Obesity stands as one of the greatest healthcare challenges of the 21st century. Obesity in reproductive-age men is ever more frequent and is reaching upsetting levels. At the same time, fertility has taken an inverse direction and is decreasing, leading to an increased demand for fertility treatments. In half of infertile couples, there is a male factor alone or combined with a female factor. Furthermore, male fertility parameters such as sperm count and concentration went on a downward spiral during the last few decades and are now approaching the minimum levels established to achieve successful fertilization. Hence, the hypothesis that obesity and deleterious effects in male reproductive health, as reflected in deterioration of sperm parameters, are somehow related is tempting. Most often, overweight and obese individuals present leptin levels directly proportional to the increased fat mass. Leptin, besides the well-described central hypothalamic effects, also acts in several peripheral organs, including the testes, thus highlighting a possible regulatory role in male reproductive function. In the last years, research focusing on leptin effects in male reproductive function has unveiled additional roles and molecular mechanisms of action for this hormone at the testicular level. Herein, we summarize the novel molecular signals linking metabolism and male reproductive function with a focus on leptin signaling, mitochondria and relevant pathways for the nutritional support of spermatogenesis.

Leptin: from discovery to energy control

Leptin was identified in 1994 by Friedman and colleagues through positional cloning [1]. Since then, the knowledge gathered on the actions of this adipocyte-derived protein has consistently grown. In brief, leptin is a 16 kDa cytokine encoded by the LEP gene, located in the chromosome 7q31.3 in humans, consisting of three exons separated by two introns, and chromosome 6 in mice [2,3]. The vast majority of leptin production is originated from adipocytes, although it is also produced in other tissues such as stomach, placenta, ovary, mammary gland, immune cells and skeletal muscle [48]. In cells other than adipocytes, leptin has been implicated in several events such as regulation of fetal development, immune responses and reproductive function [911]. Thus, leptin has pleotropic effects in several biological systems and more importantly, those actions seem to be at least partially independent of body weight regulation [1214].

Under physiological conditions, leptin is secreted by white adipose tissue cells and enters the blood-stream. Therefore, circulating leptin concentrations are suggested to be directly related to the amount of adipose tissue depots in each individual [15,16]. As a result, leptin levels are an indicator of the body fat reserves. This information is then communicated to specific brain regions that regulate energy homeostasis [17]. In summary, higher circulating leptin levels convey information to the central nervous system of high levels of adipose tissue depots, which triggers a reduction in food intake and promotes energy consumption. In contrast, a decrease in leptin-circulating levels promotes food intake and reduces energy consumption [18,19]. In overweight and obese individuals, this mechanism often becomes dysregulated and is clinically characterized by leptin resistance. In that condition, high circulating leptin levels are present but are unable to trigger the expected physiological response [20,21]. Although partially identified, the specific molecular mechanisms responsible for the secondary and acquired leptin resistance developed as a consequence of weight gain remain a matter of discussion. Leptin's saturable transport to the central nervous system was one of the first mechanisms identified. Afterwards, genetic alterations, such as polymorphisms of leptin receptor (LepR), were also proposed as responsible for leptin resistance. More recently, endoplasmic reticulum (ER) stress and negative regulation of leptin receptor signaling are on the spotlight as possible causes for the development of leptin resistance. Still, the systemic consequences of leptin resistance are well recognized as a major contributor to obesity-related health co-morbidities.

Leptin receptor and mechanisms of leptin action

Leptin's physiological effects are exerted through specific receptors. Identified in 1995 [22], LepR was isolated from mouse choroid plexus and due to strong similarities of its extracellular ligand-binding domain with the gp130 signal-transducing subunit, it is considered to be part of the large family of class I cytokine receptors. LepR is activated via ligand-induced conformational changes, forming homodimers even in the absence of leptin [23]. There are several LepR isoforms (LepRa to LepRf), all products of the LEPR gene. The different isoforms result from alternative mRNA splicing or post-translational modifications [24]. In general, LepR isoforms share identical extracellular domains (consisting of over 800 amino acids), while their intracellular domains are truncated at different lengths, which is characteristic of each isoform [25]. Secreted isoforms of LepR are characterized by possessing only the extracellular domain. This can result from alternative mRNA splicing or proteolytic cleavage products of membrane-bound isoforms of LepR. In humans, opposite to what is observed in mice, the secreted LepR isoform (LepRe) is exclusively generated through proteolytic cleavage of membrane-bound isoforms of LepR, a process known as shedding, thus being an excellent surrogate marker for the number of membrane-anchored leptin receptors [26]. Under physiological conditions, this process inherently occurs. However, this phenomenon can also be induced by different stimuli, including lipotoxicity and apoptosis [27,28]. Soluble isoforms can have either antagonistic or agonistic effects, although the former prevails [2931].

The short isoforms (LepRa, LepRc, LepRd and LepRf) possess a similar extracellular domain and a transmembrane domain consisting of 34 amino acids, as well as the first 29 amino acids from 32 to 40 amino acids in total, in the intracellular domain [32]. Owing to lack of intrinsic tyrosine kinase activity, LepR binds to cytoplasmic kinases, specifically Janus kinase 2 (JAK2) [33]. The first 29 amino acids of the LepR intracellular domain contain a box 1 motif and a JAK2. However, since the short isoforms have shorter intracellular domains, they lack the box 2 and box 3 motif and thus the ability to induce the pivotal signal transducer and activator of transcription (STAT), a characteristic of leptin signaling. Nonetheless, these isoforms are able to recruit JAKs and activate certain signaling cascades. More recently, studies highlight their involvement in leptin signaling cascades through MAPK signaling pathways [34].

LepRb, also known as long isoform of LepR, is the fully functional isoform of the leptin receptor. The major difference between LepRb and the short isoforms resides in the intracellular domain, composed of over 300 amino acids [25]. Similar to what happens in short isoforms, this domain contains a box 1 motif and a JAK2. It also contains two motifs (box 2 and box 3) that are crucial to modulate the STAT signaling pathway, a characteristic of leptin signaling [35]. This isoform is essential to the conventional signaling of leptin, as reported using animal models that lack this receptor, such as db/db mice. Animals with homozygous LepR spontaneous mutation (and thus lacking the LepRb isoform) are hyperleptinemic, hyperphagic, obese, leptin resistant and present a severe reproductive dysfunction [36]. These animals also display a phenotype similar to db3J/db3J mice, a model that is null for all known isoforms of the LepR. Expression of an LepRb transgene in these mice normalizes body weight, food intake, glucose tolerance and interestingly, restores male fertility [37]. Fortunately, mutations in the LEPR and LEP gene are extremely rare among obese human individuals [38,39].

Obesity: a major cause for (in)fertility

Nowadays, chronic metabolic diseases, such as overweight and obesity, are well recognized as healthcare problems and major disruptors of human reproductive health [40]. Obesity is characterized by a strong hormonal dysregulation, particularly in the leptin–ghrelin axis, which has a major impact in energy homeostasis. Positive energy balance, a characteristic of obesity, leads to the growth of adipose tissue. This growth can occur by an increase in the size of adipocytes (hypertrophy) and/or by the increase in the number of fat cells (hyperplasia) [41]. In the case of positive energy balance, the body responds by increasing adipocyte size to store the excess of energy. However, this situation is revertible with lifestyle change which can promote fat utilization. Adipogenesis also occurs in obesity via recruitment and differentiation of adipocyte precursors, which means that individuals with hyperplastic obesity have an increase in adipocyte number due to the life span of fat cells (2–10 years) [42]. An increase in fat cells results in higher production of leptin which is accompanied by the increase in inflammatory mediators, a characteristic of obesity [43]. Defined by a body mass index (BMI) above 30 kg/m2, obesity epidemic has been spreading worldwide entailing severe health consequences such as cardiovascular diseases, type 2 diabetes, several cancers and subfertility [44]. According to the World Health Organization, in 2014 more than 1.9 billion adults were overweight (defined as BMI over 25 kg/m2), including 38% of the adult male European population. Furthermore, it is estimated that nearly three million people per year die from health complications associated with obesity, which places the disease in the top 10 of worldwide death causes. Several studies have shown a strong correlation between increased obesity prevalence and decline of sperm counts in the last few decades, which represents a major public health problem [45,46]. A recent study showed that sperm concentration has decreased nearly 53% between 1973 and 2011 with total sperm count depicting a similar trend, with a 60% decrease in men from the U.S.A., Europe, Australia and New Zealand [47]. Furthermore, an inverse correlation exists between total sperm count and BMI [46]. Obese men have reduced sperm concentration and total sperm count, which is likely to account for the fertility problems that overweight and obese men face [48]. Additionally, obesity is also associated with lower free testosterone levels and a greater decline in age-related testosterone production [49]. In addition, decreased testosterone by reduced lipolysis also facilitates abdominal fat accumulation, thus creating a feedforward loop that further exacerbates obesity [50,51]. However, despite intense focus of the scientific community in this topic, the mechanisms underlying energy homeostasis and reproductive capacity are not yet fully understood.

