most vertebrates are unable to grow and reach their normal adult form without the thyroid hormone [ 1 , 2 ] . however , various subsequent studies revealed that thyroid hormone plays a key role in rat testis development [ 47 ] . mammalian testis is a target of thyroid hormone action and altered thyroid function which is known to affect testicular functions [ 8 , 9 ] . thyroid hormone is well known as a physiological modulator of oxidative stress [ 10 , 11 ] . previous studies on the role of thyroid hormone in testis are mainly focused on histological and physiological aspects resulting into reproductive failure [ 6 , 8 , 1214 ] . altered thyroid function during early stages of development and maturation may adversely affect testicular growth and physiology [ 79 ] ; particularly the sertoli cells , which play a major role in spermatogenesis and are the main cell type in the testis which expresses t3 receptors . it is well established that the formation of normal numbers of sertoli cells is a key factor in determining testis size , germ cell numbers per testis , and sperm production rate in adulthood in a range of mammals , including humans . in our earlier reports , it was demonstrated that experimentally hypothyroidism modulates several oxidative stress and antioxidant defence parameters in mitochondria and postmitochondrial fractions in adult rat testis [ 18 , 19 ] . we also reported about the effects of transient and persistent hypothyroidism on testicular antioxidant defence system in mature rats to know the role of hypothyroidism - induced oxidative stress in testicular development and maturation . however , information about the role of deprivation of thyroid hormone in early developmental growth and function of testis in relation to antioxidant status in immature rats is inadequate . in rat , spermatogenesis reaches the pachytene stage at 20 days of age . at 25 days of age , round spermatids appear , and at the age of 50 days , mature spermatozoa are released into seminiferous tubular lumen [ 20 , 21 ] . fetal type leydig cells disappear soon after birth ( during first 2 weeks after birth ) and are replaced by adult type leydig cells [ 22 , 23 ] . sertoli cell proliferation reaches its maximum level just before birth and ceases by the age of 3 weeks . hence , four weeks or around 30 days of age , testis is undergoing critical stages of development and thyroid hormone is playing a key role during this stage . in our earlier studies , it was reported that transient hypothyroidism ( from day 1 of neonatal age till day 30 ) modulated testicular antioxidant defence status as well as functions in adult stage . moreover , it was also shown that germ cells in these rats were under oxidative stress and had poor antioxidant defence system . so , it is interesting to know about the testicular antioxidant defence status and oxidative stress parameters along with testicular physiology at the 30 days of age , that is , before puberty . the objective of the present study is to investigate the modulation of antioxidant defence status in neonatal persistent hypothyroid rats before their sexual maturation . in addition , we have tried to identify the specific cell populations in testes vulnerable to degeneration during neonatal hypothyroidism in immature rats . male pups obtained from breeding were made hypothyroid from day 1 of neonatal age till day 30 of postnatal age . hypothyroidism was induced in neonates by feeding the lactating mother with 0.05% 6-n - propyl-2-thiouracil ( ptu ) through the drinking water [ 7 , 18 , 25 ] . from the day of parturition till weaning ( 25 day postpartum ) , the pups received ptu through mother 's milk ( or ) drinking water and then directly from drinking water containing 0.05% ptu for the remaining period of experimentation . adult male wistar rats ( rattus norvegicus ) were divided into two groups containing 15 animals each . group - ii rats were treated with ptu from day 1 postpartum to day 30 postpartum . animal care , maintenance , and experiments were conducted under the supervision of the institutional animal ethics committee ( iaec ) regulated by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india . on the 31st day postpartum , body weight of animals in group - i and ii was recorded ; the animals were sacrificed by decapitation , trunk blood was collected and allowed to clot and then centrifuged to obtain sera . the serum levels of total t3 , t4 , tsh , and testosterone were measured by using elisa kits ( monobind , inc . , costa mesa , ca , usa , and equipar diagnostici , italy ) . testes and accessory sex organs , that is , seminal vesicle , ventral prostate and epididymis were removed , cleaned in cold 0.15  mol / l nacl ( normal saline ) , pat dried , and weighed . testes from three animals were pooled to one sample and five such samples were taken for the study . the whole procedure of tissue processing was done as described earlier by sahoo et al . a 20% ( w / v ) homogenate of testis was prepared in 50  mm phosphate buffer , ph 7.4 , containing 0.25  m sucrose . the crude homogenate ( ch ) was centrifuged at 600  g for 10  min to precipitate nuclei and other