Reactive oxygen species and male reproductive hormones

Over the past 40 years, reports regarding the decline in semen quality [1‐4] and its probable consequences on male fertility have encouraged studies about the effects of environment and lifestyle factors on the male reproductive potential. Reactive oxygen species (ROS) produced by exogenous and endogenous factors are highly reactive oxygen derivatives with half-lives in the nano- to milliseconds range. These molecules reportedly play a key role in altering male reproductive functions [5, 6]. Lifestyle modifications, technological advancements, escalating levels of pollution, alcohol consumption, smoking of cigarettes and vaping, and physical stress are among the prime exogenous causes of ROS production [7‐9]. Also, multiple mechanisms involving metabolism in the cell membrane, mitochondria, peroxisomes, and endoplasmic reticulum can produce endogenous ROS [7, 9].

Antioxidants defend against excessive ROS levels through enzymatic (superoxide dismutase, catalases, and peroxidases) and non-enzymatic (vitamins, steroids etc.) mechanisms [7, 10]. In cases where the imbalance between oxidants (ROS) and antioxidants leans towards the oxidants, oxidative stress (OS) occurs, which puts the cells and the body under stress. As a result, excessive ROS can induce lipid peroxidation, disrupt DNA, RNA as well as protein functions in the spermatozoa and other testicular cells [10].

High ROS levels can increase the possibility of infertility not only directly by inducing OS, but also indirectly by acting through the hypothalamic axes of hormone release [11‐13]. ROS reduce male sex hormone levels and disrupt the hormonal balance that regulates male reproductive functions [14], and thus causes infertility. These “endocrine disruptors” not only interfere in the communication between testis and the hypothalamic-pituitary unit, they also disrupt the cross-talk between the hypothalamic-pituitary-gonadal (HPG) axis with other hypothalamic hormonal axes [15, 16]. The testis, as the primary male sex organ, is not only concerned with spermatogenesis, but also with the secretion of several hormones [17] which are required for regulation of gonadotropin secretion, spermatogenesis, formation of male phenotype during sexual differentiation, and normal sexual behaviour [18]. Hence, by interfering with normal hormonal release, ROS disrupt these essential reproductive functions.

Therefore, this review precisely elucidates (a) the role of ROS, generated by various exogenous and endogenous factors, in disrupting hormone secretion by interfering in the endocrine pathways, as well as in their cross-talk, (b) hormonal regulation of the oxidative status of male reproduction, and (c) a possible mechanism of action of ROS-induced disruption of the male reproductive hormonal profile.

The gonadotropin releasing hormone (GnRH) secreted by the hypothalamus regulates the release and secretion of gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from anterior pituitary that in turn regulate testicular functions [17]. These gonadal steroids as well as the pituitary gonadotropins, via feedback regulatory mechanisms, further establish physiological homeostasis and maintains normal reproductive functions [14, 17, 19]. FSH receptors are located on the membrane of Sertoli cells, while those of LH are on the Leydig cells. They coordinate to synthesize testosterone, maintain normal spermatogenesis, sperm health and density [19‐21].

Moreover, other hormones like estradiol (E2) and prolactin (PRL) also take part in the management of male reproductive function. E2, produced both by the testis and via the peripheral conversion of androgenic precursors, is a potent inhibitor of LH and FSH [18, 19] (Fig. 1). PRL-inhibiting GnRH secretion via modulation of dopaminergic pathway may also reduce LH and testosterone level and thus is associated with hypogonadism [22]. Dehydroepiandrosterone (DHEA) is another male reproduction ameliorating, steroid hormone secreted by the adrenal cortex [23, 24]. Inhibin A and B, dimeric hormones produced by Sertoli cells, exhibit negative feedback on FSH secretion and thus also on testicular functions [25]. Moreover, melatonin (MLT), a tryptophan-derived hormone of the pineal gland, positively regulates gonadotropin and testosterone secretion, and thus aid male reproductive functions [26, 27]. Anti-Mullerian hormone (AMH), a dimeric glycoprotein hormone produced in embryonic Sertoli cells, is structurally related to inhibin and is responsible for regression of Mullerian ducts during the first 8 weeks of embryogenesis. It reflects Sertoli cell functions and is inhibited by testosterone under the influence of LH [28‐30]. Interactions between the hypothalamo-pituitary-thyroid (HPT) and HPG axes potentially influence testicular development, mostly by the participation of thyroid hormones and FSH [31].

Thus, besides the central control through the HPG axis, the major male reproductive hormones act either individually or via the cross-talks among different endocrine axes to influence male reproductive functions. Consequently, any disruption to these networks may adversely affect male fertility.