Leptin resistance and metabolic disorders

The discovery of leptin in the early 1990s raised high expectations and attention due to its potential for solving a long-time problem: the obesity epidemic, already a matter of concern at that time. The ob/ob mice model, obese, hyperphagic, with low spontaneous locomotor activity and infertile was critical for leptin discovery. However, in contrast with the effects observed in those rare obese patients with congenital leptin deficiency, the prospects of the widespread use of leptin for obesity treatment in the clinical setting were underwhelming [52,53]. Obese individuals often present high leptin concentrations in proportion to their increased body fat mass when compared with lean counterparts, suggesting that leptin is a bystander and thus other mechanisms must be involved in the development of obesity [54]. Since then, the focus has switched from leptin as a putative anti-obesity therapeutic agent and shifted to address the mechanisms responsible for leptin resistance and how could these be tackled. Nonetheless, the pharmacological relevance of leptin is an established reality not only for the treatment of congenital leptin deficiency, but also in conditions characterized by the secondary leptin deficiency, such as generalized lipodystrophy, that was granted approval for leptin treatment from the US Food and Drug Administration in 2014 [55].

Leptin-resistance concept has arisen after the demonstration that leptin administration failed to decrease body weight and food intake in obese individuals. Thus, leptin resistance can be defined as the inability of exogenous leptin to trigger the expected biological effects. Owing to the vast network involved in leptin signaling, it is hard to pinpoint the exact mechanisms responsible for the development of leptin resistance. Nonetheless, there are several working hypotheses to explain the diminished leptin response, such as a defective leptin transport, a diminished expression of leptin receptors and endoplasmic reticulum stress [5659].

Suppression of leptin receptor signaling

After leptin resistance was described, the vast majority of studies focused on unraveling putative negative feedback mechanisms that could impair LepR signaling. As there are several molecular pathways involved in leptin signaling, which can also involve negative feedback or counter regulatory mechanisms, studying this mechanism has a high degree of complexity. For instance, the suppressor of cytokine signaling 3 (SOCS3), a cytokine-inducible negative regulator of cytokine signaling, is a target gene for leptin that provides a negative feedback regulatory mechanism to prevent overactivation of the LEPRb pathways. SOCS3 expression is induced by STAT3, which in turn binds to Tyr985, switching off leptin signaling [60]. Selective inactivation or haploinsufficiency of SOCS3 gene in neurons induces a greater response to exogenous leptin and even resistance to diet-induced obesity (DIO), resulting in a greater suppression of appetite and body weight loss [61,62]. Additionally, mice with leptin resistance have increased hypothalamic SOCS3 expression, which blocks leptin signaling in mammalian cell lines [63]. Overexpression of SOCS3 in proopiomelanocortin (POMC) neurons also leads to impairment of STAT3 and mammalian target of rapamycin (mTOR) signaling, being both events associated with obesity and leptin resistance [64].

Endoplasmic reticulum stress

Leptin signaling dysfunction seems to be associated with obesity-related inflammation via ER stress, through activation of specific inflammatory signaling pathways. ER is responsible for the correct folding of newly synthesized proteins and their transport into the Golgi apparatus. Failure in the folding of new proteins causes an accumulation of unfolded proteins, leading to ER stress, which in turn triggers the unfolded protein response [65]. ER stress has been identified as a hallmark of insulin resistance. Owing to the common features shared between leptin and insulin resistance, it was suggested that ER stress could be a link between obesity and leptin resistance [66].

Overexpression of molecules associated with the unfolded protein response was reported in the hypothalamus of C57BL/6 mice fed a high-fat diet [57]. Furthermore, central administration of an ER stress inducer inhibits leptin anorexigenic effects, through the inhibition of STAT3 activation in hypothalamic neurons [67]. Indeed, inhibition of leptin signaling was reported after exposure of LepRb-expressing 293 cells to an inducer of unfolded protein response for 4 h which were then treated with leptin for 45 min [57]. Inflammatory signaling pathways are involved in the relationship between ER stress and impaired LepR signaling. ER stress induced by high-fat diet seems to be mediated through lipotoxicity, due to abnormal accumulation of lipids in hypothalamic neurons [6870]. Remarkably, lipotoxicity also causes proteolytic cleavage of the extracellular LepR domain, which can lead to a decrease in the availability of leptin receptors, thus contributing to leptin resistance [27]. Recently, a link between mitochondria–ER interaction and leptin resistance has also been described. Hypothalamic POMC neurons are key players in leptin signaling. In DIO mice, mitochondria–ER interactions in POMC neurons are decreased [71]. Mitofusin-2 (MFN2) is a mitochondrial membrane protein involved in mitochondrial fusion and responsible for mediating mitochondrial–ER interactions [72]. Specific ablation of MFN2 in POMC neurons severely decreases these interorganelle interactions and results in ER stress, leading to leptin resistance. Interestingly, pharmacological reversion of ER stress through chemical chaperones in mice knockout for MFN2 in POMC neurons improves these conditions [71]. Furthermore, ablation of Mitofusin-1 and MFN2 in agouti-related peptide (AgRP) neurons results in alterations in mitochondria size and density with improvements in body weight of mice fed with a high-fat diet [73]. These results establish mitofusins as major players in the regulation of the whole-body energy metabolism by changing and mediating the mitochondrial–ER axis. Altogether, these studies point towards an important role carried out by ER stress in the development of leptin resistance. Still, further studies are needed to clarify the exact role of ER stress on leptin resistance.

Leptin-resistance hallmarks

As referred, the concept of leptin resistance can be compared with that of insulin resistance on obese states [74]. Interestingly, leptin resistance is not ubiquitous. Namely, leptin resistance is selective for the central nervous system control of energy homeostasis that becomes unable to suppress food intake and prevent weight gain, while at the same time retains other systemic effects [74]. For instance, leptin retains the ability to increase blood pressure. In fact, high blood pressure is a hallmark of leptin action in obese individuals [75,76]. Thus, when studying leptin resistance, one should caution that leptin can exert different functions in different systems and not extrapolate a system-specific mechanism as a general trait. Two types of leptin resistance can be defined: central leptin resistance and peripheral leptin resistance. Interestingly, the development of leptin resistance is a continuous process and from observations in mice, it can be summarized in three different stages: first, a high-fat diet triggers an increase in adipose tissue. However, at this stage, mice are still sensitive to peripheral leptin injections [77]. In a medium stage, mice lose peripheral leptin sensitivity while retaining central leptin sensitivity, since an intracerebroventricular injection of leptin results in a decrease in food intake [78]. In the later stages, central leptin sensitivity is also lost resulting in leptin resistance [59,78]. These stages characterize leptin resistance as a gradual process. Interestingly, concerning central leptin resistance, different areas of the hypothalamus lose their sensitivity to leptin at different times, which indicates that certain hypothalamic areas are more sensitive to leptin than others [79,80]. Furthermore, this also indicates that the transport across the blood–brain barrier is not the only mechanism involved in the development of central leptin resistance. However, in all cases, hyperleptinemia has shown to be required for the development of central leptin resistance. Interestingly, a recent study by Ottaway and colleagues [81] has shown that intraperitoneal or intracerebroventricular administration of an LepR antagonist in DIO mice triggered a decrease in body weight and food intake comparable to the same extent to the results obtained in lean mice administrated with an LepR antagonist. This indicates that despite the development of leptin resistance, DIO mice still retain endogenous leptin action. The identification of the mechanisms responsible for persistence of obesity despite endogenous leptin action may represent an important step to develop a future therapy to counteract co-morbidities associated with DIO.

Leptin interaction with the male hypothalamic–pituitary–gonadal axis

Leptin contribution for the reproductive function is not merely restricted to the regulation of whole-body energy homeostasis. In addition, leptin has other pleiotropic actions that include direct and indirect interactions with the hypothalamic–pituitary–gonadal (HPG) axis. HPG axis has a pivotal role in the control of the testis and testicular cells function including Leydig cells, responsible for testosterone production, and Sertoli cells (SCs) that are key players in spermatogenesis [82]. In fact, the link between leptin and reproductive function is quite evident. First of all, leptin's primary role is related with whole-body energy status and reproductive function is a highly energy-demanding process. Additionally, leptin showed to have a physiological role by triggering the onset of puberty in a setting of congenital leptin deficiency and to rescue infertility in the ob/ob obese male mice (lacking endogenous leptin), definitely emerging as a key element connecting whole-body energy status and male reproductive function [8385].

