Mechanisms of Obesity-induced Male Infertility

Karen P Phillips; Nongnuj Tanphaichitr


Expert Rev Endocrinol Metab. 2010;5(2):229-251. 

In This Article

Mechanisms of Obesity-induced Male Infertility


Hypogonadism in males encompasses disrupted testicular functions, including deficient steroidogenesis and/or spermatogenesis. Hypogonadism is classified by the origin of dysfunction, either at the testis or within the HPT axis. Primary hypogonadism results from a testicular deficit (Figure 6A) and may be caused by genetic diseases, including Klinefelter's syndrome or 5α-reductase deficiency, congenital abnormalities, including cryptorchidism or testicular feminization, or testicular insults, such as trauma, mumps orchitis, radiation or chemotherapy,[1] testicular heat stress[37] or varicocele (associated with impaired testicular venous drainage).[38] Primary testicular deficit is also classified as hypergonadotropic hypogonadism and is clinically characterized by hypogonadism (low free and/or total testosterone and low sperm production) with increased FSH and LH levels, caused by inadequate testosterone levels to provide negative feedback to the HPT axis. Hypogonadism may also be caused by deficits within the hypothalamus or pituitary, a so-called secondary hypogonadism, also termed hypogonadotropic hypogonadism (Figure 6B). This is characterized by low–normal FSH and LH levels and subsequently low testosterone, and is featured in men with Kallman's syndrome, pituitary disorders and serious illnesses, such as AIDS.[39] We will discuss several hypogonadal mechanisms with either testicular or hypothalamic origins that explain the association between obesity and male hypogonadism.

Figure 6.

Hypogonadism. (A) Hypergonadotropic hypogonadism. Also known as primary hypogonadism, reduced testosterone levels and impaired spermatogenesis are caused by testicular deficit. Reduced testosterone levels leads to attenuation of the testosterone-induced negative feedback loop to the secretory activities of the hypothalamus and pituitary. Increased amounts of GnRH and FSH/LH are thus secreted. (B) Hypogonadotropic hypogonadism. Secondary hypogonadism is caused by deficit at the hypothalamus and/or pituitary. FSH and LH are, thus, secreted at reduced levels, which also lead to decreased stimulation of Leydig cells to secrete testosterone.
FSH: Follicle-stimulating hormone; GnRH: Gonadotropin-releasing hormone; LH: Luteinizing hormone.

Adipocyte-derived Estrogen & Hypogonadism Adipocyte-derived estrogens in obese men provide feedback inhibition to the HPT axis, modulated by the presence of estrogen receptors (ER-α and -β) localized to the hypothalamus and pituitary, shown in mouse[40] and rat.[41,42] A plausible biological mechanism for obesity-induced hypogonadotropic hypogonadism may result, in part, from increased feedback inhibition of the HPT axis due to high serum levels of estrogens in obese males.[43] This may lead to a hypogonadal–obesity cycle,[44] wherein an increased adipose tissue mass represents a significant peripheral source of estrogens, which, in turn, suppress the HPT axis and increase central adiposity (Figure 7). The subsequent reduction in circulating testosterone leads to increased deposits of visceral/abdominal adipose tissue[45–47] and subfertility, whereas the increased production of circulating estrogens supports differentiation of adipocytes.[10]

Figure 7.

Hypogonadal–obesity cycle: peripheral estrogen synthesis. Adipocytes express the enzyme aromatase that converts androgens (T and androstendione) to estrogens (17β-E2 and estrone). Adipose tissue represents a rich source of peripheral estrogens that regulate testicular function. In response to an increased adipose tissue mass, peripheral E2 is elevated, which, in turn, modulates the differentiation of adipocytes and suppresses the Hypothalamus–pituitary–testis axis (hypogonadism). Reduced T contributes to abdominal adipose tissue distribution.
E2: Estradiol; T: Testosterone.

Obesity-induced hypogonadism in males may be treated by weight loss, which should reduce estrogen levels to normal and alleviate the HPT feedback inhibition. Roux-en-Y gastric bypass surgery, one option for the treatment of morbid obesity, has been shown in one study to seemingly reverse abnormal reproductive hormonal profiles, such that total testosterone is increased and serum estradiol is decreased.[48] Other treatment options for hypogonadism include supplementation with alternative or recombinant gonadotropins (e.g., human chorionic gonadotropin [hCG] and recombinant FSH), which stimulate testicular function, including testosterone production.[49] Finally, aromatase inhibitors, letrozole and anastrozole, can be used to prevent enzymatic conversion of androgens to estrogens in adipocytes and other tissues,[49,50] thereby reducing serum estradiol and suppression of the HPT axis by interrupting the hypogonadal–obesity cycle.[46]

Inter-relationship between Hypogonadism & Non-insulin-dependent Diabetes Mellitus Insulin resistance, together with obesity, are cardinal features of metabolic syndrome.[2] However, it is important to note that not all obese individuals will develop non-insulin-dependent diabetes mellitus (NIDDM) or Type 2 diabetes, a disease that also manifests in normal-weight individuals.[51] Consideration must, therefore, be granted to the differential impacts of adiposity and insulin resistance in the context of fertility in the obese male.

