The role of leptin in the male reproductive system
瘦素在男性生殖系统中的作用

Leptin

Leptin is a protein hormone produced from white adipose tissue by the Ob gene. Like many other hormones, leptin is secreted in a pulsatile fashion at higher levels in the evening and early morning hours (15). It is released into the bloodstream and binds to its receptors in the hypothalamus, creating a feeling of satiety, and therefore it was previously called the “satiety hormone” (1, 16, 17). Leptin maintains its functions by binding to specific leptin receptors (Ob-Rs) expressed in peripheral tissues, as well as in the brain. There are several isoforms of Ob-Rs. The Ob-Ra isoform (short leptin receptor isoform) plays an important role in the transport of leptin across the blood-brain barrier (BBB) (18). The Ob-Rb isoform (long leptin receptor isoform) is strongly expressed and mediates signal transduction in the hypothalamus, a region important for the regulation of neuroendocrine and energy homeostasis (19, 20). Leptin is also secreted in small quantities from gastric mucosa, brown adipose tissue, bone marrow, striated muscle cells, mammary gland, ovaries, brain tissue, lymph tissue, placenta, and spermatozoa (21, 22, 23). Leptin regulates its vital functions by binding to ob-Rs, which are found on neuronal and non-neuronal cells. The most important roles of leptin are to regulate fat storage, energy consumption, neuroendocrine function, immunity, reproduction, bone metabolism, angiogenesis, inflammation, growth hormone secretion, and improvement in insulin sensitivity (24, 25, 26).

Leptin receptors

Leptin receptors are divided into six types, one of them is long (ob-Rb), four short (ob-Ra, ob-Rc, ob-Rd, ob-Rf), and one soluble (ob-Re), according to the length of their domains inside the cell (27). The long, active isoform of Ob-Rb is expressed primarily in the hypothalamus, and plays an important role in the regulation of endocrine organs and energy homeostasis. Also, it has been reported that leptin receptors located in the uterine artery during the ovarian cycle and pregnancy regulate angiogenesis in uterine artery endothelial cells (28). Ob-Rb is found in all immune cells related to adaptive and innate immunity (29, 30, 31). Another study demonstrated that leptin/ob-Rb signaling plays an important role in the pathogenesis of obesity-associated neutrophilic airway inflammation in women by promoting M1 macrophage polarization (32). Lack of full-length Ob-Rb receptor in obese rats and db/db mice induces the development of early obesity phenotype. In db/db mice, the presence of a short Ob-Ra isoform with limited activity causes morbid obesity, diabetes, and developmental disorders in adolescence. Furthermore, the db/db mouse phenotype lacks leptin receptors but exhibits a significantly higher blood leptin concentration (33). The cytoplasmic domains of the long receptor contain segments capable of activating the JAK-STAT3 pathway and are found largely in the hypothalamus, and in small amounts in the lungs, pancreas, muscles, ovaries, testes, blood, kidney, heart, BBB, and sperm (2, 4). The cytoplasmic domains of the short receptor lack the segments that activate the JAK/STAT3 pathway, but it can activate leptin signals via the adenosine monophosphate kinase (AMPK) pathways, and it is found in the liver, pancreas, gonad, and BBB (4, 34). The soluble receptor lacks both cytoplasmic and membrane segments and plays a role as a leptin-binding protein in blood circulation and regulates its bioavailability and is also found in the seminal plasma (35, 36). The majority of Ob-R isoform receptors are intracellular, with only 5-25% found on the cell surface. After ligand binding, the receptors are internalized into endosomes via clathrin-coated vesicles. The receptor is broken down or recycled to the cell membrane. A decrease in Ob-Rb expression is much greater than changes in Ob-Ra expression, and the short isoform Ob-Ra is recycled much more rapidly to the cell membrane (37, 38, 39).
瘦素受体根据它们在细胞内的结构域的长度分为六种类型，其中一种是长的 （ob-Rb），四种是短的 （ob-Ra， ob-Rc， ob-Rd， ob-Rf），一种是可溶的 （ob-Re 27 ）。Ob-Rb 的长活性亚型主要在下丘脑中表达，在内分泌器官的调节和能量稳态中起重要作用。此外，据报道，在卵巢周期和怀孕期间，位于子宫动脉中的瘦素受体调节子宫动脉内皮细胞的血管生成 （ 28 ）。Ob-Rb 存在于与适应性免疫和先天免疫相关的所有免疫细胞中 （ 29 ， 30 ， 31 ）。另一项研究表明，瘦素/ob-Rb 信号转导通过促进 M1 巨噬细胞极化，在女性肥胖相关中性粒细胞气道炎症的发病机制中起重要作用 （ 32 ）。肥胖大鼠和 db/db 小鼠缺乏全长 Ob-Rb 受体会诱导早期肥胖表型的发展。在 db/db 小鼠中，存在活性有限的短 Ob-Ra 亚型会导致青春期病态肥胖、糖尿病和发育障碍。此外，db/db 小鼠表型缺乏瘦素受体，但表现出显著较高的血液瘦素浓度 （ 33 ）。长受体的细胞质结构域包含能够激活 JAK-STAT3 通路的片段，主要存在于下丘脑中，少量存在于肺、胰腺、肌肉、卵巢、睾丸、血液、肾脏、心脏、BBB 和精子中 （ 2 ， 4 ）。 短受体的胞质结构域缺乏激活 JAK/STAT3 通路的片段，但它可以通过单磷酸腺苷激酶 （AMPK） 通路激活瘦素信号，存在于肝脏、胰腺、性腺和 BBB （ 4 ， 34 ）。可溶性受体缺乏细胞质片段和膜片段，在血液循环中作为瘦素结合蛋白发挥作用并调节其生物利用度，也存在于精浆中 （ 35 ， 36 ）。大多数 Ob-R 亚型受体位于细胞内，只有 5-25% 存在于细胞表面。配体结合后，受体通过网格蛋白包被的囊泡内化到内体中。受体被分解或循环到细胞膜上。Ob-Rb 表达的降低远大于 Ob-Ra 表达的变化，并且短亚型 Ob-Ra 循环到细胞膜的速度要快得多 （ 37 、 、 38 39 ）。