HPG axis plays a paramount role in the control of testicular function. Gonadotropin-releasing hormone (GnRH), a hormone released by the hypothalamus, stimulates the pituitary to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which control testicular steroidogenesis and spermatogenesis, respectively. It is well established that the hypothalamus, known to regulate energy balance and reproductive function, is the primary target site for leptin action due to its ability to modulate the expression of several hypothalamic neuropeptides [86]. Leptin has a stimulatory effect on LH pulsatility in several species, which indicates that GnRH neurons are a downstream target of leptin [87,88]. However, despite LepRb being highly expressed in the hypothalamus, these receptors are not expressed in GnRH neurons [89]. Instead, LepRb is expressed in several different tissues and organs which could indicate that the ability of leptin to regulate reproduction is not restricted to a single cell type or organ. However, re-expression of LepR in the brain of mice without LepRb restored male fertility, evidencing the central role of brain in the regulation of fertility by leptin-dependent mechanisms [90]. Since GnRH neurons do not possess LepR, leptin actions in these cells seem to be mediated by intermediate neurons. Kisspeptin emerged as a major candidate for this role since it acts as excitatory stimuli upstream of GnRH in the HPG axis (Figure 1A). Interestingly, in contrast with GnRH neurons, kisspeptin neurons located in the arcuate nucleus (ARC) express LepRb [89]. Additionally, leptin administration was able to promote hypothalamic Kiss1 mRNA in various cell lines and in streptozotocin-induced diabetic male rats [91,92]. Depolarization of kisspeptin neurons was also observed in the ARC after leptin administration and leptin-deficient ob/ob mice show reduced Kiss1 mRNA in the ARC, which is partly restored after leptin treatment [93,94]. Altogether, leptin activates GnRH neurons through kisspeptin neurons in the ARC, indirectly modulating the HPG axis. Furthermore, a complex interplay between anorexigenic, orexigenic and kisspeptin neurons for regulation of energy balance seems to occur. This information is then conveyed to the hypothalamus that responds through GnRH production, regulating several physiological functions.

Leptin actions in the HPG axis and male reproductive tract, specifically in Sertoli cells.

Figure 1.
Leptin actions in the HPG axis and male reproductive tract, specifically in Sertoli cells.

(A) Leptin produced in the adipocytes travels to the hypothalamus, where it participates in the maintenance of whole-body energy homeostasis. ROS are closely linked with leptin, with several studies highlighting their importance for leptin actions. Higher levels of leptin also have an inhibitory effect on steroidogenesis. (B) Simplified representation of Sertoli cell metabolism. Leptin has shown the ability to modulate acetate and the glycolytic profile of Sertoli cells. Several studies have also reported leptin's involvement in mitochondrial biogenesis. ▾: Reactive oxygen species. →: stimulation. ┤: inhibition. GnRH: gonadotropin-releasing hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; GLUT: glucose transporter; LepR: leptin receptor; LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase.

Figure 1.
Leptin actions in the HPG axis and male reproductive tract, specifically in Sertoli cells.

(A) Leptin produced in the adipocytes travels to the hypothalamus, where it participates in the maintenance of whole-body energy homeostasis. ROS are closely linked with leptin, with several studies highlighting their importance for leptin actions. Higher levels of leptin also have an inhibitory effect on steroidogenesis. (B) Simplified representation of Sertoli cell metabolism. Leptin has shown the ability to modulate acetate and the glycolytic profile of Sertoli cells. Several studies have also reported leptin's involvement in mitochondrial biogenesis. ▾: Reactive oxygen species. →: stimulation. ┤: inhibition. GnRH: gonadotropin-releasing hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; GLUT: glucose transporter; LepR: leptin receptor; LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase.

Onset of puberty is an important event in the organism gated by multiple metabolic factors that inform the reproductive system about the whole-body energy status, so that the pulse of GnRH can be triggered. Leptin-deficient individuals are infertile and fail to advance into puberty unless treated with exogenous leptin [95]. In male rats, leptin administration causes a dose-dependent stimulation of GnRH production [96] and, in male humans, leptin values peak right before puberty [97]. This peak precedes the rise in free testosterone observed during puberty. Interestingly, women have higher concentrations of circulating leptin than men. Differences in fat distribution between sexes can explain some of these differences. However, women have higher concentrations of circulating leptin per unit of fat mass than men, which suggests that circulating concentrations of gonadal steroids are behind the sexual dimorphism of leptin levels [98,99]. In fact, leptin has an inhibitory effect in testosterone production, while estrogens stimulate leptin release from adipocytes, which could explain the decrease in serum leptin concentrations after puberty in men, whereas in women higher concentrations of leptin are found in the later stages of puberty (Figure 1A) [100,101]. Altogether, these findings clearly advocate for a crucial role for leptin in the onset of puberty, with leptin indirectly affecting GnRH production.

Exogenous leptin administration stimulates the HPG axis in ob/ob mice, leading to normalization of sperm parameters and fertility [102]. Leptin administration to animals with hypogonadotropic hypogonadism induced by dietary restriction increases LH secretion and, consequently, testosterone levels thus reversing this state [103]. Moreover, morbidly obese men also present low circulating gonadotropins and testosterone levels, a hypogonadotropic hypogonadism for which evidence suggests to be a consequence of leptin resistance [104,105].

In fact, leptin has a direct influence on the pituitary gonadotropes, of the pars tuberalis and pars distalis, which express LepR [106]. Leptin stimulates GnRH release in hypothalamic explants, and FSH and LH release from the anterior pituitary of adult male rats in vitro. However, in vivo, leptin stimulates LH but not FSH release [107]. Leptin-induced gonadotropin release is suggested to be mediated by nitric oxide, as a nitric oxide synthase competitive inhibitor is able to suppress leptin-induced LH release. Leptin seems to act both at hypothalamic and pituitary levels by stimulating nitric oxide release, presumably through activation of receptors on both locations, which results in the release of GnRH and LH, respectively [108]. Furthermore, both gonadotropes and somatotropes also locally produce leptin when stimulated by GnRH and neuropeptide Y (NPY) [109,110].

Leptin actions in the male reproductive function

Through the past decades, leptin emerged as a key player for a normal reproductive function. In fact, leptin effects in female fertility were the first to be described [11]. However, leptin role as a master regulator of male fertility is still a matter of debate. Besides the above-mentioned actions in the hypothalamus, leptin also acts at the gonadal level, both in the ovaries and in the testis.

In males, hyperleptinemia has been proposed as a negative modulator of steroidogenesis and spermatogenesis [111]. In the human testis, Leydig cells are responsible for androgen production under pituitary LH control. These cells are paramount for the establishment and maintenance of the male reproductive potential. High leptin levels negatively affect serum testosterone levels [112]. In the testis, leptin is suggested to act as a steroidogenesis-inhibiting factor, which can provide an explanation for the decreased testosterone production observed in obese men with high leptin levels (Figure 1A) [113]. In fact, leptin inhibitory effect in steroidogenesis was also observed in other peripheral tissues such as the ovaries and adrenal glands [114,115]. Further contributing to decreased testosterone levels observed in obese men is the aromatization of testosterone to estradiol in peripheral fat tissues which is also increased [116]. Moreover, hyperleptinemia in mice was shown to decrease the number of Leydig and germ cells, the diameter of seminiferous tubules, weight and volume of the testes, while the offspring number was also reduced [117].

Leptin can cross the blood–testis barrier (BTB) being present in seminiferous tubules [118] and in human seminal plasma. However, leptin's entrance into the BTB occurs through leakage, in contrast with the brain where it occurs through a saturable transport system [118]. LepR is also expressed in spermatozoa, a clear indication of a putative role for leptin on sperm function [119]. In fact, obese and often hyperleptinemic individuals and DIO rats exhibit altered spermatic parameters with reduced sperm motility, concentration and viability, which leads to a reduction in the fertility potential of these individuals [120,121].

