I Chowdhury & K P Joy* Department of Zoology, Banaras Hindu University, Varanasi , India Received 27 May 1999; revised 23 March 2000

Transcription

1 Indian Journal of xperimental Biology Vol. 38 July 2, pp ffects of administration of testosterone on some biochemical correlates in seminal vesicle of Heteropneustes fossilis (Bloch) during preparatory phase: A study correlating changes in plasma testosterone level and testis activity I Chowdhury & K P Joy Department of Zoology, Banaras Hindu University, Varanasi 221 5, India Received 27 May 1999; revised 23 March 2 In the catfish H. fossilis 1 administration of testosterone (.25,.5, I and 2 Jlg/g body weight for 2 days) during midpreparatory phase (March) increased plasma testosterone, gonadosomatic index, seminal vesicle-somatic index and concentrations of total proteins, fructose and hexosamines in seminal vesicle (SV) and testis in a dose-related manner. In the lowest dosage (.25 Jlg) group, only the hexosamine and SV protein levels were significantly high. Glucose level decreased in a dose-related manner, the decrease being not significant in the.25 Jlg group. The results indicate that testosterone stimulates SV and testicular secretions of total proteins, hexosamines and fructose in catfish. Decrease in glucose content suggests its conversion into fructose under testosterone stimulation. The male reproductive system of catfish is characterized by the presence of one or more pairs of seminal vesicle (SV) lobes which open to the common sperm dud 4. The SVs showed marked annual variations in their structure and secretory activity that can be correlated with the testis 1 ' 2.4. The secretory activity of SV is stimulated by androgens 5 7. In mammals, the epididymis and accessory sex organs secrete a variety of chemical substances like proteins, fructose, hexosarnines, sialic acid, etc., into the lumen which facilitate various functions such as capacitation, nutrition and transport of spermatozoa 8 1 These chemical correlates have been used as sensitive parameters to assess androgenic actions on the accessory sex glands. But in fishes, there are no studies involving the use of such sensitive markers to evaluate androgenic influences on testicular and SV metabolism except for a recent study by Singh and Joy 7 in the catfish arias batrachus. In both C. batrachus 7 and H. foissilis 11, total proteins, hexosamines and fructose showed significant annual variations with their concentrations increasing during gonadal recrudescent phase and decreasing during gonadal quiescent phase with the ex ception of glucose 11 Furthermore, the concentrations of these variables except glucose can be positively correlated with plasma testosterone titre 11. Although the functional androgen responsible for the stimulation of spermatogenesis and secondary sexual characters in many teleosts including catfish is not testosterone but its metabolites like 11-ketotestosterone 12-18, testosterone was shown to stimulate spermatogenesis in hypophysectomized Carassius auratus 19, Fundulus 15 and H. fossilis 5 2, possibly through its conversion into active androgens. Therefore, in the present study testosterone has been used to induce hyperandrogenism in the catfish H. fossilis to demonstrate androgenic control of SV and testicular function using biochemical parameters such as total protein, hexosamine, fructose and glucose. Materials and Methods Male H. fossilis (25-35 g body weight) were collected locally in Varanasi in early preparatory phase (last week of February 1997). They were acclimatized for 15 days under natural photoperiod and temperature (L:D 11.2: 12.8; ± 6.53 C in February and L:D 11.6:12.4; 24.2 ± 7.7 C in March) and fed goat liver during acclimatization and experiments. The acclimatized fish were divided into 6 groups of 5-55 each. Group 1 (initial control) fish were sacrificed at the start of the experiment. Fish were weighed and blood was collected by caudal puncture in heparinized tubes. The blood samples were centrifuged at 3 rpm for 15 min at 4 C to separate plasma. Plasma was stored at -2 C for testosterone RIA. The fish were sacrificed by decapitation; testis and seminal vesicle (SV) were dissected out, weighed, and stored at -2 C for biochemical analysis. Group 2 (parallel control) fish were injected with.1 ml propylene glycol as vehicle

