Aging, Mitochondria and Male Reproductive Function - IngentaConnect

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Aging, Mitochondria and Male Reproductive Function Sandra Amaral and João Ramalho-Santos* Center for Neuroscience and Cell Biology, Department of Zoology, School of Science and Technology, University of Coimbra, 3004-517 Coimbra, Portugal Abstract: The rise in life expectancy over the last century, together with higher maternal and paternal ages and have highlighted the issue of reduced fertility with advancing age. Aging of the male reproductive system is incited by multifactorial changes at molecular, cellular and regulatory levels, and individual characteristics are highly variable, although strongly influenced by lifestyle and environmental factors. Damage accumulated with age leads to progressive deregulation of the hypothalamic-pituitary-gonadal axis and of local auto/paracrine interactions, thereby inducing changes in target organs such as the testis, penis and prostate. Elderly human males produce less testosterone, have fewer motile sperm and a higher incidence of erectile dysfunction and prostate disorders, all of which contribute to lower fertility. Cellular aging can manifest itself at several levels. Aging cells progressively accumulate “waste” products, resulting in a decreased functionally. Changes to mitochondria are among the most remarkable features observed in aging cells and several theories place mitochondria at the hub of cellular events related to aging, namely in terms of the accumulation of oxidative damage to cells and tissues, a process in which these organelles may play a prominent role, although alternative theories have also emerged. Furthermore, mitochondrial energy metabolism is also crucial for male reproductive function and mitochondria may therefore constitute a common link between aging and fertility loss.

Keywords: Age, mitochondria, oxidative stress, male reproductive function, infertility. 1. AGING Aging can be defined as a time-dependent general decrease of physiological functions of an organism, associated with an increasing risk of morbidity and mortality [1-3]. There is a strict difference between non-dividing and dividing cells of multicellular organisms. While the former are unable to maintain their structure and functionality over time, contributing to the degeneration of the whole organism, dividing cells remain highly functional, even at the end of a normal lifespan. This can be explained by the existence of a constant and renewable pool of stem and progenitor cells. In contrast, long-lived postmitotic cells are only rarely replenished from stem cells, a fact that can determine some of the profound alterations brought about by age [4,5]. Cellular aging can manifest itself at several levels. Indeed, aging postmitotic cells progressively accumulate “waste” products, resulting in decreased functionally and progressive degeneration. Aged cells are often morphologically larger than young counterparts, a situation that may result from an overload of “waste” material and consequent compensatory increase in size of the remaining functional structures. At the membrane level age-induced damage to lipids and proteins leads to changes in membrane fluidity and consequent disturbances in molecular transport, membrane permeability, and other related functions. Furthermore, a wide array of changes can also be detected at an intracellular level. The nuclei of aged postmitotic cells are characterized by increased heterochromatin, as well as by damage to both DNA and nuclear proteins. Another interesting aspect is the accumulation of aberrant proteins within *Address correspondence to this author at the Center for Neuroscience and Cell Biology, Department of Zoology, School of Science and Technology, University of Coimbra, 3004-517 Coimbra, Portugal; E-mail: [email protected] 1874-6098/09 $55.00+.00

aged cells that mirrors reactive oxygen species (ROS)induced damage and incomplete digestion of altered proteins. However, the most notable features observed in aging cells are changes to mitochondria. Senescent mitochondria exhibit structural deterioration that ranges from swelling and loss of cristae to complete destruction and homogenization of the matrix and mitochondrial membranes, resulting in the formation of amorphous electron-dense material. These senescent mitochondria are frequently enlarged, sometimes excessively, and this is the reason why they are often called “giant” mitochondria. Mutations in mtDNA and changes to mitochondrial proteins also progressively increase with age [4,5]. In the past decade many molecular mechanisms of aging have been proposed, including cumulative damage by reactive oxygen species (ROS), telomere shortening in replicating cells, genome instability, mutations or altered expression of specific genes, epigenetic modifications and cell death [1]. 2. AGING, OXIDATIVE MITOCHONDRIA