Leptin and LepR expression in testis

The first demonstrations of LepR expression in murine testes, specifically in sperm and Leydig cells, occurred soon after leptin was discovered [122]. Afterwards, several studies have shown that LepR expression in testis is a trait shared among different species, although its function and involvement in reproductive processes such as spermatogenesis and steroidogenesis is not yet enlightened [123125]. In male reproductive tract, LepR was identified in the testis, epididymis and seminiferous tubules [123]. Specifically in humans, LepR was identified in testicular somatic cells, germ cells and spermatozoa [125127]. Interestingly, LepRa, a short isoform with a role that remains unknown, is abundantly expressed in testicular cells [128]. Conversely, some studies reported that LepRb, the fully functional signaling isoform of LepR, is absent from the testicular tissue. Yet, other studies report that, in testis, LepR is operative, with the JAK/STAT pathways involved in leptin signaling being fully functional [117,122]. This could be due either to the fact that LepR activation in the testicular tissue may involve cell-specific signal transduction pathways or to the fact that LepR short isoforms still convey the conventional signaling potential and are still able to recruit JAKs and activate certain signaling cascades, which could explain leptin signaling occurring without the participation of the long isoform of LepR. Still, there is very little information concerning LepR short isoforms and the signaling cascades involved, which represents an obstacle when trying to unveil the role for leptin signaling in the testis.

Leptin and Leydig cells

Leptin connection to Leydig cells started to be in the spotlight after the disclosure of leptin inhibitory effect on testosterone production. LepR was identified in Leydig cells of several species including humans [112,129]. Leydig cells are located between the seminiferous tubules and are responsible for testosterone production, which is the primary male sex hormone that, in turn, is pivotal for a normal spermatogenesis. In rats, leptin inhibits testicular testosterone secretion in a manner that is dependent of the sexual maturation state, which renders Leydig cells as candidate mediators for leptin effects in the testis [101]. Indeed, it was later demonstrated that LepR expression is a feature of mature Leydig cells and is fully functional in adult animals, whereas the receptor is not functional in prepubertal individuals [129]. Furthermore, exposure of rodent Leydig cells to increasing leptin concentrations led to a dose-dependent inhibition of human chorionic gonadotropin-stimulated testosterone production [112], indicating that these cells are the likely target for leptin-mediated inhibition of testosterone production. Additionally, in men, leptin levels are inversely correlated with testosterone levels, which strongly suggest that leptin can modulate testicular steroidogenesis, probably through a direct action on Leydig cells [130]. Recent studies have also shown that hyperleptinemia down-regulates cAMP-dependent activation of the steroidogenic acute regulatory protein (StAR) and the rate-limiting steroidogenic enzyme cytochrome P450 family 11 subfamily A member 1 (P450SCC) levels in Leydig cells. These findings suggest that the high leptin levels found in obese individuals down-regulate the STAT transcriptional activity, leading to lower expression levels of cAMP-dependent steroidogenic genes involved in testosterone production [131]. AMP-activated protein kinase (AMPK) pathway has also been implicated in the modulation of Leydig cell steroidogenesis and could be involved in mediating leptin effects [132]. In the presence of high leptin levels, AMPK pathway may be up-regulated and this up-regulation inhibits StAR expression [133,134]. Furthermore, activated STAT transcription factors have also shown to down-regulate StAR and P450SCC expression in Leydig cells [135]. Still, further studies are needed to define more precisely how leptin influences and modulates Leydig cell function and what are the consequences of those processes for the male reproductive potential.

Leptin, Sertoli and germ cells

In contrast with the volume of data available concerning leptin and Leydig cell interaction, few data are available regarding the impact of leptin and associated signaling pathways on SCs and its relevance for spermatogenesis. Several studies have shown that in infertile male ob/ob mice, Sertoli and germ cells present a condensed nucleus with high levels of DNA fragmentation. The number of spermatogonia, spermatocytes and spermatids is also lower in ob/ob mice when compared with wild-type animals [136], highlighting the importance of leptin for spermatogenesis.

SCs have been denominated as ‘the nurse cells’ due to their role in providing structural and nutritional support to germ cells and are also responsible for the seminiferous tubule formation. SCs also have unique metabolic features, exhibiting a ‘Warburg-like’ metabolism, similarly to what is observed in cancer cells, thus favoring a high glycolytic flux to support the nutritional needs of germ cells. Indeed, germ cell metabolism is entirely dependent on SCs that are the responsible for lactate production, an essential substrate for germ cell development. Moreover, SCs are also highly susceptible to internal and external factors such as hormones [137]. As LepR was identified in SCs, these can be targeted by leptin to become the mediators of the effects of this hormone on male fertility [113,126].

In cancer cells, leptin was recently shown to modulate mitochondrial dynamics, biogenesis and functioning. A concentration of leptin found in obese patients was able to shift ATP production from glycolysis to mitochondria, which led to a decrease in the production of lactate by cancer cells [138]. Owing to the similarities shared between cancer cells and SC metabolism, one can hypothesize that the same mechanism may occur in SCs. In this case, high leptin concentrations could shift ATP production to mitochondria, diminishing lactate production. Lactate acts as an anti-apoptotic to male germ cells in a dose-dependent manner and high lactate concentrations are required to sustain spermatogenesis and normal testicular function [139]. These findings could represent a novel mechanism whereby leptin regulates germ cell apoptosis through mitochondria and SCs, shifting ATP production. This would culminate in decreased sperm quality and consequently the decreased fertility potential as observed in hyperleptinemic individuals. Still, studies are needed to confirm this hypothesis and to clarify the exact role of leptin concerning SC's metabolic status. In a recent study, different leptin concentrations were shown to modulate human SC's acetate production and glycolytic profile highlighting leptin's ability to interfere with SC's metabolism, which could have a potential impact in spermatogenesis (Figure 1B) [126].

LepR expression was also reported in germ cells and spermatozoa of several species including in humans [127,140142]. However, there is a lack of consensus as other studies refuted the presence of this receptor in male germ cells [143]. Still, in pig spermatozoa, LepR is located in the acrosome region, suggesting that leptin could be involved in capacitation process. In fact, in a study by Aquila and colleagues [144], leptin enhanced both capacitation indexes and acrosin activity. Furthermore, the same authors reported that leptin triggers STAT3 and induces the activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and MAPK pathways in spermatozoa. Concerning human spermatozoa, a study by Lampiao and du Plessis [145] has shown that leptin enhances sperm motility, acrosome reaction and nitric oxide production. However, this effect was not observed in a similar experiment by Li and colleagues [146], which illustrates the difficulties in reaching a consensus regarding the effect of leptin on human sperm quality. In vivo, intraperitoneal administration of leptin in adult rats decreased sperm count and increased the amount of abnormal sperm [147]. Another study in rats reported an increase in reactive oxygen species (ROS) and sperm DNA fragmentation after administration of 10 and 30 µg/kg of leptin during 7 and 15 days. Differences in sperm parameters in leptin-treated rats compared with controls were also observed [148]. Similar effects were reported by Almabhouh and colleagues [149] with an increase in abnormal spermatozoa and DNA fragmentation accompanied with lower sperm count in rats treated with 60 µg/kg body weight in 0.1 ml 0.9% saline of leptin for 42 days. Altogether, these results advocate for a clear role for leptin in spermatozoa, although the exact mechanisms are still unknown. In human spermatozoa, leptin levels in seminal plasma were positively correlated with sperm concentration and motility but not with the BMI, which could advocate for leptin's direct effects in spermatozoa, probably through its membrane receptor [150]. Interestingly, a recent study has observed that in patients with varicocele and leucocytospermia, seminal plasma levels of leptin and spermatozoa apoptosis rates were significantly increased when compared with seminal plasma levels of leptin in control patients. A positive correlation was also found between leptin and ROS levels and also between leptin levels and spermatozoa apoptosis rate which suggests leptin as a spermatozoa pro-apoptotic factor in these patients, possibly through ROS generation [151]. Recently, leptin's involvement in capacitation and hyperactivation in equine spermatozoa was also reported; however, the rate of post-in vitro fertilization with leptin alone was null compared with the post-in vitro fertilization rates obtained with follicular fluid (51%) and progesterone (46.15%) [141]. Leptin was also able to improve sperm cryopreservation, through activation of certain antioxidant enzymes [152], illustrating that in non-pathological conditions, it may be involved in the homeostasis of ROS scavenging. These findings suggest that leptin has a role in sperm capacitation and survival but not in sperm penetration. However, the molecular mechanisms behind these actions are still unknown.

Overview of leptin signaling and its relevance in the male reproductive tract

Leptin signaling is mainly mediated by the long isoform of leptin receptor, LepRb. This isoform is expressed in specific areas of the central nervous system involved in the regulation of feeding and energy expenditure [153]. After being secreted by the white adipocytes, leptin is released into the bloodstream and circulates linked to LepRe [154]. At the central nervous system, leptin surpasses the blood–brain barrier through a saturable process. Once in the basal hypothalamus, leptin binds to the extracellular domain of LepRb counteracting orexigenic factors, such as NPY and AgRP, and enhancing the actions of the anorexigenic peptide alpha-MSH/proopiomelanocortin (α-MSH/POMC) and cocaine- and amphetamine-regulated transcript, thus inhibiting appetite [155,156]. Leptin binding to the LepRb causes a conformational change in the receptor that, in turn, enables JAK2 activation, which phosphorylates other tyrosine residues located in the LepRb–JAK2 complex triggering several downstream signaling pathways [157]. So far, three tyrosine residues were identified in the intracellular domain of LepRb, Tyr985, Tyr1077 and Tyr1138. Phosphorylation of each one of these residues sets in motion a different set of downstream signaling proteins.

