Testicular Aging: An Overview of Ultrastructural, Cellular, and Molecular Alterations

Testicular Aging: An Overview of Ultrastructural, Cellular, and Molecular Alterations Abstract The trend in parenthood at an older age is increasing for both men and women in developed countries, raising concerns about the reproductive ability, and the consequences for the offspring’s health. While reproductive activity in women stops with menopause, a complete cessation of the reproductive potential does not occur in men. Although several studies have been published on the effects of aging on semen parameters and spermatozoa DNA integrity, literature on impact of aging on the testis, particularly cellular, and molecular alterations, has been, so far, limited and controversial. This work discusses the current knowledge on testicular aging in humans and other mammals, covering topics from tissue ultrastructure, to cellular and molecular alterations. Aging affects male reproductive function at multiple levels, from sperm production and quality, to the morphology and histology of the male reproductive system. The morphological and functional changes that occur in the testes result in variations in the levels of many hormones, changes in molecules involved in mitochondrial function, receptors, and signaling proteins. Despite knowing that these age-related alterations occur, their real impact on male fertility and reproductive health are still far from being fully understood, highlighting that research in the field is crucial. Testis, Reproductive decline, Male (in)fertility In modern societies, the tendency to delay parenthood due to personal choices and socioeconomic factors is increasing (1). This trend raises many concerns about how the natural aging process may affect fertility and about what risks and consequences it may bring to the offspring. Reproductive decline occurs in a species- and gender-specific manner. In women, reproductive activity ends with menopause, and the negative impact of the advanced maternal age is well documented. In men, this process is gradual and a complete cessation of reproductive capacity does not occur (2). Reproductive decline in men with advanced age may result from a combination of morphological and molecular alterations in the reproductive organs, often due to age-related diseases or adverse environmental factors that cause secondary defects in the reproductive organs through life (3). Most studies are focused either on basic seminal parameters such as concentration, motility, and morphology, or on reproductive outcomes. Increased paternal age negatively affects sperm parameters, sperm DNA integrity, telomere length, chromosomal structures, and epigenetic factors (4). Additionally, advanced paternal age has been associated with lower pregnancy rates, higher risk of pregnancy loss, and with negative childhood health outcomes, particularly with higher incidence of congenital birth defects and disorders like achondroplasia, autism, schizophrenia, trisomy, and some types of cancers (1). Recently, our group used an omics approach to identify differentially expressed proteins and genes from spermatozoa and seminal plasma samples across several conditions affecting the infertile aging male (5). Although the alterations that occur in semen parameters and the impact of advanced paternal age in the offspring’s health are well studied and summarized in recent reviews (6,7), the impact of age on the male gonads (testes) are still poorly understood and the information is often controversial. In men, spermatogenesis and steroidogenesis, the two primary functions of the testis, are not completely independent. For instance, testosterone is important for normal spermatogenesis maintenance, but other hormones and molecules are involved in this process (3). Despite both processes are affected by the environment and some diseases, they need to be considered separately. This article reviews the current knowledge on testicular aging in human and other mammalian species. The changes in testicular size and weight, the morphological, and molecular alterations associated with aging and the impact of age in the endocrine and exocrine functions of the testes are discussed. The study of the age-associated alterations that occur in the testes may possibly explain the further alterations observed in spermatozoa and this information may be used to develop strategies and new therapies to overcome the reproductive decline. A Brief Overview of the Testicular Structure Testes are ovoid structures housed in separate compartments within the scrotum. They are surrounded by the tunica albuginea and the projections of this layer divide the testis in conic lobes. The testicular parenchyma is composed of 1–3 highly convoluted seminiferous tubules in each lobe where the production of spermatozoa occurs (spermatogenesis), and by interstitial tissue that surrounds the tubules, containing the Leydig cells that secrete testosterone (steroidogenesis). The seminiferous tubules are connected by short straight tubules, the tubuli recti, to the rete testis, which is composed of labyrinthine spaces located in the mediastinum testis. From here, spermatozoa enter the ductuli efferentes that lead into the epididymis. The seminiferous epithelium is composed of sustentacular Sertoli cells and a stratified layer of developing male germ cells. Adjacent Sertoli cells form tight junctions, constituting the blood–testis barrier that protects germ cells from recognition and attack by the immune system. The germinal cells present in the stratified germinal epithelium are defined according to their maturation state, from the basement membrane to the testicular lumen, in: spermatogonia (A-dark and A-pale), spermatocyte (primary and secondary), and spermatids (Figure 1). Figure 1. View largeDownload slide Testes. Cross-section showing the location of the seminiferous tubules, the vas deferens and the epididymis as well as the tunica albuginea. Schematic cross-section of a testicular tubules illustrating the germ cells at different stages of maturation within a somatic Sertoli cell. Leydig cells and vasculature are present in the interstitium. Major morphological, cellular, and ultrastructural alterations associated with age in testis are indicated. Figure 1. View largeDownload slide Testes. Cross-section showing the location of the seminiferous tubules, the vas deferens and the epididymis as well as the tunica albuginea. Schematic cross-section of a testicular tubules illustrating the germ cells at different stages of maturation within a somatic Sertoli cell. Leydig cells and vasculature are present in the interstitium. Major morphological, cellular, and ultrastructural alterations associated with age in testis are indicated. Impact of Age on Testicular Volume/Weight Testicular morphology and function are affected by multiple factors including age, malnutrition, and illness (8). Additionally, the volume/size of the testis, that is an important indicator of the integrity of the germinal epithelium, varies a lot amongst fertile male (9). Most of the studies performed in humans have showed a decrease in testicular volume in older men (8,10–14). For instance, Johnson et al. (14) described that testicular volume increased during puberty, peaked at age 30 and decreased significantly after 60 years of age. Testicular volume in aged men is positively associated with serum levels of some testicular hormones, such as inhibin B and testosterone (12). Since inhibin B is secreted by Sertoli cells and testosterone by Leydig cells, the reduction in the levels of these hormones may reflect the number of those cells. Johnson et al. (15) reported that despite the weight of the entire testicle being similar among young and old men, the weight of the tunica albuginea increased by 29% in older men, indicating a decrease in the testicular parenchyma. Future studies should take into consideration that the age-associated decrease on testicular volume may result from a combination of factors, including the reduction in the number and/or volume of testicular cells, and hormonal and connective tissue alterations. Similar alterations were reported in mice, with the testes of older animals being consistently smaller than those of younger animals (16,17). However, in studies conducted with other species, like hamsters (18,19), neither weight or volume of the testes changed progressively with age. These studies highlight that there is a species-dependent effect of aging in testicular characteristics and, more than descriptive measurements, there is an urgent need for mechanistic studies that may explain the specific effects of aging in each species. Furthermore, differences observed may arise from the natural aging process of each species and from the life expectancy and healthy associated to animals in captivity rather than wild ones, which constitute a huge limitation. Finally, when using animal models for aging studies, the chronological scale is particularly relevant. Aging and Testicular Morphology Histomorphometric and ultrastructural studies on human testes detected age-related changes, which were variable among individuals (20,21). These alterations included narrowing of tubular diameter, thickening of basal membrane and fibrosis, tubular sclerosis, reduction in the number of Sertoli and spermatogenic cells, vacuolization, and multinucleation of cells. Most of these studies were performed in testicular tissue obtained by biopsy, autopsy or following surgical removal of the testes. It is important to consider that biopsies of testes are mainly obtained in cases of diseases, such as prostate cancer, or in patients who requested assisted reproductive technology (ART), which may be an important source of bias (3). Consequently, these pathologies may exacerbate the underlying alterations, precluding a clear association between these findings and age. Thus, the most reliable information on the effects of testicular aging on morphology arises from small studies of postmortem fixed testis from men who died suddenly, rather than those who died due to chronic terminal illness (8,22). The major cellular and ultrastructural alterations in testes are summarized in Figure 1. Changes in Testicular Cell Population Leydig cell Age-related modifications on Leydig cells populations are still controversial. Although some studies show that the number of Leydig cells is diminished in aged individuals (22,23), investigations with the opposite results also exist (24,25). A comparison between men aged 20–48 and 50–76 years old showed that the average total number of Leydig cells was reduced by 44% in the oldest group (22). However, Ichihara et al. (25) observed that the number of Leydig cells in paired testes doubled with age, although the average volume of those cells decreased. Is important to consider that, when observing a smaller testis with seminiferous tubule involution, it may seem to have more Leydig cells than a normal-sized testis, despite the number of those cells actually remaining the same. In rats, a reduction of the total cell volume of Leydig cells accompanied by the deficient ability to produce testosterone in response to LH was reported but no changes in the total number of these cells during aging were observed (26,27). The ultrastructure of Leydig cells also seems to change with age. In humans, while some cells maintain their normal appearance, others acquire intranuclear Reinke crystals or paracrystalin inclusions, multiple vacuoles, lipofuscin granules, and lipid droplets in the cytoplasm (21,28). These cells also show signs of dedifferentiation and involution with poorly developed endoplasmic reticulum and mitochondria, as well as multinucleation (with two or three nuclei) (21,28). The accumulation of lipofuscin pigment was also identified in Leydig cells in old mice (29), cat (30), and horse (31) and in aged rats the volume of smooth surfaced endoplasmic reticulum per Leydig cell increased compared with young adult rats (25). Sasano and Ichijo (32) evaluated the vascular patterns on testes of men aged between 20 and 89 years old and showed a reduction in testicular perfusion and an increase in arteriosclerotic lesions of testicular arterioles with increasing age. A few years later, Regadera showed a correlation between tubular atrophy and testicular arteriosclerosis in the human testis (33). The interference with blood supply may, at least partially, explain the reduction in Leydig cell number and function. In fact, the decrease in blood supply