key: cord-0014057-q63edbhy
authors: Gianzo, Marta; Subirán, Nerea
title: Regulation of Male Fertility by the Renin-Angiotensin System
date: 2020-10-26
journal: Int J Mol Sci
DOI: 10.3390/ijms21217943
sha: 25448f590b63d49c47861386c4ff8821ae508566
doc_id: 14057
cord_uid: q63edbhy

The renin-angiotensin system (RAS) is a peptidic system known mainly for its roles in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, several tissues and cells have been described to possess an intrinsic RAS that acts locally through different paracrine and autocrine mechanisms. In the male reproductive system, several components of this system have been observed in various organs and tissues, such as the testes, spermatozoa and seminal fluid. Some functions attributed to this local RAS are maintenance of seminal plasma electrolytes, regulation of steroidogenesis and spermatogenesis, and sperm functions. However, their specific actions in these locations are not fully understood. Therefore, a deep knowledge of the functions of the RAS at both the testicular and seminal levels could clarify its roles in male infertility and sperm physiology, and the different RAS elements could be used to design tools enabling the diagnosis and/or treatment of male infertility.

According to the World Health Organization (WHO), infertility is defined as the inability to achieve pregnancy after a year of normal sexual relationships without the use of any contraceptive method. Infertility is one of the most prevalent chronic health disorders involving young adults, caused by advanced parental age and our lifestyle among other factors [1] . Suffering one in six couples of reproductive age, this disease has a current estimated prevalence of 14% worldwide and it affects both men and women; in fact, 50% of cases are due to the female partner, and the other 50% are due to the male partner [1, 2] .

Male infertility is mainly caused by anatomical defects, genetic diseases and injuries, as well as testicular sperm and hormonal dysfunction [3] [4] [5] . The production of fully functional spermatozoa capable of movement and fertilization is under strict endocrine, paracrine and autocrine regulation. Deregulation of this hormonal control or alterations in the different key cellular communication systems can cause male infertility [4, 5] . However, the precise causes of male infertility remain unexplained in approximately 30% of cases, as many reproductive defects cannot be detected with current diagnostic methods [2] .

RAS is a communication system that is reported to play a key role in the regulation of reproductive function in both males and females [6, 7] . In the female reproductive system, the RAS is involved in many physiological and physiopathological processes, such as oocyte maturation and quality control, endometrial lining production and/or hormone production, polycystic ovary syndrome, ovarian hyperstimulation syndrome and ovarian or endometrial cancer [7] . In this review, we focus on providing an update of recent important findings of the role of RAS in male reproductive function, which is capable of the regulation of male fertility at multiple levels [6, 8, 9] . In fact, multiple RAS family members are expressed on different male reproductive tissues, such as the testes and semen, where they regulate male fertility, acting synergistically with and/or independently of systemic RAS [6] .

The RAS is a peptidic system with endocrine characteristics known mainly for its importance in the maintenance of blood pressure (BP) and electrolyte and fluid homeostasis [6] . This system is canonically considered to be a circulating hormonal system that exerts its functions through angiotensin II (Ang II) and aldosterone. This pathway is activated when renin is released by the kidneys into the bloodstream in response to decreased BP, sympathetic nervous system activation and/or sodium depletion [6] . This renin acts on angiotensinogen (AGT) of hepatic origin, forming angiotensin I (Ang I). The inactive Ang I peptide is hydrolysed by the angiotensin-converting enzyme (ACE), giving rise to the peptide with the highest activity in this system, Ang II, which ultimately exerts its action by interacting with Ang II type 1 and 2 receptors (AT1R and AT2R) [6, 10] . Ang II signalling produces several responses, such as vasoconstriction of the peripheral circulation, increased sympathetic system activity, release of vasopressin, increased cardiac chronotropism, and release of aldosterone from the adrenal cortex, which can restore blood volume and pressure. In turn, Ang II itself inhibits the synthesis and release of renin, regulating the activation of the RAS [6] .