Tyr1138 phosphorylation recruits STAT3 that is later phosphorylated by JAK2. After this, STAT3 dimerizes and translocates to the cell nucleus, where it acts as a transcription factor of certain genes such as SOCS3, an inhibitor of LepR signaling. SOCS3 is a pivotal part of the negative feedback loop mechanism that regulates leptin signaling, binding to Tyr985 and inhibiting JAK2, restraining its activity (Figure 2) [158]. In fact, SOCS3 hypothalamic overexpression has been suggested as one possible mechanism for the development of leptin resistance, a hallmark of obesity [159]. In adipose tissue, this seems to be the primary self-regulatory mechanism between leptin and its receptor through which leptin levels are kept within normal ranges. Evidence for this mechanism was also found in the prostate [160]. However, there are no reports that this self-regulatory mechanism occurs within the testis.

Representation of leptin signaling pathways.

Figure 2.
Representation of leptin signaling pathways.

Circulating leptin bounds to the long isoform of leptin receptor (LepRb), which activates JAK2 tyrosine kinase, triggering a cascade of downstream signaling pathways. Activated JAK2 induces the phosphorylation of Tyr1138 and Tyr1077 located in the LepRb, leading to the activation of STAT3 and STAT5. Phosphorylation of Tyr985 recruits SHP2 and GRB2, which ultimately results in the activation of ERK signaling. On the other hand, leptin also activates PI3K pathway and mTOR signaling resulting in the inactivation of FoxO1 and activation of p70s6k kinase, respectively. In contrast, leptin signaling inhibits AMPK activity through phosphorylation of serine 491 by p70s6k kinase. →: stimulation; ┤: inhibition. JAK2: janus kinase 2; STAT: signal transducer and activator of transcription; SOCS3: suppressor of cytokine signaling 3; POMC: proopiomelanocortin; AGRP: agouti-related peptide; SHP2: tyrosine–protein phosphatase non-receptor type 11; RSK: ribosomal s6 kinase; mTORc1: mammalian target of rapamycin complex 1; p70s6k: ribosomal protein S6 kinase beta-1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT: protein kinase B; FoxO1: forkhead box protein O1.

Figure 2.
Representation of leptin signaling pathways.

Circulating leptin bounds to the long isoform of leptin receptor (LepRb), which activates JAK2 tyrosine kinase, triggering a cascade of downstream signaling pathways. Activated JAK2 induces the phosphorylation of Tyr1138 and Tyr1077 located in the LepRb, leading to the activation of STAT3 and STAT5. Phosphorylation of Tyr985 recruits SHP2 and GRB2, which ultimately results in the activation of ERK signaling. On the other hand, leptin also activates PI3K pathway and mTOR signaling resulting in the inactivation of FoxO1 and activation of p70s6k kinase, respectively. In contrast, leptin signaling inhibits AMPK activity through phosphorylation of serine 491 by p70s6k kinase. →: stimulation; ┤: inhibition. JAK2: janus kinase 2; STAT: signal transducer and activator of transcription; SOCS3: suppressor of cytokine signaling 3; POMC: proopiomelanocortin; AGRP: agouti-related peptide; SHP2: tyrosine–protein phosphatase non-receptor type 11; RSK: ribosomal s6 kinase; mTORc1: mammalian target of rapamycin complex 1; p70s6k: ribosomal protein S6 kinase beta-1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT: protein kinase B; FoxO1: forkhead box protein O1.

Phosphorylated STAT3 also participates in the transcriptional regulation of neuropeptides mediated by leptin and involved in appetite regulation. Specifically, the pathway LepRb → STAT3 regulates anorexigenic POMC and orexigenic AgRP levels in hypothalamus, one of the key functions through which STAT3 regulates energy balance. However, concerning the orexigenic neuropeptide NPY, which is also involved in growth and reproductive function inhibition, STAT3 has not shown the same effects, suggesting that LepRb → STAT3 signaling is insufficient to cause leptin-mediated suppression of NPY and thus other signaling pathways are suggested to be involved [161].

Tyr1077 phosphorylation by JAK2 promotes the recruitment and phosphorylation of STAT5, although it is suggested that Tyr1138 is also necessary for STAT5 activation [162]. STAT5 signaling is involved in the regulation of energy homeostasis. In fact, mice with STAT5 deletion in the brain developed severe obesity accompanied with hyperphagia, hyperleptinemia and insulin resistance with alterations in the regulation of energy expenditure [163]. Additionally, several studies have associated STAT5 with the reproductive function. In fact, concerning female reproduction, exogenous prolactin administration induces STAT5 activation in areas of the brain also related with fertility [164]. Furthermore, STAT5 has an important role in the sexual dimorphism that exists in the liver [165]. However, a study with three different mice knockout populations (STAT3, STAT5 or both STAT3/STAT5) revealed that despite increased body weight in STAT3 and in STAT3/STAT5 knockout mice, fertility parameters and puberty onset presented no differences relatively to the control group [166]. These findings indicate that LepRb → STAT3 and LepRb → STAT5 pathways are not crucial for regulating fertility, suggesting the involvement of other pathways.

Finally, protein tyrosine phosphatase 2 (SHP2) is recruited to phosphorylated Tyr985 (Figure 2). Activation of hypothalamic extracellular signal-regulated kinase (ERK) ensues, mediating some of the physiological effects of leptin action [167]. ERK signaling involvement in energy homeostasis was first suggested when ERK activation was observed in the ARC and other areas of the brain during fasting, a state reversed after food intake [168]. Pharmacological blockade of this signaling results in an inverse phenotype compared with normal leptin effects, reversing the weight-reducing effects of leptin, cementing hypothalamic ERK as crucial in the control of energy homeostasis [169]. Still, besides Tyr985 phosphorylation, other mechanisms seem to be involved in leptin-induced ERK activation, probably through some of the short isoforms of LepR whose main function and mode of action are still undisclosed [170]. Initially suggested as an inhibitor of leptin signaling, SHP2 was shown to be essential for a correct leptin signal transduction. Indeed, deletion of SHP2 from POMC neurons and forebrain promotes early onset obesity and turns the body prone to DIO [171]. These data suggest that LepRb → SHP2  → ERK signaling pathway is crucial in the maintenance of energy homeostasis, contrary to the first concepts and suggestions. However, SHP2 is involved in several signaling pathways, such as JAK/STAT signaling pathways and the insulin receptor signaling pathway among others, which makes it very difficult to assess the effective role of SHP2 in leptin signaling [172]. Besides the involvement in ERK signaling, Tyr985 also serves as a binding site for SOCS3, involved in the negative feedback loop that suppresses leptin signaling [173]. Leptin signaling mediated through the long isoform of LepR also involves other signaling pathways, such as those involving AMPK, PI3K–AKT (protein kinase B) and mTOR.

AMPK signaling and male reproductive potential

AMPK is an enzyme that functions as an intracellular energy sensor integrating several nutritional and hormonal signals in the brain [174]. AMPK is constituted by a catalytic subunit α that is present in two isoforms, α1 and α2, and two regulatory subunits β and γ. When the body is under metabolic needs (i.e. when cellular AMP:ATP ratio is increased after a decrease in ATP levels), AMPK is activated through phosphorylation of the α-subunit in Thr172 (Figure 2). Energy homeostasis is then restored, with AMPK switching on ATP-generating pathways and switching off ATP-depleting pathways. In the hypothalamus, AMPK is phosphorylated during fasting but suppressed in situations of feeding [175]. Following leptin administration, reduced AMPK activity can be observed in several areas of the brain such as ARC and paraventricular nucleus [176]. However, leptin-mediated regulation of AMPK is tissue dependent, with different responses in the muscle, liver and testicular cells [177179]. AMPK activity phosphorylates downstream targets, such as acetyl-CoA carboxylase, inhibiting them, a scenario that is inversed following leptin administration. Additionally, leptin administration inhibits AMPK activity, which in turn activates downstream targets of AMPK, such as acetyl-CoA carboxylase, observed in the hypothalamic neurons. This allows fatty acid biosynthesis and reduces food intake [180]. Several reports have revealed that leptin modulates AMPK activity through phosphorylation of specific sites in one of the two AMPK catalytic subunits (α2). Administration of leptin in wild-type mice decreased AMPK-α2 activity with increased AMPK-α2 phosphorylation specifically at Ser491. Phosphorylation of this specific serine by p70s6 kinase (p70s6k), a downstream target of mTOR, has been pointed out as the key mechanism in leptin-mediated inhibition of AMPK [181]. Furthermore, mice where AMPK-α2 subunit was knockdown showed no alterations in appetite or body weight after leptin administration [182].