and thus, in oxygen supply and LH levels that are responsible for the stimulation of Leydig cells, may also be responsible for the decrease in testosterone production in aged males. Sertoli cells Sertoli cells are known as the testicular “nurse cells”, since the interactions between these supporting somatic cells and the germ cells are crucial for normal spermatogenesis (34). They physically support the developing germ cells during their maturation, providing nutrients and protection from immune attack (35). Thus, functional or structural age-related changes in these cells could have several effects in spermatogenesis. It has been suggested that Sertoli cells are amongst the most vulnerable to age-related dysfunctions within the male reproductive system (36). In fact, multiple alterations associated with aging have been observed in the Sertoli cell population of different mammalian species. For instance, in human aged testes, a decrease in the number of Sertoli cells has been consistently reported. In men older than 50 years old, the number of Sertoli cells per gram of parenchyma and per testis is reduced when compared with men younger than 30 years old (14,37,38). This reduction was linked with a decrease in seminiferous epithelial volume (21). Still, a recent study showed that the germ cell/Sertoli cell ratios were maintained, with the exception of the spermatids and elongated spermatids to Sertoli cell ratios that were lower in the elderly men (39), which points to a parallel reduction of the number of germ cells, more pronounced in the spermatids and elongated spermatids. Moreover, multiple ultrastructural and histological alterations have been described in the remaining Sertoli cells of aged individuals. In senile rats, it has been reported that Sertoli cells lacked the thin extensions of cytoplasm (pseudopodia) that engulf germ cells and residual bodies, which might account for the failure on spermatogenesis (particularly at the stage of spermatids) and on the phagocytosis of residual bodies (40). Sertoli cells of aged rats also showed a loss of the organelles cyclical variations. The cells’ nucleus is prone to become more irregularly shaped and lose their typical localization, being present at various levels of the seminiferous epithelium (41). Enlarged vesicles are found at the base of older Sertoli cells and cytoplasmic vesicles that are common around the area of the nucleus of rat Sertoli cells tend to be longer in the older animals (40). This vesicle enlargement seems to be directly related with the age-associated reduction of the total area of endoplasmic reticulum of Sertoli cells. Moreover, in aged rats, the endoplasmic reticulum was found to be a loose, vesiculated network and not the typical elaborate, tubular and anastomotic network seen in Sertoli cells of young animals (41). A similar trend was reported in Sertoli cells of older men, which exhibited odd-shaped nuclei, vesiculated endoplasmic reticulum, and irregular lysosomes (39). Some of these Sertoli cell lysosomes are large, oddly shaped, containing lipidic inclusions and are scattered all over the cytoplasm, in contrast to the distinct membrane-bound lysosomes and dense core bodies described in young individuals. They encompass large, empty intercellular spaces, which were possibly previously occupied by developing germ cells and/or could result from the loss of ability of these aged cells to break down waste products (41). The presence of these vacuoles seems to reflect the diminished biological functions of Sertoli cells with aging (21). In fact, the increased phagocytosis of degenerating germ cells gives rise to lipid droplets in Sertoli cells’ cytoplasm, which can result in the excessive accumulation of both electron-lucent membrane-bound lipids and electron-dense lipid inclusions in older individuals. This accumulation makes Sertoli cells incapable of eliminating these lipid bodies (42). Other Sertoli cell abnormalities such as loss of polarity, dedifferentiation, and multinucleation were also reported in aging males (28,43). Mitochondrial metaplasia was also described in Sertoli cells of aging testes with oxidative stress being proposed as a mechanism for these changes (28,44). Sertoli cell junctions also seem to lose their characteristic appearance. In Sertoli cells of aged individuals, tight junctions, the structural basis for the blood–testis barrier, are rarer and are frequently replaced by focal contact points (21,39,41). This tight junction degeneration suggests the presence of a damaged sertoli/blood–testis barrier in the old individuals, compromising the specific microenvironment in which spermatogenesis occurs (39,41). Germ cells Despite some reports suggesting that apoptosis can be the principal cause of germ cell loss (45), the mechanisms underlying the age-associated atrophy of the seminiferous epithelium are still not fully understood. Impaired spermatogenesis with loss of germ cells has been observed in older men and other mammals (17,46), especially during spermatocytogenesis, meiosis, and spermiogenesis. This phenomena was characterized by the disarrangement of spermatogenic cells and releasing of premature spermatids from the seminiferous epithelium into the tubular lumen (39). The changes in germinal cells start with the spermatids and progressively affects less mature cell types until it originates a complete sclerosed tubule (16,21), which consists of a thickened tunica propria with abundant collagen and a few myoid cells (21,42). Thus, the number of germinal cells in the seminiferous tubule usually decreases as age increases in many mammals (18–20,47), resulting in a reduced diameter of seminiferous tubules (16) and epithelium vacuolization (48). Johnson et al. (14) reported that older men have reduced volume of seminiferous epithelia associated with lower daily sperm production and that the efficiency of sperm production decreases with age. With aging, the number of spermatids (32) and spermatogonia, including A-pale and A-dark spermatogonia, per unit section of the testes (49,50) significantly decreases. Age-related alterations of spermatogenesis in men between 65 and 93 years of age were reported by Holstein (51), and included reduction in A-dark and A-pale spermatogonia and arrested spermatocytes I with large nuclei in more than 70% of testicular sections, and many types of malformations of spermatids. Conversely, Johnson et al. (52) observed that young and old men have similar numbers of A-dark and A-pale spermatogonia per unit weight of testicular parenchyma and that the similar rates in germ cell degeneration at meiosis suggests that the difference in sperm production rates with age may occur in meiotic prophase, during the transition of leptotene to pachytene primary spermatocytes (52). Morphologically, multinucleated spermatocytes (53) and spermatids (42,54) in aged human testes have been reported. The formation of these kind of cells may result from the fusion of cell membranes of neighboring spermatocytes and spermatids and, as a consequence, cells undergo degeneration (53,54). Other ultrastructural alterations in spermatids comprise acrosome malformation, redundant nuclear membranes, intra-nuclear inclusions, excessive droplets in the cytoplasm, and irregular configuration of the nuclei (42). Intra-nuclear inclusions were also seen in spermatocytes and spermatogonia, as well as spirals of endoplasmic reticulum in cytoplasm (42). Morphological characteristics such as condensation of the chromatin and nuclear fragmentation in germ cells of older men support the hypothesis that the loss of germ cells observed during aging constitutes the best explanation for the low sperm counts observed in older men. On the other hand, the several ultrastructural alterations that take place may explain the higher index of teratozoospermia that these men presented. Alterations in Basement Membrane The basement membrane, a thin sheet-like extracellular structure that compartmentalize tissues, plays an important role in the maintenance of structural and functional integrity of tissues. In testes, the seminiferous tubule basement membrane is especially important during spermatogenesis (55). Damage of this structure has been associated with many testicular pathological conditions such as cryptorchidism and orchitis as well as with the aging process (56). However, literature on the morphological alterations of the basement membrane during testicular aging is contradictory. A few old studies in humans suggested that there were no alterations in this structure with aging (14,24).Conversely, thickening and herniation of this structure with dilatation of the seminiferous tubules have been described in aged males (22,32,39), mice (16), and other mammals (30,57). A statistically significant increase in the human basement membrane thickness correlated with age was found in mesenchymal (capillaries) and epithelial (testes) tissue (58). A histomorphometric study performed in testis in men from 29 to 102 years old revealed that, in cases of arrested spermatogenesis, a thickening of the basal membrane also occurs (20). Surprisingly, a more recent study (59) showed that basement membrane thickness apparently decreased with age in men, contradicting the other previous studies. The differences showed in this study may be due to the use of immunomarking techniques using specific antibodies for the epithelial basement membranes, instead of fixing silver salts on the reticulin fibers or due to the use of measurement software on digitized microscopic images, avoiding human error, making for a more accurate analysis. On the other hand, this study used surgical samples of testicular tissue from patients with prostate adenocarcinoma, which must be accounted for an important source of bias. Regardless, the clinical significance of alterations of this structure, remains unknown. Modifications in Vasculature The testicular artery is responsible for the blood supply of the testis and the epididymis, and several blood vessels are present in the interstitium of the seminiferous tubules. Paniagua et al. (21,42) showed that the pattern of testicular involution observed in aged men was similar to the one observed after induced ischemia. This pattern is influenced by alterations in testicular perfusion caused by vascular changes, mainly in the most distal regions of artery supply. The decrease of testicular perfusion is also caused by atherosclerotic alterations in testicular arterioles (32). Observations of the human testicular microvasculature revealed increased coiling of interlobular arteries with age, together with the degradation of the peritubular capillary network, resulting in a considerable reduction of blood flow (60). These changes are not exclusive to humans, occurring in other species with senescence, where a progressive decrease in capillary density, partial, or complete occlusion of the lumen and thickening of vessel walls were observed (61,62). The deficient irrigation related to vascular changes might be an explanation for the involution of the seminiferous tubules and consequently, the lower testicular volume observed in aged males. Alterations in Connective Tissue In human testes, the tunica albuginea thickens and the parenchyma decreases in volume (63). Johnson et al. (15) showed increased fibrosis and weight of the tunica albuginea with age. This feature is so consistent that it was proposed as a metric to estimate age (3). Lopez-Marambio and Hutson (64) found that in elderly men the tunica vaginalis became adherent to the posterior surface of the testes adjacent to the body of the epididymis, giving the appearance that the testes was retroperitoneal. Other testicular feature that appears to change with age is the testicular tunica adventitia, that becomes thicker (30). Increasing fibrosis of the tunica propria and tunica albuginea, may be part of a vicious cycle, where oxygen deprivation due to vascular changes may have an important role (see Section “Modifications in vasculature”). The modifications on these structures are, however, far from being completely understood and consequently, their involvement and consequences in the decline of male reproductive capacity are still unknown. Aging and Testicular (dys)fuction Aging has a well-documented effect on every level of the hypothalamic–pituitary–testicular axis (HPTA) (Figure 2), that is responsible for regulating testicular function (3). The testis has two main functions—spermatogenesis (exocrine function) and steroidogenesis (endocrine