural photothennal conditions (14:lO,L:D, 302 5OC).The animals were randomly divided in four equal groups. The toads were fed with ant eggs every alternate day and subjected to different treatment for 6 days as follows: Group I treated with vehicle (10% alcohol ) for 6 days (control) : Group 2 treated with corticosterone (3rg/O.lml 10% alcohol/ 1OOg b.w): Group 3 treated with another vehicle (lO%propylene glycol);Group 4 treated with metapyrone (2OOpgIO.1ml 10% propylene glycoU1OOgb.w) corticosterone (Sigma Chemical, USA) dissolved in 1O”h alcohol and metapyrone ( Ciba-Geigy, Switzerland, Basel) dissolved in propylene glycol were given subcutaneously to two different groups for 6 days at 1790 every evening. The animals were sacrificed 24 hours after the last injection. The pithed toad was operated with 2-3 cm incision in the abdominal wall. Chest was opened and blood was collected from the pumping heart. Testes were dissected out, treamed off fat, weighed and kept at -2ooC for studying enzyme activities. Blood samples were centrifuged and serum was preserved at -20” C for the estimation of corticosterone and testosterone. Serum corticosterone was determined by spectrofluorometry according to the methods of Glick (11) and Silber (12). The fluorescence was measured in a spectrofluorometer at 462 nm (excitation) and 516 nm (emission) by setting the instrumental sensitivity to an arbitrary point at 60 with high standard (0.6pg of corticosterone per 0.5 ml of distilled water). Adrenal ( A5-3 6 -HSD) was measured according to the method of Talalay (13). The adrenal glands were scrapped from the kidney, weighed and homogenized at a tissue concentration of 10 mglml in the fluid containing 20% spectroscopic grade glycerol, 5@ potassium phosphate and 1pM EDTA. The activity of testicular 176-HSD was measured according to the method of Jarabak (14). In the same homogenizing fluid, testes, obtained from different animals at a concentration of 50mg/lml were homogenized and centrifuged at 10,000 g for 30 min in a cold centrifuge.The supematant (Iml) was mixed with 440 ,u mol sodium pyrophosphate buffer (pH 10.2) 25 mg crystalline BSA and 0.3pmol testosterone, making the incubation mixture in a spectrophotometer at 340 nm against a blank with NADP. One unit of enzyme activity was equivalent to a change in absorbency of 0.001 mirrl at 340 nm. Radioimmunoassay of testosterone was carried out using testosterone lz51RIA kii (ICN Biochemical Inc., Diagnostic Division, Costa Mesa, CA 92626, USA) . Radioactivity was determined by the gamma Scintillation Counter at Bose Institute, Calcutta, India. All samples were run in duplicate in a single assay to avoid interassay variations. The interassay coefficient of variations for testosterone was 6.5% . For histological study of the testes, one testis from each animal (10 animals from each group) was fixed in Bouin’s fixative and used for embedding in paraffin wax. A section (5~ m thick) was cut from central portion of each testis stained with haematoxylin and eosin. For quantitative assesment of spermatogenesis, the process was divided into the following five stages. Stage 0, primary spermatogonia, in the resting phase; Stage I, small cell nests of secondary spermatogonia containing 10 cells; Stage II large cell nests of secondary sparmatogonia containing more than ten cells Stage Ill, primary spermatocytes; Stage IV, secondary spermatocytes( 15).

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Results Adultmale toads given corticosterone or metapyrone for 6 days and killed 24 hours later showed

no significant change in body weight in comparison with control animals (Fig. 1). Administration of corticosterone decreased adrenal A5-36 -HSD and testicular 17P_HSD activities along with the levels of serum testostemne (Fig. 1) while serum level of corticosterone was significantly higher than the control (Fll.1). On the other hand administratiin of metapyrone decreased adrenal As-BP -HSD and testicular 17p -HSD activities, serum levels of testosterone and corticosterone in comparison with vehicle treated toads (Fii.2). Corticosterone treatment for 6 days significantly decreased counts of germ cell nests representing primary (stage 0) and secondary (stages I & II ) spermatogonia and primary (stage Ill) and secondary (stage IV) spermatocytes in the testis of lall animals in comparison to the vehicle treated control (Table Ia 8 Fig.4)and metapyrone treatment showed significant decreased count of primary spermatocytes (stage III) of all the animals compared to vehicle treated control group (Table I b & Fig. 5).

TABLE I a Effect of corticosterone on spermatogenesis ( nest/tubule ) after 6 days during breeding season in toad.Values are mean _+ SEM. n=10 animals in each group. ANOVA followed by student ‘t’ test. Group

Treatment

0 Stage

stage I

1.

Control( O.lml 10 % alcohoV100g.b.w.)

2.

3~gcorticosteroneI 0.669.151' O.bP+J.Wl 0.1ml10 % alcohoU 100g.b.w) l

l

1.1650.096 1.1220.22

Stage11

Stage Ill

0.6O+JJ.o72.23.19

Stage IV 0.7ofl.04

0.24+J.O80' 0.969.163* 0.339.075"

p~0.01whencomparedtocontrol

* p
TABLE I

b

Effect of metapyrone on spermatogenesis ( nest/tubule ) after 6 days during breeding season in toad. Values are mean _+SEM.n= 10 animals in each group. ANOVA followed by student ‘ t’ test. Group 3.

