Lifestyle factors
-
Lifestyle factors that significantly promote ROS generation and oxidative stress include cigarette smoking, alcohol abuse, narcotics, obesity, sedentary lifestyle, and diet
[123][176][123,176].
According to a study conducted by Aboulmaouahib
[177], smoking contributes more to infertility. Cigarette smoke has stable and unstable free radicals within its particles, including toxic, carcinogenic, and mutagenic substances
[178]. As well as directly producing reactive oxygen radicals, smoking may also indirectly enhance OS by decreasing antioxidant defense systems
[179][180][179,180]. Cigarette smoke enhanced the generation of O
2•
− and H
2O
2, resulting in damaging the cells’ lipid membranes, proteins, enzymes, and deoxyribonucleic acid (DNA)
[181]. Following a 13-week treatment, cigarette smoke reduced the population of Leydig cells in male rats and increased lipid peroxidation compared to the control
[182]. Nicotine, found within cigarettes, downregulates the expression of nuclear receptor subfamily 5 group A member 1 (NR5A1), P450scc/CYP11A1, as well as 3-beta-hydroxysteroid dehydrogenase 1 (3β-HSD1) and steroidogenic factor 1
[183][184][183,184]. Additionally, nicotine (50 µM) prevented the growth of immature Leydig cells, induced apoptosis, and obstructed the mitochondrial membrane potential, as well as reduced the rate at which cellular enzymes responsible for steroidogenesis and steroid biosynthesis (steroidogenic enzymes) responded to the external stimulus for gene expression (downregulation) in vitro
[184]. Furthermore, an in vivo study demonstrated that that Leydig cells exposed to nicotine showed reduced levels of LH and FSH, Leydig cell numbers, and steroidogenesis
[184]. In addition, smoking can stimulate proinflammatory leukocytes, which elevate levels of ROS with the response of neutrophils, macrophages, and eosinophils to the inflammation resulting from smoking
[185]. Nevertheless, the pathway of smoking cytotoxicity is complex due to the fact that tobacco smoke has different chemical compounds, such as nicotine (C
10H
14N
2), tar, carbon monoxide (CO), and heavy metals
[69].
Alcohol intake enhances the formation of ROS by boosting the activity of cytochrome P450 enzymes, altering specific metal levels (especially free iron or copper ions), and eventually, lowering antioxidant levels, thereby resulting in oxidative stress
[186]. Chronic alcohol intake decreases blood testosterone, LH, and FSH levels by interfering with the neural and endocrine systems’ interconnections
[187][188][187,188]. In another study, the chronic intake of alcohol resulted in low levels of testosterone and progesterone levels, whilst increasing LH, FSH, and prolactin levels
[189]. Chronic alcohol intake also significantly increased thiobarbituric acid-reactive substances (TBARS), superoxide dismutase, and glutathione S-transferase, with a decrease in GSH, ascorbic acid, catalase, glutathione reductase, and glutathione peroxidase
[187]. The detrimental effect of alcohol on the serum testosterone levels may be due to increased oxidative stress, which can harm Leydig and supporting Sertoli cells, as well as impair the HPG axis
[187].
The use of numerous recreational drugs, such as cannabis, opioids, and narcotics, may impair male fertility, as they cause hypogonadism by interfering with the HPG axis
[123][190][123,190]. Additionally, recreational drug use negatively affects Leydig cells’ functions
[191]. For instance, cannabis sativa extract (marijuana) is the most commonly used illegal substance
[192]. Cannabis primarily modulates reproductive functions via the endogenous endocannabinoid system (ECS), mainly anandamide and 2-arachidonoylglycerol, that act via the cannabinoid receptors CB1 and CB2
[123][192][193][123,192,193]. Acute or chronic administration of Δ
9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the major cannabinoids present in cannabis, had no effect on testosterone production or the spatial distribution of Leydig cells compared to controls in male rats
[194]. Various studies on human males showed no difference in serum testosterone levels among marijuana users compared to non-marijuana users
[195][196][197][195,196,197]. Likewise, a systematic review indicated a non-significant relationship between long-term marijuana usage and HPG axis hormones
[198]. On the contrary, acute (˂10 joints per week) and chronic (>10 joints per week) consumption of cannabis may lower LH and testosterone levels
[199]. Furthermore, THC significantly reduced testosterone levels in testis microsomes and murine Leydig cells
[200][201][200,201]. Additionally, a reduction in the expression of LH receptors on the testis as well as the activity of 3-HSD was noted in mice fed with cannabis
[202]. Cocaine is an alkaloid derived from the leaves of many species of the Erythroxylaceae family
[192]. Chronic consumption of cocaine reduced the free testosterone concentration
[203], but remained unchanged after intravenous low-dose injection
[204], in men. In a rat model, chronic administration of cocaine did not cause any change in testosterone, FSH, and LH levels
[205]. On the other hand, intraperitoneal injection of low-dose cocaine increased testosterone levels, while the LH level remained unchanged; however, high doses caused no change in testosterone levels
[206]. Further studies are necessitated to understand the effect of the various recreational drugs on Leydig cells and the male reproductive functions, as well as their mechanisms of action.