This traditional concept of the RAS has been revised due to the discovery of new RAS family members, which has provided evidence of the existence of new non-canonical pathways of this system ( Figure 1 ). Principally, the field was redefined by the discovery of ACE2, an ACE homologue that cleaves Ang II to generate angiotensin 1-7 (Ang 1-7), which activates the Mas receptor (MasR). Later, the list of pathway members was extended when another fragment of Ang II, Ang 3-8 (also called Ang IV), was observed; Ang 3-8 acts on its own receptor, insulin-regulated aminopeptidase (IRAP, also called AT4R) [11] . Finally, the (pro)renin receptor (PRR), on which prorenin and renin act directly, was described [12] . The appearance of all these new RAS components has implied that this response system involves complex interplay among various angiotensin receptors and their signalling pathways, as well as receptor-like activity of RAS enzymes that can promote Ang II-independent intracellular enzymatic pathways [11] .

In recent years, attention has also been paid to evidence of a widespread local tissue RAS. This new concept emerged after discovery of the existence of different RAS family members in several tissues that are able to regulate several specific functions, working synergistically with or independently of the systemic RAS [6, 11] . These local RASs are mainly characterized by (1) the presence of different RAS components, such as AGT and converting enzymes; (2) local synthesis of Ang II and other angiotensins; and (3) the presence of the specific receptors. However, the local production of bioactive peptides is not necessarily dependent on local expression of all components of the local tissue RAS, since components from the circulation, such as renin, can also be taken up [7] . In fact, it has been observed that local RASs are present in a wide range of systems, organs, and tissues, such as the kidneys, brain, cardiovascular system, pituitary gland, adipose tissue, skin, adrenal gland, and female and male reproductive systems; these RASs play important roles in many physiological processes, such as, cell growth, extracellular matrix formation, vascular proliferation, endothelial function and apoptosis [11, 13] . Furthermore, local RASs can also interact with other signalling pathways, including those involving tachykinins, enkephalins, nitric oxide (NO), prostaglandins or cellular phosphatases [6, 13] . Along with the local synthesis and uptake of RAS elements by tissues, there is increasing evidence that both synthesis and uptake also occur at the cellular level, which suggests a new mechanism of action for a physiological system called the intracellular RAS [14] . This new intracellular system is characterized by the presence of different RAS components inside the cell and by the synthesis of Ang II at an intracellular site. The concept of this system is based on 1) observation of the existence of diverse isoforms of AGT and renin (as a result of glycosylation and alternative splicing, respectively) and different forms of ACE (intracellular and secreted), 2) the existence of alternative enzymes for the synthesis of Ang II (such as cathepsin and chymase), and 3) intracellular detection of these components under particular cellular conditions. In addition, these components must be able to mediate biological effects from an intracellular location to be functionally relevant [15] . In fact, changes in cell structure and gene transcription have been reported to induce the mobilization of intracellular calcium deposits and stimulate the growth of different cell types have been reported [13, 15] . In spite of that, the functional roles of the intracellular RAS in physiology and pathophysiology have not yet been fully elucidated.

Nowadays, RAS has been described as a complex system composed by the canonical axis as well as the non-canonical axes (Figure 1 ), whose components are widely expressed in all cell types and organs in humans. Therefore, RAS plays important roles in numerous physiological events, such as renal, neuronal, cardiac, pancreatic, vascular, adrenal, pituitary, cognitive, ageing-related, inflammatory and reproductive processes [16] . Notably, one of the most relevant functions of the RAS is its participation in various processes related to male fertility [7, 8, 17, 18] . Along with the local synthesis and uptake of RAS elements by tissues, there is increasing evidence that both synthesis and uptake also occur at the cellular level, which suggests a new mechanism of action for a physiological system called the intracellular RAS [14] . This new intracellular system is characterized by the presence of different RAS components inside the cell and by the synthesis of Ang II at an intracellular site. The concept of this system is based on (1) observation of the existence of diverse isoforms of AGT and renin (as a result of glycosylation and alternative splicing, respectively) and different forms of ACE (intracellular and secreted), (2) the existence of alternative enzymes for the synthesis of Ang II (such as cathepsin and chymase), and (3) intracellular detection of these components under particular cellular conditions. In addition, these components must be able to mediate biological effects from an intracellular location to be functionally relevant [15] . In fact, changes in cell structure and gene transcription have been reported to induce the mobilization of intracellular calcium deposits and stimulate the growth of different cell types have been reported [13, 15] . In spite of that, the functional roles of the intracellular RAS in physiology and pathophysiology have not yet been fully elucidated.