Leptin signaling pathways
瘦素信号通路

Leptin causes JAK/STAT3 signal activation by binding to long receptors (ob-Rb) with intracellular signaling capabilities. JAK2 phosphorylates Tyr985, Tyr1138, and Tyr1077 tyrosine localize in the intracellular domain. Two units of STAT3 bind to phosphorylated tyrosine residues and are phosphorylated to form the STAT3 dimer. The dimer migrates to the nucleus and binds to target genes. If this signal occurs in the hypothalamus, the dimer activates cocaine- and amphetamine-regulated transcript (CART) and pro-opiomelanocortin (POMC) neurons and inhibits agouti-related peptide (AGRP) and neuropeptide Y (NPY) neurons. Moreover, the dimer causes transcription of suppressors of cytokine signaling 3 (SOCS3) which prevents excessive activation of leptin by inhibiting JAK2, so this protein is part of the negative feedback mechanism (35, 40).
瘦素通过与具有细胞内信号传导能力的长受体 （ob-Rb） 结合来引起 JAK/STAT3 信号激活。JAK2 磷酸化 Tyr985、Tyr1138 和 Tyr1077 酪氨酸位于细胞内结构域中。两个单元的 STAT3 与磷酸化的酪氨酸残基结合，并被磷酸化形成 STAT3 二聚体。二聚体迁移到细胞核并与靶基因结合。如果该信号发生在下丘脑，二聚体会激活可卡因和苯丙胺调节转录物 （CART） 和促阿片黑皮质素 （POMC） 神经元，并抑制刺豚鼠相关肽 （AGRP） 和神经肽 Y （NPY） 神经元。此外，二聚体引起细胞因子信号转导 3 （SOCS3） 抑制因子的转录，从而通过抑制 JAK2 来防止瘦素的过度激活，因此该蛋白是负反馈机制的一部分 （ 35 ， 40 ）。