The Western diet is characterized by energy-dense, refined, and nutritionally deficient foods, such as high-energy sugars, trans-fatty and hydrolyzed fatty acids, omega-6 polyunsaturated fatty acids, and processed foods, as well as a reduction in the intake of fruits and vegetables, omega-3 polyunsaturated fatty acids, important micronutrients, antioxidants, and phyto-compounds
[207]. Obesity, caused by relative overnutrition and a sedentary lifestyle, has emerged as a serious public health concern in recent decades
[208]. A high-fat diet (HFD) is the most common cause of obesity
[208]. Obesity alters various components of HPG in men, lowering testosterone synthesis and thereby impairing sperm production
[209][210][211][209,210,211]. In one study, HFD reduced the steroidogenic capacity of Leydig cells of rats and serum testosterone levels
[208]. In another study, HFD decreased IL-1β levels and increased testosterone in mice treated at the immature stage (TIS) but had the opposite effect in mice treated at the mature stage (TMS). Furthermore, IL-1β reduced testosterone secretion by downregulating P450scc and P450c17 gene expression. In addition, HFD reduced the number of macrophages in the testis as well as the expression of inflammasome-related genes and proteins in mice TIS
[208]. IL-1β, which is found in testicular macrophages and/or Leydig cells, is a proinflammatory cytokine that promotes the release of several cytokines/chemokines, including IL-6, IL-8, IL-10, IL-13, MCP-4, and TNF-α
[212]. Excess adipose tissue increases insulin resistance and plays a significant role in the development of oxidative stress, affecting reproductive pathways and sperm function
[213]. In addition, excess adipose tissue increases the activity of aromatase, an enzyme responsible for converting testosterone to estrogen, that consequently results in a decreased testosterone level and impairment of the spermatogenesis
[214]. Taken together, modifications of the respective lifestyle factors that minimize ROS formation and oxidative stress may enhance the functioning of Leydig cells, thereby improving sperm function and male fertility.
5. The Effects of Oxidative Stress on Leydig Cell Functions
Oxidative stress arises due to the imbalance between the generation of oxygen-containing free radicals and cellular antioxidants, thus overpowering the scavenging capability of the intracellular antioxidant defense system. It results in Leydig cell lipid peroxidation, lipoprotein injury, misfolded proteins, DNA fragmentation, inhibition of steroidogenic enzymes, and contributes to male infertility
[215].
Lipid peroxidation negatively affects the structure and integrity of Leydig cells by transforming the permeability of membranes, which results in defective membrane receptors, reduced membrane-bound enzyme activities, and high rigidity of the Leydig cells’ membrane, ultimately reducing the membrane’s fluidity
[8]. Free radicals induce lipid peroxidation, which in turn activates peroxide-metabolizing enzymes. The activity of peroxide-metabolizing enzymes can be reduced by suppressing gonadotropins through one of the following: the testosterone- or the gonadotropin-releasing hormone antagonist treatment. However, gonadotropin suppression results in sex cell atrophy, which might elevate degradation of lipids within seminiferous tubules and simultaneously reduce the Leydig cell number
[216].
Oxidative stress also decreases testosterone production from damaged Leydig cells or causes injury to parts of the endocrine system, such as the adenohypophysis
[129]. Under normal conditions, mitochondrial respiration, catalytic reactions of the steroidogenic cytochrome P450 enzymes, and steroid synthesis produce ROS in high concentrations. The excessive production of ROS induces OS, which impairs steroid production and causes injury to the Leydig cell mitochondrial membrane
[7]. Should toll-like receptors expressed in Leydig cells fail to activate, testosterone production may be impaired
[217]. Ultimately, programmed cell death of Leydig cells results from acute toxic disruptions of Sertoli cells and disturbs the testes’ microvascular networks, affecting the secretion of testosterone. Furthermore, the dysfunction of seminiferous tubules corresponds with the reduced Leydig cell number
[218]. Leydig cells have receptors for insulin-like growth factor binding protein I (IGF-I) and platelet-derived growth factor A (PDGF-A). An insufficiency of IGF-1 reduces the testes and lowers the levels of serum testosterone testes’ size, lowering serum testosterone levels
[219], while the deficiency of PDGF-A leads to a continuous decrease in testes’ size, dysfunction of germ cell genes, which induces a complete altered spermatozoa development (spermatogenesis arrest), and the absolute absence of matured Leydig cells
[218].
Furthermore, mitochondrial DNA (mtDNA) is susceptible to oxidative damage, even at low production levels of oxygen-containing free radicals, due to it being located near the sites of ROS generation. During mitochondrial respiration, several reactive species are produced, which could lead to mutations such as base substitutions, missense mutations, and deletions
[68]. The mutations impair the mitochondria’s capability to perform their vast number of metabolic roles, such as synthesizing ATP via the process of electron transport-linked phosphorylation. The repair process of mtDNA decelerates due to continuous subjection to oxidative harm. Moreover, oxidative harm can increase the likelihood of mitochondrial permeability transition pores. The damage can, therefore, activate an apoptosis cascade that promotes Leydig cell apoptosis, resulting in fewer Leydig cells, and subsequently leading to insufficient testosterone levels
[42], impairments of spermatogenesis, and the synthesis of immature spermatozoa, with a loss of sperm mobility, viability, and capacity for fertilization
[220].