function) which are not completely independent (3). In normal conditions, the hypothalamus release gonadotrophin releasing hormone (GnRH) which stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells stimulating testosterone biosynthesis (steroidogenesis). FSH is recognized by FSH receptors in Sertoli cells having an important role in spermatozoa production (spermatogenesis) (Figure 2). These functions are affected by age resulting in alterations in hormonal levels during senescence in humans and other animals, comprising lower circulating androgens (3,65). However, lower androgen levels in aging men are not linked to a complete cessation of reproductive capacity, contrary to what happens in women with menopause and androgen deficiency is not only associated with advanced age (2). Figure 2. View largeDownload slide Hypothalamic–pituitary–testicular/gonadal axis. The release of gonadotrophin releasing hormone (GnRH) by the hypothalamus stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells and stimulates the biosynthesis of testosterone. On the other hand, FSH is recognized by FSH receptors in Sertoli cells that has an important role in spermatogenesis. Figure 2. View largeDownload slide Hypothalamic–pituitary–testicular/gonadal axis. The release of gonadotrophin releasing hormone (GnRH) by the hypothalamus stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells and stimulates the biosynthesis of testosterone. On the other hand, FSH is recognized by FSH receptors in Sertoli cells that has an important role in spermatogenesis. A comparative study between the effects of age on the testicular steroidogenic activity of the Brown Norway rat and the Sprague-Dawley rat showed an age-associated decrease in serum and testicular testosterone in both models (66). In fact, the ability of Leydig cells to produce testosterone is reduced in older Brown Norway rats, resulting in reduced serum testosterone levels (67,68). The Brown Norway rat is already identified as the best available model to study the male reproductive aging given that, as in men, both primary and secondary testicular failure occur (68,69). A comparison of plasma testosterone levels on various species are summarized in Table 1. Table 1. Comparison of the Levels of Plasma Testosterone (ng/ml) in Various Species From Infancy to Senescence Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Note: GLC = gas-liquid chromatography; RIA = radioimmunoassay. View Large Table 1. Comparison of the Levels of Plasma Testosterone (ng/ml) in Various Species From Infancy to Senescence Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Note: GLC = gas-liquid chromatography; RIA = radioimmunoassay. View Large Testicular Exocrine System It is known that androgen stimulation of Sertoli cells is fundamental for the initial induction of spermatogenesis. In humans, few studies that compare endogenous steroid levels in testicular tissue from young and elderly men are available. Takahashi et al. (76) measured the concentration of nine endogenous steroids in men from 25 to 35 years old with oligozoospermia and varicocele and in men aged 61–85 years old with prostatic cancer and showed that the levels of all steroid hormones declined with age. A more recent study conducted by Carreau et al. (77) also evaluated the intra-testicular levels of steroids (pregnenolone, progesterone, DHEA, DHEAS, testosterone, and estradiol) in aged men, but no significant differences between the various age groups tested were found. The reduction in testicular testosterone production with age can have a big impact in spermatogenesis by affecting the function of Sertoli cells. Nevertheless, this decline often appears in older men with associated age-related diseases related to hormonal alterations. It is well established that the plasma levels of FSH (follicle-stimulating hormone), the second major stimulus for spermatogenesis, increase with age in older men, associated with a decrease in testicular size and spermatogenic efficacy (3,78). Additionally, increasing levels of FSH in elderly men has been linked to germ cell degeneration during meiosis, affecting sperm concentration (79). In men, serum inhibin levels decline with aging, with older individuals presenting significantly lower serum concentrations of this glycoprotein already at the age of 40 (80). Inhibin is a hormonal glycoprotein secreted by Sertoli cells in response to the stimulation by FSH, which negatively feedbacks on the pituitary gland to specifically suppress the production and secretion of FSH. This hormone is often used as a functionality marker for Sertoli cells and for the impairment of spermatogenesis (81). The decrease observed in the secretion of inhibin by Sertoli cells led to the suggestion that the testicular endocrine functions decline as soon as the fourth decade of life, and that Sertoli cell function declines earlier than that of other somatic testicular cells (80). In fact, the expression of the receptor for the hormone serotonin, an important player on germ cell–Sertoli cell interactions (82), is lost in Sertoli cells from rats older than 24 months (44), further confirming this suggestion. Testicular Endocrine System Steroidogenesis occurs in testicular Leydig cells and is mainly regulated by the LH secreted by the pituitary gland. In turn, LH secretion is induced by gonadotrophin releasing hormone (GnRH) released from the hypothalamus (Figure 2). In men, one of the most relevant age-related changes is the decline in testosterone levels, particularly plasma levels (3,83). Although some studies examining men of different ages failed to detect alterations in testosterone levels in aged heathy men (84), the majority showed that the plasma testosterone decline with increasing age (85–87). There is no consensus about the mechanism that cause testosterone age-related decline, but it has been associated with the three levels of the HPTA (45,87) or with the waning of testicular function (88). The decreased number of Leydig cells (22) and impaired testicular perfusion due to atherosclerosis, previously discussed, as well as reduced release of testosterone upon human Chorionic Gonadotropin (hCG) stimulation (89), support a primarily testicular cause for low testosterone levels (3). However, lower levels of testosterone in aged males should not be strictly associated with aging since other factors, such as lifestyle and pathologies, can be involved. This information highlights the need to understand how men with decreased testosterone levels would benefit from hormonal replacement. Like testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, cortisol, and estrone showed a significant longitudinal decline with age. On the contrary, dihydrotestosterone and pituitary gonadotropins levels raised longitudinally (90). It was shown that serum LH levels normally rise with aging, possibly as a response to the decline in testosterone, which supports the hypothesis of secondary hypogonadism (78). In men older than 60 years, it was reported that the production of LH increases with age (85). In rats, serum and pituitary prolactin levels and serum estradiol levels increased with age whereas serum and pituitary FSH and LH levels decreased (72). Other factors, besides aging, may influence the plasma and testicular levels of testosterone, including hereditary, environmental (stress, obesity), psychosocial (depression, drugs, smoking), and socio-economic factors (84,91). Therefore, the association of testosterone levels with age is not always linear. Furthermore, the decreased concentration of serum testosterone levels in aged males has been linked not exclusively to andropausal symptoms such as poor libido, erectile dysfunction, fatigue, and deterioration of cognitive functions which can explain the infertility experienced by these couples, but also with the functionality of androgen-sensitive somatic tissues (3,83,92). The hormonal variations related to age in men are summarized in Table 2. When interpreting the data, attention should be paid to the fact that, until recent years, most of the studies that measure circulating hormones used steroid immunoassays, which are known to have important bias and inaccuracies. Table 2. Age-Related Changes in Hormonal Levels in Human Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Note: AMH = anti-mullerian hormone; DHT = dihydrotestosterone; DHES = dehydroepiandrosterone; DHEAS = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; INSL3 = insulin-like peptide 3; InhB = inhibin B; LH = luteinizing hormone; SHBG = sex hormone-binding globulin. View Large Table 2. Age-Related Changes in Hormonal Levels in Human Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Note: AMH = anti-mullerian hormone; DHT = dihydrotestosterone; DHES = dehydroepiandrosterone; DHEAS = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; INSL3 = insulin-like peptide 3; InhB = inhibin B; LH = luteinizing hormone; SHBG = sex hormone-binding globulin. View Large Molecular Changes in Aged Testes In the past decade, several molecular mechanisms of aging have been proposed, including damage by reactive oxygen species (ROS) produced during aerobic metabolism, implicating mitochondria in the aging process (97). Indeed, mitochondrial dysfunction was already described as being involved in testicular aging (97). Aging was also associated with an increase in oxidative stress and free radical production, due to the alterations in the enzymatic activity of anti-oxidant molecules such as glutathione-s-transferase (GST), an enzyme that protects cellular components from electrophilic and oxidative attack in testis (98,99), glutathione peroxidase (GPx) (99,100), and superoxide dismutase (SOD), especially in Leydig cells (99–101). Several testicular age-related alterations, including the decrease in steroidogenic capacity, have also been associated to an increase in tissue inflammation. Cyclooxygenase‐2 (COX2) expression is induced by cytokines and growth factors, particularly at sites of inflammation and has been associated with aged-Leydig cells phenotype since these cells express higher levels of this inducible isoenzyme (102,103). Moreover, pharmacological inhibition of COX2 increases StAR expression, a Leydig cell’s marker, and consequently steroidogenesis (103). However, a recent study showed that the levels of COX2 decreased, particularly in Leydig cells and macrophages, in rats with accelerated senescence when compared with the control animals (104). In long-lived mice, the expression of CD68 and CD163, macrophages markers, was reduced by half in testicular interstitium (104). Thus, the actual role of inflammation in reproductive aging is poorly understood and needs further investigation. The smooth muscle located in tunica albuginea is responsible for testicular capsular contraction that is involved in the transport of non-motile sperm to the epididymis. With aging, the capsule gets thicker, as already explained above, and the response to norepinephrine and prostaglandin becomes progressively higher, suggesting that neuro-humoral agents may have an important role in the maintenance of testicular capsular contractions (105). The main molecular age-related alterations identified in the different testicular cells are described below and summarized in Table 3. Table 3. Major Molecular Alteration Occurring in Testes with Age Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Note: Ach = acetylcholine; AM = adrenomedullin; cAMP = cyclic adenosine monophosphate; CGRP = calcitonin gene-related peptide; COX2 = cyclooxygenase‐2; GST = glutathion-S-transferase; LH = luteinizing hormone; MAPK = mitogen-activated protein kinase; NE = norepinephrine; PGA2 = prostaglandin A2; RLF = relaxin-like factor; UCP2 = uncoupling protein 2; 11β-HDS = 11beta-hydroxysteroid dehydrogenase; UCHL1 = ubiquitin carboxy-terminal hydrolase L. View Large Table 3. Major Molecular Alteration Occurring in Testes with Age Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Note: Ach = acetylcholine; AM = adrenomedullin; cAMP = cyclic adenosine monophosphate; CGRP = calcitonin gene-related peptide; COX2 = cyclooxygenase‐2; GST = glutathion-S-transferase; LH = luteinizing hormone; MAPK = mitogen-activated protein kinase; NE = norepinephrine; PGA2 = prostaglandin A2; RLF = relaxin-like factor; UCP2 = uncoupling protein 2; 11β-HDS = 11beta-hydroxysteroid dehydrogenase; UCHL1 = ubiquitin carboxy-terminal hydrolase L. View Large Leydig Cells The aged Leydig cells are characterized by the reduced ability to produce testosterone in response