2 714 INDIAN 1 XP BIOL, JULY 2 control. Groups 3,4,5, and 6 were injected with testosterone (Sigma) intraperitoneally daily for 2 days in dosages of.25,.5, I and 2 11glg body weight, respectively. Testosterone was dissolved in propylene glycol. After termination of the experiments, fish in all the groups were weighed, plasma collected and sacrificed by decapitation. The tissues (SV and testis) were removed quickly, weighed and stored at -2 C for biochemical analysis. Study parameters-seminal vesicle-somatic index (SVSI) and gonado-somatic index (GSI) were expressed as the weight of the SV and testis in g percent of body weight, respectively. Plasma testosterone was assayed by an quate RIA125I testosterone diagnostic kit procedure (Binax, Portland, USA). The minimum sensitivity of the assay was 18 pg I mi. Intra- assay and inter- assay coefficients of variation were 3.33 and 5.96 %, respectively. Total protein levels in the SV and testis was determined by the method of Lowry et al., 21. Hexosamine was measured by the procedure of lson and Morgan 22, as modified by Davidson 23. Fructose concentration in the SV and testis was measured according to the method of Mann 24. Glucose was measured by a standard glucose test reagent (acetic acid 94% and o-toluidiene 6% ). The details of the procedure were described previousl/ 11. Statistical analysis-data were expressed as means ± SM and analyzed by a one way analysis of variance (ANOV A), followed by Newman-Keuls' test. Differences were considered significant at P <.1 for ANOVA and P <.5 for Newman- Keuls' test. Results and Discussion A comparison of initial control and vehicleinjected parallel control data show that significant temporal changes were noticed in the levels of plasma testosterone, SV proteins and glucose, and both SV and testicular hexosamines and fructose (P <.5, Newman-Keuls' test). The administration of testosterone produced an overall significant effect on plasma testosterone level (Fig. 1; F = 12.97, P <.1, one way ANOVA). Testosterone level was significantly higher in.5, 1 and 2!lg groups compared to the vehicle control group (P <.5, Newman-Keuls' test; for other comparisons, see Table 1). The injected dosage range was effective to induce hyperandrogenism in the fish in a dosedependent manner. In a similar study in C. batrachus, testosterone administration caused elevations of both plasma testosterone and estradiol-17 levels 7. In this study also,.25!lg steroid injection elevated plasma gonadotropin-11 level (positive feedback) and other Table!-Comparison of group means by Newman-Keuls' multiple test Vehicle Testosterone dosages Plasma Testosterone SVSI GSI Protein sv Testis Hexosamines sv Testis Fructose sv Testis Glucose sv Testis Mean values of all parameters except glucose are arranged in ascending orders and of glucose in a descending order. Groups that are not significantly different are underscored.

3 CHOWDHURY & JOY : TSTOSTRON, SMINAL VSICL TC OF HTROPNUSTS I Testoste; 8 ' 7. 6 NS c: e Q) Q) :: 2 IC Veh Testosterone in)jg I g body weight Fig. 1-ffect of testosterone injections on plasma level of testosterone in H. fossilis (mean ± S, n=5). Comparisons with the vehicle control group are shown. P <.5; NS- not significant (one- way ANOVA, Newman-Keuls' test). IC Veh Testosterone in )l9 I g body weight Fig. 2-ffect of testosterone treatment on seminal vesicle somatic index (SVSI) and gonadosomatic index (GSI) in H. fossilis (mean ± S, n=5). Comparisons with the vehicle control group are shown. P <.5; NS- not significant (one- way ANOVA, Newman-Keuls' test)..

4 716 INDIAN J XP BIOL, JULY s:; 'ijj 6 5 G>., = c: 2 Q. IC Veh Testosterone in pg I g body weight Fig. 3- ffect of testosterone treatment on seminal vesicle (SV) and testicular concentrations of total proteins in H. fossilis (mean± S, n=5). Comparisons with the vehicle control group are shown. P <.5 ; NS-not significant (one -way ANOVA, Newman-Keuls' test) c 'Qi G) G) r::. CQ 2 >< G) J.:: 1 IC Veh Testosterone in )19 I g body...eight Fig. 4-ffect of testosterone treatment on seminal vesicle (SV) and testicular concentrations of hexosamines in H. fossilis (mean ± S, n=5). Comparisons with the vehicle control group are shown. P <.5 (one- way ANOV A, Newman-Keul s' test).