Despite being already relatively well described for female reproduction, relevance of AMPK signaling in male reproduction is still a gray area of knowledge [183]. AMPK subunits are expressed in the rat testis, both in somatic and in germ cells, where AMPK-α1 subunit is predominant [184]. Additionally, AMPK subunits are also expressed in gonads of a wide variety of species [185,186]. Administration of an AMPK activator increases SCs lactate production in a dose and time dependent manner, due to an increase in glucose transporter 1 (GLUT1) expression and glucose uptake [179]. Additionally, mice knockout for spermatozoa AMPK-α1 subunit present decreased motility and an overall lower fertility rate. Knockout AMPK-α1 mice also revealed a decrease in spermatozoa mitochondrial activity and morphological defects accompanied with higher production of testosterone due to hyperactive Leydig cells [187]. Following this line of thought, SC-specific AMPK-α1 subunit knockout mice show reduced fertility coupled with abnormal spermatozoa. SC's metabolism was also highly dysregulated, with a lower ATP and higher lactate production. These changes were also corroborated by differences in mitochondrial biomarkers. Additionally, the characteristic junctions between SCs that form the BTB, protecting germ cells, were also dysfunctional [188]. Concomitantly, AMPK activation by adenosine in SCs was shown to be involved in lactate production and in BTB maintenance [189].

In spermatozoa, AMPK is found in different locations depending on the species [190192]. In humans, AMPK protein is found in the acrosome, the midpiece and in the tail of the flagellum. However, AMPK's active form (phosphorylated AMPK at the Thr172) is mainly found in the apical part of the acrosome region and in the flagellum [190]. Interestingly, in chicken, AMPK is found in the acrosome, the midpiece and in the flagellum, suggesting a role for AMPK in the modulation of the flagellum motility, which correlates with AMPK function as an energy sensor [191]. This AMPK distribution profile indicates that AMPK function in the acrosome reaction and motility of the spermatozoa differs between species. These findings point towards an important role for AMPK on modulating the nutritional support of spermatogenesis and influencing the preservation of an adequate microenvironment necessary for a successful spermatogenesis, which can mediate the effects of leptin in male reproductive tract, and hence male fertility.

PI3K–AKT signaling

LepRb activation phosphorylates several members of the insulin receptor substrate (IRS) family with the assistance of SH2B1, an activity enhancer of JAK2, which forms a complex with JAK2, SH2B1 and IRS, that promotes the activation of leptin-dependent downstream pathways (Figure 2) [193,194]. IRS family members are then phosphorylated by JAK2, which leads to PI3K activation. First reports associating this pathway with leptin signaling were observed in IRS2-deficient mice that presented a phenotype with several traits of obesity, such as high leptin levels, reduced energy expenditure and increased food intake [195]. Then, studies showed that suppressing hypothalamic PI3K pathway inhibited the anorexigenic effects of leptin, consolidating previous findings [196,197]. In fact, deletion of SH2B1 results in leptin resistance and morbid obesity in mice, while SH2B1 restoration in SH2B1 knockout mice recovers normal function, also improving leptin-dependent JAK2 downstream pathways and correcting the overexpression of orexigenic neuropeptides in the hypothalamus [193,198].

PI3K seems to modulate the effects of leptin in POMC neurons as the use of PI3K inhibitors blocks the effects of leptin [199]. However, this signaling does not seem crucial for energy homeostasis, as mice with impaired PI3K signaling on POMC neurons show normal body weight. In addition, PI3K catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), whereas phosphatase and tensin homolog (PTEN) catalyze the opposite reaction [200]. PIP3 activation leads to activation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) and AKT that inhibits the transcription factor forkhead box O1 (FoxO1) (Figure 2). FoxO1 is a downstream factor of PI3K–AKT signaling that mediates the anorexigenic effects of leptin, up-regulating the transcription of AgRP/NRY neurons and inhibiting POMC neurons [201203]. In the case of nutrition privation, FoxO1 translocates from the cytoplasm to the nucleus acting in orexigenic and anorexigenic neurons to increase food intake. Mice, where FoxO1 action in POMC neurons is severely limited, present oversensitivity to the effects of leptin, which illustrates FoxO1 importance regulating leptin-mediated effects in energy homeostasis [204]. In addition, FoxO1 also has negative effects on STAT3 signaling in both orexigenic and anorexigenic neurons, as hypothalamic overexpression of FoxO1 results in a loss of leptin function, specifically due to the arrest of STAT3-stimulated POMC promoter activity. This occurs due to FoxO1 inhibition of the interaction between STAT3 and SP1 transcription factor, which disrupts the POMC transcriptional activity mediated by leptin [205].

Similar to AMPK signaling, PI3K–AKT signaling pathway has some well-established functions in female reproductive tract and its involvement in female reproduction has been described [206]. Concerning male reproduction, some links between this pathway and the levels of key hormones required for a normal male reproductive function, such as FSH, relaxin and estradiol, have been established. A study by Khan and colleagues [207] based on the administration of FSH, an essential hormone in spermatogenesis, has shown to amplify PI3K–AKT signaling mediated by insulin-like growth factor 1 (IGF-1) in immature rat SCs, which could be an intracellular mechanism required for the proliferation and differentiation of SCs. Furthermore, in mature rat SCs, FSH administration increased phosphorylated AKT levels. Administration of a PI3K inhibitor partially inhibited FSH ability to stimulate SC's lactate production that was coupled with a decrease in the ability of FSH to stimulate lactate dehydrogenase activity and glucose uptake [208]. However, FSH action on phosphorylated AKT levels and mature rat SC's metabolism has shown to be independent from IGF-1 regulation, as previously proposed in immature rat SCs by Khan and colleagues [209]. Further studies have confirmed PI3K–AKT signaling pathway's involvement in the stimulation of lactate production by FSH [210]. These studies indicate that PI3K–AKT pathway has a clear and significant role on the action of FSH in SCs, specifically in modulating glucose uptake and lactate production, which is crucial for germ cell development and spermatogenesis. PI3K–AKT pathway is also involved in the proliferation of SCs, as the administration of thyroid hormone inhibits SC proliferation through suppression of PI3K–AKT signaling [211]. An opposite result is observed following estradiol administration, which promotes SC proliferation and strengthens the hypothesis for a role of this pathway in SC proliferation, an event that is fundamental for the adult reproductive potential [212]. In seminiferous tubules, insulin and IGF-1 stimulated calcium uptake and glucose and amino acid transport. Administration of a PI3K–AKT inhibitor has nullified these effects, suggesting the involvement of this pathway in these hormonal effects [213]. Overall, there is compelling evidence that this pathway, which is modulated by leptin, has a crucial role in several processes that determine the success of spermatogenesis and the reproductive health of males.

mTOR signaling at the interplay between leptin signaling and male reproduction

mTOR is constituted by a conserved large serine/threonine kinase associated with several other proteins giving form to two distinct complexes, mTORC1 and mTORC2. Rapamycin, an mTOR inhibitor, acts differently on those mTOR complexes being mTORC1 the most prone to rapamycin action. mTOR is a central regulator of several cellular processes, including protein and lipid synthesis, cell growth, proliferation, insulin signaling and autophagy [214]. In the hypothalamus, mTOR is activated through PI3K–AKT signaling [215].

Similar to what was described for several players of the other signaling pathways discussed above, mTOR and p70s6k are expressed in AgRP/NPY and in POMC neurons [216]. Deletion of p70s6k greatly diminishes leptin effects in mice, failing to reduce food intake [217]. Moreover, p70s6k activity in the hypothalamus is also related with hypothalamic leptin sensitivity. Rats expressing active p70s6k in the hypothalamus present a greater reduction in food intake and body weight than those expressing a negative dominant p70s6k [217]. Furthermore, leptin administration increases mTOR hypothalamic activity and administration of mTOR inhibitor, rapamycin, decreases the expected effect of leptin [216]. Exposure to a high-fat diet also decreases hypothalamic mTOR signaling, indicating a potential role for this pathway in the development of leptin resistance and weight gain [218]. Altogether, these results point towards an important role for mTOR in leptin signaling, although the molecular mechanisms remain unknown.