to LH (91). Several cellular changes have been identified in the steroidogenic pathway of aged Leydig cells in humans that result in the reduction of testosterone production. Cyclic nucleotides, cAMP and cGMP, are intracellular secondary messengers that play a central role in signal transduction cascades involved in testosterone production. Aging modulates the expression of genes responsible for cGMP production and degradation, which results in an increase in cGMP in Leydig cells, which can be a mean to overcome the reduction in testosterone levels (117). Additionally, several studies suggested a reduction in cAMP-signaling in aged cells, consistent with a reduce in testosterone production in aged rats in response to LH (118). Previous investigations also reported alterations in the expression profiles of 45 genes (119) and a decrease in LH receptor levels (72) in aged rats. The secretion of the insulin-like factor 3 (INSL3), a small peptide hormone secreted by mature Leydig cells, is an important indicator of the differentiation status and the number of Leydig cells present in testes. The levels of this peptide hormone decline gradually with aging reflecting the functional capacity of this type of cells. Supporting this data, in old rats INSL3 expression also seems to be down-regulated with age (110,120). The expression levels of LH receptors in Leydig cells also decreases with age in Fisher 344 rats (72). The response of Leydig cells to LH depends not only on the number of receptors and the circulating concentration of glucocorticoids, but also on 11β-HSD activity (109). With increasing age, the expression of this enzyme is reduced and consequently, a reduction in testosterone production is observed (109). The aging impact in the number and volume of Leydig cells and the molecular alterations described in this section may explain, in part, the reduction in testosterone levels often observed in older men. However, it is important to keep in mind that these alterations may result from a combination of elements, including environmental factors that can affect the testes and, particularly, this type of cells. Sertoli Cells Sertoli cells secretions and metabolites are crucial for the normal occurrence of spermatogenesis. The expression of some of these products is dependent on an intricate network of signaling molecules and hormones (34,121). It has been reported that aging is able to alter the production of some important secretions by the Sertoli cell and even its molecular components, such as transcripts, cytoskeleton proteins, and secretory proteins. Indeed, the synthesis and secretion of two of the major products of Sertoli cells, transferrin and cathepsin L (also called cyclic protein-2, CP-2) is affected in older individuals, while that of others, such as sulphated glycoprotein-2 (SGP-2, also called Clusterin), remains unaffected (41,46,112). Transferrin is a major secretory protein of Sertoli cells, being involved in the transport of iron in cells of the seminiferous epithelium. In brown Norway rats, the total testicular content of transferrin mRNA remained unchanged up to 18 months of age, but dramatically increased by the age of 2 years. In opposition, the mRNA CP-2 testicular content of older rats was shown to be decreased (46,112). The stage-specific expression of CP-2 in the seminiferous epithelium suggested that the presence of the active cathepsin L facilitates the intense reorganization of the seminiferous epithelium during germ cell development (122), enhancing the degradation of dying or dead germ cells (112). These age-associated alterations of transferrin and CP-2/cathepsin L levels in aged rats led to the suggestion that age-related changes in Sertoli cell gene expression reflect a primary dysfunction of aging and not an indirect effect due to germ cell loss (46). Moreover, age-associated alterations in the cytoskeletal components of Sertoli cells, namely F-actin, vimentin, and cytokeratin, were described in aged BDF1 mice. In old mice, F-actin was distributed at the inter-Sertoli cells junctions and at the luminal portion of the Sertoli cell cytoplasm, while in young mice it was only detectable at the junctions between adjoining Sertoli cells (43). Similarly, vimentin was recognized only around the nucleus in Sertoli cell of old animals, and not in the Sertoli cell trunk as seen in young mice. Instead, in old mice testes, sheet-like organizations of vimentin were detected near the luminal surface parallel to the basement membrane (43). On the other hand, cytokeratin expression pattern was dramatically altered in the mice Sertoli cells. Up until 27 months of age, no cytokeratin expression was detected in Sertoli cells. Only in older mice, which exhibited thinner seminiferous epithelia, the expression of cytokeratin was consistently detected within the Sertoli cell cytoplasm, being one of the most characteristic occurrences of age-associated changes in Sertoli cells (43). Additionally, age-associated accumulation of intracellular amyloid fibrils in Sertoli cells has also been reported (113). This accumulation is concurrent with the buildup of lipofuscin-loaded lysosomes, as well as of damaged mitochondria, in Sertoli cells. The activity of the enzymes that degrade lipofuscin and damaged mitochondria (by autophagy) is diminished, leading to their accumulation. Dysfunctional mitochondria produce more ROS and lipofuscin accumulation sensitizes cells to ROS-induced damage, which then becomes a self-amplifying, vicious cycle in these cells (123). Germ Cells With aging the number of germ cells in the seminiferous tubule decreases in humans and most mammals (18–20,47) and several ultrastructural alterations take place. Indeed, spermatogenesis gradually decreases in older men (124). To investigate whether this reduction resulted from apoptotic events, Barnes et al. (125) studied rats from different age groups and observed that apoptotic cell death of testicular germ cells significantly increases with age but a significant decrease in apoptotic cells/seminiferous tubule was observed. However, an increase in apoptotic metaphase spermatocytes at tubule stage XIV in mice with 24 months of age when compared with 6-months-old mice was observed (126). Like Leydig cells, the decreased number of germinal cells in aged testes was accompanied by changes in gene expression profile, particularly in the expression of spermatogonia markers (114). An investigation conducted in mice that compared the gene expression in four age groups (6, 21, 60 days, and 8 months) revealed that certain genes were specifically expressed in older mice and some genes were up- (Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, SELP, and Trio) and down-regulated (Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1) (115). Additionally, 16 proteins were identified as dramatically increased and 21 were decreased in aged human testes compared to the young ones, especially proteins involved in oxidative stress (124). These proteins can constitute valuable markers of reproductive aging. Summary and Future Perspectives Advanced paternal age is a common phenomenon in modern societies. Social-economic factors and personal choices contribute to this tendency. This evidence highlights the need to discuss and investigate the effect of advanced age in the male reproductive system, in semen parameters and the impact on the offspring’s health. As described in the above sections, the testes, as all the other systems in the body, suffer several complex alterations that might be an important piece to explaining the reproductive decline observed in aged men. Aged testes undergo profound histological and morphological alterations, including tissue atrophy, leading to a reduced functionality. In addition, several molecular alterations were reported in all testicular cell types. These age-related changes are usually so small that men are generally fertile until old age. Importantly, the pathophysiology of age-impact in testes may result from specific effects of age alone or other conditions, such as vascular diseases, infection on reproductive glands, and exposure to environmental chemicals. Additionally, the impact of age is not exclusive to humans but was also present in many mammalian species, reflecting the universality of this phenomenon. Some of them, such as mice and rat, can be used as animal models to study the aging phenomenon (69). Nevertheless, despite rodent models being very useful in reproductive biology, we must be aware of their limitations. In fact, the aging process is very different between species and the presence of specie-specific mechanisms should be considered when taking conclusions. Particularly relevant, there are also serious doubts that human aging, which takes decades, can be realistically studied using rodents, which have a much shorter life cycle. Finally, some important questions remain unanswered and should be addressed by future research in this field. Are the alterations in the composition of cell populations the main responsible for the decreased testicular volume with age, or this happens only due to an increased basement membrane and tunica albuginea thickness? Are the levels of steroid hormones the key to these changes or are their clinical impact irrelevant? Is the decrease in the amount of different cell types a result of the molecular changes that occur or do they precede them? Are the changes in testicular vascularization the main responsible for most of the testicular age-related alterations? These and other questions should be investigated and the identification of the sequence of events might be important to better understand this phenomenon. Indeed, the understanding of the mechanisms of testicular aging and reproductive decline can be very useful to better understand some cases of male infertility and to finally address the question—How old is too old to be a father?. Funding This work was supported by FEDER Funds through Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through FCT–Foundation for Science and Technology under the project PTDB/BBB-BQB/3804/2014. This study was also supported by Institute for Biomedicine—iBiMED (UID/BIM/04501/2013 and POCI-01-0145-FEDER-007628) and by individual grant from FCT of the Portuguese Ministry of Science and Higher Education to JVS (SFRH/BPD/123155/2016). Conflict of Interest None declared. References 1. Sharma R, Agarwal A, Rohra VK, Assidi M, Abu-Elmagd M, Turki RF. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring. Reprod Biol Endocrinol . 2015; 13: 35. doi: 10.1186/s12958-015-0028-x Google Scholar CrossRef Search ADS PubMed  2. Tenover JS. Declining testicular function in aging men. Int J Impot Res . 2003; 15 ( Suppl 4): S3– S8. doi: 10.1038/sj.ijir.3901029 Google Scholar CrossRef Search ADS PubMed  3. Handelsman DJ. Aging in the hypothalamic-pituitary-testicular axis. In: Neill JD, ed. Knobil and Neill’s Physiology of Reproduction . 3rd ed. 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Abstract