cellular debris . the resulted supernatant was again centrifuged at 10,000  g for 20  min to separate mitochondria . the mitochondrial pellet was washed thrice in 50  mm phosphate buffer , ph 7.4 ( 10,000  g for 5  min each ) , and finally suspended in the same buffer to obtain mitochondrial fraction ( mf ) . for lipid peroxidation ( lpx ) , lpx was measured in ch and mf by monitoring the formation of thiobarbituric acid - reactive substances ( tbarss ) following ohkawa et al . the assay was performed in presence of 0.02% ( w / v ) butylated hydroxytoluene to suppress artefactual peroxidation during heating . malondialdehyde ( mda ) was used as the standard and tbars were expressed in terms of mda equivalents as nmol tbars formed / mg protein . protein carbonyl ( pc ) content was estimated in testicular ch , mf , and pmf following levine et al . for measuring protein - sh content , pmf and mf samples were first precipitated in ice - cold 5% trichloroacetic acid ( tca ) containing 0.01  m hcl and centrifuged at 1000  g for 15  min . protein precipitates dissolved in 8  m guanidine hydrochloride were then used to measure thiol content and presented as mol / mg protein [ 29 , 30 ] . total glutathione content was measured in the supernatant of tca - precipitated testicular pmf and mf samples by enzymatic recycling procedure and gssg was measured after masking gsh with 2-vinylpyridine [ 31 , 32 ] . total gsh equivalents , gsh and gssg contents were expressed as nmol / g tissue . hydrogen peroxide ( h2o2 ) content was determined in testicular pmf and mf following horseradish peroxidase - dependent oxidation of phenol red . for measuring the activity of catalase ( cat ) , pmf treated with ethanol ( 0.17  m ) and triton x-100 ( 1% ) was directly used . the enzyme activity was measured following decomposition of h2o2 at 240  nm as described earlier   and expressed as nkat / mg protein . superoxide dismutase ( sod ) activity in pmf ( for cu / zn - sod ) and mf ( for mn - sod ) of testis was determined following modified nitrite method . one unit of enzyme activity was defined as the amount of enzyme capable of inhibiting 50% of nitrite formation under assay conditions . glutathione peroxidase ( gpx ) activity was assayed in pmf and mf by measuring oxidation rate of nadph in presence of hydroperoxide , gsh , and glutathione reductase ( gr ) [ 19 , 36 ] . total and selenium - dependent gpx activities were estimated by using cumene and tert - butyl hydroperoxides , respectively . the difference between total glutathione peroxidase and selenium - dependent glutathione peroxidase ( se - d - gpx ) activities represents the selenium - independent glutathione peroxidase ( se - i - gpx ) activity [ 19 , 37 ] . glutathione reductase ( gr ) activity in testicular pmf and mf was assayed by measuring oxidation rate of nadph in presence of gssg . in testicular pmf and mf , glutathione - s - transferase ( gst ) activity was measured following change in absorbance of the conjugated product of gsh and 1-chloro , 2 , 4-dinitrobenzene ( cdnb ) at 340  nm . gpx , gr , and gst enzyme activities were expressed as nkat / mg protein . glucose-6-phosphate dehydrogenase ( g6pd ) activity was assayed in pmf by measuring nadph formation from glucose-6-phosphate along with nadp   and expressed as nkat / mg protein . following sacrifice , testis tissues were immediately fixed for histological studies in freshly prepared sublimate formal , dehydrated in graded ethanol series , cleared in xylene , and embedded in paraffin wax . all data represent means  standard deviation and were subjected to unpaired student 's t - test to find out the level of significance between control and experimental rats . the weight gain in group - ii rats on 31st day of age was almost 50% less than group - i control rats ( table 1 ) . the weight of testis , seminal vesicle , and ventral prostate ( g/100  g body wt ) decreased significantly in ptu - treated rats of group - ii ( table 2 ) . the serum t3 , t4 , and testosterone level decreased significantly whereas the level of tsh increased by several folds in group - ii rats in comparison to control rats in group - i ( table 3 ) . marked decrease in seminiferous tubule diameter was observed in neonatal persistent hypothyroid 30-day - old rats ( group - ii ) as compared to the corresponding controls ( table 4 ) . there was a significant decrease in spermatogonia ( 24% ) and spermatocytes ( 79% ) accompanied by complete absence of round spermatids in group - ii testis ( table 4 ) . however , a significant increase in the sertoli cell number in group - ii rat testis was marked ( table 4 ) . the level of endogenous lipid peroxidation showed a decrease by 13.6% and 64% in crude and mitochondrial fractions , respectively , in response to 30 days persistent ptu treatment ( group - ii ) in comparison to control ( group - i ) rats ( figure 1 ) . a significant elevation in protein carbonyl content was recorded in the crude homogenate ( by 35.6% ) , mitochondrial ( 78% ) , and postmitochondrial fraction ( 22% ) of testis of group ii hypothyroid rats when compared to the controls ( group i , figure 2 ) . hydrogen peroxide content remained unaltered in pmf and decreased ( by 15% ) in mf of group - ii ptu - treated rats ( figure 3 ) . the protein - sh content decreased significantly by 14% and 24% , respectively , in mitochondrial and postmitochondrial fractions of testis of group - ii rats ( figure 4 ) .   