Oxidative Impairments in Macromolecules in Immature Mammalian Testis under Experimentally Induced Diabetes: Biochemical and Functional Consequences

Chandrashekar, K. N. (2009) Oxidative Impairments in Macromolecules in Immature Mammalian Testis under Experimentally Induced Diabetes: Biochemical and Functional Consequences. PhD thesis, University of Mysore.

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Abstract

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and protein with an increased risk of vascular complications. The incidences of diabetes in young men/children are increasing alarmingly over the past decade exposing them to a greater risk of developing the macrovascular and microvascular complications during their prime of their life. Sexual dysfunction is frequently associated with diabetes in men and infertility is a common outcome in diabetic men. Although oxidative stress has been implicated as the primary causative factor, the underlying biochemical mechanism/s that leads to testicular dysfunctions is poorly understood. Further, most of what we know about diabetes associated reproductive dysfunctions in animal models is based on experimentally-induced diabetes in adult rodents (rats/ mice) by either Streptozotocin (STZ) or Alloxan. As a result of which there is a big lacuna in our understanding of the vulnerability of prepubertal testis to diabetes. Given the scenario that diabetes is becoming prevalent in children and adolescents and is likely to increase in phenomenal proportions, it is pertinent to obtain comprehensive insight into the biochemical /.functional impairments and their subsequent implications on steroidogenesis /spermatogenesis during the reproductive period. Hence, in the present study, attempts have been made to obtain evidence in favor of our central hypothesis that “Immature and developing testis is likely to be more susceptible to oxidative stress under diabetic conditions and the biochemical and functional alterations may have far reaching consequences (transient or permanent) resulting in sub-fertility or infertility”. The basic objectives envisaged were (i) To understand the nature, pattern and progression of diabetes induced oxidative damage in immature and developing testis (ii)To elucidate the biochemical and physiological implications in testis and its impact on functional development of testis (iii) To understand the spectrum of oxidative impairments in spermatozoa and its correlations with reproductive outcome and infertility and iv) to ascertain Abstract whether these oxidative impairments are amenable for amelioration by phytochemicals/nutrients. Streptozotocin administration (STZ, 90mg/kg bw) to prepubertal rats caused marked hyperglycemia together with marked increase in oxidative stress response. The oxidative induction was robust in testis compared to other somatic organs (viz., liver and brain) as evidenced by elevated levels of ROS and MDA, perturbations in the activities of antioxidant enzymes / non-enzymic antioxidants. Further, studies carried out to ascertain vulnerability pattern among two age group PP rats to diabetes induced oxidative stress revealed that the susceptibility increased with downward shift in age (4wk-old > 6wk-old) as evidence by enhanced levels of ROS and MDA, perturbations in antioxidant defenses and functional enzyme activities in both cytosol and mitochondria. Testis mitochondria of diabetic rats also exhibited altered activities of TCA cycle and oxidative phosphorylation enzymes together with loss in membrane potential and mitochondrial membrane leakage. Collectively, these findings clearly demonstrate increased susceptibility of 4wk- old prepubertal testis to diabetes induced oxidative stress. In prepubertal diabetic rats, the oxidative impairments were discernable in all the major testicular subcellular organelles viz., cytosol, mitochondria and microsomes as evidenced by enhanced levels of ROS and MDA, perturbations in the activities of antioxidant enzymes and non-enzymic antioxidants, elevation in protein carbonyl content and in vital functional enzymes. In testis cytosol, the oxidative response induction was evident during both acute (<5d) and progressive phase (7/14d) while both mitochondria and microsomes exhibited marked oxidative alterations only during progressive phase. Among diabetic PP rats, the activities of functional enzymes such as SorDH and LDH were consistently reduced throughout. However, the activities of ADH and AR were reduced during progressive phase. Diabetic testis further showed robust increase in ascorbate levels, non-protein thiols were significantly elevated at all sampling days in contrast to the decreased tocopherol levels evident only during progressive phase. Further, a progressive decrease in the activities of mitochondrial TCA cycle enzymes such as citrate synthase, succinate dehydrogenase and malate Abstract dehydrogenase were also evident in testis of diabetic PP rats. Further, diabetic testis mitochondria showed a preferential increase in the activity of NADH-cyt C reductase compared to succinate-Cyt C reductase accompanied by loss of mitochondrial potential/integrity. These changes were accompanied by altered activities of cytochrome-p450 enzymes in testis microsomes both during acute and progressive phase. These findings clearly suggest that testis of prepubertal rats is indeed subjected to significant oxidative stress during conditions of diabetes, and the testicular impairments observed during diabetes is a cumulative effect of the oxidative responses elicited by different organelles to varying degrees. Further, data obtained from flow cytometric analysis of diabetic prepubertal testis over one spermatogenic cycle (56days) revealed complete loss of germinal cell population and other cell types. These changes were accompanied by elevated activities of Caspase-3 and increased incidence of DNA fragmentation. Collectively, these observations clearly demonstrates involvement of apoptosis mediated cell death as the underlying mechanism for the higher incidence of testicular degeneration observed during diabetes that leads to decreased fertility. Results of ameliorative studies with supplementation of D-Aspartic acid (D-Asp, i.p), testosterone (TP, i.p) and Withania somnifera (WS, oral) showed varying degree of protection in testis of diabetic PP rats. Supplementation with TP or WS significantly offset the STZ-induced oxidative impairments as evidenced by reduction in blood glucose levels, lowered levels of ROS/ MDA, restoration of protective thiols and antioxidants defenses in testis (cytosol/mitochondria) of PP rats. In the interactive model, D-Asp administered diabetic rats showed significantly reduced levels of oxidative stress markers together with upregulation in antioxidant defenses and the major steroidogenic enzyme, 3β-hydroxy steroid dehydrogenase (3β-HSD) suggesting the potential of D-Asp to attenuate testicular oxidative stress during diabetic conditions.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Diabetes mellitus, prepubertal testis, testicular dysfunctions, oxidative stress, biochemical/functional impairments
Subjects:	600 Technology > 01 Medical sciences > 04 Diabetes Mellitus
Divisions:	Dept. of Biochemistry
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Mar 2011 04:44
Last Modified:	09 May 2012 05:41
URI:	http://ir.cftri.res.in/id/eprint/9939

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