Indian Journal of Experimental Biology Vol. 42, August 2004, pp. 8 16-822

Effect of different intensities of swimming exercise on testicular oxidative stress and reproductive dysfunction in mature male albino Wistar rats

I Manna, K Jana & P K Samanta*

Dept. o f Su rgery & Radiology, West Bengal Uni versity of Animal & Fishery Sciences, Ko lkata 700 037. India

Received 10 September 2003; revised 6 April 2004

Swimming exerc ise fo r I, 2 and 3 hr for 5 days/week for consecuti ve 4 weeks, results in a significant reduction in testicul ar, epididymal, prostetic, seminal vesic le somatic indices; epididymal sperm count, sperm motili ty ; preleptotine spermatocytes, mi d pachytene spermatocytes and stage 7 spermatids; plasma levels o f testosterone, lu te ini zing hormone; testicular /15

, 3~-hydroxyste ro i d dehydrogenase, 17~-hydroxysteroid dehydrogenase; testicul ar supero>.ide di smutase, ca ta lase, glutathi one perox idase, glutathione-s- transferase and glutathione along with sig nifi cant e le vation in malondialdehyde in male albino rats. However, no signifi cant change was noted in fin al body weight, spermatogonia-A and plasma level of fo ll ic le stimulating hormone. The results that ox idati ve stress develops with the increasing o f exercise intensity, which may interfere in male reproductive acti vi ties.

Keywords: Exercise, Oxidati ve stress, Spermatogenesis, Steroidogenesis, Rat

Phys ical acti vity has been recognized as an important li fes tyle factor, which contributes to good health and delays the onset of many diseases later in life. It is well establi shed that mitochondri a generate reacti ve oxygen species (ROS) and that the extent of thi s production is a direct functi on of the rate of oxygen utili zati on 1• Because oxygen utilization increases 10 to 15 fo ld during exercise, it seems likely that free radical production will also be enhanced. Numerous studies have been published inves ti gating thi s concept2

. In addition, there have been several tudies that examined antiox idant defense systems, which appear to increase during regul ar exercise3

.

Intensive exercise can result dysfunction in the male reproducti ve sys tem3

.4 . Some research findings in thi s area have shown that chroni c exercise training lowers the levels of testosterone along with other reproducti ve hormonal abnormalities5

·6

. However, tes tosterone plays a major ro le in the development and maturation of sperm during the process of spermatogenesis and maintenance of testosterone levels within the Serto li cells is essential fo r the development of adequate numbers of mature, vi able

*Address for correspondence: 64/1/1 4, Belgachi a Road, Kolkata 700 037, India Phone: +033-2556 8329; Fax : +033-2557 1986 E- mail: samanta_pk2002 @yahoo.co. in

sperm that are necessary for a male to be fertil e7• ROS

are reported to damage almost all cellular macro­molecules including membrane po lyunsaturated fatty ac ids (PUFA), containing impairment of cellular function 8

. Testicular membrane is rich in poly­unsaturated fatty acids and is hig hly susceptible to oxidati ve stress .

The present study has been to find out the effect of chronic intensive exercise on male reproducti ve system in rats with testicular ox idative stress and antioxidant defense systems as parameters.

Materials and Methods Animals-Sexually mature 3-month o ld male

Wi star strain albino rats (24), weighing 127.95±4.05 g at the beginning of the experiment were used. They were housed in a temperature contro lled room at

25°±2°C, 60% RH with a 12: 12 L:D cycle 15 days prior to the experiment and thereafter during the whole period of experiment. Body weight of the animals was maintained among the groups by proper diet [dietary composition (egg albumin-420, corn starch-86, sucrose-240, cellulose-40, sa1t-80, coconut o il-70, oligoelements-16, vitamin B complex- 1.5) g kg- 1

] and water ad Libitum. All ani mals had their body weight recorded weekly. The principles of Laboratory Animal care (NIH publication No 85-23, revised 1985) were fo llowed throughout the experimental

MANNA et. al.: EXERCISE & OXIDATIVE STRESS IN RATS 817

schedule. The ex periment was duly approved by the Animal Ethics Committee of the Institute.