STRESS

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There are several theories that place mitochondria at the hub of cellular events during the aging process, although recent alternatives have called at these hypotheses into question. Early in 1956, Harman proposed that free radicals produced during aerobic metabolism cause cumulative oxidative damage that ultimately results in aging and death, the so-called oxidative stress theory [6]. Later, the idea was extended, to suggest that mitochondria are the main target of free-radicals leading to a vicious cycle in which damaged mitochondria produced progressively increased amounts of ROS, in turn leading to a progressive augmentation of damage that culminates in the process of aging, the mitochondrial theory of aging [7,8]. In the past few years, © 2009 Bentham Science Publishers Ltd.

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this theory of aging has been solidified by extensive molecular and cellular studies of aging [9-12]. Recently, the mitochondrial lysosomal axis theory has again involved mitochondria in the process of aging [4]. Therefore, mitochondria have been suggested as an active participant in the aging process. However, although correlations between oxidative stress and aging are evident, the exact causal nature of these correlations is disputed. Alternative theories either question the validity on oxidative damage accumulation with aging, or link it with an increased propensity for degenerative disorders (e.g. cancer, neurodegenerative diseases, etc) that, although related with a diminished lifespan and with the quality of life, are not necessarily directly involved with aging itself [for review see 106-108]. Clearly the immerging scenario is more complex than predicted by initial hypotheses, it seems unlikely that one sole aspect being studied will provide conclusive answers, and a multifactorial approach must thus be integrated and acknowledged in future studies. At any rate it has been shown that mitochondrial oxidation is the major source of oxidative lesions that accumulate with age [13]. As the major intracellular source of ROS, mitochondria are also vulnerable to direct attack by ROS. In fact, the levels of ROS-modified proteins and lipid peroxides in mitochondria seem to increase with age [14-16]. It should be noted that mitochondrial membrane lipids, particularly cardiolipin, are at high risk of oxidative damage, due to high levels of fatty acid unsaturated bonds. Since cardiolipin content in the inner mitochondrial membrane is essential for mitochondrial bioenergetics, oxidative stress and aging are associated with decreased activities of inner membrane proteins, which are themselves also susceptible to oxidative stress [17-19]. Other components of the oxidative (OXPHOS) system, such as the adenine nucleotide transporter (ANT), ATP synthase, and the matrix enzyme aconitase are also highly sensitive to oxidative stress [20-22]. The transport of metabolites to and from mitochondria is also affected by aging, as is the case of malate import, which has been demonstrated to decrease with age [16]. Furthermore mitochondrial DNA is highly sensitive to oxidative stress [23-25] for a variety of reasons: (1) the mitochondrial genome is present in numerous copies (2) mtDNA lacks histones and other DNA associated proteins making it readily accessible to oxidative damage, (3) mtDNA encodes thirteen polypeptides which are critical for the function of electron transport chain (ETC) complexes and (4) mtDNA is near the ETC where ROS production occurs, and is therefore an easy target for ROS-induced damage [26]. Given that most of the mitochondrial genome codes for genes that are expressed, while nuclear DNA contains sequences that are not transcribed, it is not surprising that damage to mtDNA could have more serious implications than the damage for nuclear DNA [27]. Another perspective is that electron transport chain-induced ROS damage to mitochondrial DNA accumulates during a lifetime [26] and may result of deficient mitochondrial biogenesis and/or turnover in the cell. Defects in mtDNA accumulated during aging may also reduce or prevent the renewal of mitochondrial proteins [9]. The transmission of these mutations by both mitochondrial and cell divisions is expected to lead to a general decline in respiratory capacity, which will not only affect the efficiency of ATP production, but will lead to augmented ROS