When leptin binds to the receptor, a second signal pathway, ERK1/2 [also known as Ras/Raf/mitogen-activated protein kinase (MAPK)], is also activated. Tyrosine-protein phosfatase (SHP2) and growth factor receptor-bound protein 2 bind to Tyr985 residue phosphorylated by JAK2. Then the enzyme, ERK, initiates a protein chain. Afterward, the activated mammalian target of rapamycin complex 1 (mTORC1) inhibits the AMPK signal. ERK also activates MAPK (3, 40). AMPK enzyme functions as an energy sensor. When the energy level inside the cell decreases and the ATP ratio increases, AMPK is activated by phosphorylation of the α subunit, allowing amino acids and glucose to enter the cell for energy synthesis. Thus, leptin must inhibit this enzyme to secure energy consumption (41, 42). AMPK is also found in the midpiece and flagellum of the sperm and plays a role in motility modulation. Therefore, in the absence of leptin expression or if mTORC1 is deleted, AMPK is not inhibited, resulting in decreased sperm motility (43).
当瘦素与受体结合时，第二个信号通路 ERK1/2 [也称为 Ras/Raf/丝裂原活化蛋白激酶 （MAPK）] 也被激活。酪氨酸蛋白磷酸酶 （SHP2） 和生长因子受体结合蛋白 2 与被 JAK2 磷酸化的 Tyr985 残基结合。然后 ERK 酶启动一条蛋白质链。之后，雷帕霉素复合物 1 （mTORC1） 的激活哺乳动物靶标抑制 AMPK 信号。ERK 还会激活 MAPK （ 3 ， 40 ）。AMPK 酶起能量传感器的作用。当细胞内的能量水平降低而 ATP 比率增加时，AMPK 被 α 亚基磷酸化激活，使氨基酸和葡萄糖进入细胞进行能量合成。因此，瘦素必须抑制这种酶以确保能量消耗 （ 41 ， 42 ）。AMPK 也存在于精子的中段和鞭毛中，并在运动调节中发挥作用。因此，在瘦素表达不存在或 mTORC1 缺失的情况下，AMPK 不会受到抑制，从而导致精子活力降低 （ 43 ）。

The third signaling pathway that may be activated by leptin is the PI3K/AKT signaling pathway. Insulin receptor substrate (IRS) is phosphorylated and activates PI3K which stimulates the protein kinase (AKT). AKT activates mTORC1 and inhibits FoxO1, which inhibits POMC neurons and activates AGRY neurons (42, 44).
可能被瘦素激活的第三条信号通路是 PI3K/AKT 信号通路。胰岛素受体底物 （IRS） 被磷酸化并激活 PI3K，从而刺激蛋白激酶 （AKT）。AKT 激活 mTORC1 并抑制 FoxO1，后者抑制 POMC 神经元并激活 AGRY 神经元 （ 42 ， 44 ）。

In a study, it was shown that the proliferation and neuronal differentiation of neural stem cells were supported by the cooperative effect of MAPK/ERK1/2, JAK2/STAT3 and PI3K/AKT signaling pathways induced by leptin (45). In addition, these signaling pathways induced by leptin play an important role in many cyclic activities, such as development, differentiation, renewal and repair. Dysregulation of these leptin-induced signaling pathways leads to pathological processes (46). In one study, dysregulation of leptin signaling in Alzheimer’s disease was reported as evidence of neuronal leptin resistance (47).
在一项研究中，研究表明，瘦素诱导的 MAPK/ERK1/2、JAK2/STAT3 和 PI3K/AKT 信号通路的协同作用支持神经干细胞的增殖和神经元分化 （ 45 ）。此外，瘦素诱导的这些信号通路在许多周期性活动中起重要作用，例如发育、分化、更新和修复。这些瘦素诱导的信号通路失调导致病理过程 （ 46 ）。在一项研究中，阿尔茨海默病中瘦素信号转导失调被报道为神经元瘦素耐药的证据 （ 47 ）。