Exercise protocol-The rats were randomly divided into following 4 groups of 6 each: (1) Contro l (CG; no exercise); (2) exercise group I (EGI; exercise for l hr/ day, 5 days/week for 4 weeks); (3) exercise group II (EGIJ ; exercise for 2 hr/day, 5 days/ week for 4 weeks) and (4) exercise group III (EGIII; exercise for 3 hr/day, 5 days/week for 4 weeks). All the rats of the exercise groups swam at the same time in separate water tanks; with a calcu lated average 300 cm2 of water surface area for each rat and a depth of 60 em. at 35° ± I 0 C. An electric hair dryer was used to dry the body immediately on removal from water.

Collection of blood and reproductive organs­An imals of both the groups were killed by li ght ether anesthesia 24 hr after the last day of exercise in order to avo id the acute effect of exercise. No food was provided for 2 hr before the decapitation. Body weigh t of the animals was recorded . An initi al 5 ml of blood from the dorsal aorta was collected and allowed to clot for 3 hr at 4°C and then centrifuged at 3000 rpm (2000 g) for 15 min at room temperature. The plasma samples were separated and stored at -20°C prior to hormone assay . The testes and the accessory sex organs were dissected out and weighed for the study of steroidogenesis, and the activities of superox ide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-s­transferase (GST) along with the levels of glutathione (GSH) and malondi aldehyde (MDA) were performed .

Epididymal sperm count and sperm motility--The epididymal sperm were collected by cutting epididy mi s into small pieces and flushing the sperm in normal saline. The sperm collected was centrifuged at 250 g for 10 min. The pellet was resuspended in 2 ml of normal saline. An aliquot of sperm suspension was homogenized for a few seconds, thereafter centrifuged at 800 g for 10 min and used for biochemical assays. Sperm motility and count were noted. The acti vities of antioxidant enzy mes, the levels of non-enzymatic antioxidants and level of lipid peroxidation were measured in epididymis and epididymal sperm of both the g roups. Epididymal sperm counts and evaluation of the motility of epididymal sperm were done by the standard method9

.

The epididymal sperm was obtained as described above and incubated at 32°C, which is the optimum temperature of rat epididymal sperm. The epididymal fluid was diluted to a volume of 5 ml of pre-washed

(32°C) normal saline. An aliquot of thi s solution was placed in Neubauer hemocytometer and motile sperm were counted under compound microscope. Non­motile sperm numbers were first determined, followed by counting of total sperm. Sperm motility was expressed as a per cent of motile sperm of the total sperm counted.

Quantitative study of spermatogenesis--Quanti­tative study of spermatogenesis was carried out at stage 7111 of seminiferous epitheli al cycle according to the method of Clermont and Morgeutaler10

. Charac­teristic cellular assoc iation present in this stage is spermatogoni a-A (ASg), preleptot ine spermatocytes (pLSc) , midpachytene spermatoeytes (mPSc), stage 7 spermatids (7Sd) and most mature step 19 spermatids (19Sd). The different nuclei of the germ cells (except step 19 spermatids, whi ch can not be enumerated precisely) were counted.

Assay of hormones by RIA-Testosterone concen­trati on in plasma samples was analyzed fo llowing the method of Jacobs 11 using a double-antibody (1251) RIA (ICN, Biochemical Inc, Diagnostic Division, Costa Mesa, CA, USA). Pl asma level of leutinizing hormone (LH) and follicle st imulating hormone (FSH) were measured by RIA 12 using reagents supplied by the rat pituitary di stribution program and NIDDK (Bethesda, MD, USA). The hormone assays were caiTied out in duplicate .

