Elsevier

Theriogenology

Volume 66, Issue 9, December 2006, Pages 2056-2067
Theriogenology

Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes

https://doi.org/10.1016/j.theriogenology.2006.06.006Get rights and content

Abstract

Diabetes mellitus is a degenerative disease that has deleterious effects on male reproductive function, possibly through an increase in oxidative stress. This study was conducted in order to clarify the mechanisms by which oxidative stress influences animal models for both type 1 (streptozotocin-treated rats, STZ) and type 2 (Goto-Kakizaki (GK) rats) diabetes. We determined the extent of lipid peroxidation, protein oxidation, lactate levels, adenine nucleotides, adenylate energy charge and the activity of glutathione peroxidase, glutathione reductase and lactate dehydrogenase, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with sperm count and motility. Sperm concentration and motility were decreased in STZ-treated rats. ATP levels were lower in rats treated with STZ for 3 months, in contrast to GK and rats treated with STZ for 1 month, suggesting an adaptative response. STZ-treated rats showed increased lipid peroxidation after 1 week and 3 months of treatment. Glutathione reductase (G-red) activity was found to be higher in GK rats. Glutathione peroxidase activity was lower in GK and rats treated with STZ for 1 month, which is in accordance with the proposal of functional recovery in these animals. We conclude that hyperglycemia has an adverse effect in sperm concentration and motility via changes in energy production and free radical management. Furthermore, both animal models, particularly GK rats and rats treated with STZ for 1 month, present some metabolic adaptations, increasing the efficiency of mitochondrial ATP production, in order to circumvent the deleterious effects promoted by the disease.

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

References (74)

  • H.U. Bergmeyer et al.

    UV-assay with pyruvate and NADH

  • J.-L. Gatti et al.

    Post-testicular sperm environment and fertility

    Anim Reprod Sci

    (2004)
  • H.-S. Hsu et al.

    Defective mitochondrial oxidative phosphorylation in varicocele-bearing testicles

    Urology

    (1995)
  • L. Lionetti et al.

    Changes in the hepatic mitochondrial respiratory system in the transition from weaning to adulthood in rats

    Arch Biochem Biophys

    (1998)
  • L.K. Kwong et al.

    Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse

    Arch Biochem Biophys

    (2000)
  • D. Harman

    Free radical theory of aging

    Mutat Res

    (1992)
  • D. Strodter et al.

    The influence of thioctic acid on metabolism and function of the diabetic heart

    Diab Res Clin Pract

    (1995)
  • I. Seghrouchni et al.

    Oxidative stress parameters in type I, type II and insulin-treated type 2 diabetes mellitus; insulin treatment efficiency

    Clin Chim Acta

    (2002)
  • G. Wu et al.

    Glutathione metabolism and its implications for health

    J Nutr

    (2004)
  • P. Maher

    The effects of aging on glutathione metabolism

    Aging Res Rev

    (2005)
  • M.F. Knapen et al.

    Glutathione and glutathione-related enzymes in reproduction. A review

    Eur J Obstet Gynecol Reprod Biol

    (1999)
  • A.A. Hassan et al.

    The effect of diabetes on sexual behaviour and reproductive tract function in male rats

    J Urol

    (1993)
  • D.F. Cameron et al.

    Sustained hyperglycemia results in testicular dysfunction and reduced fertility potential in BBWOR diabetic rats

    Am J Physiol

    (1990)
  • G.Y. Jiang

    Practical diabetes

    (1996)
  • J.R. Gavin et al.

    Report of the expert committee on the diagnosis and classification of diabetes mellitus

    Diab Care

    (1997)
  • W.J. Malaisse

    Insulin release: the fuel concept

    Diab Metab

    (1983)
  • H.C. Lee et al.

    Mitochondrial gene transfer ribonucleic acid (tRNA) Leu (UUR) 3243 and (tRNA) Lys 8344 mutation and Diabetes mellitus in Korea

    J Clin Endocrinol Metabol

    (1997)
  • V. Jakus et al.

    Advanced glycation end-products and the progress of diabetic vascular complications

    Physiol Res

    (2004)
  • R.G. Ahmed

    The physiological and biochemical effects of diabetes on the balance between oxidative stress and antioxidant defense system

    Med J Islamic World Acad Sci

    (2005)
  • S. Taysi et al.

    Lipid peroxidation, some extracellular antioxidants and antioxidant enzymes in serum of patients with rheumatoid arthritis

    Rheumatol Int

    (2002)
  • M. Gul et al.

    Cellular and clinical implications of glutathione

    Indian J Exp Biol

    (2000)
  • M.F. Polat et al.

    Oxidant/antioxidant status in blood of patients with malignant breast tumour and benign breast disease

    Cell Biochem Funct

    (2002)
  • J. Yang et al.

    Antioxidant enzyme levels in oral squamous cell carcinoma and normal human oral epithelium

    J Oral Pathol Med

    (2002)
  • M. Sasvári et al.

    Time dependent changes in oxidative metabolism during chronic diabetes in rats

    Acta Biologica Szegediensis

    (2003)
  • R.J. Aitken et al.

    Generation of reactive oxygen species, lipid peroxidation, and human sperm function

    Biol Reprod

    (1989)
  • C.S. Chen et al.

    Hydroxyl radical induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm

    Biochem Mol Biol Int

    (1997)
  • S.H. Kao et al.

    Multiple detection of mitochondrial DNA associated with the decline of motility and fertility of human spermatozoa

    Mol Hum Rep

    (1998)
  • Cited by (147)

    • Heat treatment reduced the expression of miR-7-5p to facilitate insulin-stimulated lactate secretion by targeting IRS2 in boar Sertoli cells

      2022, Theriogenology
      Citation Excerpt :

      Thus, these data suggest that heat treatment increases insulin sensitivity in SCs by modulating miR-7-5p/IRS2/PI3K/Akt axis. Glucose metabolism disorder in SCs caused by insulin dysfunction might be the important reason of infertility in male diabetic patients [43,44]. Diabetic patients usually need to use exogenous insulin or insulin analogues to maintain the balance of blood glucose level [45,46].

    • Male Sexual and Reproductive Health

      2022, Comprehensive Pharmacology
    View all citing articles on Scopus
    View full text