Non-insulin-dependent diabetes mellitus is generally characterized by obesity, insulin resistance, hypogonadism, low sex-hormone binding globulin (SHBG) and reduced free and total testosterone.[2,52] However, it is unclear which of these parameters are causal factors and which are independent. Hypogonadism, for example, has been demonstrated in several studies to predict NIDDM risk (e.g., Multiple Risk Factor Intervention Trial cohort study,[53] Massachusetts Male Aging Study,[54] Rancho Bernardo Study,[55] Kuopio Ischaemic Heart Disease Risk Factor Study [KIHD][56–58]). Men with NIDDM are more likely to exhibit hypogonadism with reduced serum testosterone and SHBG.[59–61] These studies are consistent with established associations between obesity and hypogonadism, and between obesity and insulin resistance. SHBG and testosterone are inversely correlated with BMI[17] and insulin,[62,63] such that obese males exhibit high serum insulin with low SHBG and bioavailable testosterone.[52] The inverse relationship between total serum testosterone and insulin appears to be independent of central adiposity, demonstrated in the Health in Men study.[64] Hyperinsulinemia featured in NIDDM patients appears to produce hypergonadotropic hypogonadism by directly reducing testicular testosterone levels.

Pitteloud and colleagues demonstrated a positive correlation between insulin sensitivity and serum testosterone levels in a cohort of 60 men, across a broad range of BMI categories.[65] In a subsequent study, Pitteloud's group reported no correlation between insulin sensitivity and LH, suggesting no hypothalamic or pituitary deficit.[66] Instead, insulin resistance, associated with hyperinsulimia, somehow directly suppressed Leydig cell testosterone production in a cohort of men with mild-to-moderate obesity.[65] In vitro, Leydig cells express insulin receptors, and insulin has been shown to induce testosterone secretion from Leydig cell cultures.[67,68] The molecular mechanism wherein insulin modulates Leydig cell steroidogenesis is unknown, with the possibility that the testis, as with other organs in individuals with NIDDM, is resistant to insulin signaling.[66]

Non-insulin-dependent diabetes mellitus and, therefore, insulin resistance, appears to be associated with 'mixed hypogonadism', reflecting the dual actions of insulin resistance at the testis (hypergonadotropic hypogonadism); and the hypothalamus/pituitary (hypogonadotropic hypogonadism). Insulin is predominantly stimulatory, acting at hypothalamic neurons to induce GnRH secretion and gonadotropin secretion from the pituitary gonadotrophs, demonstrated in vitro.[69] In obese men with NIDDM, insulin resistance may blunt the normal, insulin stimulation of the HPT axis.[66] More studies are needed to identify the mechanisms of HPT insulin signaling and the consequences of insulin resistance to the normal endocrine function of these glands.

Although reproductive hormonal profiles have been fairly well established for men with NIDDM, semen quality has been examined in only a few studies. Generally, sperm count is normal or increased, with decreases in sperm motility[70] and semen volume,[71] and increases in abnormal sperm morphology.[72] Unfortunately, many published studies of 'diabetic men' do not disaggregate the insulin-dependent diabetes mellitus (IDDM) and NIDDM groups; a critical reporting gap in the literature as it appears that NIDDM may be more of a concern with respect to male reproduction. The pathology of IDDM (autoimmune and insulin deficiency) is different from NIDDM (insulin resistance). The stimulatory effects of insulin on the HPT axis in IDDM patients are continued through exogenous insulin, the standard intervention. Insulin resistance in NIDDM patients, however, leads to aberrance in testicular cell signaling and subsequent hypogonadism. With so few studies examining the association between semen quality and insulin resistance, it is impossible to profile 'typical' semen parameters of a NIDDM man. More studies using NIDDM men and animal models are urgently needed to verify the causal effect of insulin resistance on hypogonadism, as well as to discern the molecular mechanisms involved.

Leptin's Roles in Hypogonadism Leptin is a 16-kDa protein hormone, encoded by the ob gene[73] and secreted by adipocytes. This adipocytokine plays a major role in energy homeostasis, including neuroendocrine regulation of bodyweight. Leptin is the endogenous agonist for the leptin receptor (Ob-R), a member of the class I cytokine receptor superfamily. Ob-R is produced as alternatively spliced forms and further classified as short (Ob-Ra, Ob-Rc, Ob-Rd and Ob-Rf), long (Ob-Rb) and secreted (Ob-Re) receptor types, all with extracellular, transmembrane and variable intracellular domains with the exception of Ob-Re.[74–76] Ob-Rb has the longest intracellular domain and is the functional isoform in the hypothalamus, whereas Ob-Ra and Ob-Rc are proposed to participate in leptin transport across the blood–brain barrier (BBB).[76,77] Ob-Re is the putative soluble leptin receptor lacking the transmembrane and intracellular domains, and proposed to buffer circulating leptin levels, thereby regulating leptin bioavailability.[78]

Leptin was initially characterized in ob/ob mice, which, because of a natural mutation, possess no ob gene and are, therefore, leptin deficient.[79] Both male and female ob/ob mice are morbidly obese and infertile.[80] Similar phenotypes are observed in Ob-R-deficient mice[81] and the Zucker fatty (fa/fa) rat.[82] Several human families have also been identified with congenital leptin deficiency owing to recessive mutations in the leptin gene (Delta133G,[83]R105W[84,85] and N103K[86]) or the leptin receptor.[87] Clinical features of human congenital leptin deficiency[84–85] include early onset of obesity, hyperphagia (overeating), hypogonadotropic hypogonadism and delayed pubertal onset. Congenital leptin deficiency caused by leptin receptor mutation is associated with a slightly less severe phenotype.[87] Recombinant leptin administration has been used successfully to mitigate some of the features of congenital leptin deficiency, resulting in weight loss and reversal of hypogonadism.[83,88,89]

In healthy men, serum leptin positively correlates with BMI and adipose mass.[90] A number of studies in overweight and obese participants report an inverse relationship between serum levels of leptin and testosterone[91–94] but no significant correlation between levels of leptin and the gonadotropins.[91,93] In contrast to the hypogonadism featured in congenital leptin deficiency cases, hypogonadism in obese men is associated with high serum leptin. Altered leptin dynamics may, therefore, contribute to male infertility via at least two mechanisms, both of which may produce hypogonadism. These include leptin resistance or leptin insufficiency at the hypothalamus and leptin modulation of testicular physiology.