Assay of testicular .15, 3{3-HSD and 17{3-HSD

activities-The levels of !15, 3B-hydroxysteroid

dehydrogenase (/15, 3B-HSD) 13 and 17B-hydroxy­

steroid dehydrogenase (17B-HSD) 14 were estimated using ti ssue homogenate taken from the left testis of each animal.

Assessment of testicular oxidative stress­Oxidative stress as indicated by the measurement of enzy matic and non-enzymatic antioxidants and the products of free radicals were estimated in testes. Antioxidant enzymes viz. superoxide di smutase (SOD) 15

; catalase (CAT) 16, glutathione peroxidase

(GPx) 17 and glutathionc-s-transferase (GST) 18. Non­

enzy matic ant ioxidant glutathione (GSH) 19 was measured in testi s. Testicular lipid peroxidation as indicated by malondi aldehyde (M DA)20 was esti­mated in all the groups. The amount of protein present in the ti ssue was measured by the Lowry 's method21

for the determination of oxidative stress parameters. Statistical analysis-The data were first converted

into mean and standard error of mean (SE). To find out the difference between the variables among the

818 INDIAN J EXP BIOL, AUGUST 2004

groups ANOV A followed by multiple comparison t­test with Bonfen·oni modification was employed22

.

Accordingly, statistical software package (SPSS) was used.

Results Effects of different intensities of swimming exercise

on body weights, reproductive organs weights, sperm count and sperm motility (Table 1)--The effect of chronic intensive exercise has shown no significant alteration in body weight among the groups. In contrast testicular, epididymal, prostatic and seminal vesicle somatic indices were decreased significantly (P<O.OS) in all the exercise groups. Sperm count and sperm motility were declined significantly (?<0.05) in all exercise groups compared to control. Moreover, severe effects were noted in EGIII compared to other exercise groups (EGI and EGII).

Effects of different intensities of swimming exercise on quantitative analysis of spermatogenesis (Table 2)--A significant reduction (P<O.OS) in the number of pLSc, mPSc and 7sd were noted in all the exercise groups. However, no significant alteration was noted in the number of ASg among the groups.

Effect of different intensities of swimming exercise on plasma levels of testosterone, LH and FSH (Fig. 1)--The levels of plasma testosterone and LH were found to reduce significantly (P<O.OS) in all the exercise groups when compared to control, whereas no significant change was observed in plasma levels of FSH among the groups.

Effects of different intensities of swimming exercise on testicular .15

, 3{3-HSD and 17{3-HSD activities (Fig. 2)--After prolonged intensive exercise, the activities of both testicular /15

, 3~-HSD and 17~-HSD were decreased significantly (P<O.OS) in all the exercise groups.

Effects of different intensities of swimming exercise on testicular oxidative stress (Table 3)--For the assessment of testicular oxidative stress, enzymatic and non-enzymatic antioxidants were measured along with products of lipid peroxidation. In all the exercise

Table 2-Effect of different intensities of swimming exercise on quantitative analysis of spermatogenesis in rats

[Values expressed in number are mean± SE from 6 rats in each group]

Group Asg pLSc mPSc 7Sd

CG 1.42" 14.9" 18.2" 40.6" ±0.26 ± 2.5 ± 2.1 ±2.7

EGI 1.40" 10.5b 14.5bb 34.5b ± 0.22 ±2.2 ±2.1 ± 2.3

EGII 1.40" 9.1 b 13.9b 30.J c ±0.27 ±2.1 ± 2.2 ±2.4

EGIII 1.41 a 8.8b 12.8 28.6c ±0.29 ±2.2 ±2.1 ±2.1

ANOV A followed by multiple comparison t test with Bonferroni modification. In each vertical column the mean with different superscript c ·b.c) differ from each other significantly, P<0.05. Asg=spermatogonia-A, pLSc=prelep­totine spermatocytes, mPSc=midpach)tene spermatoeytes, 7SD=stage 7 spermatids, CG=control group, EGI=exercise group I, EGII=exercise group II, EGIII=exercise group III.