Reactive oxygen species (ROS), which are short-lived, unstable, and highly reactive species containing at least one oxygen atom, are able to snatch electrons from other molecules to achieve an electronically-stable state. In this process, the other molecule loses an electron following which a new radical is formed. Subsequently, this radical reacts with another neighbouring molecule, thus passing on the radical status via a reaction called ‘radical-chain reaction’ until two radicals react with one another forming a stable bond. These reactions amplify the degree of alterations in the cellular structures [32‐34].

Human spermatozoa contain abundant mitochondria, particularly in its midpiece [35]. An NADH-dependent oxidoreductase (in the inner mitochondrial membrane) and NAD (P) H-oxidase (in the plasma membrane) are two main sources of superoxide (O₂^●-) [32, 33, 36]. The majority of ROS generated in human spermatozoa is O₂^●- which is a product of oxidative phosphorylation by addition of an electron to intracellular oxygen and is created between complex I and III of the electron transport chain [37]. H₂O₂ is an uncharged, membrane permeable molecule which has been found to be the major initiator of peroxidative damage of the plasma membranes of germ cells [34]. In the presence of transition metals, such as iron (Fe³⁺) and copper, O₂^●- and H₂O₂ can generate the extremely reactive OH^• through the Haber-Weiss reaction, which consist of a reduction of ferric (Fe³⁺) to ferrous ion (Fe²⁺) [38]. In a subsequent second step, called Fenton reaction, Fe²⁺ is oxidized by H₂O₂ to Fe³⁺ whereby hydroxide (OH⁻) and the most reactive hydroxyl radical (OH^•) are formed. Furthermore, O₂^●- has the ability to interact with nitric oxide (NO) to form peroxynitrite (ONOO⁻), subsequent reactions of which may lead to either apoptotic or necrotic cell death [39]. In the male reproductive tract, ROS finally can be generated by one of these sources according to the above-mentioned mechanisms.

In order to produce the immense amount of energy needed for motility, spermatozoa possess numerous mitochondria in the mid-piece of the flagellum. In the mitochondria, disruption of the membrane potential leads to electron leakage in the electron transfer chain and subsequently produces ROS. The Ca²⁺-dependent NADPH oxidase, called NOX5 (encoded by the NOX5 gene) was initially detected in the human testis, but was also found to be present in the acrosomal and mid-piece regions of human spermatozoa [40]. NOX5 is a major generator of ROS and could subsequently induce OS. This enzyme is activated when Ca²⁺ binds to its cytosolic N-terminal EF-hand and causes conformational changes to the cell through OS [41]. Moreover, during spermatogenesis, the developing spermatozoa extrude their cytoplasm. When spermiogenesis is disrupted and/or excess cytoplasm is not completely extruded (excess residual cytoplasm), the excess cytoplasm will be retained around the mid-piece. Since cytoplasm contains the enzymatic machinery to produce ROS, any hindrance in the elimination of excess cytoplasm would trigger the production of intrinsic amounts of ROS in excess, which, in turn, would lead to oxidative damage of the plasma membrane and sperm DNA [42].

The prostate and seminal vesicles are the major sources of peroxidase-positive leukocytes (polymorphonuclear leukocytes (50 ∼ 60%) as well as macrophages (20 ∼ 30%)) [43, 44]. Inflammatory responses trigger these cells to generate ROS about 100-times more than it is produced under normal conditions [34, 45, 46]. This elevated ROS production is a part of the natural defense mechanisms of these cells, whereby NADPH-production through the hexose monophosphate shunt is elevated. Leukocyte participation in inflammation is closely connected with the accompanying leukocytospermia [47], a condition defined by the World Health Organization (WHO) as semen samples containing more than one million peroxidase-positive leukocytes per milliliter of semen [48]. Varicocele, a condition caused by an abnormal dilation of veins in the pampiniform plexus surrounding the spermatic cord [49], is also associated with elevated levels of seminal ROS [50].

ROS generation, which can be elicited through various exogenous and endogenous pathways, may adversely affect the male reproductive potential by interfering with the endocrine axes both individually and via their cross-talks (Table 1).

OS that occurs due to either the enhanced production of ROS or reduced availability of antioxidants may cause lipid peroxidation in Leydig cells and germ cells, damage to lipoproteins, protein aggregation and fragmentation, and steroidogenic enzyme inhibition [10]. Testicular OS causes a reduction in testosterone production, either as a result of the injury to the Leydig cells or to other endocrine structures like the anterior pituitary [201, 202]. Reportedly, normal steroidogenesis also generates ROS, which are largely produced by mitochondrial respiration and the catalytic reactions of the steroidogenic cytochrome P450 enzymes [186]. ROS generated in this way, in turn, have been identified to inhibit subsequent steroid productions, and to damage mitochondrial membranes of spermatozoa [203]. OS is associated with increased numbers of immature spermatozoa via an indirect effect on the male hormone production that is correlated with spermatogenesis [204, 205].