Abstract The trend in parenthood at an older age is increasing for both men and women in developed countries, raising concerns about the reproductive ability, and the consequences for the offspring’s health. While reproductive activity in women stops with menopause, a complete cessation of the reproductive potential does not occur in men. Although several studies have been published on the effects of aging on semen parameters and spermatozoa DNA integrity, literature on impact of aging on the testis, particularly cellular, and molecular alterations, has been, so far, limited and controversial. This work discusses the current knowledge on testicular aging in humans and other mammals, covering topics from tissue ultrastructure, to cellular and molecular alterations. Aging affects male reproductive function at multiple levels, from sperm production and quality, to the morphology and histology of the male reproductive system. The morphological and functional changes that occur in the testes result in variations in the levels of many hormones, changes in molecules involved in mitochondrial function, receptors, and signaling proteins. Despite knowing that these age-related alterations occur, their real impact on male fertility and reproductive health are still far from being fully understood, highlighting that research in the field is crucial. Testis, Reproductive decline, Male (in)fertility In modern societies, the tendency to delay parenthood due to personal choices and socioeconomic factors is increasing (1). This trend raises many concerns about how the natural aging process may affect fertility and about what risks and consequences it may bring to the offspring. Reproductive decline occurs in a species- and gender-specific manner. In women, reproductive activity ends with menopause, and the negative impact of the advanced maternal age is well documented. In men, this process is gradual and a complete cessation of reproductive capacity does not occur (2). Reproductive decline in men with advanced age may result from a combination of morphological and molecular alterations in the reproductive organs, often due to age-related diseases or adverse environmental factors that cause secondary defects in the reproductive organs through life (3). Most studies are focused either on basic seminal parameters such as concentration, motility, and morphology, or on reproductive outcomes. Increased paternal age negatively affects sperm parameters, sperm DNA integrity, telomere length, chromosomal structures, and epigenetic factors (4). Additionally, advanced paternal age has been associated with lower pregnancy rates, higher risk of pregnancy loss, and with negative childhood health outcomes, particularly with higher incidence of congenital birth defects and disorders like achondroplasia, autism, schizophrenia, trisomy, and some types of cancers (1). Recently, our group used an omics approach to identify differentially expressed proteins and genes from spermatozoa and seminal plasma samples across several conditions affecting the infertile aging male (5). Although the alterations that occur in semen parameters and the impact of advanced paternal age in the offspring’s health are well studied and summarized in recent reviews (6,7), the impact of age on the male gonads (testes) are still poorly understood and the information is often controversial. In men, spermatogenesis and steroidogenesis, the two primary functions of the testis, are not completely independent. For instance, testosterone is important for normal spermatogenesis maintenance, but other hormones and molecules are involved in this process (3). Despite both processes are affected by the environment and some diseases, they need to be considered separately. This article reviews the current knowledge on testicular aging in human and other mammalian species. The changes in testicular size and weight, the morphological, and molecular alterations associated with aging and the impact of age in the endocrine and exocrine functions of the testes are discussed. The study of the age-associated alterations that occur in the testes may possibly explain the further alterations observed in spermatozoa and this information may be used to develop strategies and new therapies to overcome the reproductive decline. A Brief Overview of the Testicular Structure Testes are ovoid structures housed in separate compartments within the scrotum. They are surrounded by the tunica albuginea and the projections of this layer divide the testis in conic lobes. The testicular parenchyma is composed of 1–3 highly convoluted seminiferous tubules in each lobe where the production of spermatozoa occurs (spermatogenesis), and by interstitial tissue that surrounds the tubules, containing the Leydig cells that secrete testosterone (steroidogenesis). The seminiferous tubules are connected by short straight tubules, the tubuli recti, to the rete testis, which is composed of labyrinthine spaces located in the mediastinum testis. From here, spermatozoa enter the ductuli efferentes that lead into the epididymis. The seminiferous epithelium is composed of sustentacular Sertoli cells and a stratified layer of developing male germ cells. Adjacent Sertoli cells form tight junctions, constituting the blood–testis barrier that protects germ cells from recognition and attack by the immune system. The germinal cells present in the stratified germinal epithelium are defined according to their maturation state, from the basement membrane to the testicular lumen, in: spermatogonia (A-dark and A-pale), spermatocyte (primary and secondary), and spermatids (Figure 1). Figure 1. View largeDownload slide Testes. Cross-section showing the location of the seminiferous tubules, the vas deferens and the epididymis as well as the tunica albuginea. Schematic cross-section of a testicular tubules illustrating the germ cells at different stages of maturation within a somatic Sertoli cell. Leydig cells and vasculature are present in the interstitium. Major morphological, cellular, and ultrastructural alterations associated with age in testis are indicated. Figure 1. View largeDownload slide Testes. Cross-section showing the location of the seminiferous tubules, the vas deferens and the epididymis as well as the tunica albuginea. Schematic cross-section of a testicular tubules illustrating the germ cells at different stages of maturation within a somatic Sertoli cell. Leydig cells and vasculature are present in the interstitium. Major morphological, cellular, and ultrastructural alterations associated with age in testis are indicated. Impact of Age on Testicular Volume/Weight Testicular morphology and function are affected by multiple factors including age, malnutrition, and illness (8). Additionally, the volume/size of the testis, that is an important indicator of the integrity of the germinal epithelium, varies a lot amongst fertile male (9). Most of the studies performed in humans have showed a decrease in testicular volume in older men (8,10–14). For instance, Johnson et al. (14) described that testicular volume increased during puberty, peaked at age 30 and decreased significantly after 60 years of age. Testicular volume in aged men is positively associated with serum levels of some testicular hormones, such as inhibin B and testosterone (12). Since inhibin B is secreted by Sertoli cells and testosterone by Leydig cells, the reduction in the levels of these hormones may reflect the number of those cells. Johnson et al. (15) reported that despite the weight of the entire testicle being similar among young and old men, the weight of the tunica albuginea increased by 29% in older men, indicating a decrease in the testicular parenchyma. Future studies should take into consideration that the age-associated decrease on testicular volume may result from a combination of factors, including the reduction in the number and/or volume of testicular cells, and hormonal and connective tissue alterations. Similar alterations were reported in mice, with the testes of older animals being consistently smaller than those of younger animals (16,17). However, in studies conducted with other species, like hamsters (18,19), neither weight or volume of the testes changed progressively with age. These studies highlight that there is a species-dependent effect of aging in testicular characteristics and, more than descriptive measurements, there is an urgent need for mechanistic studies that may explain the specific effects of aging in each species. Furthermore, differences observed may arise from the natural aging process of each species and from the life expectancy and healthy associated to animals in captivity rather than wild ones, which constitute a huge limitation. Finally, when using animal models for aging studies, the chronological scale is particularly relevant. Aging and Testicular Morphology Histomorphometric and ultrastructural studies on human testes detected age-related changes, which were variable among individuals (20,21). These alterations included narrowing of tubular diameter, thickening of basal membrane and fibrosis, tubular sclerosis, reduction in the number of Sertoli and spermatogenic cells, vacuolization, and multinucleation of cells. Most of these studies were performed in testicular tissue obtained by biopsy, autopsy or following surgical removal of the testes. It is important to consider that biopsies of testes are mainly obtained in cases of diseases, such as prostate cancer, or in patients who requested assisted reproductive technology (ART), which may be an important source of bias (3). Consequently, these pathologies may exacerbate the underlying alterations, precluding a clear association between these findings and age. Thus, the most reliable information on the effects of testicular aging on morphology arises from small studies of postmortem fixed testis from men who died suddenly, rather than those who died due to chronic terminal illness (8,22). The major cellular and ultrastructural alterations in testes are summarized in Figure 1. Changes in Testicular Cell Population Leydig cell Age-related modifications on Leydig cells populations are still controversial. Although some studies show that the number of Leydig cells is diminished in aged individuals (22,23), investigations with the opposite results also exist (24,25). A comparison between men aged 20–48 and 50–76 years old showed that the average total number of Leydig cells was reduced by 44% in the oldest group (22). However, Ichihara et al. (25) observed that the number of Leydig cells in paired testes doubled with age, although the average volume of those cells decreased. Is important to consider that, when observing a smaller testis with seminiferous tubule involution, it may seem to have more Leydig cells than a normal-sized testis, despite the number of those cells actually remaining the same. In rats, a reduction of the total cell volume of Leydig cells accompanied by the deficient ability to produce testosterone in response to LH was reported but no changes in the total number of these cells during aging were observed (26,27). The ultrastructure of Leydig cells also seems to change with age. In humans, while some cells maintain their normal appearance, others acquire intranuclear Reinke crystals or paracrystalin inclusions, multiple vacuoles, lipofuscin granules, and lipid droplets in the cytoplasm (21,28). These cells also show signs of dedifferentiation and involution with poorly developed endoplasmic reticulum and mitochondria, as well as multinucleation (with two or three nuclei) (21,28). The accumulation of lipofuscin pigment was also identified in Leydig cells in old mice (29), cat (30), and horse (31) and in aged rats the volume of smooth surfaced endoplasmic reticulum per Leydig cell increased compared with young adult rats (25). Sasano and Ichijo (32) evaluated the vascular patterns on testes of men aged between 20 and 89 years old and showed a reduction in testicular perfusion and an increase in arteriosclerotic lesions of testicular arterioles with increasing age. A few years later, Regadera showed a correlation between tubular atrophy and testicular arteriosclerosis in the human testis (33). The interference with blood supply may, at least partially, explain the reduction in Leydig cell number and function. In fact, the decrease in blood supply and thus, in oxygen supply and LH levels that are responsible for the stimulation of Leydig cells, may also be responsible for the decrease in testosterone production in aged males. Sertoli cells Sertoli cells are known as the testicular “nurse cells”, since the interactions between these supporting somatic cells and the germ cells are crucial for normal spermatogenesis (34). They physically support the developing germ cells during their maturation, providing nutrients and protection from immune attack (35). Thus, functional or structural age-related changes in these cells could have several effects in spermatogenesis. It has been suggested that Sertoli cells are amongst the most vulnerable to age-related dysfunctions within the male reproductive system (36). In fact, multiple alterations associated with aging have been observed in the Sertoli cell population of different mammalian species. For instance, in human aged testes, a decrease in the number of Sertoli cells has been consistently reported. In men older than 50 years old, the number of Sertoli cells per gram of parenchyma and per testis is reduced when compared with men younger than 30 years old (14,37,38). This reduction was linked with a decrease in seminiferous epithelial volume (21). Still, a recent study showed that the germ cell/Sertoli cell ratios were maintained, with the exception of the spermatids and elongated spermatids to Sertoli cell ratios that were lower in the elderly men (39), which points to a parallel reduction of the number of germ cells, more pronounced in the spermatids and elongated spermatids. Moreover, multiple ultrastructural and histological alterations have been described in the remaining Sertoli