Table !-Effect of different intensities of swimming exercise on body weight, testicular, epididymal, prostatic a d seminal vesicle somatic indices, epididymis, epididymal sperm count and sperm motility in rats

[values are mean± SE from 6 rats in each group]

Group mw· FBW• TS!t ESit Psrt SVS!t ESC .. SM 1

CG 127.5" J39.9a 1.41 a 0.606" 0.475" 0.472a 4.09" 81.66a

±4.1 ±3.4 ±0.06 ±0.04 ±0.03 ±0.02 ±0. 13 ± 9.43

EGI 128.1a 136.7" 1.25b 0.39b 0.37b 0.35b 3.5b 66.9b

±4.2 ± 2.1 ±0.05 ±0.04 ±0.03 ±.03 ±0.25 ±8.6

EGII 127.9a 138.2a 1.19b 0.33b 0.34b 0.34b 3.34b 56.7b

± 3.8 ±2.4 ±0.06 ± 0.03 ±0.04 ±0.03 ±0.19 ±9.2

EGIII 128.3a 136.1 a 1.08° 0.24c 0.25b 0.32b 3. 12c 42.34c

±4.1 ±2.2 ±0.04 ±0.04 ±0.03 ± 0.02 ±0.21 ± 8.67

ANOV A followed by multiple CL'Inparison t test with Bonferroni modification. In each vertical column the mean with different superscript ("· b. c) differ from each other significantly, P<0.05. IBW=initial body weight, FBW=final body weight, TSI= testicular somatic index, ESI=epididymal soma~ic index, PSI= prostatic somatic index, SVSI= seminal vesicle somatic index, ESC=epididymal sperm count, SM=sperm motility, CG:' control group, EGI= exercise group I, EGII= exercise group II, EGIII= exercise group III, *: g; t:g%; **: countxl08 epididymis' 1';!: %

MANNA et. a/. : EXERCISE & OXIDATIVE STRESS IN RATS 819

groups, a significant reduction (P<0.05) in the activities of testicular antioxidant scavenger enzymes like SOD, CAT, GPx and GST as well as non enzymatic antioxidant GSH were noted. Testicular lipid peroxidation, as estimated by the levels of MDA was elevated significantly (?<0.05) in all the exercise groups. However, significant (?<0.05) inter group variations were noted in CAT, GST, GSH and MDA levels among the exercise groups.

Discussion The present experiment enlights the effect of

oxidative stress on male reproductive system imposed by chronic heavy exercise in albino rats. In testicular

5 25 a

a

4 ~ CG 20 ,-., b b b b b b ] CJ EGI 0~ § EGII !:l .__ .... [] EGII ;.. ~ 2

'"' a

8 Vl

"' Q::;

0

Testosterone LH FSH

Fig. !-Effect of different intensities of swimming exercise on plasma levels of testosterone, LH and FSH in rats. [Each value represents mean ± SE from 6 rats in each group. ANOV A followed by multiple comparison t test with Bonfserroni modification . In each vertical column the mean with different superscript("· b) differ from each other significantly, P<0.05 . CG= control group, EGI= exercise group I, EGII= exercise group II, EGIII= exercise group III.]

steroidogenic events, f..5, 3~-HSD and 17~-HSD play

a key regulatory role23. The inhibition in steroidogenic

enzyme activities in the experimental rats after chronic exercise may be a result of low plasma levels of LH as this is a prime regulator of testicular steroidogenic enzyme activities24

. However, no significant change was noted in plasma level of FSH. Moreover, inhibition of testicular steroidogenic enzyme activities in experimental rats after intensive exercise may be due to elevation in testicular MDA products of lipid peroxidation, as mitochondrial and microsomal steroidogenic enzyme activities in testi s