Leptin - hypothalamus - adipocyte axis

When we eat food, the energy obtained may be greater than the energy consumed. To maintain the balance of this energy, fatty acids and glucose in the blood are stored as triglycerides in adipocyte droplets within the white adipose tissue. After about two hours, fat mass increases and leptin is released. Leptin and insulin in the blood both bind to cognate receptors on the hypothalamus, inhibiting anabolic reactions by inhibiting neuropeptides, such as NPY and AGRP and initiating catabolic reactions by stimulating neuropeptides likely POMC and CART. POMC is cleaved by proteolytic enzymes into adrenocorticotropic, β-lipotropic, and α-melanocyte stimulating hormone (α-MSH). These hormones and CART reduce the appetite and increase energy expenditure. After energy consumption and increased lipolysis, fat mass decreases and leptin release stops. In this way, leptin plays a role in maintaining energy balance and regulating body mass (Figure 1) (4, 48, 49). As a result, blood leptin levels are positively correlated with bodily fat mass (2, 6). When a mutation occurs in the Ob gene, the energy balance may be disturbed leading to increased food intake and potentially resulting in severe obesity (Figure 1) (16).

Leptin resistance

Leptin resistance is a major biological factor in cases of obesity. Leptin resistance, in which the body becomes insensitive to leptin, will prevent the feeling of satiety and lead to increased food intake. SOCS3 and protein tyrosine phosphatase 1B (PTPB1), which are part of the negative feedback mechanism after leptin is expressed, inhibit JAK2 phosphorylation, preventing leptin overactivation. When leptin expression increases significantly in obese men, SOCS3 and PTPB1 concentrations increase significantly, permanently inhibiting leptin expression (50, 51).

All excess fatty acids combine with glycerol and are stored in adipocyte tissue in the form of triglyceride. When some people eat too much, for unknown reasons these fatty acids turn into diacylglycerol, ceramide, or acetyl-CoA and are stored in different locations, such as the liver, kidney, or hypothalamic neurons leading to lipotoxicity. In the hypothalamic neurons, these molecules cause stress of the endoplasmic reticulum. PTPB1, which is located on the surface of the endoplasmic reticulum, and SOCS3 expressions increase, permanently inhibiting leptin expression. Furthermore, endoplasmic reticulum stress in POMC neurons leads to incorrect or absent folding of MSH, so appetite is not reduced and energy is not consumed (52, 53). Moreover, in obese persons, matrix metalloproteinase 2 is activated in the hypothalamus. This enzyme cleaves leptin receptors and leads to inhibition of leptin expression (54).

Another reason for leptin resistance may be the incapacity of leptin to cross the BBB. If leptin cannot pass through the BBB, it will not reach the hypothalamus and exert its effects. A high triglyceride level inhibits this crossing of the BBB (35, 55). Triglycerides may cross the BBB and regulate central leptin receptor resistance (55). The relationship between leptin and triglycerides is not fully known, but in obese rats, fasting reduced triglyceride levels and increased leptin transport across the BBB and satiety increased triglyceride levels and reduced leptin transport across the BBB, so it is thought that the leptin transporter may have a regulative site controlled by the triglyceride (55, 56). A study showed that leptin resistance protected mice from hyperoxia-induced acute lung injury (57). In addition, in the presence of low gene expression or gene mutation in leptin receptors, leptin resistance occurs (9). Blood-testicular barrier (BTB) does not play a role in leptin resistance, indicating that Sertoli, Leydig, and germ cells are exposed to high concentrations of leptin (35).

Interaction of leptin and the male HPG axis

Leptin regulates neural pathways that have multidirectional effects, linking energy storage with other physiological activities. It plays a main and important role in regulating reproductive function and securing the vital energy needed for it (3, 36). The leptin released from adipose tissue travels through the blood and reaches the hypothalamus by transport across the BBB. It stimulates POMC, CART, and kisspeptin neurons by binding to the ob-Rb in the hypothalamic paraventricular and arcuate nuclei and inhibits NPY and AGRP, which suppress gonadotropin production. POMC, CART, and kisspeptin stimulate gonadotropin-releasing hormone (GnRH) that transfers to the anterior lobe of the pituitary gland and triggers the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH bind to their receptors in the testis inducing steroidogenesis and spermatogenesis (Figure 2) (12, 58).