Leptin Resistance or Leptin Insufficiency at the Hypothalamus Consider the perplexing presence of the same phenotype (obesity and hypogonadotropic hypogonadism) in individuals with high serum leptin due to obesity, and in humans/animals with leptin deficiencies due to mutations. Leptin resistance has been proposed to explain this apparent paradox in the former group, referring to the inability of leptin to act at the hypothalamus, either due to reduced levels of bioavailable leptin (leptin insufficiency)[95] or impaired leptin signaling,[96] thereby mirroring the leptin deficiency present in animal models and congenital genetic conditions.[97] There does not appear to be a model for central leptin excess, in spite of the increased serum leptin levels exhibited by morbidly obese individuals.

Peripheral adipocyte-derived leptin circulating in the bloodstream can cross the BBB via a saturable transport system[95] to act centrally via Ob-Rb in the brain, particularly at the hypothalamus.[98,99] Saturation of this BBB transport system is believed to produce central leptin insufficiency, as leptin is present in large quantities in plasma without corresponding leptin action at the hypothalamus.[97] Impaired central leptin action was first considered following reports that the ratios of cerebrospinal fluid leptin to plasma leptin are reduced in obese individuals compared with lean counterparts.[95] Thus, in spite of large circulating leptin plasma levels in obese individuals, cerebrospinal fluid leptin levels are not proportionately high.[95,100] As peripheral leptin is ordinarily released in a pulsatile fashion, any number of factors, including circadian rhythm, meal spacing and aging could disrupt this initial leptin pulsatile signal and produce leptin receptor downregulation, thereby attenuating leptin transport at the BBB.[101] Reduced hypothalamic leptin stimulation may produce the morbidly obese phenotype in humans, as these individuals lack leptin-induced suppression of appetite or stimulation of energy expenditure,[81] thereby producing a phenotype similar to the ob/ob mouse.[102] The molecular mechanisms of leptin transport saturation at the BBB are not well characterized; however, triglyceride inhibition/reduction of leptin transport at the BBB has been proposed.[99] Alternatively, leptin resistance, characterized by deficits in Ob-R signaling pathways, may impair leptin regulation of eating and energy expenditure. Candidate signaling pathways include SOCS3 and STAT3, both downstream of Ob-Rb.[103] Although impairments in leptin signaling may well accompany central leptin insufficiency, perturbed signaling as a basis for leptin resistance remains to be elucidated.[101] As described previously, ob/ob mice are obese, infertile and exhibit hypogonadism; however, this phenotype can be reversed, including restoration of fertility, by treatment with exogenous leptin.[102,104,105] Gonadotropins and sex-steroid hormones are low in ob/ob mice, consistent with hypogonadotropic hypogonadism and a role for leptin in the regulation of the HPT axis.[102] Leptin acts indirectly to regulate gonadotropin secretion in the hypothalamus by modulating kisspeptins in the arcuate nucleus (Figure 8A).[106] Kisspeptins are proteins encoded by the Kiss1 gene, transcribed as KiSS1 mRNA and act via a G-protein-coupled receptor (GPR54)[107] to stimulate GnRH release, thereby triggering the gonadotropin cascade.[108] Peripheral leptin, once transported across the BBB to the hypothalamus, binds leptin receptors in the forebrain. Arcuate nucleus neurons in the mouse express both Ob-Rb and KiSS-1 mRNA in approximately 40% of cells, suggesting that so-called KiSS1 neurons (kisspeptins-expressing neurons) are direct targets of leptin. Further, the numbers of KiSS1 neurons are decreased in the hypothalamus of ob/ob mice, indicating that leptin regulates KiSS1 neurons, and indirectly gonadotropin release.[108,109] Modulation of the gonadotropin levels is almost certainly limited to an indirect role of leptin, since leptin receptors are not present on rat GnRH neurons. Supporting these data is the observation of normal fertility in transgenic mice lacking GnRH neuronal leptin receptors.[110]

Figure 8.

Leptin modulation of hypothalamic–pituitary–testis axis. (A) Normal leptin action. Leptin provides a physiological link between energy expenditure and reproduction by stimulating expression of KiSS, located in the hypothalamic arcuate nucleus. KiSS neurons stimulate GnRH release and trigger the gonadotropin cascade and, in turn, testicular steroidogenesis and spermatogenesis. Leptin may also negatively regulate steroidogenesis through direct actions on Leydig cells. (B) Leptin resistance. In obese men, leptin is unable to cross the BBB and stimulate release of GnRH via KiSS neurons, thereby resulting in hypogonadism. With insufficient gonadotropic stimulation, testicular dysfunction occurs. Higher circulating leptin levels may also impair testicular function through inhibition of steroidogenesis. Dotted lines indicate proposed mechanisms and solid lines indicate established mechanisms.
Arc: Arcuate nucleus; BBB: Blood–brain barrier; FSH: Follicle-stimulating hormone; GnRH: Gonadotropin-releasing hormone; KiSS: Kisspeptin; LH: Luteinizing hormone; T: Testosterone.