'i:' 50

· ~ CG ~ EGII

~ 45 D EGI [] EGll :::1 a ~ 40

·.;:; bD 35

.g 30

·c: 2s 2-.2:! 20 ., ;;. 15 ~ 0: 10 a ~ 5

a

~ o.l-..a.:::.CL.:....:..~::::::I-.:.:.:i.l--.....l:ll::.l....;...l==~ s

D., 3~-HSD 17~-HSD

Fig. 2-Effect of different intensities of swimming exercise on testicular tJ.5, 3~-HSD and 17~-HSD activities in rats. [Each value represents mean ± SE for 6 rats in each group. ANOV A followed by multiple comparison t test with Bonfserroni modification. In each vertical column the mean with different superscript ("· b) differ from each other significantly, P<0.05. CG= control group, EGI= exercise group I, EGII= exercise group II, EGIII= exercise group III]

Table }---Effect of intensive swimming exercise on testicular lipid proxidation and antioxidant system in rats

[Values are mean± SE from 6 rats in each group]

Group soo· CAT• GPx· GST• GSHt MOAt

CG 0.54" 4.41" 37.2" 4.7" 311.7" 3.08"

±0.09 ±0.4 ±6.6 ±0.2 ±12.4 ±0.43

EGJ 0.37b 3.72b 29. lb 3.4b 271.5b 4.67b

±0.08 ±0.6 ± 8.5 ±0.3 ± 10.2 ±0.3

EGII 0.32b 2.65< 27.7b 2.8< 268.2b 5.7<

±0.08 ±0.7 ±7.6 ±0.3 ±10.3 ±0.4

EGJII 0.35b 2.21 < 24.4b 2.6< 244.8< 6.24d

±0.07 ±0.5 ± 8.8 ±0.2 ± 10.4 ±0.3

ANOVA followed by multiple comparison t test with Bonfserroni modification. In each vertical column the mean with different superscript ("· b. c. d) differ from each other significantly, P<0.05 . SOD=superoxide dismutase, CAT= catalase, GPx= glutathione peroxidase (GPx), GST= glutathione-s-transferase, GSH= glutathione, MDA= malondialdehyde, CG= control group, EGI= exercise group I, EGII= exercise group II, EGIII= exercise group III. •: Unit/mg protein; t: nmoUmg protein

820 INDIAN J EXP BIOL, AUGUST 2004

are reduced in the presence of these products25· 26. Free radicals cause cytotoxicity, one of the manifesta­tions of which can be observed through lipid peroxidations25. Induction of membrane damage through lipid peroxidation by UV irradiation has been observed27

. In the present study, the chronic intensive exercise elevated the levels of MDA. The elevation in tes ticular free radicals in the experimental rats has been supported by the diminution in the activities of testicul ar SOD, CAT and GPx as these are important scavenger enzymes against free radicals in male gonad28

. Moreover, low plasma levels of testosterone in the experi mental rats also strengthen the idea about the inhibitory effect of chronic intensive exercise on testicular steroidogenesis.

Further, the low activities of testicular !::.5, 3~-HSD and 17~-HSD has been supported by diminution in plasma testosterone levels in this experiment as tes tos terone is the prime androgen in male testis29 . Moreover, inhibition in testicular steroidogenesis is aga in confirmed in the present study by the significant diminution in epididymal somatic index, prostatic somatic index and seminal vesical somatic index in experimental group as the growth of the accessory sex organs are purely under the control of plasma testosterone30. The inhibition in gonadal steroido­genesis in stress condition is in consistence to others, where gonadal steroid synthesis significantly interferes in mammals in stress condition25·31.

32. In addition to that, a significant inhibition in testicular somatic index in experimental animals also supported the low level of plasma testosterone and LH in stress condition as testicular growth is dependant upon the plasma level of LH2532. Indeed, the actual causes for the diminution in plasma testosterone and LH levels in this experiment are not clear but this could occur due to hyper activation of hypophysical-adreno­cortical axis29 . As it has been well established that the stress full condition also stimulates this axis32.