cells of aged individuals. In senile rats, it has been reported that Sertoli cells lacked the thin extensions of cytoplasm (pseudopodia) that engulf germ cells and residual bodies, which might account for the failure on spermatogenesis (particularly at the stage of spermatids) and on the phagocytosis of residual bodies (40). Sertoli cells of aged rats also showed a loss of the organelles cyclical variations. The cells’ nucleus is prone to become more irregularly shaped and lose their typical localization, being present at various levels of the seminiferous epithelium (41). Enlarged vesicles are found at the base of older Sertoli cells and cytoplasmic vesicles that are common around the area of the nucleus of rat Sertoli cells tend to be longer in the older animals (40). This vesicle enlargement seems to be directly related with the age-associated reduction of the total area of endoplasmic reticulum of Sertoli cells. Moreover, in aged rats, the endoplasmic reticulum was found to be a loose, vesiculated network and not the typical elaborate, tubular and anastomotic network seen in Sertoli cells of young animals (41). A similar trend was reported in Sertoli cells of older men, which exhibited odd-shaped nuclei, vesiculated endoplasmic reticulum, and irregular lysosomes (39). Some of these Sertoli cell lysosomes are large, oddly shaped, containing lipidic inclusions and are scattered all over the cytoplasm, in contrast to the distinct membrane-bound lysosomes and dense core bodies described in young individuals. They encompass large, empty intercellular spaces, which were possibly previously occupied by developing germ cells and/or could result from the loss of ability of these aged cells to break down waste products (41). The presence of these vacuoles seems to reflect the diminished biological functions of Sertoli cells with aging (21). In fact, the increased phagocytosis of degenerating germ cells gives rise to lipid droplets in Sertoli cells’ cytoplasm, which can result in the excessive accumulation of both electron-lucent membrane-bound lipids and electron-dense lipid inclusions in older individuals. This accumulation makes Sertoli cells incapable of eliminating these lipid bodies (42). Other Sertoli cell abnormalities such as loss of polarity, dedifferentiation, and multinucleation were also reported in aging males (28,43). Mitochondrial metaplasia was also described in Sertoli cells of aging testes with oxidative stress being proposed as a mechanism for these changes (28,44). Sertoli cell junctions also seem to lose their characteristic appearance. In Sertoli cells of aged individuals, tight junctions, the structural basis for the blood–testis barrier, are rarer and are frequently replaced by focal contact points (21,39,41). This tight junction degeneration suggests the presence of a damaged sertoli/blood–testis barrier in the old individuals, compromising the specific microenvironment in which spermatogenesis occurs (39,41). Germ cells Despite some reports suggesting that apoptosis can be the principal cause of germ cell loss (45), the mechanisms underlying the age-associated atrophy of the seminiferous epithelium are still not fully understood. Impaired spermatogenesis with loss of germ cells has been observed in older men and other mammals (17,46), especially during spermatocytogenesis, meiosis, and spermiogenesis. This phenomena was characterized by the disarrangement of spermatogenic cells and releasing of premature spermatids from the seminiferous epithelium into the tubular lumen (39). The changes in germinal cells start with the spermatids and progressively affects less mature cell types until it originates a complete sclerosed tubule (16,21), which consists of a thickened tunica propria with abundant collagen and a few myoid cells (21,42). Thus, the number of germinal cells in the seminiferous tubule usually decreases as age increases in many mammals (18–20,47), resulting in a reduced diameter of seminiferous tubules (16) and epithelium vacuolization (48). Johnson et al. (14) reported that older men have reduced volume of seminiferous epithelia associated with lower daily sperm production and that the efficiency of sperm production decreases with age. With aging, the number of spermatids (32) and spermatogonia, including A-pale and A-dark spermatogonia, per unit section of the testes (49,50) significantly decreases. Age-related alterations of spermatogenesis in men between 65 and 93 years of age were reported by Holstein (51), and included reduction in A-dark and A-pale spermatogonia and arrested spermatocytes I with large nuclei in more than 70% of testicular sections, and many types of malformations of spermatids. Conversely, Johnson et al. (52) observed that young and old men have similar numbers of A-dark and A-pale spermatogonia per unit weight of testicular parenchyma and that the similar rates in germ cell degeneration at meiosis suggests that the difference in sperm production rates with age may occur in meiotic prophase, during the transition of leptotene to pachytene primary spermatocytes (52). Morphologically, multinucleated spermatocytes (53) and spermatids (42,54) in aged human testes have been reported. The formation of these kind of cells may result from the fusion of cell membranes of neighboring spermatocytes and spermatids and, as a consequence, cells undergo degeneration (53,54). Other ultrastructural alterations in spermatids comprise acrosome malformation, redundant nuclear membranes, intra-nuclear inclusions, excessive droplets in the cytoplasm, and irregular configuration of the nuclei (42). Intra-nuclear inclusions were also seen in spermatocytes and spermatogonia, as well as spirals of endoplasmic reticulum in cytoplasm (42). Morphological characteristics such as condensation of the chromatin and nuclear fragmentation in germ cells of older men support the hypothesis that the loss of germ cells observed during aging constitutes the best explanation for the low sperm counts observed in older men. On the other hand, the several ultrastructural alterations that take place may explain the higher index of teratozoospermia that these men presented. Alterations in Basement Membrane The basement membrane, a thin sheet-like extracellular structure that compartmentalize tissues, plays an important role in the maintenance of structural and functional integrity of tissues. In testes, the seminiferous tubule basement membrane is especially important during spermatogenesis (55). Damage of this structure has been associated with many testicular pathological conditions such as cryptorchidism and orchitis as well as with the aging process (56). However, literature on the morphological alterations of the basement membrane during testicular aging is contradictory. A few old studies in humans suggested that there were no alterations in this structure with aging (14,24).Conversely, thickening and herniation of this structure with dilatation of the seminiferous tubules have been described in aged males (22,32,39), mice (16), and other mammals (30,57). A statistically significant increase in the human basement membrane thickness correlated with age was found in mesenchymal (capillaries) and epithelial (testes) tissue (58). A histomorphometric study performed in testis in men from 29 to 102 years old revealed that, in cases of arrested spermatogenesis, a thickening of the basal membrane also occurs (20). Surprisingly, a more recent study (59) showed that basement membrane thickness apparently decreased with age in men, contradicting the other previous studies. The differences showed in this study may be due to the use of immunomarking techniques using specific antibodies for the epithelial basement membranes, instead of fixing silver salts on the reticulin fibers or due to the use of measurement software on digitized microscopic images, avoiding human error, making for a more accurate analysis. On the other hand, this study used surgical samples of testicular tissue from patients with prostate adenocarcinoma, which must be accounted for an important source of bias. Regardless, the clinical significance of alterations of this structure, remains unknown. Modifications in Vasculature The testicular artery is responsible for the blood supply of the testis and the epididymis, and several blood vessels are present in the interstitium of the seminiferous tubules. Paniagua et al. (21,42) showed that the pattern of testicular involution observed in aged men was similar to the one observed after induced ischemia. This pattern is influenced by alterations in testicular perfusion caused by vascular changes, mainly in the most distal regions of artery supply. The decrease of testicular perfusion is also caused by atherosclerotic alterations in testicular arterioles (32). Observations of the human testicular microvasculature revealed increased coiling of interlobular arteries with age, together with the degradation of the peritubular capillary network, resulting in a considerable reduction of blood flow (60). These changes are not exclusive to humans, occurring in other species with senescence, where a progressive decrease in capillary density, partial, or complete occlusion of the lumen and thickening of vessel walls were observed (61,62). The deficient irrigation related to vascular changes might be an explanation for the involution of the seminiferous tubules and consequently, the lower testicular volume observed in aged males. Alterations in Connective Tissue In human testes, the tunica albuginea thickens and the parenchyma decreases in volume (63). Johnson et al. (15) showed increased fibrosis and weight of the tunica albuginea with age. This feature is so consistent that it was proposed as a metric to estimate age (3). Lopez-Marambio and Hutson (64) found that in elderly men the tunica vaginalis became adherent to the posterior surface of the testes adjacent to the body of the epididymis, giving the appearance that the testes was retroperitoneal. Other testicular feature that appears to change with age is the testicular tunica adventitia, that becomes thicker (30). Increasing fibrosis of the tunica propria and tunica albuginea, may be part of a vicious cycle, where oxygen deprivation due to vascular changes may have an important role (see Section “Modifications in vasculature”). The modifications on these structures are, however, far from being completely understood and consequently, their involvement and consequences in the decline of male reproductive capacity are still unknown. Aging and Testicular (dys)fuction Aging has a well-documented effect on every level of the hypothalamic–pituitary–testicular axis (HPTA) (Figure 2), that is responsible for regulating testicular function (3). The testis has two main functions—spermatogenesis (exocrine function) and steroidogenesis (endocrine function) which are not completely independent (3). In normal conditions, the hypothalamus release gonadotrophin releasing hormone (GnRH) which stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells stimulating testosterone biosynthesis (steroidogenesis). FSH is recognized by FSH receptors in Sertoli cells having an important role in spermatozoa production (spermatogenesis) (Figure 2). These functions are affected by age resulting in alterations in hormonal levels during senescence in humans and other animals, comprising lower circulating androgens (3,65). However, lower androgen levels in aging men are not linked to a complete cessation of reproductive capacity, contrary to what happens in women with menopause and androgen deficiency is not only associated with advanced age (2). Figure 2. View largeDownload slide Hypothalamic–pituitary–testicular/gonadal axis. The release of gonadotrophin releasing hormone (GnRH) by the hypothalamus stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells and stimulates the biosynthesis of testosterone. On the other hand, FSH is recognized by FSH receptors in Sertoli cells that has an important role in spermatogenesis. Figure 2. View largeDownload slide Hypothalamic–pituitary–testicular/gonadal axis. The release of gonadotrophin releasing hormone (GnRH) by the hypothalamus stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH is recognized by LH receptors in Leydig cells and stimulates the biosynthesis of testosterone. On the other hand, FSH is recognized by FSH receptors in Sertoli cells that has an important role in spermatogenesis. A comparative study between the effects of age on the testicular steroidogenic activity of the Brown Norway rat