Despite the energy requirements of spermatogenesis and the fact that male reproduction heavily depends on metabolic processes including insulin and glucose metabolism, studies associating mTOR with male reproduction are scarce. Nevertheless, there are some evidence that mTOR signaling influences SCs function and male fertility. First associations of mTOR with fertility began after reports that males under rapamycin treatment, used as an immunosuppressant for transplants, exhibited a lower sperm count and a decrease in sperm motility, vitality and viability [219]. Later studies also confirmed a decrease in testosterone levels with an increase in FSH and LH levels after rapamycin treatment [220]. Normalization of sperm and hormone parameters was achieved after switching rapamycin to another immunosuppressant drug, suggesting a role for mTOR signaling in the HPG axis that consequently affected male fertility [221]. mTOR inactivation by rapamycin in vitro and in vivo led to spermatogonia proliferation arrest. In addition, in these cells, phosphorylation levels of p70s6k were also down-regulated after inhibition by rapamycin, suggesting a role in spermatogenesis for this intervenient in mTOR signaling [222]. Mice with defective mTORC1 also presented spermatozoa with decreased motility and an augment in the number of spermatozoa destroyed and absorbed in the epididymis. Furthermore, an impaired cellular metabolism with a dysregulated protein secretion in epidydimal epithelial cells was also observed, further indicating a role for mTOR in preserving sperm physiology and epididymis function [223]. SCs express mTORC1 and mTORC2 where each complex has different functions. Furthermore, mTOR complexes are also expressed in both germ and Leydig cells. mTORC1 is involved in the FSH-mediated proliferation of SCs that is regulated through the PI3K–AKT pathway as discussed above [224]. In addition, mTORC1 is also involved in the modulation of the nutritional support of spermatogenesis provided to germ cells by SCs and in the redox balance of in vitro cultured human SCs [225]. In turn, mTORC2 is involved in the maintenance and control of the BTB during spermatogenesis [226]. Interestingly, mTORC1 has an opposite effect on BTB maintenance, as ribosomal protein s6 stimulates its opening, compromising BTB integrity [227]. mTOR knockout in SCs promotes a loss of cell polarity resulting in an increase in abnormal spermatozoa and overall lower sperm quality. This suggests a pivotal role for mTOR signaling in these cells as its disruption leads to a deficient spermatogenesis [228]. The evidence shows that mTOR is involved in the control of spermatogenesis, testis physiology and SC's metabolism, which are key steps that determine the reproductive potential of males. However, further studies are needed to clarify the exact contribution of mTOR signaling on the role of leptin in SCs and thus in male reproductive health.

Mitochondria in male reproduction: a connection with leptin

Mitochondria are pivotal organelles present in almost all eukaryotes, being responsible for multiple important functions such as oxidative phosphorylation (OXPHOS) and the tricarboxylic acid cycle, also known as Krebs cycle. Frequently referred as the powerhouse of the cell, due to its role in the production of all ATP needed to sustain life, mitochondria are also heavily linked with the production of ROS, specifically during OXPHOS [229]. First named in 1898, curiously due to their identification during spermatogenesis, mitochondria are complex in form and structure. Mitochondria are constituted by more than a thousand proteins, varying between cell and tissue types, being the majority encoded by nuclear genes and a smaller number, 13, encoded by the maternally inherited mitochondrial DNA (mtDNA) [230]. Owing to their importance in cellular processes, they are constantly monitored which requires bidirectional pathways mediating the cross-talk between mitochondria and the nucleus response, accordingly to developmental and environmental changes. Recent studies have switched focus from the traditional roles of mitochondria in OXPHOS and Krebs cycle to other functions such as the production of ROS and their emerging role in cell signaling in both normal and pathological conditions, production of steroid hormones that are important in several processes, epigenetics and the central role in several signaling pathways [231233].

The role of mitochondria in male reproduction is heavily tied with sperm, with several proposed roles for mitochondria in sperm maturation, capacitation and motility. Furthermore, mitochondria provide energy for sperm survival and in sperm quality control through the modulation of apoptosis [234]. In fact, the presence of mitochondria in male germ cells and the fact that there are several testis-specific mitochondrial protein isoforms corroborate the importance and involvement of this organelle in testicular metabolism [235,236]. Testicular mitochondria seem to morphologically differ according to the cell type in focus, which could be due to mitochondrial fusion and fission. In fact, somatic and germ cells have distinct metabolic activities that then translate into different mitochondrial contributions not explained due to substrate availability [237,238]. One of the key functions of mitochondria in spermatogenesis is the regulation of apoptosis to ensure that a viable number of germ cells can be supported by the existing number of SCs. Several studies have shown that the deletion of pro-apoptotic proteins causes an increase in germ cells and testicular tumorigenesis [239,240]. In fact, leptin exerts anti-apoptotic activities in a variety of cell types and systems. Several studies in cardiomyocytes, using cell lines and primary cultures, reported a protective effect from induced apoptosis due to pretreatment with leptin which caused lower cytochrome c release, reduced translocation of pro-apoptotic Bax protein to the mitochondrial membrane and reduced the decrease in Bcl-2 levels [241243]. Leptin's involvement in mitochondrial biogenesis could thus influence the role of this organelle in controlling germ cell numbers through apoptosis, a hypothesis that remains to be tested. Interestingly, in human sperm, mtDNA copy number is present in lower quantities than in oocyte [244]. Low mtDNA copy numbers are important to a correct sperm function. In fact, sperm from men with oligozoospermia and asthenozoospermia contain high quantities of mtDNA [245]. It seems that the decrease in mtDNA copy numbers found in normal men is caused by the down-regulation of mitochondrial biogenesis during spermatogenesis [246]. This could indicate that the reduced numbers of mtDNA in sperm present in normal men is an evolutionary adaptation, which decreases sperm susceptibility to ROS damage that occurs during OXPHOS. ROS are highly reactive molecules that can oxidize several cell constituents such as DNA and proteins, compromising the integrity of the cell [247]. Thus, ROS levels must be kept in safe levels, and otherwise, an imbalance between ROS and the antioxidant defenses (such as catalase and superoxide dismutase) can occur. This condition of oxidative stress is a common characteristic of several pathologies including cancer and interestingly, insulin resistance [248,249]. ROS unbalance could have a detrimental effect in sperm function and overall sperm quality. In a study by George and colleagues [250], mice with a mutation in the inner mitochondrial membrane peptidase 2-like presented impaired processing of signal peptide sequences from mitochondrial cytochrome c1 and glycerol phosphate dehydrogenase 2, which caused oxidative stress, impaired spermatogenesis and subfertility due to excessive ROS production. Furthermore, the higher quantities of mtDNA found in oligozoospermic and asthenozoospermic men could explain the defects in sperm number and function and consequently male infertility that is a characteristic of these conditions. One of mtDNA copy number regulators is the mitochondrial transcription factor A (TFAM). This factor regulates the transcription of mtDNA and the expression of germ cell-specific TFAM isoforms occurs during spermatogenesis, in mice and humans [246]. TFAM could be involved in the regulation of mtDNA copy number during spermatogenesis; however, further studies are needed to support and prove this hypothesis.

As referred, mitochondria have also an important role in sperm function. In fact, after spermiogenesis most of mitochondria are lost. The remaining mitochondria are found near the midpiece of the spermatozoa [251]. Following the principle that if they are there it is because they have a purpose, otherwise they would be lost together with most of the cytoplasm in the end of spermiogenesis, they seem to be involved in ATP production. In fact, mitochondrial inhibition impairs sperm activity and mitochondrial parameters also positively correlate with sperm quality [252,253]. The administration of mutant mtDNA in mice also resulted in male infertility with similar results being obtained in humans [252,254]. However, sperm motility seems to be dependent on several energy sources, with sperm cells exhibiting an enormous versatility in their metabolism being capable of modulating their energy production according to the available substrates present in the female reproductive tract.