Leptin affects the testis through the HPG axis and by binding directly to its receptors in the testis and sperm (59). Kisspeptin functions as a stimulator of steroidogenesis. It has been shown that an interaction between leptin and sex hormones can trigger KISS-1/GPR54 signaling to GnRH neurons, suggesting novel mechanisms regulating the onset of puberty (60). Leptin levels peak before puberty, and with the increase in kisspeptin levels, leptin has been reported to be critical for the onset of puberty in males. Studies conclusively showed that kisspeptin neurons are not direct targets of leptin at the onset of puberty. Leptin signaling in kisspeptin neurons occurs only after the completion of sexual maturation and may experience a critical window of sensitivity to the influence of metabolic factors that may alter the onset of fertility (61). Furthermore, altering neonatal leptin fluctuation may alter the timing of pubertal onset and have long-term effects on reproductive and hypothalamic expression of metabolic neuropeptides (62). It also provides the energy and the availability of fats needed for puberty, where some studies have shown that the lack of leptin in boys leads to a delay in puberty (63, 64).

Leptin, sertoli, and germ cells

Sertoli cells are supporting cells found in the epithelium of the seminiferous tubules that have an important role in regulating spermatogenesis. The Sertoli cell contains glucose transporters (GLUTs) and ob-Rb. Glucose enters the Sertoli cell and converts to pyruvate via phosphofructokinase. Pyruvate is converted to alanine through alanine aminotransferase, to lactate through lactate dehydrogenase, and, in mitochondria, to acetyl-CoA via pyruvate dehydrogenase. Through monocarboxylate transporters, lactate passes into the adluminal space and enters the germ cells. Lactate is an important energy source for the germ cell and functions as an anti-apoptotic factor through an unknown mechanism. Acetyl-CoA is then converted into acetate that also enters germ cells, but its role is still unknown (Figure 3). However, acetate is considered the most important carbon source for the synthesis of lipids and cholesterol that are necessary for germ cell division and spermatogenesis. When the leptin level increases in the Sertoli cell, lipolysis increases, and acetate is consumed to synthesize lipids again. It was also observed that when the leptin level increased, lactate was not produced. Therefore, germ cells are damaged and their concentration decreases (65, 66, 67). Thus, leptin triggers the production of factors necessary for spermatogenesis both through the HPG axis and by binding to its receptors in Sertoli cells (68). Moreover, considering the glycolytic flow suitability of Seroli cells, it has been reported that leptin affects mitochondrial physiology in human Sertoli cells and that leptin plays a role in glycolysis (68). Leptin also directly affects germ cells by binding to its receptors in these cells, as it phosphorylates STAT3, which supports stem cell renewal, proliferation, and differentiation (69).

In a study, Sertoli cells and peritubular myoid cells together form the testis microenvironment (TME). It has been shown that the differentiation of Leydig stem cells is severely impaired as a result of the loss of TME (70). This study was supported by other studies suggesting that cells within the TME are involved in the release of paracrine factors, which are very important for stimulating the differentiation of Leydig cells (71). In a study published in 2022, the important role of leptin, which is secreted by the TME and serves as a paracrine factor, on human Leydig cell differentiation and function was detected (72). In the same study, it was shown that low-level leptin treatment in cells taken from male testis biopsies with azoospermia can also increase testosterone levels and Leydig cell differentiation (72).