in mitochondrial fraction of testis of group - ii , rats , total gsh equivalent , oxidized , and reduced glutathione contents and gsh to gssg ratio declined , respectively , by 50% , 16.7% , 67% , and 61% ( table 5 ) . on the other hand , the group - ii testicular postmitochondrial total gsh equivalent and reduced glutathione contents did not alter with 23.5% decrease in gssg activity and 27% elevation in gsh to gssg ratio ( table 5 ) . the mn - sod activity was elevated by 20% in mf whereas cu / zn - sod activity was increased by 30% in pmf of testis of group - ii rats ( figure 5 ) . in the postmitochondrial fraction of testis , the cat activity was increased by 83% in response to ptu treatment in group - ii rats in comparison to control rats ( group - i ) ( figure 6 ) . in group - ii rat however , in postmitochondrial fraction , total gpx and se - i - gpx declined , respectively , by 6.7% and 22.35% with 9.5% elevation in se - d - gpx activity ( figure 7 ) . the gr activity increased significantly by 56% and 55% , respectively , in mitochondrial and postmitochondrial fractions of testis of group - ii rats in response to ptu treatment for 30 days ( figure 8) .   the gst activity decreased by 59% in mitochondrial fraction and elevated by 42% in postmitochondrial fraction of testis in response to ptu treatment in group - ii rats when compared to control rats ( group - i , figure 9 ) . an increase in enzyme activity in post mitochondrial fraction of testis was recorded in group - ii rats by 73% ( figure 10 ) . thyroid hormones are reported to play a critical role in growth , differentiation , maturation , and metabolism in vertebrates . essential role of thyroid hormone in male sexual maturation , and reproduction was reported earlier [ 4 , 6 , 42 ] . both hyperthyroidism and hypothyroidism states alter testicular antioxidant defence parameters in adult rats [ 7 , 18 , 19 , 25 , 4345 ] . however , the effects of persistent hypothyroidism on neonatal rats before their puberty need to be studied . the present study reports the effect of neonatal persistent hypothyroidism induced by ptu treatment on the testicular antioxidant system and spermatogenic function in rats before puberty . the efficacy of the treatment was confirmed by a dip in t3 and t4 levels and a consequent increase in tsh level in serum of neonatal persistent hypothyroid rats . ptu is well known to decrease the conversion of peripheral t4 to t3 and thereby reduces serum t3 concentration . a significant reduction in the body weight was observed in ptu - treated rats when compared to control rats as reported earlier in case of neonatal hypothyroidism . the lower h2o2 contents in hypothyroid rats might be due to lower superoxide production in hypothyroid state due to hypometabolic rate . the hypothyroid rat testis resulted in a reduction in lipid peroxidation , which might be as a result of metabolic depression due to hypothyroid condition which serves as a protective factor to prevent lipid peroxidation [ 18 , 47 , 48 ] . in another study , ( 2005 )   also observed a low lipid peroxide content in testicular and renal tissues of hypothyroid rats . on the contrary , oxidative stress is marked as the increase in protein carbonyl contents in crude and mitochondrial fraction of hypothyroid rat testis in the present study . ( 2008 ) , who observed an increased level of carbonylation of protein in crude homogenate of testes of hypothyroid rats . superoxide dismutase ( sod ; ec 1.15.1.1 ) constitutes the first line of coordinated enzymatic defense against ros by dismutating o2 into o2 and h2o2 . catalase ( cat ; ec 1.11.1.6 ) and glutathione peroxidase ( gpx ; ec 1.11.1.9 ) are most crucial for detoxifying h2o2 , thereby preventing the generation of hydroxyl radical by the fenton reaction . selenium - dependent glutathione peroxidases ( se - d gpxs ) are the major selenoprotein - containing gene family in mammals . among the different types of selenium - dependent hydroperoxide - reducing isozymes , phospholipid hydroperoxide glutathione peroxidase ( ph - gpx / gpx-4 ; ec 1.11.1.12 ) and classic cellular glutathione peroxidase ( cgpx / gpx-1 ; ec 1.11.1.9 ) other gpx activities in mammalian systems are selenium independent . the se - independent gpx ( se - i gpx ) , component of gst alfa class ( accession : ipr003080 gst_alpha ) , is responsible for gpx activity in testis . a significant elevation in total sod and cat activities in response to neonatal persistent hypothyroidism and a simultaneous decrease in total gpx , se - d gpx ( gpx-1 and gpx-4 ) , and se - i gpx in the pmf of testis in experimental group suggested that sod and cat have predominant role to fight oxidative stress than gpx in hypothyroid rats . the majority of the cytosolic gpx in rat testis existed as selenium- and nonselenium - dependent gpx which is present in the leydig cells . gpx is primarily responsible