Low level of LH and testosterone may interfere in spermatogenesis process33. The concentration of testosterone in the circulation is a function of the amount of hormone entering (testicular production and secretion) and the amount leaving (metabolic clearance) the blood pooL This process is affected by any change in the physiological state that alters metabolic turnover of the hormone34. In fact, testosterone secretion is affected mainly by testicular blood flow through the testicular area, since testosterone is lipid soluble and thus freely diffusible.

Further, the testicules apparently have little or no storage capacity for testosterone. Testicular blood flow moreover is a function of the levels of vascular vasoconstnctiOn or vasodilatation6·35. Therefore, anything that influences vascular tone can affect the rates of testosterone secretion by increasing sympa­thetic nervous system activitl. Chronic intensive swimming exercise-induced spermatogenic disorders has been reflected by the diminution in the number of different generati on of germ cells at stage VII in spermatogenic cycle. Stage VII of seminiferous epitheli al cycle was selected as quan titative study of spermatogenesis , because all varieties of germ cells are present at this stage 10

. Th is inhibition in spermatogenesis may be due to low level of LH and tes tosterone33. It has been observed that endurance exercise reduces the blood flow to the testicles and causes low level of testosterone secretion thus affecting some degree of spermatogenesis6. In the present study, swimming exercise was employed in order to minimize the changes in co re and testicular temperature in these animals (compared to running exercise). Hence, the changes that were noted in testicular functions following the exercise are less likely to be due to abnormally high testicular temperatures.

Beside this hormonal alterations, the spermatogenic inhibition in experimental rats (may be due to lipidperoxidation, indicated by the elevated levels of MDA as it is a product of lipid peroxidation) exert detrimental effect on steroidogenesis and spermato­genesis36. Diminution in testicular somatic index and relative wet weights of accessory sex glands in the experimental rats also supports the inhibition in testicular steroidogenesis37

. As body growth was not altered significantly in the experimental rats with respect to control, these adverse effects of intensive exercise on male reproductive organs may be due to oxidative stress caused by the increased production of free radicals. The elevation in the levels of MDA further supports the diminution in the activities of antioxidant scavenger enzymes like SOD, CAT, GPx and GST in testicular tissues of rats25·28 . The microsomal steroidogenic enzyme activities in testis were reduced as the level of MDA (breakdown products of lipid peroxidation) e levated38. SOD protects the cell against spontaneous lipid peroxidation and ROS generation. SOD and CAT act synergistically to remove superoxide anion (02"·) generated by NADPH-oxidase in the celL These play

MANNA et. a/.: EXERCISE & OXIDATIVE STRESS IN RATS 821

an important role in decreasing oxidative stress and membrane lip id peroxidation38

. GPX, selenium containing antioxidant enzyme with g lutathione as the electrone donar, removes peroxyl radicals (ROO") from various peroxides including H20 2 (ref. 38). Anti­oxidant enzymes such as GSTs, a family of enzymes, are able to detox ify electrophoratic compounds by catalyzing the fo rmation of g lutathione conjugates39

.

Mammal GSTs are also involved in the transport of sexual steroids and could pl ay a key role in the physiological action of sex steroids. GSH is the most abundant non-protein thiol fou nd virtually in all mammali an cells. GSH serves multiple roles in cellular antiox idant defenses38

. The most important antioxidant function of GSH is to remove hydrogen peroxide and organic peroxides. Therefore, any decline in the level of GSH indicates the increased production of free radicals38

, as found in the present study.

The overall results of the study indicate that oxidati ve stress is imposed on male reproductive system by chronic intensive exercise. It is suggested that with the increas ing intensity of exercise the detrimental effect of oxidative stress elevated gradually. Long-term exposure to such types of stress may interfere in testicular steroidogenesis and spermatogenesi s, which may lead to disorders in male reproductive systems.