Nowadays, RAS has been described as a complex system composed by the canonical axis as well as the non-canonical axes (Figure 1 ), whose components are widely expressed in all cell types and organs in humans. Therefore, RAS plays important roles in numerous physiological events, such as renal, neuronal, cardiac, pancreatic, vascular, adrenal, pituitary, cognitive, ageing-related, inflammatory and reproductive processes [16] . Notably, one of the most relevant functions of the RAS is its participation in various processes related to male fertility [7, 8, 17, 18] .

Renin/ACE/Ang II/AT1R/AT2R axis is a well-known pathway as it is considered the canonical or systematic pathway, which is mainly formed by AGT; renin; ACE; Ang I and Ang II and their receptors, AT1R and AT2R. Angiotensin III (angiotensin (2) (3) (4) (5) (6) (7) (8) or Ang III) is produced from Ang II through the action of the enzyme aminopeptidase A (APA) and exerts its actions through AT1R and AT2R [6, 10] . Traditionally, the main functions of this axis were considered to be the maintenance of BP and electrolyte and fluid homeostasis. Other functions have since been attributed to the axis at the local and intracellular levels, which differ depending on the receptor that triggers the signal [6, 15] . AT1R is definitively known to operate through various signalling mechanisms, such as by increasing the intracellular levels of Ca 2+ (by increasing the influx of extracellular Ca 2+ and the mobilization of intracellular Ca 2+ ); activating various kinase pathways, including the mitogen-activated protein (MAP) kinase pathway; and activating the epidermal growth factor receptor (EGFR) in the plasma membrane. In contrast, the effects of AT2R stimulation are mediated mainly by phosphatases [19] . Therefore, the effects of AT2R-and AT1R-mediated signalling have been considered to be antagonistic [6, 10, 19] . AT1R has thus been associated with physiological responses such as vasoconstriction, the inflammatory response, cell proliferation or oxidative stress, while AT2R has been associated with processes such as vasodilation; apoptosis; and anti-inflammation, anti-cell proliferation and anti-oxidative stress processes [19] .

The proteolysis cascade of ACE2/Ang(1-7)/MasR non-canonical axis starts with ACE2. This enzyme cleaves Ang II to directly generate Ang (1-7), which exerts its action by binding to MasR. Additionally, Ang I can be cleaved by many other peptidases, especially neutral endopeptidase (NEP), which can generate Ang (1-7). Finally, this heptapeptide can also be generated via the hydrolysis of Ang I by ACE2 to form angiotensin (1-9) (Ang (1-9)), which is subsequently cleaved by NEP or ACE. However, it is important to highlight that this pathway seems to be catalytically less efficient than the ones mentioned above [20] . Different studies have observed antagonistic effects between the proteins Ang II and Ang (1-7) as well as among the receptors on which they act, the G-protein coupled receptors (GPCRs) MasR and AT1R [7] . Indeed, the beneficial effects of this axis encompass various biological processes, such as vasodilation and the stimulation of bradykinin and NO release [20] .

The Ang IV/AT4R-IRAP axis is another non-canonical axis. In this signalling pathway, Ang III is transformed into angiotensin (3) (4) (5) (6) (7) (8) , also called angiotensin IV (Ang IV), by the action of aminopeptidase N (APN) and aminopeptidase B (APB). Ang IV ultimately acts through binding to AT4R/IRAP [21] . This axis plays key roles in the regulation of cognitive functions such as learning and memory, renal metabolism, cardiovascular damage, modulation of glucose uptake into cells, and regulation of the growth of several cell types [13] .

Finally, the (pro)renin/PRR axis has been described after discovery of the existence of the renin receptor (PRR) [12] , which acts independently of the classical axis after activation by renin itself and its precursor, (pro)renin [12, 22] and it has been linked to cardiovascular, renal and degenerative diseases [22] .