Leptin and Leydig cells

Leydig cells are interstitial cells located between the seminiferous tubules in the testes which are responsible for spermatogenesis, the biosynthesis and secretion of androgens, and maintaining secondary sexual characteristics in males. Leydig cells express ob-Rb. Leptin triggers the production of testosterone both through the HPG axis and by binding to its receptors in Leydig cells (25, 73). In a study, leptin was identified as an important paracrine factor released by cells within the TME, modulating Leydig cell differentiation and testosterone release from mature Leydig cells (74). When LH binds to its receptors on the Leydig cell, cAMP levels increase which causes dissociation of the catalytic unit by binding to PKA. This unit enters the nucleus and phosphorylates the GATA4 transcription factor, allowing the expression of the steroidogenic acute regulatory protein (StAR) that transfers cholesterol from the outer membrane of the mitochondria to the inner membrane to produce testosterone (74, 75). Normal levels of leptin are involved in stimulating StAR transcription factors via the PI3K/AKT and ERK1/2 pathways (76).

In order not to produce excessive amounts of steroids, cAMP is converted into AMP by phosphodiesterase. AMP activates AMPK that stops steroidogenesis by inhibiting transcription factors stimulating steroidogenesis and activating transcription factors inhibiting steroidogenesis (77). Since leptin inhibits AMPK when leptin expression is absent, AMPK is not inhibited, and sustained AMPK activity inhibits StAR expression, leading to a decrease in testosterone production (3, 42, 48, 76). A study showed that high leptin levels lead to decreased expression of cAMP-dependent steroidogenic genes (STAR and CYP11a1) in MA-10 Leydig cells (78). Furthermore, another study showed that leptin inhibits the division of prepubertal Leydig cells through a cyclin D-independent mechanism and that cyclin D1 may play a role in leptin-induced differentiation of Leydig cells (79).

The role of leptin on male reproductive function

Since leptin hormone acts by crossing the BTB, it is present in the testicular fluid and seminal plasma and has receptors in spermatozoa, sperm, germ cell, somatic cell, epididymis, Leydig cell, Sertoli cell and epithelial cells of seminal vesicles and prostate (11, 25, 80, 81). Leptin induces FSH and LH release via the HPG axis. Therefore, leptin plays a role in the production of testosterone in Leydig cells and androgen binding protein, testicular fluid, inhibin, activin, and factors necessary for spermatogenesis in Sertoli cells (Figure 2) (12, 82). In an in vitro study, it was shown that leptin application reduced oxidative stress and apoptosis of sperm and positively affected mitochondrial function and energy source (83). Therefore, when leptin is absent or present at very low concentration due to being underweight, the level of steroid hormones decreases, germ cell apoptosis and the expression of pro-apoptotic genes in the testes increase (84) and vacuolization occurs in Sertoli cells (8, 85, 86). In the absence of leptin in ob/ob mice, fertility was restored with leptin therapy (87). Furthermore, when leptin concentration is elevated, the rate of apoptosis of all testis cells and the number of abnormal spermatazoa increases, sperm motility, concentration, and progressive motility decrease, and the BTB is disrupted, especially in the VIII of seminiferous epithelium stages, which is restructured for the pre-leptotene spermatocytes to pass through to enter the stage of meiosis (84, 87, 88). Another study indicates significant morphological, hormonal and enzymatic changes in leptin-deficient mouse testes. Alterations in the enzymatic steroidogenic pathway and enzymes involved in spermatic activity support insights into the fertility failures of these animals (85). In addition, a study has shown that leptin deficiency in mice was associated with impaired spermatogenesis, increased germ cell apoptosis, and upregulated expression of pro-apoptotic genes within the testes (86). It has been reported that dysfunction of spermatogenesis in infertile men associated with varicocele was associated with an increase in leptin concentration and leptin receptor expression, and leptin had local effects on the function of testicles and spermatogenesis (89). Furthermore, a study reported that leptin and leptin receptor expression in the testicles of fertile and infertile patients is due to a systemic effect related to the central neuroendocrine system, androgen levels, or spermatogenic presence, rather than a direct effect on testicular tissue (16).