Leptin actions seem to be linked with mitochondria in several aspects. Numerous studies have shown that leptin actions in the hypothalamus are partially mediated by the production of ROS [255257]. An increase in ROS activates POMC neurons triggering leptin-associated effects, whereas suppression of ROS increases the activity of NPY/AgRP neurons [256]. ROS levels in POMC neurons seem to be partly mediated by peroxisomes, which are intracellular organelles known to be involved in the catabolism of very long chain fatty acids and reduction in ROS [258]. In fact, a higher number of peroxisomes were observed in POMC neurons from DIO mice when compared with lean mice. Peroxisome proliferation in the hypothalamus could be explained by the metabolic stress that ER is under in obese individuals, a well-known factor of leptin resistance. This could represent a novel mechanism of leptin resistance through the decreased activity of POMC neurons by peroxisomes, even in the presence of high leptin concentrations, leading to a diminished leptin effect. Proliferation of peroxisomes is mediated, in part, by a peroxisome proliferator-activated receptor-γ (PPAR-γ). PPAR-γ is a nuclear receptor that functions as a transcription factor regulating the expression of target genes [259]. The administration of a PPAR-γ agonist in lean mice increases the number of peroxisomes in POMC neurons accompanied by a decrease in the number of ROS and an increase in food intake [256]. Furthermore, administration of a PPAR-γ antagonist in DIO mice leads to the opposite with a lower number of peroxisomes in anorexigenic neurons, higher ROS levels and a reduction in daily food intake. PPAR-γ is highly expressed in ejaculated spermatozoa [260]. It is also expressed in somatic and germ cells, where it seems to be involved in regulating the patterns of expression of lipid metabolic genes with the ultimate goal of providing energy for spermatogenesis [261]. Furthermore, PPAR-γ has shown to have important roles in sperm capacitation, increasing the motility of capacitated spermatozoa and sperm metabolism [262]. PPAR-γ also shares signaling pathways with leptin, as PPAR-γ promoter region is downstream of the JAK/STAT pathway involved in leptin signaling [263]. Altogether, these findings demonstrate an important role of PPAR-γ regulating whole-body energy balance through changes in POMC neurons to leptin sensitivity mediated by ROS and in reproductive control, with important functions in sperm development.

Leptin can also modulate mitochondrial dynamics and biogenesis in various systems. Singh and colleagues [264] have shown that leptin administration in the liver of ob/ob mice decreases basal metabolic rate due to a reduction in mitochondrial volume density. Additionally, leptin also decreased protein levels of several substrate oxidation system components. Furthermore, lower mRNA levels of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and TFAM together with lower mitochondrial DNA content and lower mitochondrial complexes activity are present in cardiomyocytes from ob/ob mice [265]. A study by Li and colleagues also demonstrated that in wild-type mice, physical activity induces mitochondrial biogenesis, through sirtuin 1-dependent PGC-1α deacetylation, which may require AMPK activation. However, in ob/ob mice, no differences occurred between pre-training and after physical activity which could indicate that leptin is somehow required and involved in mitochondrial biogenesis. Furthermore, treatment of a myoblasts cell line with leptin resulted in increased AMPK phosphorylation and PGC-1α deacetylation [266]. In another study, wild-type and ob/ob mice were administrated with leptin. Before leptin administration, the liver and oxidative soleus muscle of ob/ob mice presented reduced expression of TFAM, a state reverted after leptin treatment which corroborates the hypothesis of leptin acting as a modulator of mitochondrial biogenesis [267]. Additionally, a recent study in a cell line of breast cancer cells also demonstrated that leptin administration up-regulated genes and proteins that are involved in mitochondrial biogenesis and dynamics such as PGC-1α and TFAM [138]. Altogether, leptin seems to be tightly involved with mitochondria at a signaling level, due to the growing importance of ROS in leptin signaling. At the same time, several findings point towards a role for leptin in the modulation of mitochondrial biogenesis, which then disrupts leptin signaling and interferes with mitochondrial functions in male reproduction.

Concluding remarks

Obesity is reaching pandemic proportions. Accompanying this trend, (in)fertility problems are poised to be one of the greatest challenges of the 21st century. In fact, the need for medically assisted reproduction is becoming predominant among couples in reproductive age. Despite being overlooked in the past decades, a relationship between obesity and fertility is sustained by the most recent studies. Obesity is heavily related with changes in the metabolic profile and reproductive processes are directly linked with the metabolic status of the individual evidencing the interconnection between these problems.

Although leptin was discovered over two decades ago, recent findings suggest that leptin could be involved in the interplay between obesity and infertility. Leptin resistance is a well-known hallmark of obesity. In fact, the first studies after leptin discovery were focused on the possible role of this hormone as a putative pharmacological target for obesity. However, there is a remarkable lack of studies focused on the possible effects resulting from the presence of high leptin levels in this condition, specifically in the peripheral tissues where leptin receptor is present and leptin-mediated effects occur. The effects of leptin in the male reproductive tract are still a matter of debate, but compelling evidence points towards a role for this hormone, either through the classic signaling pathway or through other signaling pathways, as discussed. Thus, leptin can be the missing link between obesity and the associated infertility problems that arise in overweight and obese individuals. In fact, leptin effects involving the reproductive function range from being able to rescue puberty and infertility in ob/ob mice, to being considered a negative factor to spermatogenesis and steroidogenesis when in high concentrations, as well as, being indirectly involved in the control of the HPG axis by regulating the release of gonadotropins. These conclusions indicate a potential important role for leptin as a novel player behind the mechanisms related with obesity-associated infertility. Recent studies have also shown that leptin modulates mitochondrial biogenesis and the production of ATP, redirecting ATP production from glycolysis to mitochondria in cancer cells. This ability to change the metabolic profile was also observed in Sertoli cells, where leptin modulates the glycolytic profile and acetate production. These findings offer new clues of the molecular mechanisms that mediate the effects of leptin responsible for the lower reproductive parameters present in obese individuals. Nevertheless, more studies are needed to test these hypotheses, particularly if there is a shift in ATP production to mitochondria in Sertoli cells which would result in lower lactate availability for germ development or modulation of the glycolytic profile of Sertoli cells, disrupting the microenvironment needed for the correct germ cell development. These novel hypotheses open exciting possibilities that need further research and deserve special merit for the years to come. However, the first steps in trying to understand the effects of leptin in the male reproductive tract are still being done and there is still much to be discovered. It is still also unclear what are the isoforms of leptin receptor present in the testicular cells and the role of the short isoforms of leptin receptor on leptin signaling in the male reproductive tract. This could open new possibilities and contribute to the discussion whether leptin directly or indirectly regulates male fertility. These issues remain a major challenge that needs to be overcome to better understand the interactions between obesity, infertility and the possible role of leptin as an intermediary in this cross-talk.

Abbreviations

     
  • AgRP

    agouti-related peptide

  •  
  • AKT

    protein kinase B

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • ARC

    arcuate nucleus

  •  
  • BMI

    body mass index

  •  
  • BTB

    blood–testis barrier

  •  
  • DIO

    diet-induced obesity

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • FoxO1

    forkhead box O1

  •  
  • FSH

    follicle-stimulating hormone

  •  
  • GLUT

    glucose transporter

  •  
  • GnRH

    gonadotropin-releasing hormone

  •  
  • HPG

    hypothalamic–pituitary–gonadal

  •  
  • IGF-1

    insulin-like growth factor 1

  •  
  • IRS

    insulin receptor substrate

  •  
  • JAK2

    janus kinase 2

  •  
  • LepR

    leptin receptor

  •  
  • LH

    luteinizing hormone

  •  
  • MFN2

    mitofusin-2

  •  
  • mtDNA

    mitochondrial DNA

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NPY

    neuropeptide Y

  •  
  • OXPHOS

    oxidative phosphorylation

  •  
  • P450SCC

    cytochrome P450 family 11 subfamily A member 1

  •  
  • p70s6k

    p70s6 kinase

  •  
  • PGC-1α

    peroxisome proliferator-activated receptor γ co-activator 1α

  •  
  • PI3K

    phosphatidylinositol-4,5-bisphosphate 3-kinase

  •  
  • PIP3

    phosphatidylinositol-3,4,5-trisphosphate

  •  
  • POMC

    proopiomelanocortin

  •  
  • PPAR-γ

    peroxisome proliferator-activated receptor-γ

  •  
  • ROS

    reactive oxygen species

  •  
  • RSK

    ribosomal s6 kinase

  •  
  • SCs

    Sertoli cells

  •  
  • SHP2

    protein tyrosine phosphatase 2

  •  
  • SOCS3

    suppressor of cytokine signaling 3

  •  
  • StAR

    steroidogenic acute regulatory protein

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • TFAM

    mitochondrial transcription factor A

Author Contribution

B.P.M., P.F.O. and M.G.A. conceived the idea. B.P.M. wrote the draft of the manuscript. All authors contributed to the final version and approved the submission of the manuscript.

Funding

This work was supported by the Portuguese Foundation for Science and Technology: B.P.M. [PTDC/BBB-BQB/1368/2014]; M.G.A. [IFCT2015, PTDC/BIM-MET/4712/2014 and PTDC/MEC-AND/28691/2017]; P.F.O. [IFCT2015 and PTDC/BBB-BQB/1368/2014] and UMIB [Pest-OE/SAU/UI0215/2014]; co-funded by FEDER funds through the POCI/COMPETE 2020.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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