for h2o2 removal in testicular mitochondria that does not contain catalase . gpx plays a crucial role in scavenging peroxyl radicals and thereby maintains functional integrity of the cell membrane , spermatogenesis , sperm morphology , and motility . in testis , ph - gpx or gpx-4 which is partially cytosolic and partially bound we observed that mitochondrial se - dependent gpx ( including gpx1 and gpx-4 ) activity was unaltered in hypothyroid rats as reported earlier by chattopadhyay et al . in fact , the role of cgpx / gpx1 in protecting testes from oxidative injury is questionable as cgpx knockout mice were found to be fertile . it has also been suggested that the metabolic pathway of testosterone biosynthesis requires protection against peroxidation and will be affected by a decrease in the gpx activity . the lower serum testosterone level in hypothyroid rats in the present study also corroborates the fact . mammalian testis contains relatively high levels of gsh , which is reported to play an important role in the proliferation and differentiation of spermatogenic cells   by protecting them from noxious effects of ros . the level of gsh is different in testicular cell types , having maximum level in sertoli and germ cells . thyroid hormone is known to play a role in triggering the biosynthesis of gsh , therefore under hypothyroid states , it is possible that there may be a fall in gsh levels contributing to susceptibility of the testes to oxidative stress . the ratio of reduced glutathione ( gsh ) to oxidized glutathione ( gssg ) in tissues is also considered as one of the important marker of oxidative stress . the decrease in its ratio and also a decrease in gsh contents in testicular mf of hypothyroid rats suggest severely hindered gsh metabolism . such type of decreased gsh contents in testis - mitochondria of hypothyroid rats was also reported earlier . moreover , declined gsh contents in germ cells were reported earlier in case of transient hypothyroid rats . the decreased protein - sh contents in both mitochondrial and postmitochondrial fractions might be due to the response against disturbed testicular redox pool in hypothyroidism . similar effects on testicular protein - sh during hypothyroidism were reported by chattopadhyay et al . glutathione - s - transferase ( gst ; ec 2.5.1.18 ) metabolizes xenobiotics by conjugating with gsh . in fact , gst - catalysed conjugation of gsh with exogenous compounds and endogenous metabolites such as 4-hydroxynonenal is regarded as major cellular defense mechanism against toxicity . the decreased activity of this enzyme in mf of testes in neonatal persistent hypothyroid rats reflects the inability of the testis to counteract oxidative stress by this pathway . glutathione reductase ( gr ; ec 1.6.4.2 ) is the enzyme responsible for maintenance of gsh by reducing gssg back to gsh . stimulated gr activity in neonatal hypothyroid rats indicated that mitochondrial metabolism attempted reduction of gssg to gsh in order to maintain redox status . such type of gr elevation was also reported in hypothyroid rat testis by sahoo et al . increase in activity of glucose-6-phosphate dehydrogenase ( g6pd ) due to neonatal hypothyroidism might be due to higher demand for nadph as gr requires this reducing equivalence to generate reduced gsh from gssg . the testis seems to be the most vulnerable during neonatal persistent hypothyroidism as evident from the significantly decreased germ cell count . they reported absence of round spermatids in 30 days hypothyroid testis and the inability of the spermatogenic cells to complete meiosis . ( 1991 )   also reported an impaired spermatogenesis and increased degeneration of germ cells in testis in response to prolonged hypothyroidism in rats . increased sertoli cell number in hypothyroid rats in the present investigation might be due to extended sertoli cell proliferation as in an earlier report , van haaster et al . ( 1992 )   and holsberger and cooke ( 2005 )   demonstrated that neonatal hypothyroidism may increase adult sertoli cell populations by extending sertoli cell proliferation . as hypothyroidism causes significant decrease in lh and fsh with a fall in serum testosterone [ 69 , 70 ] , this might lead to a disruption of the spermatogenic and steroidogenic processes . moreover , it has been reported that neonatal hypothyroidism adversely affects leydig cell proliferation , differentiation , along with impaired steroidogenesis [ 71 , 72 ] . hampered of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of sertoli cells . a compromised antioxidant defence system marked by increased protein carbonylation , disturbed redox status during neonatal hypothyroidism , might have contributed to poor growth and development of testis by affecting spermatogenesis and steroidogenesis in rats before puberty as indicated by reduced germ cell number , complete absence of round spermatids , decreased seminiferous tubule diameter , and decreased testosterone level . such type of altered testicular physiology by hypothyroidism is reflected in adulthood with hampered fertility as evidenced by reduced total viable germ cells   and sperm counts .