The interaction of leptin and obesity on the male reproductive system

Leptin has many effects on the reproductive system, and studies have shown that it provides a link between infertility and obesity. The obese male body is resistant to leptin. When leptin is not expressed, AMPK increases, leading to StAR production decreases, and thereby testosterone production decreases. Obesity also reduces the expression of steroidogenic factor-1 which is necessary to produce StAR and P450 side-chain cleavage enzyme which is involved in the synthesis of testosterone (3, 76, 90, 91).

When leptin concentration increases, it decreases the activities of antioxidant enzymes in the cytosol and mitochondria via the PI3K/AKT signaling pathway and increases respiratory chain enzymes in the mitochondria. When the level of antioxidant is decreased in the cytosol, oxidative stress occurs and activates the pro-apoptotic molecules BAX and BAK that enter the mitochondria by changing the permeability of the outer mitochondrial membrane. The increased ROS in the mitochondria crosses into the cytosol and damages DNA. In addition, apoptosis-inducing factor and serine protease high-temperature requirement A2 (HtrA2) from mitochondria pass to the cytosol and cause cell apoptosis by breaking DNA. HtrA2 also separates the cytoskeleton and other cell substrates (Figure 4) (2). Protamine replaces histone during spermiogenesis. This is an important process for protecting the DNA because protamine is capable of packing longer sections of DNA than histone. In an unknown way high leptin levels in sperm reduces the replacement of histone by protamine, so that a smaller number of unpackaged DNA fragments are packaged. Consequently DNA is easily vulnerable to ROS damage (Figure 4) (2, 59, 92). Thus, ROS decreases the concentration of sperm and increases the percentage of abnormal sperm (25, 93). In addition, ROS causes apoptosis of Leydig cells, Sertoli cells, and especially germ cells by damaging DNA, and in so doing also reduces sperm concentration and increases the percentage of abnormal sperm. Moreover, ROS disrupts the tight junction-related proteins (occludin, claudins, and ZO-1), disrupting the BTB. This also causes germ cell damage and negative changes in sperm parameters (88, 94).

Since too much fat accumulates around the testis, the scrotal temperature (hyperthermia) increases, causing ROS to increase (95). In obesity, when adipocytes enlarge, the blood supply to them decreases, causing hypoxia and an increase in the accumulation of macrophages in the adipose tissue, which leads to adipocyte inflammation. Under normal conditions, a small amount of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) is produced from adipose tissue, and during inflammation the levels of these proteins increase significantly, also causing ROS (96, 97, 98). The increased ROS oxidizes unsaturated fatty acids in the plasma membrane of the sperm, which leads to formation of malondialdehyde that causes DNA fragmentation and so the concentration of healthy sperm in obese men decreases. Moreover, ROS changes the phospholipid membrane of mitochondria and inhibits oxidative phosphorylation. Hence ATP and then the activity of mitochondria decreases, resulting in decreased sperm motility and progressive motility (99, 100, 101). ROS has been shown in a number of studies to disrupt the tight junction-related proteins of the BTB causing damage to germ cells and increasing the rate of apoptosis in Sertoli and Leydig cells (102, 103, 104).

In the adipocyte cell, testosterone is produced and converted to estradiol by an aromatase. Estradiol inhibits the HPG axis through a negative feedback mechanism and stimulates the proliferation of adipocyte cells. In obese men, increased adipose tissue produces high levels of estradiol which in turn inhibits the HPG axis completely. Therefore testosterone production in the testicle is greatly reduced. Moreover, because testosterone reduces triglyceride accumulation and increases lipolysis in visceral adipose tissue by inhibiting lipoprotein lipase activity, a lack of testosterone leads to increased accumulation of these tissues which causes more estradiol production (105, 106, 107). Furthermore, obese men have low levels of inhibin B, sex hormone-binding globulin, FSH, LH, and androgen receptors (59, 91, 104, 108).