Testicular stress induced by leptin-modulated reductions in circulating gonadotropins, established antiapoptotic agents,[111] can induce apoptosis.[112] FSH, a prosurvival factor in rat testis, upregulates expression of antiapoptotic protein Bcl-w in rat Sertoli cells, spermatogonia and spermatocytes, but not spermatids, in vitro.[113] Germ cell death is a normal event during spermatogenesis and may serve to regulate the size of the germ cell population;[112,114] however, in the event of central leptin insufficiency and reduced gonadotropins release, testicular apoptosis may be pathological. This has been demonstrated in leptin-deficient ob/ob mice that exhibit upregulation of nine testicular pro-apoptotic genes involved in both intrinsic and extrinsic apoptosis pathways.[115] Abnormal spermatogenesis and infertility in these mice is likely due to inadequate gonadotropin support of spermatogenesis and accelerated germ cell death by apoptosis.[115,116]

Deficiency in hypothalamic leptin signaling resulting in hypogonadotropic hypogonadism is observed in ob/ob mice and humans with leptin deficiency. It would appear that central leptin insufficiency is the causal mechanism of deficient hypothalamic leptin signaling and, thus, the underlying cause of hypogonadotropic hypogonadism (Figure 8B). The stimulation of KiSS1 neurons by leptin provides a link between energy homeostasis and reproduction. Moderate elevations in serum leptin levels due to seasonal weight gain in response to changing environmental conditions may signal reproductive opportunity.[80] While moderate fluctuations in leptin may prove physiologically relevant to seasonal breeders and other animals, high serum leptin levels in the obese human male may be detrimental, as this leads to saturation of the BBB transport system and central leptin insufficiency.

Leptin Modulation of Testicular Physiology Leptin is found in human and rodent Sertoli cells, Leydig cells, seminiferous tubules and germ cells[117,118] and is able to cross the testis–blood barrier (TBB),[119] suggesting both testicular and peripheral sources of leptin may be involved in reproduction. Unlike the BBB's saturable transport system for leptin, the TBB system is nonsaturable, enabling leptin to 'leak' across the barrier between the blood and the testicular interstitium and traverse the Sertoli cell barrier, dividing the interstitium from the seminiferous tubule fluid. The mechanism at the Sertoli cell barrier has not been elucidated as saturable or leakage.[119] Thus, as serum leptin levels increase, intratesticular leptin levels would be expected to similarly increase with leptin action limited by receptor expression in the testis. Ob-R have been localized to isolated Sertoli and Leydig cells, and testicular germ cells in rodents,[119–122] and in seminiferous tubules in humans.[117,123,124] Together, these results strongly suggest that leptin directly modulates testicular functions.[80,102]

Leptin is a negative regulator of testicular steroidogenesis; it acts directly on testicular Leydig cells. In response to LH, Leydig cells activate PKA-dependent gene expression, which triggers steroidogenesis (i.e., the production of testosterone).[125] Leydig cells in the rat[121,126] and human,[117] but not mouse,[127] express leptin receptors, thus providing a mechanism for direct leptin modulation of Leydig cell functions. In rat Leydig culture, leptin suppresses hCG-stimulated testosterone secretion, supporting a role for leptin in the negative regulation of steroidogenesis.[126,128,129] Leydig cells exhibit differential sensitivity to leptin, according to the developmental expression profiles of Ob-R, such that embryonic and adult but not prepubertal rat Leydig cells demonstrate leptin suppression of hCG-induced testosterone secretion.[121] In an elegant experiment designed by Tena-Sempere and colleagues,[122] hCG-stimulated rat testicular samples exposed to increasing doses of human recombinant leptin decreased expression of steroidogenic enzymes mRNAs and, subsequently, reduced testosterone secretion. Leptin is also able to decrease hCG-induced expression levels of steroidogenic factor (SF)-1, steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) in a dose-dependent manner.[122]

Drawing from the findings reported by the teams of Caprio and Tena-Sempere, a model of leptin regulation of Leydig cell steroidogenesis is proposed (Figure 9). Leptin signaling via Ob-R is relatively well characterized[76] and is mediated by the JAK–STAT pathway,[130–132] with JAK2[133] and STAT3, primarily described.[134] STAT3 ultimately regulates expression of steroidogenic genes including SF-1, StAR and P450scc.[135] Alternatively, steroidogenic gene expression can be regulated via the PI3K/Akt or MAPK cascades, triggered by leptin binding to Ob-R.[76] Leptin's repression of steroidogenic gene expression, particularly StAR, the rate-limiting step in steroidogenesis[136] would, therefore, counteract LH-mediated testosterone production. Leptin's negative regulation of steroidogenesis provides subtle control over reproductive functions and represents yet another mechanism for leptin in reproduction.

Figure 9.

Model of leptin action in the Leydig cell. In response to LH, Leydig cells activate PKA-dependent gene expression and downstream steroidogenesis pathways, including production of testosterone. Leptin-bound Ob-Rb captures JAK2, a non-receptor tyrosine kinase, which phosphorylates STAT3 and Y on the Ob-Rb. Phosphorylated STAT3 dimers translocate to the nucleus and negatively regulate expression of steroidogenic genes, including SF-1, StAR and P450scc. Leptin-inhibition of gene expression may also occur via recruitment of SHP-2 to phosphotyrosine residues on the Ob-Rb. SHP-2 recruits Grb2 and leads to activation of the MAPK pathway (Ras, Raf, MEK and ERK). Leptin-dependent reduction of testicular expression of StAR, the rate-limiting step in steroidogenesis at the mitochondria, would negatively regulate steroidogenesis and, thereby, counteract LH-mediated activation of Leydig cells. Dotted lines indicate proposed pathways and solid lines indicate established pathways.
LH: Luteinizing hormone; Ob-Rb: Leptin receptor; P450scc: Cytochrome P450 cholesterol side-chain cleavage enzyme; SF-1: Steroidogenic factor-1; StAR: Steroidogenic acute regulatory protein; Y: Tyrosine residue.