Amaral and Ramalho-Santos

production, thus resulting in a overall increase in mitochondrial DNA mutations and oxidative stress, creating a vicious cycle. Additionally, increased ROS may onset cytochrome c release from the mitochondria activating the apoptotic cell death pathway, via the permeability transition pore (PTP) [26]. Age-related mitochondrial oxidative stress seems to have a key role in promoting the intrinsic pathway of apoptosis, where it is considered an early event. In fact, aging and apoptosis share many features, such as decreased mitochondrial membrane potential, higher lipoperoxide levels, glutathione oxidation, and mitochondrial DNA oxidative damage, mainly due to an increase in oxidative stress common to both conditions [12, 28]. All the above-mentioned factors contribute to a reported gradual decline in mitochondrial respiratory function with age in both humans and laboratory animals. Respiratory control, oxidative phosphorylation efficiency, the rates of resting (State 4) and ADP-stimulated (State 3) respiration and the activities of the respiratory enzyme complexes all decline with age in various human tissues [29,30], although again it should be noted that this some of this data has been called into question, or alternatively interpreted [106-108]. Regardless, aging was also associated with decreased expression of the LON protease (implicated in mitochondriogenesis), ATP synthase subunits, NADP transhydrogenase, and finally, with decreased expression of genes involved in synthesis of fatty acids and cholesterol, and in those involved in protein turnover [10]. Additionally, decreased ATP synthesis due to an age-dependent decline of mitochondrial respiratory function may have a negative impact on the amount of proteins in the cell. Thus, age-associated mitochondrial dysfunction could potentially lead to an energy crisis that would affect cellular homeostasis, thereby resulting in age-related degenerative complications. Moreover, the reduced capacity of antioxidant systems in aged cells also contributes to increased mitochondrial damage [9]. The protonmotive force set up across the inner membrane also influences mitochondrial ROS production, in such a way that the mild uncoupling caused by activation of UCPs might lower the protonmotive force, attenuate mitochondrial ROS production, and protect against ROS-related cellular damage [31]. A handful of studies have examined the effects of UCP deletion or overexpression on aging and lifespan. Fridell and collaborators observed that Drosophila lifespan can be lengthened by approximately 10-30% by over expression of human UCP2 in the nervous system, and that the production of ROS was reduced in hUCP2 expressing flies [32]. On the other hand, the same group described that UCP5 Knockout flies live longer during caloric restriction, but had reduced survival during starvation [33]. In the same vein, the deletion of UCP4-like protein in the nematode C. elegans, increased ATP levels but had no effect on lifespan when compared to controls [34]. In addition, some studies in rats also provide evidence on the role of UCPs on lifespan and aging. A correlation between proton leak and aging has been established due to the observation of an age-dependent increase in proton leak rate and a decrease in ATP turnover reactions in mouse hepatocytes [35]. Recently, Gates and colleagues also report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in mouse models [36]. Moreover, the over expression

Aging, Mitochondria and Male Reproductive Function

of UCP3 in rats has been shown to blunt age-induced increase in ROS formation [37]. However, oxidative stress impacts not only mitochondria but also extra-mitochondrial structures, with effects on lipids, proteins and DNA [38-40]. Changes in degradation pathways promote accumulation of damaged proteins, macromolecules and organelles with age [9]. Cells are endowed with several mechanisms to degrade their components. Short lived proteins are mainly degraded by calpains, (cytosolic calcium-dependent cysteine proteases) and proteasomes (multicatalytic proteinase complexes), while most long-lived proteins, other macromolecules and all organelles are degraded by autophagy. Autophagy is a well organized and probably specific intralysosomal degradation pathway that has been demonstrated to be down regulated in aged cells. Consequently there will be a lengthening in the halflives of long lived proteins, lipids and organelles leading to a situation where cells would be forced to perform their functions under sub-optimal conditions. In the case of mitochondria decreased autophagic capacity in aged cells will lead to a decline in organelle turnover and accumulation of damaged mitochondria with age. Senescent mitochondria will produce gradually less ATP and more ROS. Enhanced oxidative stress, in turn, will complicate mitochondrial turnover even more and may induce apoptotic cell death [4,5,9]. In conclusion, several line of evidence suggest that mitochondrial function may be impaired in aging, as judged by a decline in membrane potential, an increase in peroxide