The establishment of male fertility comprises a series of intricate and highly structured steps that depend on complex orchestration of communication systems, especially the RAS. In fact, numerous components of this system have been described in many organs and tissues of the male reproductive tract, including the epididymis [23, 24] , vas deferens [25] , prostate [26] , seminal fluid [27] [28] [29] , testes [9, 30] , and spermatozoa [8, [31] [32] [33] . In addition, accumulating data from in vitro, animal and clinical studies have indicated that this peptide system is involved in the correct functioning of the human male reproductive system and is frequently altered or deregulated in pathological conditions [8, 18, 22, 29, [31] [32] [33] .

The main functions of the testes are the formation of spermatozoa, or spermatogenesis, and the production of testosterone, or steroidogenesis. Considering the importance and complexity of these processes, it is easy to understand the strict endocrine, paracrine and autocrine regulation to which they must be subject and how their deregulation can contribute to male infertility [4, 5] . The first point of hormonal regulation of testicular function is the hypothalamic-pituitary-testicular axis [5] . Several members of the RAS are present in the testes, such as renin and Ang II, and are regulated by sex hormones as well as by gonadotropins [30, 34] (Figure 2 ). Likewise, various paracrine and autocrine mechanisms modulate testicular function at the local level by acting on different types of cells present in this tissue, such as Sertoli, Leydig and/or spermatogenic cells [4] . At this point, the local RAS becomes involved; this RAS is isolated from the plasma RAS by a testicular blood barrier that protects male fertility from substances such as AT1R blockers and ACE inhibitors [22] . Within this local system, numerous researchers have found evidence of the synthesis and presence of components of different axes of the RAS. processes, it is easy to understand the strict endocrine, paracrine and autocrine regulation to which they must be subject and how their deregulation can contribute to male infertility [4, 5] . The first point of hormonal regulation of testicular function is the hypothalamic-pituitary-testicular axis [5] . Several members of the RAS are present in the testes, such as renin and Ang II, and are regulated by sex hormones as well as by gonadotropins [30, 34] (Figure 2 ). Likewise, various paracrine and autocrine mechanisms modulate testicular function at the local level by acting on different types of cells present in this tissue, such as Sertoli, Leydig and/or spermatogenic cells [4] . At this point, the local RAS becomes involved; this RAS is isolated from the plasma RAS by a testicular blood barrier that protects male fertility from substances such as AT1R blockers and ACE inhibitors [22] . Within this local system, numerous researchers have found evidence of the synthesis and presence of components of different axes of the RAS. At the testicular level, after gonadotropin stimulation, the levels of Ang II and renin increase, while the expression of the genes encoding both Ang II receptors as well as the levels of the receptors decrease. On the other hand, after hypophysectomy, the levels of renin decrease, but the mRNA expression of AT1R and AT2R increases. At the cellular level, in Leydig cells, exogenous administration of gonadotropins increases the levels of Ang I and II and renin, whereas oestrogen treatment or hypophysectomy decreases renin levels.

3.1.1. The Renin/ACE/Ang II/AT1R/AT2R Axis All members of this axis have been found in the testes of various mammals, including humans ( Table 1 ). The initial studies carried out in this regard showed that the major elements of this axis, AGT [34] and renin [35] [36] [37] , are present in Leydig cells. At first it was thought that these proteins originated in the systemic RAS, since the testicular levels of AGT and renin and subsequently ACE, Ang II and its receptors increase at the beginning of puberty with the appearance of LH and FSH in the bloodstream [8, 38, 39] . Later, it was observed that their transcripts are synthesized in Leydig cells At the testicular level, after gonadotropin stimulation, the levels of Ang II and renin increase, while the expression of the genes encoding both Ang II receptors as well as the levels of the receptors decrease. On the other hand, after hypophysectomy, the levels of renin decrease, but the mRNA expression of AT1R and AT2R increases. At the cellular level, in Leydig cells, exogenous administration of gonadotropins increases the levels of Ang I and II and renin, whereas oestrogen treatment or hypophysectomy decreases renin levels.