It has been reported that systemic hormones (FSH, LH, testosterone, E2, PRL) may regulate seminal total antioxidant capacity (TAC) [206, 207]. A positive relationship between PRL or free T4 (fT4) and a negative correlation between gonadotropins or gonadal steroids with TAC have also been shown [22]. It is evident that some hormones like testosterone and MLT may act as antioxidants to protect sperm and other testicular cells from damage induced by ROS [208, 209]. Other metabolites of the steroidogenic pathway like DHEA are reported to enhance the level of cellular antioxidants, but the proper mechanism is still unclear [210]. Direct and indirect relationships between testosterone and antioxidant levels like selenium and/or CoQ10 and between testosterone and zinc in infertile men, respectively, have been observed [207, 211]. CoQ10 can decrease FSH and LH levels [212]. A negative relationship has been found between serum level of testosterone, E2, fT4 and sperm DNA damage [213, 214]. Also, the antioxidant inhibition could affect triiodothyronine (T3), thyroxine (T4), neurotransmitter noradrenaline and increase sperm DNA damage [215]. Intramuscular or subcutaneous injection of highly purified FSH to idiopathic infertile men reduces ROS production [216] and the subsequent sperm DNA damage [217]. Although it has been reported that testosterone could produce DNA fragmentation in Sertoli and germ cells by stimulating caspase activities in Sertoli cells [218], long-term effects of antioxidants can alter FSH, testosterone, and inhibin B levels [219].

Innumerable exogenous and endogenous factors, as discussed above, can produce ROS in the male reproductive system by disrupting the balance of oxidants and antioxidants. Following the generation of ROS, the HPA axis becomes activated and releases corticosterone (in animals) and cortisol (in humans) in response to stress. These stress hormones, through the cross-talk between the HPG and HPA axes, negatively affect LH secretion from the anterior pituitary. Decreased LH fails to stimulate Leydig cells to produce enough testosterone. Decreased FSH diminishes the release of androgen-binding protein (ABP) from the Sertoli cells, and thus, an overall decline in circulating testosterone occurs during severe OS.

ROS also affect HPT axis to reduce T3 and T4 secretion. Decreased T3 reduces the levels of the StAR mRNA and protein in Leydig cells, as well as testosterone production [220]. Increased OS also decreases the secretion of insulin from the pancreas which further negatively affects T3 release from the thyroid gland and thereby testosterone biosynthesis.

Conditions such as obesity not only involve the HPA and HPT axes, it also includes several metabolic hormones that manifest ROS-induced alterations in male reproductive functions. Obesity-induced ROS can affect adipocytes to secrete more leptin, which together with insulin, negatively regulate T3-release and thereby inhibit testicular functions. Leptin, secreted by adipocytes also inhibit GnRH release from the hypothalamus.

Testicular E2 and inhibin are produced intensely during OS, which then inhibit testosterone release. Following ROS exposure, aromatase activity increases which result in more E2 production. ROS exposure is also reported to increase PRL secretion from anterior pituitary which causes decreased GnRH release. Infections in the reproductive tract can lead to the production of pro-inflammatory cytokines (TNF-α, IL-1b, and IL-6) which again inhibit both GnRH release and testosterone secretion.

Thus, through its actions on an individual hormonal axis and/or by disrupting the cross-talk among different endocrine systems, ROS can lead to decreased testosterone production as the outcome of endocrine disruption. Decreased testosterone fails to regulate spermatogenesis properly to produce enough mature spermatozoa. It also fails to maintain the normal growth of accessory reproductive organs which play crucial roles in sperm maturation. As a prime regulator of male reproductive behaviour, testosterone deficiency may lead to suppressed sexual behaviour among men. Thus, by disrupting the endocrine reproductive functions, ROS may result in male infertility (Fig. 1).

This review summarizes the alterations of the reproductive endocrinological status by numerous endogenous and exogenous sources of ROS. Pivotal hormonal regulators of male reproductive functions can be affected by the disruption of the balance between ROS production and the antioxidant defence mechanism in the male reproductive system. Uncontrolled generation of ROS may directly damage reproductive tissues or can interfere with the normal regulatory mechanisms of the HPG axis and its crosstalk with other endocrine axes, to adversely affect male reproductive functioning, thereby inducing male infertility.