4.

Treatment

OStage

Control(O.lml10% propy1enegiyu-N 1.12+0.31 100 g.b.w.) 200pgmetapyroneI 0.71+0.182 0.1ml10 %alcohol/ 100 g.b.w) 'p
Stageti

StageIll

StageIV

1.10~0.31 05~0.06

2.3kO.14

0.80_+0.06

Stage1

0.94_+0.148 0.48+0.06 1.6kO.21'

0.64+0.08

-

60

60

-

=2

0

-

54 E a3

1-

-

-

7

g:

-

6

I

l

II

Serum+e&terone

Serum Corticosterone

Effect~&ostrone( 3 @ 100 gm b.w.) on body wt. , adrenal@-3/I -HSD, testicular17 B_HSD,Serum corlicosterone,serum testosteronein tcsdduringbreedingseason. Values are mean : SEM, n=l0 animals in each group,ANOVA followedby students w test P< 0.01, w P < 0.001, whencomparedwiththe controlgroup.

Adrenal b- 3fb HSD

0

10

20

5”m

60

-

m

Testicular 17&HSD

m

Sody Weight

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Fig.3 Testicular section of vetchicle treated control toad showing all the stages of spermatogenesis . X 100.

Fig.4 Testis from corticosterone treated toad showing decreased number of spermatogenic cell nests at different stages. X 100.

Fig.5 Section of testis from metapyrone treated toad showing decreased cell nests of primary spermatocytes. X 100 .

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Discussion The present experiment provides new observations regarding the effects of corticosterone and its blocker, metapyrone on adrenal and testicular activity during the breeding season in toad. Corticosterone administration to normal toads results in the inhibition of A5-3P_HSDin the adrenal gland. Our previous observations showthat administration of corticosterone in normal rats decreases A5-3p -HSD is serum level of ACM and adrenal A5-3p -HSD activity (16). Since the enzyme concerned with steroid hormone synthesis in the adrenal gland of rat (17, 18) and ACTH regulates AS-39-HSD activity in adrenocortical steroidogenesis, a fall of serum corticosterone level and corticosterone treated toads might be a reflection of reduced ACM secretion by negative feedback of corticosterone in toad. Moreover, corticosterone or metapyrone administration decreases testicular 17P_HSDactivity and serum testosterone level. Testicular 173 -HSD also plays an important role in steroid hormone synthesis in testis producing testosterone from A4 - andro-stenedione and activity of the enzyme is regulated by LH(19, 20). So the reduced level of serum testosterone and testicular 17P_HSD activity may be due to reduced pituitary gonadotropin secretion in corticosterone or metapyrone treated toads. The quantitative analysis of the different spermatogenic stages of the testis reveals that both corticosterone and metapyrone reduce the number of spermatogonia and spermatocytes. The mechanism of actions of corticosterone and metapyrone in pituitary-adrenal-gonadal system in toad can not be determined from the present experiment. Studies on the relationship between adrenocortical and gonadal actiiities in bird reveal both parallel as well as antagonistic relationship (21). Reduction in testicular volume in Indian Garden lizard (Calores versicolor) after corticosterone administration during the breeding phase has been suggested to be due to negative feedback effect of elevated circulating corticosterone at the hypothalamo-hypophyseal gonadal axis resulting testicular regression(8). On the otherhand, inhibitory effects of dexamethasone, an inhibitor of ACTH release, on ovulation in the domestic hen (22) indicated a favourable influence of cotticosterone on gonad. Beneficial effect of corticosteroids in inducing the release of gonadotrophins has also been observed in rat (23). However, a chronic hypercotiicalism in Leghorn chicks decreases the weight of the testes (24). Several investigators have reported that corticosterone inhibits LH secretion in rats (25, 26). Corticosterone has been shown to metabolize to androgen (27) which by negative feedback may decrease pituitary gonadotropin release. ln vitro study with the rat pituitary cells also shows that inhibition of LH secretion by corticosterone is possibly due to decrease in arachidonic acid release (26). In the present study it seems that exogenous corticosterone increases the high circulating levels of endogenous cotticosterone to a level capable of reducing LH secretion by negatiie feedback in toads, although the mechanism involved in LH secretion remain to be determined. Further, metapyrone which blocks the synthesis of corticosterone by inhibiting the 11 -beta -hydroxylase enzyme (10) may cause an increased secretion of ACTH (28,29). Prolonged ACTH administration causes testicular and ovarian atrophy in sexually matured chickens (30). It has been reported that ACTH acts directly to inhibitgonadotropinseccmtii in adrenalectomized rats (31). Testicular inhibitii in the present study in toads after metapyrone administration is, perhaps a retlection of an increased ACTH secretion.

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In summary, it is evident that optimal level of adrenocotical hormone is required in toad for normal testicular activity during the breeding season. Either hypercorticalism by corticosterone or hypocorticalism after metapyrone administration inhibits testicular activity in toad.

Acknowledgment We are thankful to Prof. B.K. Dutta and Dr. S. Bhattacharyya, Department of Chemical Engineering, Calcutta University, for their kind Co-operation and constant encouragement in this work.

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