Leptin's modulation of male infertility also involves direct action of leptin on the sperm itself. Studies in mice indicate that leptin and its receptor are expressed in specific types of male germ cells, implicating leptin in cell proliferation and differentiation. Leptin protein and mRNA expression are present in gonocytes from neonatal mice, spermatogonia from 10-day-old mice and in spermatocytes from adult mice.[118] Neonatal and adult mice express Ob-Ra, Ob-Rb and Ob-Re in the testes,[118,120] suggesting leptin normally functions in an autocrine or paracrine manner to regulate spermatogenesis. It remains to be elucidated whether leptin exclusively acts as a positive or negative regulator of spermatogenesis, or whether leptin's role is more refined, dependent on the receptor isoforms expressed at different stages of spermagogenesis. Leptin signaling via STAT3 suggests a role in the proliferation of undifferentiated germ cells;[120] consistent with reports that stem cell STAT3 phosphorylation prevents differentiation and enables continuous stem cell renewal.[137] Leptin STAT3 signaling may enable undifferentiated germ cells to replicate without loss of potency while triggering later-stage spermatocytes to undergo development and differentiation.[118,120] In obese males, serum leptin levels are elevated, which may lead to downregulation of testicular Ob-R, previously demonstrated in vitroin the rat.[138] Downregulation of leptin receptor expression would disrupt autocrine/paracrine testicular signaling and perhaps spermatogenesis. Taken together, the localization studies demonstrating testis and spermatocyte-specific leptin and leptin receptor expression,[117,123,124] along with the studies of testicular leptin signaling,[122] suggest that elevated serum leptin levels exhibited by obese males are likely to perturb normal testicular physiology.

Ejaculated sperm are not able to fertilize the egg and must undergo further structural and functional changes during capacitation (Figure 3 & 4). The purpose of seminal plasma leptin is still unclear,[117,123,139–143] and is required to modulate sperm capacitation and the acquisition of hyperactive motility. Human seminal plasma leptin levels are positively correlated with serum leptin levels[117,139–141] but inversely correlated with serum testosterone and normal sperm parameters.[123,139,142] Seminal plasma leptin levels tend to be disproportiately lower than serum leptin levels[139] but are still positively correlated.[139,142] Obese men would be expected to have greatly increased seminal plasma levels of leptin, relative to their lean counterparts; however, very few studies have reported seminal plasma leptin levels in morbidly obese men, and the capacitation/acrosome reaction status of their sperm has not been investigated.

A role for leptin in sperm capacitation has been proposed by the Aquila research team, drawing upon their studies in humans and boars. Leptin and leptin receptor expression are localized exclusively on the plasma membrane overlying the acrosome in boar sperm, consistent with their putative regulations of sperm fertilizing ability.[135,144] Leptin has been localized to midpiece/equatorial segment in uncapacitated human sperm, undergoing an overall decrease in expression and more uniform localization in capacitated sperm,[141,145] with leptin receptor confirmed at the tail region in ejaculated spermatozoa.[145,146] These expression patterns support a physiological role for leptin, perhaps in the modulation of human sperm motility. Following acute leptin exposure (30–60 min), uncapacitated boar and human sperm undergo leptin-enhanced cholesterol efflux and protein tyrosine phosphorylation,[135,141] both hallmark steps in capacitation.[143] Furthermore, boar sperm acrosin activity (acrosomal trypsin-like enzyme) is stimulated by leptin.[135] It is important to note that leptin's stimulation of these capacitation-related events in both studies[135,141] is not dose dependent, with very low concentrations of leptin (0.63 nM human leptin and 1–10 nM porcine leptin) producing the greatest response. This may be explained by receptor downregulation at higher leptin levels[138] and may hold significance for obese men who would be expected to exhibit seminal plasma leptin levels in the highest ranges.

A model of capacitation signaling crosstalk emerges, wherein leptin signaling via the leptin receptor induces tyrosine phosphorylation downstream of MAPK pathway activation (Figure 4). The process of capacitation includes changes to the sperm membrane and the acquisition of hyperactivated motility. Molecular events include HCO3-dependent increase in intracellular cAMP, subsequent activation of PKA and, ultimately, tyrosine kinase activation and protein tyrosine phosphorylation, necessary for acquisition of hyperactivated sperm motility.[6,7] Leptin activation of prosurvival pathways may lead to the activation of ERK1/2 signaling,[135,140] representing capacitation signaling crosstalk. It is intriguing to speculate that acrosomal leptin receptor expression is associated with cholesterol efflux and acrosome reaction, whereas tail leptin receptor expression in human sperm may reflect leptin's modulation of hyperactivated sperm motility.

