Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes
Introduction
About 90% of diabetic patients have disturbances in sexual function, including a decrease in libido, impotence and infertility, in the latter case due to testicular dysfunction associated with sustained hyperglycemia [1], [2]. Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both [3]. Type 1 diabetes is the consequence of an autoimmune-mediated destruction of pancreatic β-cells, leading to insulin deficiency, while type 2 diabetes is characterized by insulin resistance and relative, rather than absolute, insulin deficiency [4]. Growing evidence indicates that oxidative stress is increased in diabetes, due to the overproduction of reactive oxygen species (ROS), and decreased efficiency of antioxidant defences [5], [6]. Oxidation of lipids, proteins and other macromolecules (such as DNA) occurs during the development of diabetes [7]. Mitochondrial DNA mutations have also been reported in diabetic tissues, suggesting oxidative stress-related mitochondrial damage [8].
Hyperglycemia leads to the increased production of free radical intermediates via at least four different routes: increased glycolysis; intercellular activation of the sorbitol (polyol) pathway; auto-oxidation of glucose and non-enzymatic protein glycation [9], [10]. To control the flux of ROS, aerobic cells have developed an antioxidant defense system, which includes enzymatic and non-enzymatic components [11]. The antioxidant system consists of low molecular weight antioxidant molecules and various antioxidant enzymes [12], [13], [14], [15]. Glutathione (GSH), the most prevalent low-molecular weight antioxidant peptide, and GSH-related antioxidant enzymes, such as glutathione peroxidase (G-Px), glutathione reductase (G-Red) and glutathione S-transferase (GST), are the most important enzymes of this cellular antioxidant system. G-Px catalyses the reduction of hydrogen peroxide and organic hydroperoxides by transferring electrons from GSH, thus forming GSSG. In turn, GSSG is then reduced back to GSH by G-Red [16]. The stability and capacity of antioxidant status during chronic diabetes seriously influences the outcome of the long-term complications caused by oxidative stress [17].
Mammalian sperm cells present a specific lipidic composition, with a high content of polyunsaturated fatty acids, plasmalogens and sphingomyelins. The lipids in spermatozoa are the main substrates for peroxidation, and Aitken et al. [18] showed that excess amounts of ROS and free radicals have adverse effects on sperm motility and fertility. Furthermore, oxidative damage to lipids and DNA of spermatozoa is associated with declining motility and diminished fertility of human sperm [19], [20], [21]. However, the mechanisms of altered spermatogenesis in diabetic men are poorly understood. In this regard, animal models for diabetes are important research tools, since they provide insights which are almost impossible to duplicate in human populations [22], [23]. In the present study, we used STZ-induced diabetic rats as models for type 1 diabetes and Goto-Kakizaki (GK) rats as models for type 2 diabetes. STZ-induced diabetic rats are obtained after selective destruction of β cells by streptozotocin (STZ), an antibiotic with diabetogenic effects. STZ-injected rats present many characteristics seen in insulin-dependent diabetic human patients, such as hypoinsulinemia, hyperglycemia, ketonuria, and hyperlipidaemia [23]. On the other hand, Goto-Kakizaki (GK) rats are currently used as an animal model for type 2 diabetes. This animal is a non-obese, spontaneously diabetic rat [22], produced by selective breeding of Wistar rats, using glucose intolerance as a selection index [24], [25], [26], [27]. GK rats exhibit a moderate but stable fasting hyperglycemia, which does not progress to a ketotic state. Furthermore, the GK rat is one of the best characterized animal models of spontaneous non-obese type 2 diabetes mellitus, since it exhibits similar metabolic, hormonal, and vascular disorders as the human disease [22]. Furthermore, in the initial stages of diabetes, GK rats do not exhibit severe complications associated with the disease, constituting an important model to study the initial events of diabetes [28].
In the present work, we have tried to elucidate the mechanisms by which diabetes and associated oxidative stress influence testicular and sperm function. For this purpose we have determined the extent of lipid peroxidation, protein oxidation, activity of glutathione peroxidase and reductase, lactate and pyruvate levels, internal and external energetic charge and lactate dehydrogenase release, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with epididymal sperm count and sperm motility.
Section snippets
Materials
All chemicals were obtained from Sigma, St. Louis, MO, USA.
GK rats
Spontaneously diabetic male GK rats, 3 months of age (n = 23), were obtained from our local breeding colony (Animal Research Center Laboratory, University Hospitals, Coimbra), established in 1995 with breeding couples from the colony at the Tohoku University School of Medicine (Sendai, Japan; courtesy of Dr. K. Susuki). Control animals were non-diabetic male Wistar rats of similar age (n = 23). Animals were kept under controlled light (12-h
Glycemia
Blood glucose (non-fasting) levels, were significantly higher in diabetic rats when compared to respective controls (p < 0.001). However, GK rats presented mild hyperglycemias (110–330 mg/dl), while STZ-treated rats presented severe hyperglycemias (>293 mg/dl), p < 0.001 (Fig. 1).
Epididymal sperm count and motility
An effect of age was noted in control Wistar rats, with 5-month-old rats (controls for rats treated with STZ for 3 months) showing higher sperm counts and motility than 3-month-old rats (controls for all other experimental
Discussion
Increasing evidence suggests that diabetes has an adverse effect on male reproductive function [2], and that oxidative stress may be involved [29]. According to Mahboob et al. [40], antioxidant enzyme-dependent defences may play an important role by scavenging free radicals produced under oxidative stress. The current study was conducted in order to clarify the relationship between oxidative stress originated by a diabetic condition, and parameters related to spermatogenesis and sperm function.
Acknowledgments
This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal (POCTI/ESP/38049/2001) and by Instituto de Investigação Interdisciplinar, University of Coimbra (III/BIO/50/2005). Sandra Amaral is a recipient of a fellowship from FCT (SFRH/BD/18734/2004). We thank Teresa Proença (University Hospitals, Coimbra) for help with pyruvate and lactate dosing, José Paulo Sousa and Sandra Gamboa for assistance with statistical analysis, Paula Mota, Ana Duarte, Pedro Oliveira and Paula
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