and the Sprague-Dawley rat showed an age-associated decrease in serum and testicular testosterone in both models (66). In fact, the ability of Leydig cells to produce testosterone is reduced in older Brown Norway rats, resulting in reduced serum testosterone levels (67,68). The Brown Norway rat is already identified as the best available model to study the male reproductive aging given that, as in men, both primary and secondary testicular failure occur (68,69). A comparison of plasma testosterone levels on various species are summarized in Table 1. Table 1. Comparison of the Levels of Plasma Testosterone (ng/ml) in Various Species From Infancy to Senescence Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Note: GLC = gas-liquid chromatography; RIA = radioimmunoassay. View Large Table 1. Comparison of the Levels of Plasma Testosterone (ng/ml) in Various Species From Infancy to Senescence Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Specie  Infancy  Puberty  Adulthood  Senescence  Assay  References  Guinea-pig  0.8  6.1  3.6  >2.2  GLC  (70)  Rat  —  1.77  —  0.83  RIA  (71)      3.7  2.5-2.2  1.1  RIA  (72)    3.2  1.8  2.2  1.1  RIA  (73)  Mice      5.2  1.8  RIA  (74)        1.12  1.17  RIA  (65)  Chimpanzee  0.13  1.78  3.97    RIA  (75)  Note: GLC = gas-liquid chromatography; RIA = radioimmunoassay. View Large Testicular Exocrine System It is known that androgen stimulation of Sertoli cells is fundamental for the initial induction of spermatogenesis. In humans, few studies that compare endogenous steroid levels in testicular tissue from young and elderly men are available. Takahashi et al. (76) measured the concentration of nine endogenous steroids in men from 25 to 35 years old with oligozoospermia and varicocele and in men aged 61–85 years old with prostatic cancer and showed that the levels of all steroid hormones declined with age. A more recent study conducted by Carreau et al. (77) also evaluated the intra-testicular levels of steroids (pregnenolone, progesterone, DHEA, DHEAS, testosterone, and estradiol) in aged men, but no significant differences between the various age groups tested were found. The reduction in testicular testosterone production with age can have a big impact in spermatogenesis by affecting the function of Sertoli cells. Nevertheless, this decline often appears in older men with associated age-related diseases related to hormonal alterations. It is well established that the plasma levels of FSH (follicle-stimulating hormone), the second major stimulus for spermatogenesis, increase with age in older men, associated with a decrease in testicular size and spermatogenic efficacy (3,78). Additionally, increasing levels of FSH in elderly men has been linked to germ cell degeneration during meiosis, affecting sperm concentration (79). In men, serum inhibin levels decline with aging, with older individuals presenting significantly lower serum concentrations of this glycoprotein already at the age of 40 (80). Inhibin is a hormonal glycoprotein secreted by Sertoli cells in response to the stimulation by FSH, which negatively feedbacks on the pituitary gland to specifically suppress the production and secretion of FSH. This hormone is often used as a functionality marker for Sertoli cells and for the impairment of spermatogenesis (81). The decrease observed in the secretion of inhibin by Sertoli cells led to the suggestion that the testicular endocrine functions decline as soon as the fourth decade of life, and that Sertoli cell function declines earlier than that of other somatic testicular cells (80). In fact, the expression of the receptor for the hormone serotonin, an important player on germ cell–Sertoli cell interactions (82), is lost in Sertoli cells from rats older than 24 months (44), further confirming this suggestion. Testicular Endocrine System Steroidogenesis occurs in testicular Leydig cells and is mainly regulated by the LH secreted by the pituitary gland. In turn, LH secretion is induced by gonadotrophin releasing hormone (GnRH) released from the hypothalamus (Figure 2). In men, one of the most relevant age-related changes is the decline in testosterone levels, particularly plasma levels (3,83). Although some studies examining men of different ages failed to detect alterations in testosterone levels in aged heathy men (84), the majority showed that the plasma testosterone decline with increasing age (85–87). There is no consensus about the mechanism that cause testosterone age-related decline, but it has been associated with the three levels of the HPTA (45,87) or with the waning of testicular function (88). The decreased number of Leydig cells (22) and impaired testicular perfusion due to atherosclerosis, previously discussed, as well as reduced release of testosterone upon human Chorionic Gonadotropin (hCG) stimulation (89), support a primarily testicular cause for low testosterone levels (3). However, lower levels of testosterone in aged males should not be strictly associated with aging since other factors, such as lifestyle and pathologies, can be involved. This information highlights the need to understand how men with decreased testosterone levels would benefit from hormonal replacement. Like testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, cortisol, and estrone showed a significant longitudinal decline with age. On the contrary, dihydrotestosterone and pituitary gonadotropins levels raised longitudinally (90). It was shown that serum LH levels normally rise with aging, possibly as a response to the decline in testosterone, which supports the hypothesis of secondary hypogonadism (78). In men older than 60 years, it was reported that the production of LH increases with age (85). In rats, serum and pituitary prolactin levels and serum estradiol levels increased with age whereas serum and pituitary FSH and LH levels decreased (72). Other factors, besides aging, may influence the plasma and testicular levels of testosterone, including hereditary, environmental (stress, obesity), psychosocial (depression, drugs, smoking), and socio-economic factors (84,91). Therefore, the association of testosterone levels with age is not always linear. Furthermore, the decreased concentration of serum testosterone levels in aged males has been linked not exclusively to andropausal symptoms such as poor libido, erectile dysfunction, fatigue, and deterioration of cognitive functions which can explain the infertility experienced by these couples, but also with the functionality of androgen-sensitive somatic tissues (3,83,92). The hormonal variations related to age in men are summarized in Table 2. When interpreting the data, attention should be paid to the fact that, until recent years, most of the studies that measure circulating hormones used steroid immunoassays, which are known to have important bias and inaccuracies. Table 2. Age-Related Changes in Hormonal Levels in Human Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Note: AMH = anti-mullerian hormone; DHT = dihydrotestosterone; DHES = dehydroepiandrosterone; DHEAS = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; INSL3 = insulin-like peptide 3; InhB = inhibin B; LH = luteinizing hormone; SHBG = sex hormone-binding globulin. View Large Table 2. Age-Related Changes in Hormonal Levels in Human Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Hormone  Younger Men  Elderly Men  Age Groups (years)  Ref.  Plasma  Total testosterone (ng/ml)  6.59  4.90  26–90  (93)  4.91 – 5.59  4.75  20–40, 40–60 and 60–80  (94)  6.35  4.04  18–40 and 70–85  (12)  4.24  3.22  60–91  (77)  4.8  4.6 and 4.9  30–40, 41–50, 51–60 and 61–70  (95)  Free testosterone (ng/dl)  10.7  5.8  26–90  (93)  Estradiol (pg/ml)  24.46  18.60  60–91  (77)  SHBG          (nmol/l)  20.7  41.5  18–40 and 75–90  (12)  (ng/ml)  2.76  7.66  60–91  (77)  FSH          (mU/l)  3.4  102  18–40 and 75–90  (12)  (mIU/ml)  8.71  10.27  60–91  (77)  LH          (ng/ml)  1.9  2.2  26–90  (93)  (U/l)  4.2  6.3  18–40 and 75–90  (12)  (mIU/ml)  10.77  8.86  60–91  (77)  Intra-testicular  Testosterone          (ng/g)  408.50  461.77  60–91  (77)  (nM)  17.5  11.8  19–50 and 70–90  (96)  Estradiol (ng/g)  20.25  16.21  60–91  (77)  Pregnenolone (ng/g)  165.54  147.79  60–91  (77)  Progesterone (ng/g)  90.70  85.82  60–91  (77)  DHEA (ng/g)  235.38  285.17  60–91  (77)  DHEAS (ng/g)  254.90  239.89  60–91  (77)  INSL3 (ng/ml)  1.8  1.0  19–50 and 70–90  (96)  AMH (pM)  44.9  27.4  19–50 and 70–90  (96)  InhB (pg/ml)  175  103  19–50 and 70–90  (96)  Note: AMH = anti-mullerian hormone; DHT = dihydrotestosterone; DHES = dehydroepiandrosterone; DHEAS = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; INSL3 = insulin-like peptide 3; InhB = inhibin B; LH = luteinizing hormone; SHBG = sex hormone-binding globulin. View Large Molecular Changes in Aged Testes In the past decade, several molecular mechanisms of aging have been proposed, including damage by reactive oxygen species (ROS) produced during aerobic metabolism, implicating mitochondria in the aging process (97). Indeed, mitochondrial dysfunction was already described as being involved in testicular aging (97). Aging was also associated with an increase in oxidative stress and free radical production, due to the alterations in the enzymatic activity of anti-oxidant molecules such as glutathione-s-transferase (GST), an enzyme that protects cellular components from electrophilic and oxidative attack in testis (98,99), glutathione peroxidase (GPx) (99,100), and superoxide dismutase (SOD), especially in Leydig cells (99–101). Several testicular age-related alterations, including the decrease in steroidogenic capacity, have also been associated to an increase in tissue inflammation. Cyclooxygenase‐2 (COX2) expression is induced by cytokines and growth factors, particularly at sites of inflammation and has been associated with aged-Leydig cells phenotype since these cells express higher levels of this inducible isoenzyme (102,103). Moreover, pharmacological inhibition of COX2 increases StAR expression, a Leydig cell’s marker, and consequently steroidogenesis (103). However, a recent study showed that the levels of COX2 decreased, particularly in Leydig cells and macrophages, in rats with accelerated senescence when compared with the control animals (104). In long-lived mice, the expression of CD68 and CD163, macrophages markers, was reduced by half in testicular interstitium (104). Thus, the actual role of inflammation in reproductive aging is poorly understood and needs further investigation. The smooth muscle located in tunica albuginea is responsible for testicular capsular contraction that is involved in the transport of non-motile sperm to the epididymis. With aging, the capsule gets thicker, as already explained above, and the response to norepinephrine and prostaglandin becomes progressively higher, suggesting that neuro-humoral agents may have an important role in the maintenance of testicular capsular contractions (105). The main molecular age-related alterations identified in the different testicular cells are described below and summarized in Table 3. Table 3. Major Molecular Alteration Occurring in Testes with Age Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Note: Ach = acetylcholine; AM = adrenomedullin; cAMP = cyclic adenosine monophosphate; CGRP = calcitonin gene-related peptide; COX2 = cyclooxygenase‐2; GST = glutathion-S-transferase; LH = luteinizing hormone; MAPK = mitogen-activated protein kinase; NE = norepinephrine; PGA2 = prostaglandin A2; RLF = relaxin-like factor; UCP2 = uncoupling protein 2; 11β-HDS = 11beta-hydroxysteroid dehydrogenase; UCHL1 = ubiquitin carboxy-terminal hydrolase L. View Large Table 3. Major Molecular Alteration Occurring in Testes with Age Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Cell/Structure  Specie  Alterations Observed with Aging  Ref.  