6. Mechanism of Action of Oxidative Stress on the Leydig Cell Functions
Endogenous and exogenous factors that increase the levels of ROS in the male reproductive tract can cause an imbalance in the synthesis of oxidants and the scavenging ability of antioxidant enzymes, consequently resulting in OS. Leydig cells produce ROS from several sources, including mitochondrial ETC, and mitochondrial and microsomal cytochrome P450 enzyme reactions
[26]. OS has been shown to suppress Leydig cell steroidogenesis via decreased transcription of the StAR protein and the subsequent transport of cholesterol into the inner mitochondrial membrane for conversion into 17-hydroxypregnenalone
[129]. ROS, specifically H
2O
2 derived from Leydig cells and testicular macrophages, cause mitochondrial dysfunction-mediated steroidogenesis to collapse via decreased transcription of steroidogenic enzymes, specifically P450 cholesterol side-chain cleavage (P450scc), 17-hydroxylase/17, 20-lyase (CYP17), and 3-hydroxysteroid dehydrogenase/54 isomerase (3-HSD)
[221]. OS in Leydig cells is also associated with lower levels of cellular antioxidant enzymes, including SOD, GPx, and CAT, as well as the onset of apoptosis
[222].
7. Impact of Oxidative Stress on Endocrine Axes
Elevated amounts of ROS may disrupt the endocrine axes (HPA, HPG, and HPT) and their crosstalk
[223]. When ROS levels increase, cells respond by releasing stress hormones, i.e., 17-deoxycortisol (in animals) and cortisol/the stress hormone (in humans), which are activated by the HPA axis. These stress hormones signal to cross-communicate between HPA and HPG axes, reducing the release of LH by the adenohypophysis
[224]. The excessive production of ROS activates HPA, which causes the hypothalamus to release CRH, that in turn activates secretion of adrenocorticotropic hormone (ACTH) by the frontal lobe of the hypophysis. ACTH activates the adrenal gland to secrete cortisol in response to OS, decreasing luteinizing hormone and follicle-stimulating hormone secretion from the adenohypophysis
[178]. A miscommunication between the HPG and HPA axes further obstructs an increased concentration of LH receptor expression and enzymes involved in steroidogenesis and steroid biosynthesis
[76]. The reduced LH release results in a failure to activate sufficient synthesis of testosterone by Leydig cells
[225], while reduced FSH negatively affects androgen-binding protein (ABP) secretion by the Sertoli cells. This causes a net decrease in the flow of testosterone due to drastic OS
[25], resulting in unregulated spermatogenesis, and may also suppress sexual behaviors
[129]. Excess ROS production has also been demonstrated to impair LH signaling by mediating oxidation-sensitive MAPK pathways and inhibiting the mitochondrial cholesterol transport
[226]. Additionally, FSH and human chorionic gonadotropin (hCG) activate the generation of ROS through cellular metabolism, negatively affecting the differentiation processes of germ cells. ROS production may be in response to LH, as Leydig cells exposed to LH had increased ROS levels and DNA damage
[41].
Leydig cell dysfunction lowers triiodothyronine (T3) production, which reduces the general testosterone level
[129]. Oxidative stress may also impact the hypothalamic–pituitary–thyroid (HPT) axis by decreasing the release of triiodothyronine (T3) and triiodothyronine (T4). A reduction in T3 lowers the levels of StAR, messenger ribonucleic acid (mRNA), and proteins in Leydig cells, and reduces the generation of testosterone
[7]. In addition, severe OS causes insufficient concentrations of insulin to be secreted. In return, the thyroid gland fails to release T3, which results in an inadequate low circulating testosterone
[25], and a failure to modulate the normal production of spermatozoa. One study indicated that insufficient levels of testosterone result in repressed sexual behavior in men, thus disturbing the endocrine and reproductive functions and contributing to male infertility
[129]. Additionally, obesity results in excessive synthesis of oxygen-containing free radicals, which activates the activity of adipocytes to generate satiety hormone (leptin) and prevents the HPC axis activity. In addition, excessive ROS decreases insulin production from the pancreas, resulting in decreased testosterone production
[178].
Consequently, the reduced testosterone fails to adequately regulate spermatogenesis, resulting in insufficient mature spermatozoa. It decreases sexual behavior as well as fails to maintain proper growth of accessory reproductive organs, which are important in sperm maturation
[129], and may ultimately negatively affect male fertility.
Medicinal plants contain phytochemicals that have been shown to have protective effects on Leydig cells against ROS-induced damage due to their antioxidant activities
[78][158][183][78,158,183]. The treatment must, however, be of an optimal dosage and duration, as excessive amounts of antioxidants can lead to reductive stress and cause adverse damage
[227]. This implies that increased concentrations of antioxidants may be cytotoxic to Leydig cell function and obstruct neutralization or scavenging of free radicals. Hence, a balance between ROS production and antioxidant levels is essential for optimal functioning of Leydig cells
[228].