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production and size of the organelles, decreases in mitochondrial protein synthesis, mitochondrial transcripts, and expression of genes involved in mitochondrial turnover [9,12]. 3. THE MALE REPRODUCTIVE SYSTEM The primary sex organs of the male reproductive system are the two testes in which sperm cells and are produced. The other structures of the male reproductive system are termed accessory sex organs and can be divided in internal and external reproductive organs. The internal accessory organs include the epidydimis, genital ducts, as well as seminal vesicles, prostate gland and bulbourethral glands. The external reproductive organs are the scrotum that encloses the testis, and the penis [41,42]. The testis contains seminiferous tubules that consist of germinal epithelium and peritubular tissue (lamina propria) [43]. The seminiferous tubule epithelium has two basic cell types: somatic and germinal cells [44]. Germ cells are at different developmental stages, including spermatogonia, primary and secondary spermatocytes and spermatids, which are located within invaginations of somatic Sertoli cells, with which they maintain an intimate and cooperative relationship [43]. Besides forming the blood-testis barrier other Sertoli cell functions include mechanical and nutritive support for the germ cells, phagocytosis, participation in spermiation, secretion of testicular fluid to enable sperm transport, production of endocrine and paracrine substances for the regu-

Fig. (1). Spermatogensesis (left) and its Hormonal regulation (right) Spermatogenesis is the process that culminates in the production of haploid sperm. It can be divided in three major phases. Spermatogoniogenesis is characterized by spermatogonial proliferation and formation of primary spermatocytes that then enter meiosis, a long phase characterized by changes in chromatin. After two cell divisions, round spermatids are formed and suffer a terminal differentiation in the phase of spermiogenesis to produce spermatozoa that, due to their unique shape, are able to leave testis during the spermiation process. This process is highly regulated. The male hypothalamus secretes GnRH that stimulates the pituitary to produce FSH and LH. FSH exerts its effects on testis by stimulating the Sertoli cells that then secrete factors such as inhibin that exerts a negative feedback on the pituitary. FSH is important for the maturation of germ cells. LH stimulates Leydig cells to produce testosterone that has a role in spermatogenesis and expression of male secondary characteristics. Additionally testosterone can provide a negative feedback to the pituitary reducing LH secretion (adapted from Shauf et al., 1990) [41].


lation of spermatogenesis and secretion of androgen binding protein (ABP) [43,45]. Testosterone-secreting Leydig cells are found in the intertubular tissue surrounding the capillaries and have a prominent role in the maintenance of spermatogenesis, the differentiation of male sexual organs and male secondary sex characteristics. Spermatogenesis takes place in the semineferous tubules and is a highly dynamic and metabolically active biological process during which haploid spermatozoa are produced through a gradual transformation of an interdependent population of germ cells. These cells sequentially migrate from the basal compartment towards the luminal regions of the seminiferous tubules, passing the blood-testis barrier. Spermatogenesis can be divided in three main phases: spermatogoniogenesis, meiosis and Spermiogenesis [43,44] (Fig. 1). Accurate spermatogenesis is dependent of several hormonal messengers acting through endocrine, paracrine, and autocrine pathways. The primary messengers are the pituitary gonadotrophins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), and the androgens, primarily testosterone [46,47]. Normal male reproductive function depends on the pulsatile secretion of LH and FSH by the pituitary under the influence of gonadotrophin releasing hormone (GnRH) release by the hypothalamus. Pulsatile LH stimulates Leydig cells to produce testosterone, that in turn exerts a negative feedback on GnRH and gonadotrophin secretion [43,47] (Fig. 1). 3.1. Testis Metabolism and Energy Requirements In the adult testis survival of germ cells is strictly dependent on carbohydrate metabolism, including both anaerobic (glycolysis) and aerobic (oxidative phosphorylation) pathways. Nonetheless, the cells in the seminiferous epithelium differ in their favoured substrates for energy supply [48-52]. In fact, during spermatogenesis, there is a considerable change in the energy metabolism of germ cells. It has been previously described by several authors that spermatogonia, mature spermatozoa, and the somatic Sertoli cells exhibit high glycolytic activity, whereas spermatocytes and spermatids produce ATP mainly by mitochondrial oxidative phosphorylation [48, 53-56, 109]. However, it is still a matter of debate if sperm obtain energy only from glycolitic pathway or if oxidative phosphorylation is also involved. Intensive research in this field has revealed contradictory results but it is believed that spermatozoa can take advantage of different energy sources, according to their availability [57, 109]. 3.2. The Remarkable Place of Mitochondria in Male Reproductive Function The morphology, localization and energy metabolism of testicular mitochondria change markedly during spermatogenesis, and at least three types of mitochondria are recognizable: the usual orthodox-type mitochondria in Sertoli cells, spermatogonia, preleptotene and leptotene spermatocytes; the intermediate form of mitochondria in zygotene spermatocytes; and the condensed mitochondria form in pachytene spermatocytes, secondary spermatocytes and early spermatids, a conformation that shifts back to the intermediate form in late spermatids and spermatozoa [56, 58, 109]. These