In summary, leptin regulation of normal male reproduction includes central (HPT) and testicular actions. In morbidly obese men, central leptin insufficiency produces hypogonadotropic hypogonadism, reducing circulating gonadotropins,[95,98,99] and subsequently inducing testicular apoptosis.[111] It is believed that high serum leptin is able to perturb testicular steroidogenesis and spermatocyte differentiation and development.[118,120] It can also be hypothesized that ejaculated sperm from obese males exhibit reduced capacitation through leptin receptor downregulation in response to high seminal plasma leptin.[138] Many knowledge gaps remain to be elucidated, including a greater understanding of BBB and TBB leptin transport, the differential roles and associated signaling pathways associated with soluble and transmembrane leptin receptor isoforms in the brain, testis and sperm, with further study required to enable the characterization of leptin's modulation for reproduction in the obese male.

Heat Stress, Hypoxia-induced Testicular Apoptosis

It is well established that the testis is sensitive to heat, evidenced by the approximately 3°C lower temperature of the scrotum compared with core body temperature, a condition critical for efficient spermatogenesis.[147] Many environmental/lifestyle factors are associated with elevated testicular temperatures including varicocele,[148,149] tight fitting undergarments, sedentary work positions, laptop position of portable computers, saunas and occupational heat exposures.[37] Although it is likely that suprapubic and inner thigh adipose tissue in obese males combined with sedentary behaviour increases scrotal temperatures,[37] there are no studies that report scrotal temperature measurements in an obese population. As increased scrotal temperature during sedentary activities is associated with impaired semen quality,[150–152] it follows that obese men would be at risk of genital heat stress and infertility. Testicular scrotal temperature measurements are difficult to obtain in humans and data regarding duration of sustained sedentary position is not often collected, underscoring the need for more well-designed testicular thermal stress research studies.

Research studies using animals provide better models and opportunity to investigate the biological effects of thermal stress on testicular functions and structure. Various experimental approaches have been used. One mechanism used to elevate testicular temperatures in rats[153] and mice[154,155] is transient exposure of the animals to temperatures exceeding 40°C. Alternatively, surgically induced cryptorchidism is used in mice to elevate testicular temperature to the body temperature.[156] In another design, mice are housed at 35–38°C over a period of several hours to induce thermal stress.[157,158] Major reproductive outcomes in responses to hyperthermia include decreased testicular weights, germ cell loss and increased rates of apoptosis. Testicular heat-stress responses involve complex signaling pathways with interconnection among hypoxia, apoptosis, gene expression and inhibition of DNA repair, which eventually culminates in altered spermatogenesis.

Elevations in temperature and other environmental stressors, including oxidative stress and chemical exposures, are known to trigger intracellular changes in gene expression, resulting in altered cell survival/apoptosis pathways. Heat stress induces increased expression of a class of 70-kDa heat-shock proteins (Hsp70), a class of chaperone proteins that help regulate protein folding and assembly.[159,160] Hsp70 also play an important role in the prevention of apoptosis.[161,162] At least two testis-specific Hsp70 have been identified. Spermatocyte-specific Hsp70.2 (mice) is continuously expressed by pachytene spermatocytes during meiosis and is not heat inducible.[159] Testis-specific Hsc70t is similarly expressed in round spermatids. Expression of inducible Hsp70 by germ cells is conflicting. Rockett and colleagues demonstrated upregulation of Hsp70-1 and Hsp70-3 in mouse spermatocytes following acute hyperthermia (10 min at 43°C).[163] Other researchers reported that testicular heat stress did not increase expression of heat shock genes at the mRNA or protein levels in mouse germ cells.[161,164] Transgenic mice designed to express constitutively active heat-shock factor (HSF1) in spermatocytes did not induce Hsp70 gene expression but did activate caspase-dependent apoptosis pathways in the absence of heat shock.[161] HSF1 is a transcription factor activated during stress (hypoxia and hyperthermia); it trimerizes, translocates to the nucleus and binds heat-shock response elements in the promoter regions of HSF1 target genes.[165,166] Hsp70 does not appear to be sufficient to prevent HSF1-induced apoptosis as mouse spermatocytes with constitutive expression of both Hsp70 and HSF1 exhibited characteristic DNA strands breaks consistent with apoptosis.[161]

The mechanism underlying heat-stress-induced apoptosis appears to be primarily regulated by HSF1 (Figure 10). Intriguingly, the endogenously established temperature 'set-point' for HSF1 activation is higher for core somatic tissues compared with the testis, consistent with external location of the male reproductive organ in mammalian species. Thus, testicular tissues are exquisitely sensitive to even mild hyperthermia through regulation by HSF1. Persistent testicular HSF1 activation is observed in cryptorchid mice and is associated with disrupted spermatogenesis.[164] HSF1 may even downregulate Hsp70 to ensure unopposed activation of apoptosis pathways. In early developmental stages of mouse spermatocytes that express HSF1, Hsp70.2 protein undergoes translocation from the cytoplasm to the nucleus. At later stages of development, germ cells exhibit HSF1-mediated repression of Hsp70.2 following hyperthermia. Widlak and colleagues propose that a reduction in both Hsp70.2 mRNA and protein levels occurs prior to heat-stress induction of apoptosis, with both events mediated by HSF1.[167] Depending on the conditions of experimentally induced testicular heat stress and the subsequent assays to detect Hsp70, HSF1's negative regulation of Hsp70 expression and nuclear translocation may explain the inconsistencies regarding spermatocyte expression of Hsp70 in the literature discussed earlier.

Figure 10.