There is a positive correlation between leptin and adipose tissue mass in normal men. In a state of positive energy balance, the body increases the size and number of adipocytes to store excess energy. Thus, the more adipose tissue in the body, the more leptin is released, and the man has no impaired reproductive function associated with leptin (6, 109). In individuals with homozygous Ob gene mutation (Ob/Ob), no leptin is produced. Therefore, the satiety signal does not interact with the hypothalamus and so the person continues to eat food and gains weight, and this person has reproductive failure associated with leptin deficiency (110). When the person continues to eat constantly, too much fat is stored, hence leptin is released at a high level. In this case, the body becomes unresponsive to leptin to protect itself from high leptin concentration, so the satiety signal is again not detected and the person continues to eat and gain excess weight, and this person has impaired reproductive function associated with leptin deficiency (7, 87, 111).

Clinical and experimental studies

Studies demonstrated that seminal plasma leptin and its receptors in the testis were elevated in a varicocele patient and this elevation was inversely correlated with sperm density, sperm motility, the weight of testis, the diameter of seminiferous tubules and the thickness of the seminiferous epithelium, and positively correlated with ROS levels and the rate of sperm apoptosis, and it was concluded that leptin was the cause of sperm apoptosis by raising ROS levels (13, 24, 89).

In patients with leukocytospermia, studies have shown that seminal plasma leptin was elevated and that this elevation was inversely associated with sperm motility, and positively associated with ROS levels, TNF-α levels, and rate of sperm apoptosis. Thus, leptin was the cause of sperm apoptosis by raising ROS and TNF-α levels. Leukocytes migrate to the inflammation area in the genital system in leukocytospermia and phagocytose the damaged cells. Leptin receptors are found in macrophages and monocytes. When inflammation occurs, leptin binds to these receptors, causing macrophages and monocytes to proliferate, produce and release IL-1 and TNF-α and initiate apoptosis. TNF-α activates the caspase system by binding to its receptors in damaged cells. When the macrophage phagocytoses apoptotic bodies, which formed as a result of apoptosis, ROS is released. ROS causes apoptosis of sperm. In this way, leptin increases the release of TNF-α and IL-1. These also increase leptin mRNA expression in adipose tissue for an unknown reason, which means that there is a positive relationship between them. Also, leptin receptors are found in neutrophils and when leptin binds to them, it causes ROS production. As a result, leptin contributes to immune responses affecting fertility (13, 112, 113, 114).

The leptin level was low in male Akita type 1 diabetic mice and leptin monotherapy was proven to rescue spermatogenesis in these mice. Akita mice have a mutation in the insulin 2 gene that results in hyperglycemia and eventually type 1 diabetes. In Akita homozygous mice, body mass index, testicular and seminal vesicle weights, LH, testosterone, leptin, and insulin levels are low and spermatogenesis is absent (14, 115). There is a relationship between insulin and leptin, as they converge at the PI3K signaling pathway in hypothalamic neurons. When they bind to their receptors, they initiate this signal and activate AKT, which stimulates mTORC1, which contributes to leptin secretion and inhibits FoxO1, which works against STAT3. In this way, insulin increases leptin secretion and expression (14, 29, 116). In adipocyte cells, insulin activates the vesicle containing GLUT4 through the same signaling pathway, causing GLUT4 to open and glucose to enter the cell. Moreover, insulin stimulates the formation of fatty acids by increasing the activity of fatty acid synthase and acetyl-CoA carboxylase through the same signaling pathway (117, 118). Every three fatty acid molecules combine with glycerol, which is synthesized from glucose, to form triglycerides, which are stored in lipid droplets. Thus, insulin stimulates leptin release by increasing lipid synthesis (119). In type 1 diabetes, the decrease in insulin causes the fatty acid storage to decrease, so less leptin is released from the adipose tissue and infertility occurs (120, 121). Low insulin causes infertility through both leptin deficiency and hyperglycemia, as hyperglycemia causes excess ROS production by various mechanisms (122, 123). Leptin monotherapy, in the absence of exogenous insulin, in homozygous Akita mice significantly improved reproductive system functions and rescued Spermatogenesis. Consequently, infertility in patients with type 1 diabetes is not due to insulin deficiency but to leptin deficiency (14). In summary, studies on leptin metabolism and molecular signaling mechanism are shown in Table 1.