structural changes during spermatogenesis mirror metabolic differentiation [58]. Mitochondria also have a role in Leydig cell sterodoigenesis, a process that involves the steroidogenic acute regulatory (StAR) protein-mediated delivery of free cholesterol to the inner membrane of mitochondria. Once there it is then converted to testosterone through a series of sterodoigenic steps catalized by different proteins [59]. Energized, polarized, and actively respiring mitochondria have been proven to be necessary for sterodoigenesis in Leydig cells [60,61]. 3.3. Mitochondria, Oxidative Stress and Infertility Oxidative stress occurs when the production of ROS overcomes the available antioxidant defenses, leading to cellular damage, and is a common pathology seen in approximately half the population of infertile men. There are two main sources of ROS in semen: leucocytes and sperm. Although the production of ROS by sperm plays a positive role in fertilization at low levels, when produced at high levels it can lead to potential toxic effects on sperm quality and function [62,63]. Additionally, environmental and lifestyle factors as well as pathologies of the reproductive system and chronic diseases are sources of sperm oxidative damage [63]. Although seminal plasma is well endowed with an array of protective antioxidants (Superoxide dismutase (SOD), Catalase (CAT), and Glutathione Peroxidase (GPX)), these defenses are less abundant in sperm and seem to be impaired in cases of male infertility [62,63]. ROS-induced damage to sperm membranes and to sperm DNA, are the two main mechanisms by which ROS can cause infertility. The first mechanism reduces sperm motility and its ability to fuse with the oocyte, while the latter compromises paternal genomic contribution to the embryo [63]. In fact, sperm are especially vulnerable to oxidative stressinduced damage due to the high portion of polyunsaturated fatty acids (PUFAs) in their membranes and also due to the low concentrations of scavenging enzymes in their cytoplasm, both contributing to the defective sperm function observed in a high percentage of infertility patients [62]. 4. AGING AND REPRODUCTIVE FUNCTION: ARE MITOCHONDRIA INVOLVED? Similarly to other aging processes in the human body, aging in the male reproductive system involves multifactorial, stochastic changes at molecular, cellular and regulatory levels [64]. Whereas female fertility ends with the onset of menopause, men do not show such a strict cessation of reproductive capacity but more a gradual decline of fertility. This is the reason why the term andropause is not appropriate, despite being widely used [65-68]. Even so, the increase in life expectancy together with higher maternal and paternal ages and progresses in assisted reproduction have refocused attention in male fertility with advancing age [65]. Characteristics of the aging male develop as a combination of morphological changes in organs, partially coupled to endocrine networks. These characteristics are highly variable individually and seem to be strongly influenced by lifestyle and environmental factors [64,65].