Testicular capsule  Rat  Increased response to NE and PGA2 and unchanged response to ACh  (105)  Testes  Mice  Increased activity of gonadal GST in old animals  98,106)    Reduced expression of CD68 and CD163, macrophages markers  (104)  Rat  Decreased mitochondria function; Increased proton leak, and expression and activity of UCP2  (107)      Increased AM levels and mRNA and increase in CGRP receptors  (108)    Human  Increased levels of COX2  (102,103)  Leydig cells  Rat  Reduced concentration of LH receptors in Leydig cells with age  (72)  Decreased expression of 11β-HDS type 2 with age  (109)  Reduced expression of RLF/INSL3 gene with age  (110)  Molecular changes in MAPK and cAMP signaling related with Leydig cells hypofunction in aging  (111)  Sertoli cells  Rat  Increased synthesis and secretion of transferrin and decreased synthesis of cathepsin L in old individuals  (41,46,112)      Loss of the expression of receptors for the hormone serotonin in rats older than 24 months of age  (44)    Mice  Alterations in cytoskeletal components (F-actin, vimentin, and cytokeratin)  (43)    Men  Accumulation of intracellular amyloid fibrils  (113)  Germ cells  Bovine  UCHL1 and SELP gene expression increases with age and the number of UCHL1 positive cells is higher  (114)  Mouse  Genes down-regulated with age: Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1 Genes up-regulated with age: Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, Selp, and Trio  (115)  Increased levels of mRNA for Cres gene and protein levels with age  (116)  Note: Ach = acetylcholine; AM = adrenomedullin; cAMP = cyclic adenosine monophosphate; CGRP = calcitonin gene-related peptide; COX2 = cyclooxygenase‐2; GST = glutathion-S-transferase; LH = luteinizing hormone; MAPK = mitogen-activated protein kinase; NE = norepinephrine; PGA2 = prostaglandin A2; RLF = relaxin-like factor; UCP2 = uncoupling protein 2; 11β-HDS = 11beta-hydroxysteroid dehydrogenase; UCHL1 = ubiquitin carboxy-terminal hydrolase L. View Large Leydig Cells The aged Leydig cells are characterized by the reduced ability to produce testosterone in response to LH (91). Several cellular changes have been identified in the steroidogenic pathway of aged Leydig cells in humans that result in the reduction of testosterone production. Cyclic nucleotides, cAMP and cGMP, are intracellular secondary messengers that play a central role in signal transduction cascades involved in testosterone production. Aging modulates the expression of genes responsible for cGMP production and degradation, which results in an increase in cGMP in Leydig cells, which can be a mean to overcome the reduction in testosterone levels (117). Additionally, several studies suggested a reduction in cAMP-signaling in aged cells, consistent with a reduce in testosterone production in aged rats in response to LH (118). Previous investigations also reported alterations in the expression profiles of 45 genes (119) and a decrease in LH receptor levels (72) in aged rats. The secretion of the insulin-like factor 3 (INSL3), a small peptide hormone secreted by mature Leydig cells, is an important indicator of the differentiation status and the number of Leydig cells present in testes. The levels of this peptide hormone decline gradually with aging reflecting the functional capacity of this type of cells. Supporting this data, in old rats INSL3 expression also seems to be down-regulated with age (110,120). The expression levels of LH receptors in Leydig cells also decreases with age in Fisher 344 rats (72). The response of Leydig cells to LH depends not only on the number of receptors and the circulating concentration of glucocorticoids, but also on 11β-HSD activity (109). With increasing age, the expression of this enzyme is reduced and consequently, a reduction in testosterone production is observed (109). The aging impact in the number and volume of Leydig cells and the molecular alterations described in this section may explain, in part, the reduction in testosterone levels often observed in older men. However, it is important to keep in mind that these alterations may result from a combination of elements, including environmental factors that can affect the testes and, particularly, this type of cells. Sertoli Cells Sertoli cells secretions and metabolites are crucial for the normal occurrence of spermatogenesis. The expression of some of these products is dependent on an intricate network of signaling molecules and hormones (34,121). It has been reported that aging is able to alter the production of some important secretions by the Sertoli cell and even its molecular components, such as transcripts, cytoskeleton proteins, and secretory proteins. Indeed, the synthesis and secretion of two of the major products of Sertoli cells, transferrin and cathepsin L (also called cyclic protein-2, CP-2) is affected in older individuals, while that of others, such as sulphated glycoprotein-2 (SGP-2, also called Clusterin), remains unaffected (41,46,112). Transferrin is a major secretory protein of Sertoli cells, being involved in the transport of iron in cells of the seminiferous epithelium. In brown Norway rats, the total testicular content of transferrin mRNA remained unchanged up to 18 months of age, but dramatically increased by the age of 2 years. In opposition, the mRNA CP-2 testicular content of older rats was shown to be decreased (46,112). The stage-specific expression of CP-2 in the seminiferous epithelium suggested that the presence of the active cathepsin L facilitates the intense reorganization of the seminiferous epithelium during germ cell development (122), enhancing the degradation of dying or dead germ cells (112). These age-associated alterations of transferrin and CP-2/cathepsin L levels in aged rats led to the suggestion that age-related changes in Sertoli cell gene expression reflect a primary dysfunction of aging and not an indirect effect due to germ cell loss (46). Moreover, age-associated alterations in the cytoskeletal components of Sertoli cells, namely F-actin, vimentin, and cytokeratin, were described in aged BDF1 mice. In old mice, F-actin was distributed at the inter-Sertoli cells junctions and at the luminal portion of the Sertoli cell cytoplasm, while in young mice it was only detectable at the junctions between adjoining Sertoli cells (43). Similarly, vimentin was recognized only around the nucleus in Sertoli cell of old animals, and not in the Sertoli cell trunk as seen in young mice. Instead, in old mice testes, sheet-like organizations of vimentin were detected near the luminal surface parallel to the basement membrane (43). On the other hand, cytokeratin expression pattern was dramatically altered in the mice Sertoli cells. Up until 27 months of age, no cytokeratin expression was detected in Sertoli cells. Only in older mice, which exhibited thinner seminiferous epithelia, the expression of cytokeratin was consistently detected within the Sertoli cell cytoplasm, being one of the most characteristic occurrences of age-associated changes in Sertoli cells (43). Additionally, age-associated accumulation of intracellular amyloid fibrils in Sertoli cells has also been reported (113). This accumulation is concurrent with the buildup of lipofuscin-loaded lysosomes, as well as of damaged mitochondria, in Sertoli cells. The activity of the enzymes that degrade lipofuscin and damaged mitochondria (by autophagy) is diminished, leading to their accumulation. Dysfunctional mitochondria produce more ROS and lipofuscin accumulation sensitizes cells to ROS-induced damage, which then becomes a self-amplifying, vicious cycle in these cells (123). Germ Cells With aging the number of germ cells in the seminiferous tubule decreases in humans and most mammals (18–20,47) and several ultrastructural alterations take place. Indeed, spermatogenesis gradually decreases in older men (124). To investigate whether this reduction resulted from apoptotic events, Barnes et al. (125) studied rats from different age groups and observed that apoptotic cell death of testicular germ cells significantly increases with age but a significant decrease in apoptotic cells/seminiferous tubule was observed. However, an increase in apoptotic metaphase spermatocytes at tubule stage XIV in mice with 24 months of age when compared with 6-months-old mice was observed (126). Like Leydig cells, the decreased number of germinal cells in aged testes was accompanied by changes in gene expression profile, particularly in the expression of spermatogonia markers (114). An investigation conducted in mice that compared the gene expression in four age groups (6, 21, 60 days, and 8 months) revealed that certain genes were specifically expressed in older mice and some genes were up- (Gpr116, Gpr146, Gpr56. Grb2, Icam1, Lims1, Lin52, SELP, and Trio) and down-regulated (Gpr107, Tyrobp, Smad4, Ms4a7, and Mrc1) (115). Additionally, 16 proteins were identified as dramatically increased and 21 were decreased in aged human testes compared to the young ones, especially proteins involved in oxidative stress (124). These proteins can constitute valuable markers of reproductive aging. Summary and Future Perspectives Advanced paternal age is a common phenomenon in modern societies. Social-economic factors and personal choices contribute to this tendency. This evidence highlights the need to discuss and investigate the effect of advanced age in the male reproductive system, in semen parameters and the impact on the offspring’s health. As described in the above sections, the testes, as all the other systems in the body, suffer several complex alterations that might be an important piece to explaining the reproductive decline observed in aged men. Aged testes undergo profound histological and morphological alterations, including tissue atrophy, leading to a reduced functionality. In addition, several molecular alterations were reported in all testicular cell types. These age-related changes are usually so small that men are generally fertile until old age. Importantly, the pathophysiology of age-impact in testes may result from specific effects of age alone or other conditions, such as vascular diseases, infection on reproductive glands, and exposure to environmental chemicals. Additionally, the impact of age is not exclusive to humans but was also present in many mammalian species, reflecting the universality of this phenomenon. Some of them, such as mice and rat, can be used as animal models to study the aging phenomenon (69). Nevertheless, despite rodent models being very useful in reproductive biology, we must be aware of their limitations. In fact, the aging process is very different between species and the presence of specie-specific mechanisms should be considered when taking conclusions. Particularly relevant, there are also serious doubts that human aging, which takes decades, can be realistically studied using rodents, which have a much shorter life cycle. Finally, some important questions remain unanswered and should be addressed by future research in this field. Are the alterations in the composition of cell populations the main responsible for the decreased testicular volume with age, or this happens only due to an increased basement membrane and tunica albuginea thickness? Are the levels of steroid hormones the key to these changes or are their clinical impact irrelevant? Is the decrease in the amount of different cell types a result of the molecular changes that occur or do they precede them? Are the changes in testicular vascularization the main responsible for most of the testicular age-related alterations? These and other questions should be investigated and the identification of the sequence of events might be important to better understand this phenomenon. Indeed, the understanding of the mechanisms of testicular aging and reproductive decline can be very useful to better understand some cases of male infertility and to finally address the question—How old is too old to be a father?. Funding This work was supported by FEDER Funds through Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through FCT–Foundation for Science and Technology under the project PTDB/BBB-BQB/3804/2014. This study was also supported by Institute for Biomedicine—iBiMED (UID/BIM/04501/2013 and POCI-01-0145-FEDER-007628) and by individual grant from FCT of the Portuguese Ministry of Science and Higher Education to JVS (SFRH/BPD/123155/2016). Conflict of Interest None declared. References 1. Sharma R, Agarwal A, Rohra VK, Assidi M, Abu-Elmagd M, Turki RF. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring. Reprod Biol Endocrinol . 2015; 13: 35. doi: 10.1186/s12958-015-0028-x Google Scholar CrossRef Search ADS PubMed  2. Tenover JS. Declining testicular function in aging men. Int J Impot Res . 2003; 15 ( Suppl 4): S3– S8. doi: 10.1038/sj.ijir.3901029 Google Scholar CrossRef Search ADS PubMed  3. Handelsman DJ. Aging in the hypothalamic-pituitary-testicular axis. In: Neill JD, ed. Knobil and Neill’s Physiology of Reproduction . 3rd ed. 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All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Apr 21, 2018

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