Model of testicular heat stress. In response to testicular heat stress, apoptosis pathways may be activated through several mechanisms. Caspase-2 perturbs the ratio of apoptotic proteins Bax:Bcl2 and directly activates apoptosis. p38MAPK (also known as MAPK14) phosphorylates Bcl2, thereby removing the inhibitory block to apoptosis. Caspase-2 and/or caspase-3 degrade ICAD, leading to activation of CAD and DNA fragmentation. Caspase-2 activates HSF1, which downregulates expression of DNA repair genes, ICAD, Hsp70, germ cell antioxidants, upregulates Hif1α, HSF1 and testicular antioxidants, and induces apoptosis. Dotted lines indicate proposed mechanisms and solid lines indicate established mechanisms.
CAD: Caspase-activated DNase; HSF: Heat-shock factor; Hsp: Heat-shock protein; ICAD: Inhibitor of caspase-activated DNase; P: Phosphorylation

Other important changes in testicular cells in response to heat stress include downregulation of DNA repair pathways and activation of p38MAPK signaling (Figure 10). Mouse testis exposed to acute heat stress (43°C for 10 min) downregulate DNA repair genes, including Ogg1 (base excision repair), Xpg (nucleotide excision repair) and Rad54 (double-strand break repair).[163] Decreased expression of poly (ADP-ribose) polymerase PARP (base excision repair/nucleotide excision repair pathways) also occurs in the rat testis in response to heat stress.[168] With DNA repair pathways disabled, apoptosis is induced via multiple mechanisms, including direct hyperthermic/hypoxic stress. Heat stress induces activation of p38MAPK (also known as MAPK14), which, in turn, phosphorylates Bcl2, thereby removing the inhibitory block to apoptosis.[169] Apoptosis is regulated, in part, by the balance between proapoptotic proteins (e.g., Bax) and antiapoptotic proteins (e.g., Bcl2). Caspase-2 is proposed to be activated upstream of p38MAPK by hyperthermia and/or reactive oxygen species produced during hypoxia. Caspase-2 may also perturb the ratio of Bax:Bcl2[170] and directly activate caspase-3, thereby promoting apoptosis.[170,171] Finally, heat-stress-induced cleavage of inhibitor of caspase-activated DNase (ICAD) via caspase-3 activation[154] or directly via caspase-2 activation[171] produces the final fragmentation of nuclear DNA. Another mechanism, yet to be defined, links hyperthermic stress with induced activation of HSF1, which, in turn, promotes apoptosis of pachytene spermatocytes.[164] Testicular heat stress is accompanied by localized hypoxia that induces oxidative stress, apoptosis and reduction in DNA repair gene expression (Figure 10). Increases in testicular cell metabolism during heat stress may be so high that testicular blood flow cannot provide sufficient tissue oxygenation, thereby creating oxidative stress to the tissue.[155] During hypoxia, hypoxia-inducible factor (Hif)-1α translocates from the cytoplasm to the nucleus to form a heterodimer with Hif-1β (also known as aryl hydrocarbon receptor nuclear translocator), now known as HIF1. The formation of the Hif-1α–Hif-1β heterodimer provides genomic protection from oxidative stress.[172] Following mild hyperthermia, the mouse testis undergoes hypoxia and oxidative stress and responds with increased expression of Hif-1α mRNA/protein in the interstitial compartment and increased expression of Hif-1α protein in the nuclei of germ cells.[155] Antioxidants, expressed during hypoxia, are downregulated in mouse germ cells following hyperthermia,[163] but not in testicular somatic cells, as whole mouse testis exhibit increased expression of testicular antioxidants (HMOX1, GPX1 and GSTA) following heat stress.[155] Thus, germ cells are particularly vulnerable to hypoxia and hyperthermia, which, in turn, induce apoptosis pathways and ultimately loss of male germ cells.

The testis exhibits germ cell stage-specific susceptibility to heat stress (Figure 11). As described previously, premeiotic germ cells express cell survival factors, including HSF1 and Hsp70, that protect against heat stress.[161,164] In response to heat stress, as spermatocytes undergo meiosis, they begin to exhibit significant DNA damage that subsequently initiates apoptosis pathways.[154] HSF1 seems to be associated with cell survival only in premeiotic and somatic cells, such that meiotic (pachytene spermatocytes and early spermatids) and postmeiotic stages are most susceptible to heat stress.[153,154,173]

Figure 11.

Spermatocytes exhibit stage-specific susceptibility to heat stress. Testicular germ cells exhibit stage-specific susceptibility to heat stress. Pre-meiotic germ cells express cell survival factors that protect them against heat stress. By contrast, meiotic germ cells (pachytene spermatocytes and round spermatids) are most susceptible to heat stress; they also have limited capacity for DNA repair.

Taken together, it can be speculated that a combination of factors, including sedentary lifestyle and suprapubic and inner thigh adipose tissue deposits, increase testicular temperatures, thereby triggering apoptosis pathways. As discussed, heat-stress-induced apoptosis of actively dividing germ cells would reduce sperm counts, thus contributing to male infertility in morbidly obese men. Animal studies also point to diminished embryo survival following paternal heat stress,[157,158,163,174] providing another mechanism by which testicular heat stress contributes to reproductive loss. There remain significant technical limitations in measuring scrotal temperatures in humans in a relatively noninvasive manner. These limitations must be overcome to further investigate the relationship between testicular hyperthermia, obesity and male infertility.

Endocrine Disruption

It has been well established that environmental chemicals (endocrine disrupters) are reproductive toxicants and can be associated with impaired semen quality and reproductive potential in animals and humans.[175–178] An endocrine disruptor is defined as:[176]

"as an exogenous agent that interferes with the synthesis, secretion, transport, binding, action or elimination of natural hormones in the body that is responsible for the maintenance of homeostasis, reproduction, development and/or behavior."