Acknowledgement Authors are thankful to Dr. J. Sengupta, Patho-Win

Laboratory, Kolkata, West Bengal, India for helping in RIA of LH and testosterone.

References I Di Meo S & Venditt i P, Mitochondria in exercise- induced

oxidative stress, Bioi Signal Recept, 10 (200I) I25. 2 Temiz A, Baskurt 0 K, Percetin C, Kandemir F & Gure A,

Leukocyte activation, ant iox idant stress and red blood cell properties after acute, exhausting exercise in rats, Clin Hem Micro, 22 (2000) 253.

3 Manna I , Jana K & Samanta P K, Effect of intensive exercise-induced testicular gametogenic and steroidogenic di sorders in mature male Wi star s train rats: A correlative approach to oxidati ve stress, Acta Physiol Scand, 178 (2003) 33.

4 Hackney A C, The male reproductive system and endurance exerc ise, Med Sci Sport Exerc, 28 ( 1996) I 80.

5 Hackney A C, Endurance exercise training and reproductive endocrine dysfunction in man: Alterations in the hypothalamic-pituitary-testicular axis, Cur Pharmacal Dis, 7 (200 1) 261.

6 Raastand T, Bjoro T & Hallen J, Hormonal responses to high and moderate- intensity strength exercise, Eur J Appl Physio/, 82 (2000) 12 I.

7 Almeida S A, Petenusci S 0 , Franci J A, Rosa De Sil va A A & Carvalho T L, Chronic immobilization-induced stress increases plasma testosterone and de lays testicular matu ra­tion in pubertal rats, Androlgia, 32 (2000) 7.

8 Radak Z, Sasvari M, Nyakas C, Pucsok J , Nakamoto H & Goto S, Exercise preconditioning against hydrogen peroxide induced ox idati ve damage in proteins of rat myocardium, Arch Biochem Biophy, 376 (2000) 248 .

9 Linder R E, Strader L F & McElroy W K, Measurement of epididymal sperm motility as a test variable in the rat, Bull Environ Con tam Toxicol, 36 ( 1986) 3 I 7.

I 0 Clermont Y & Morgentaler H, Quantitat ive study of sper­matogenesis in hypophysectomized rat, Endocrinology, 57 (1955) 369.

II Jacobs L S, Prolac tine, in Methods of hormone radio­immunoassay, edited by B M Jaffe and H R Bhronan, (Academic Press, New York) 1974, 199.

12 Moudgal N R & Madhawa Raj H G, Pituitary gonodotroph, in Methods of hormone radioimmunoassay, ed ited by B M Jaffe & H R Bhronan (Academic Press, New York) 1974, 67.

13 Talalay P, Hydroxysteroid dehydrogenase, in Methods in enzymology, edited by S P Colowick & N 0 Kaplan (Academic Press, New York) 1962,512.

14 Jarabak J, Adams J A, Willi ams-Ashman H G & Talaly P, Purificat io n of 17!)-hydroxystero id dehydrogenase function, J Bioi Chem, 237 ( 1962) 345.

15 Paoletti F & Mocal i A, Determination of superoxide dismutase activity by purely chemi cal system based o n NAD(P)H ox idation, in Methods in enzymology, edited by I Packer & A N Galzer (Academic Press, San Diago) 1999, 209.

16 Beers R F & Sizer I W, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J Bioi Chem, 195 ( 1952) I 33.

17 Paglia D E & Valentine W N, Studies on quantitative and qualitati ve characterization of erythrocyte g lutathi one peroxidase, J Lab Clin Med, 70 ( 1967) 158.

18 Habig W H, Pabst M J & Jacoby W B, Gl utatione-s­transferase: The first steps in marcapturic acid formation , J Bioi Chem, 249 (I 974) 7130.

19 Ellman G L, Ti ssue sulphydryl groups, Arch Biochem Biophys, 82 ( 1959) 70.

20 Ohkawa H, O hi shi W & Yagi K, Assay for lipid peroxidation in animal ti ssue by thiobarbituric acid reaction, Anal Biochem, 95 ( 1979) 35 1.