Aging is associated with a gradual and progressive decline in testosterone levels. This decline begins about the age of 30 and decreases progressively as men age [69-71]. Clinical symptoms, such as decreased bone and muscle mass, abdominal obesity and decreased sexual body hair and beard growth are associated with the gradual decline in testosterone as well as other non-specific symptoms, including nervousness, irritability, psychological depression, impaired memory, fatigue, insomnia, hot flushes, periodic sweating and loss of sexual vigor [64,66, 72-74]. Due to this reduction in testosterone levels, testosterone replacement therapy in older men has been the target of intensive research in the last years. However, the risk/benefit assessment is still a debated issue [66,67,73-75]. The age-related decline in serum testosterone may be explained by changes in all the components and regulatory levels of the hypothalamus-pituitary-gonadal axis. Impairment of testicular steroidogenesis, diminished testosterone secretion by Leydig cells, as well as changes other in hormone production and in feedback mechanisms may be involved [64,65,72]. Compared with young men, healthy older men with low serum testosterone levels demonstrate an abnormal LH pulse frequency, reduced LH pulse amplitude, and more disorderly LH secretion, suggesting an ageassociated impairment of the hypothalamic GnRH pulse generator [76-80]. Androgen receptor expression in the hippocampus and the number of androgen binding sites in genital skin is also decreased in older compared to young men [81-83] Age-related hypothalamic pituitary dysfunction may also lead to a reduction in growth and thyroid hormone secretion and an increase in glucocorticoids, all of which will have a detrimental role in sterodoigenesis [72]. Moreover, an age-related decrease of dehydroepiandrosterone (DHEA), a precursor of estrogenic steroids, has also been observed with aging [84,85]. In addition, the levels of many biological factors that influence sterodoigenesis are altered, including cytokines, interleukins, transforming growth factor- b1, tumor necrosis factor and ROS all of which are increased with aging [72]. Alterations in testicular function and histomorphology also contribute to partial primary hypogonadism in elderly men, such as reduction in testicular perfusion, thickening, widening and hernia-like protrusions of the basal membrane of the seminiferous tubules, decrease in Leydig cell number, increased accumulation of lipofuscin in Leydig cells [64,65], decline in Leydig cell mitochondrial steroidogenesis due to several factors (see next section), and a blunted rise in testosterone, upon stimulation by human chorionic gonadotrophin (hCG) [86,87]. The extent of age-induced damage to testicular and sperm function is still a hotly debated issue. Nonetheless, several studies have described age-related declines in semen volume, sperm count and motility and an increase in sperm morphological anomalies [64,88]. This is correlated with a decrease in antioxidant enzymatic capacity and increased production of ROS in sperm with aging [89]. Additionally, the observed age-related decline in Sertoli cell number and function [90] and the disruption in communication between Sertoli and germ cells [91] may also be involved. It also seems that men contribute to the reduced fertility of couples around the age of 50 and to diminished fecundity a few years


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later [64]. Additionally, male age seems to be associated to a greater probability of birth defects [64,65]. In what concerns sexual function, and not ignoring the array of factors that may play a role (such as psychological issues and medication), there is evidence to show that sexual function (including sexual thoughts, enjoyment and performance) decrease with age [73,88]. Erectile dysfunction (ED), i.e. the inability to achieve or maintain an erection suitable with the sexual act, is strongly associated with age and agerelated disorders, being mainly common in men over the age of 50 [64,73]. Homeostasis of the erectile process is dependent on the presence of androgens that are decreased in aging. In addition, ED is also caused by atherosclerosis of the penile arteries, or may arise due to reduced nitric oxide (NO) production as a consequence of aging [64]. The prostate is also susceptible to age-induced changes. Benign prostatic hyperplasia (BPH) and prostate cancer are classical age-related diseases and due to their high frequency will become increasing clinical and socio-economic problems, as the trend to an increase in life expectancy continues in western populations [64,65,73]. Interestingly, and in a different vein, Schmit and collaborators, found that male mice housed with females remain fertile longer than male mice housed alone and that fertility became significantly reduced 6 months sooner for males housed alone than for males housed with females. Additionally, they also observed that abnormal spermatogenesis occurred sooner in isolated males [92]. 4.1. Are Mitochondria a Link between Aging and Loss of Fertility? Considering age-associated mitochondrial dysfunction, as well as the role of mitochondria and ROS in reproductive function, it can be hypothesized that mitochondria are a common link between age and age-related loss of male fertility. Several studies have emerged recently that support this hypothesis. In fact, analysis of testicular mitochondria has shown a decrease in mitochondrial function with age, including changes in fatty acid composition, possibly affecting fluidity and mitochondrial complex activity [93], correlated also with an increase production of superoxide anion, lipid peroxidation and with reduced activity of the antioxidant enzyme SOD [94]. In agreement with this data, Sahoo and colleagues observed increased levels of lipid peroxidation and H2O2 production, as well as decreased GSH content, which was accompanied by a decline in activities of antioxidant enzymes with advancing age. It was suggested that the antioxidant defense profile of testicular mitochondria exhibit age-related alterations which might play a critical role in regulating physiological functions [95]. In fact, it had been previously described that the balance between pro and antioxidative agents were altered in aging testis mitochondria, particularly with a shift in the glutathione redox state towards the pro-oxidizing condition [96]. Recently, we have examined the effects of age on the bioenergetic characteristics of testicular mitochondria isolated from rats of different ages. We observed that mitochondrial respiratory and phosphorylative function correlate with the rat reproductive cycle, exhibiting a peak of