Baillie-Hamilton proposed that the obesity epidemic over the past 40 years may also be related to the increased use of industrial chemicals with demonstrated endocrine-modulating activity.[179–181] Male infertility and obesity may therefore share an environmental etiology caused by perturbations in normal endocrine pathways.

The Barker hypothesis relates poor fetal nutrition to adult-onset diseases including coronary heart disease, Type 2 diabetes and metabolic dysfunction,[182] and has formed the basis for the developmental origins of health and disease paradigm, which similarly posits a correlation between perinatal health and the eventual development of chronic diseases (Figure 12).[183,184] Toxicologists have also identified neonatal development as a 'critical window of exposure', such that chemical exposures (e.g., endocrine disrupters) have been linked to adult-onset reproductive cancers.[177,185,186] Taken together, these models support the extreme sensitivity of the neonatal period to environmental influences, and as proposed by Baillie-Hamilton[179] and Newbold and colleagues,[187] the models provide an explanation for fetal origins of adult obesity risk.

Figure 12.

Fetal origins of adult-onset diseases.

The intersection between estrogenic and adipogenic pathways has been best examined in a series of studies defining adipogenesis in penile corpus cavernosum induced by activation of ER-α by potent estrogen disrupter, diethylstilbestrol (DES) (Figure 13A).[188–190] Neonatal exposure to DES (3–4 mg/kg) generates adult rats that are infertile with gross penile abnormalities, including the appearance of adipocytes in the cavernous spaces of the corpora cavernosa penis. Similarly, histological changes to the penis along with male infertility were observed following postnatal exposures to DES (1 mg/kg).[188,189] Goyal and colleagues have hypothesized that estrogen-dependent differentiation and proliferation of stromal cells occur following estrogen exposure during a critical development period (1–12 postnatal days).[190] This transformation replaces endothelial and smooth muscle cells in the corpora cavernosa with adipocytes, resulting in grossly abnormal morphology of the penis and infertility. Transgenic mice lacking ER-α (α-ERKO) are 'resistant' to neonatal DES exposure and do not exhibit penile deformities. These studies thereby confirm a role for ER-α in DES-mediated abnormalities of the corpora cavernosa in both rats and mice.[191] Furthermore, this developmental disruption has been recently elucidated as a 'biological overlap' between PPAR-γ and ER-α (Figure 13B & C).[192]

Figure 13.

ER-α- and PPAR-γ-dependent gene expression. (A) ER-α. Following ligand (estrogens) binding, ER-α forms a homodimer that together with transcription factors, assemble as a pre-initiation complex at the ERE present in the promoter region of estrogen-responsive genes. (B) PPAR-γ. Once activated by ligand (fatty acids, putative obesogens), PPAR-γ forms a heterodimer with RXR and binds as a complex to PPRE in target gene promoter regions. (C) DES-mediated ER-α and PPAR-γ signal crosstalk. ER-α may also bind the PPRE consensus sequence. DES-binding to ER-α induces adipogenic effects in the rodent penis, possibly through PPAR-γ signal crosstalk. (D) PPAR-γ and ER-α signal crosstalk. Similarly, PPAR-γ may bind the ERE consensus sequence. Although the consequences on male reproduction are unknown, such signal crosstalk may represent yet another mechanism by which putative obesogens including phthalates, organotins and phytoestrogens may disrupt endocrine function.
DES: Diethylstilbestrol; ER: Estrogen receptor; ERE: Estrogen response element; PPRE: PPAR response elements; RXR: Retinoid X receptor.

The PPAR family includes three isotypes PPAR-α, -β and -γ. PPAR-γ, a nonsteroidal nuclear receptor, regulates proliferation and differentiation of adipocytes through the promotion of genes involved in fatty acid storage and the repression of genes necessary for adipocyte lipolysis. Once activated by ligand (fatty acids, putative obesogens), PPAR-γ forms a heterodimer with retinoid X receptor and binds as a complex to PPAR response elements in target gene promoter regions (Figure 13B).[193] ER-α and PPAR-γ pathways exhibit several instances of 'signal crosstalk', including shared coactivators involved in adipocyte differentiation such as the recently identified constitutive coactivator of PPAR-γ.[193] ER-α and PPAR-γ may each bind directly to either PPAR response elements[194] or estrogen response elements (Figure 13C & D).[195–197] It is the latter mechanism that is proposed to trigger DES-induced differentiation of stromal cells and subsequent penile abnormalities in rodents.[192]

Modulation of adipocyte function and differentiation is now recognized as a subset of endocrine disruption. Such environmental chemicals have been termed 'metabolic disrupters'[198] or 'obesogens'.[199] Obesogenic pathways proposed include nuclear receptors involved in lipid metabolism, such as PPAR-γ, liver X receptor and farnesoid X receptor.[200] Several xeno-PPAR-γ ligands have been identified, including phthalate monoesters,[201] such as di(2-ethylhexyl) phthalate[202] and its metabolite mono(2-ethylhexyl)phthalate,[198] organotin compounds triphenyltin and tributyltin,[200,203–206] dioxins[207] and phytoestrogen genistein,[208] many of which have established toxicity to the reproductive system.[178,209] Studies performed in laboratory and wildlife animals provide additional evidence for crosstalks between endocrine and adipogenic pathways, further implicating that environmental contaminants, which can be endocrine disrupters, probably also modulate adipocyte differentiation and contribute to reproductive pathologies (Figure 13C & D).


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