2 1 Lowry 0 H, Rosenbrough N J, Farr A L & Randall R J, Protein measurement with the folinphenol reagent, J Bioi Chem, 193 ( 1951) 265.

22 Das D & Das A, Statistics in biology and psycho logy (Academic Publisher, Calcutta) 1998.

23 Murono E P & Payne A H, Testicular maturation in the rat, in vivo effect of gonadotropins steroidogenic enzymes in hypophysectomized immature rats, Bioi Reprod, 20 ( 1979) 9 11.

24 Shaw M J, Georgapoul s L E & Payne A H, Synergistic effect

o f FSH and LH and testicular tJ.5, 3!)-hydroxysteroid dehydrogenase isomerase, applicati on of a new method for the separation of testic ular compartments, Endocrinology. 104 (1979) 9 12.

25 Debnath D & Mandai T K, Study of quinalphos (an envioronmental oestrogenic insecticide) formulation (Ekalux

822 INDIAN J EXP BIOL, AUGUST 2004

25 E.C.)-induced damage of the testicular tissues and antioxidant defence systems in Sprague-Dawley albino rats, J Appl Toxicol, 20 (2000) 197.

26 Halliwell B & Gutteridge J M C, Role of free radicals and catalytic metal ion in human disease, Method Enzymol, 186 (1989) I.

27 Mandai T K, Ghosh S, Sur P & Chatterjee S N, Effect of led and sunlight induced lipid peroxidation in liposomal branes, lnt J Rad Bioi, 33 (1978) 75.

28 Chainy G B N, Samantaray S & Samanta L, Testost·erone­induced changes in testicular antioxidant system, Androlgia, 29 (1997) 343.

29 Rivier C & Vale W, Effects of corticotropin-releasing factor, neuro-hypophyseal peptides, and catecholamins on pi tuitary function, Fed Proc, 44 (1985) 189.

30 Edman C D, The effect of steroid on endometrium, Semin Reprod Endocrinol, I (1983) 179.

31 Demura R, Suzuki T, Nakamura S, Komatsu H, Odagiri E & Demura H, Effect of immobilization stress on testosterone and inhibin in male rats, J Androl, 10 ( 1989) 210.

32 Kulin H E & Reiter E 0, Gonadotrophins during childhood and adolescence, Pediatrics, 51 (1973) 260.

33 Chowdhury A K, Dependence of testicular germ cells on hormone, a quantitative study in hypophysectomized testo­sterone treated rat, J Androl, 82 ( 1979) 33 1,

34 Griffin J E & Wilson J D, Disorders of the testis and the male reproductive tracts, in Williams's textbook of endocrinology, edited by J D Wilson (Saunders, Philadelphia) 1992, 799.

35 Hackney A C, Sinning W E & Bruot B C, Hypothalamic­pituitary-testicular axis functi on in endurance trained male, lnt J Sport Med, II ( 1990) 298.

36 Ghosh D, Biswas N M & Ghosh P K, Studies on the effect of prolactin treatment on testicular steroidogenesis and gametogenesis in lithium-treated rat, Acta Endocrinol, 125 (1991) 313.

37 Trasler J M, Hales B F & Robaire B, Chronic low dose cyclophosphamide treatment of adult male rats: Effect on fertility , pregnancy outcome and progeney, Bioi Reprod, 34 (1986) 275.

38 Powers S K, Ji L L & Leeuwenburgh C, Exercise trai ning­induced alterations in skeletal muscle antioxidant capacity: A brief review, Med Sci Sports Exerc, 31 (1 999) 987.

39 Hemachand T, Gopalakrishnan B, Salunke D M, Totey S M & Shaha C, Sperm plasma-membrane-assoc iated glutathione­s-transferase as gamete recognition molecules, J Cell Sci, 115 (2002) 2053.