Fig. (2). Effects of age at cellular level, particularly at the mitochondria where remarkable changes occur (right). In fact, the increased production of ROS leads to mDNA damage and consequently, to a general decline in mitochondrial function that, in a vicious cycle-like manner, results in more oxidative stress. Mitochondrial function decline and oxidative stress response in aging seems to have implications for fertility, mainly due to the resultant energy crisis. An increase in proton leak, mediated by UCP2, may represent an adaptive strategy to overcome the increased production of ROS (see text). Therefore mitochondria seem be a common link between aging and the age-related decline in reproductive function.

functionality in fully adult animals and being depressed in older animals and [97]. Similar effects were also reported in the cat [98]. Although a declining mitochondrial function in older animals was observed, there was also evidence of the triggering of protective mechanisms, namely the increase in mitochondrial UCP2 content and function, which can promote proton leak, and attenuate ROS levels by causing a controlled decrease in mitochondrial membrane potential. It was therefore suggested that age-induced alterations in reproductive function may be caused by testicular mitochondrial dysfunction that may lead to a decline in ATP synthesis and a consequent energy crisis affecting the maintenance of testicular homeostasis [97] (Fig. 2). Moreover, mitochondria from Leydig cells are altered during the aging process, a phenomenon that might be related with the decrease in testosterone production. The number of Leydig cell mitochondria seems to also be reduced in the aging male [99]. Moreover, the protein levels of StAR were observed to be reduced [100] as well as expression and activity of steroidogenic enzymes [101]. Using a long-term suppression of steroidogenesis model, Chen and Zirkin

observed the abolishment of age-associated alterations indicating that sterodoigenic activity and associated ROS production are probably the main cause for these effects [102]. These results match with previous work that, in a similar model, observed an increase in lipid peroxidation after steroidogenesis resumption, with the steps regulated by P450 enzymes as the most likely sites of free radical generation [103]. Subsequently Chen and coworkers observed agerelated changes in the production of ROS by the mitochondria of Leydig cells, with those of old Leydig cells producing significantly greater levels than those of young Leydig cells. Absolute mitochondrial volume was reduced in old cells. The results are consistent with the proposal that mitochondrially-derived ROS may play a role in the irreversible decline in the ability of old Leydig cells to produce testosterone [104]. In accordance, Luo and collaborators, observed an age-related decrease in the antioxidant machinery of Leydig cells, a situation that can contribute to the increased oxidative damage previously observed [105]. In conclusion, it is clear that mitochondrial dysfunction is negatively correlated with, and may affect, several reproduc-


tive function parameters. Further studies will pinpoint the exact mechanisms, which govern age-dependent changes in male fertility.

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ACKNOWLEDGEMENTS Given that the available literature in this field is vast, in some cases we have employed reviews instead of original articles. We apologize to all authors whose work was not directly cited. S. Amaral would like to thank Cláudio Batista and Ângela Amaral for continuous encouragement and support. J. Ramalho-Santos would like to thank all lab members for helpful discussions as well as the Fulbright Foundation for support.

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