5 CHOWDHUR Y & JOY : TSTOSTRON, SMINAL VSICL TC OF HTROPNUSTS :c 7 'Gi 3: ) 6 ; ; 4.= ) 3 -u 2 u IC Veh Testosterone in )J9 I g body 'Neight Fig. 5- ffect of testosterone treatment on seminal vesicle (SV) and testicular concentrations of fructose in H. fossilis (mean ± S, n=5). Comparisons with the vehicle control group are shown. P <.5; NS- not signifi cant (one-way ANOV A, Newman-Keuls' test). 8 7 :, 6 "iii 3: > :::1 Ul 5 g 4.,... :::1.= 3 > Ul CJ :::1 2 a 1 IC Veh Testosterone in)lg I g body weight Fig. 6- ffect of testosterone treatment on seminal vesicle (SV) and testicular" concentrations of glucose in H. f ossilis (mean± S, n=5). Comparisons with the vehicle control group are shown. P <.5; NS- not signi ficant (one-way ANOV A, Newman-Keuls ' test).

6 718 INDIAN J XP BIOL, JULY 2 dosages decreased it (negative feedback) in a dose dependent manner 25. Therefore, the increase in plasma testosterone level in the 1 and 2!lg groups might be caused by the administered steroid itself. The testosterone level in 2!lg group (8.93 ng I ml plasma) was within the maximal limit of the steroid level (9.33 ng I ml) measured in plasma in prespawning phase of the annual reproductive cycle 11. The steroid treatment resulted in dose-dependent increases in the weight (Fig.2) of SV (SVSI, F = 28.59, P <. I, one way ANOV A) and testis (GSI, F = 7.96, P <. I, one way ANOV A). The increases were not significant in the.25!lg dosage group but were significantly higher in the higher dosage groups (see Table 1 for grotipwise comparisons). The increase in SVSI and GSI indicate an overall sttmulatory effect of the androgen treatment on these organs as has been d. I.. 2o 26 reporte 111 t 11s spectes ear t ter an d m C. batrachu/. In testosterone treated H. fossilis, the concentrations of proteins (Fig. 3; SV: F = 19.7; testis: F = 12.74; P <.1, one way ANOVA), hexosamines (Fig. 4; SV: F = 36.72; testis: F = 29.36; P <.1, one way ANOV A) and fructose (Fig. 5; SV: F = 1.78; testis: F = 6.44; P <.1, one way ANOV A) increased in a dose-dependent manner. In the low dosage group (.25!lg I g body weight), the changes were insignificant for fructose and testicular proteins. In the remaining dosage groups, the changes were significant (except for testicular fructose in the.5!lg group) and maximal in the highest dosage (2!lg ) group (see Table I for group comparisons). Proteins, hexosamines and fructose are major components of the epididymis and accessory sex gland secretions in mammals 8 ' 9 ' 24 and C. batrachui. In C. batrachus, a dose-dependent increase of total proteins, fructose and hexosamines were reported similar to the present observations. Thus, the positive correlation between testosterone and these variables observed during the recrudescent phase can be explained on the basis of the stimulatory effect of the steroid on the secretions of these variables. Cyproterone acetate (antiandrogen) treatment inhibited the concentrations in a duration-dependent manner in both castrated and sham castrated (testisintact) C. batrachui 7, indicating the specificity of the testosterone I androgen response. Glucose concentration, on the other hand, was decreased after the testosterone treatment in a dosedependent manner (Fig. 6; SV: F = ; testis: F = 7.29; P <. I, one way ANOVA). The decrease was insignificant in the.25!lg group and maximal in 2!lg group (Table I for other group comparisons). The annual pattern of glucose maintains an inverse relationship with fructose in both SV and testis during the reproductive cycle of the catfish. The decrease of glucose in response to the testosterone treatment suggests its conversion into fructose, as in mammals 9 ' 1. These data show that fructose is the main seminal sugar during the recrudescent phase and glucose in the quiescent phase 11. The differential patterns of the sugar levels are androgencontrolled9'1. In conclusion, the concentrations of proteins, hexosamines and fructose responded positively, and glucose negatively to hyperandrogeni sm induced by testosterone treatment. They can be used as sensitive markers to evaluate androgen actions in the gonads. That the SV and testicular correlates responded similarly to the steroid treatment suggests basic similarities in the regulatory mechanism of these metabolites. Acknowledgement I Chowdhury is thankful to the University Grants Commission of India, New Delhi for a research fellowship. 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