3.1.1. The Renin/ACE/Ang II/AT1R/AT2R Axis All members of this axis have been found in the testes of various mammals, including humans ( Table 1 ). The initial studies carried out in this regard showed that the major elements of this axis, AGT [34] and renin [35] [36] [37] , are present in Leydig cells. At first it was thought that these proteins originated in the systemic RAS, since the testicular levels of AGT and renin and subsequently ACE, Ang II and its receptors increase at the beginning of puberty with the appearance of LH and FSH in the bloodstream [8, 38, 39] . Later, it was observed that their transcripts are synthesized in Leydig cells [6, 34, 40] , indicating their local production and the linked regulation between the systemic and local RAS [22] . In addition, it has been found that increases in renin levels cause increases in testosterone synthesis [38] ; however, further studies are necessary to elucidate whether such increases in testosterone are due to direct action on the PRR or signalling through the canonical pathway. Likewise, Ang I has also been found in the testes, mainly in Leydig cells [41] , serving as a substrate for ACE. In fact, the testes also contain much higher concentrations of ACE (also known as ACE1) than other organs [6, 42] . Interestingly, in the testes, two isoforms of this enzyme, somatic ACE (sACE) and testicular or germinal ACE (tACE or gACE), have been observed. Even though both are transcribed from the same gene, they differ structurally by the presence/absence of a 66-amino acid sequence [43] and consequently present different molecular structures. sACE is formed by two identical subunits, whereas tACE is formed by a single subunit [44] [45] [46] [47] and both exhibit comparable enzymatic activity [6] . The transcription of the testicular form is tissue-specific and occurs as a result of alternative splicing, alternative transcription initiation, and alternative polyadenylation [9, 48, 49] . The most notable differences between the isoforms are their locations; while tACE is present only in male germ cells [9, 50, 51] , being found in high concentrations during spermiogenesis [6, 9] , sACE is expressed in other testicular cells, such as Leydig cells and endothelial cells of the testicular interstitial tissue [9] , and in a soluble form in seminal plasma [42] . Although the concentration of sACE in the testes is among the highest in all organs the specific role of sACE in the testes remains unknown. sACE-deficient mice are fertile and the testes might be the sources of this enzyme in seminal fluid [6] , where it protects sperm during and after transfer to females [52] .

The Ang II peptide is present in both germ cells [35] and in Leydig cells [41] . The presence of a blood-testicular barrier, together with the fact that all the components necessary for the production of this protein (renin, ACE, AGT and Ang I) are found within Leydig cells, supports the intracellular synthesis of this peptide. Ang II has been reported to be capable of inhibiting adenylate cyclase activity in rat Leydig cells, reducing basal and gonadotropin-stimulated cAMP and testosterone production [53, 54] . On the other hand, it has also been proposed that Ang II may play a role in testicular growth and/or differentiation [55] . Finally, the Ang II receptors AT1R and AT2R have also been detected in rat, monkey and human testes, specifically in Leydig cells [53, 56, 57] . However, the protein levels of both receptors, as well as the levels of their respective mRNAs, depend on age; for example, the expression of AT2R predominates in the first days of life but gradually decreases until the fourth week of life, leaving AT1R to be almost exclusively expressed [40] . It is unknown whether these receptors play a role in the regulation of testicular formation [6] . Notably, two subtypes of AT1R, AT1AR and AT1BR, have been described in the testes of mice but not in those of humans, although no specific functions have been established for either of these subtypes [21] . The presence of AT1R has also been observed in rat and human seminiferous tubules, specifically in sperm cells at different maturation stages (spermatogonia and spermatids), suggesting that this receptor could be involved in spermatogenesis [57] . Finally, Ang II, through its binding to AT1R, may interfere with testosterone production [55] .

Additional studies have shown that Ang II is transformed into Ang III by the action of APA, after which Ang III exerts its action by binding to AT1R and AT2R [6, 10] . The APA enzyme has been detected in rat testis homogenates, showing high enzymatic activity. It has been proposed that this enzyme, like Ang II, is involved in inhibiting steroidogenesis through production of Ang III and activation of AT1R [58] .

Taken together, these findings affirm that this canonical axis modulates steroidogenesis. Specifically, Ang II and III, through binding to AT1R, negatively regulate testosterone production. Moreover, AT1R may be involved in the spermatogenesis process. Although future studies are necessary, it has been suggested a positive regulation of steroidogenesis through the AT2R, considering the contrasting effects of AT1R and AT2R. All the principal components of the axis ACE2/Ang (1-7)/ MasR have been detected in rat, mouse, and human testes [32] . Single-cell RNA sequencing data on human testes showed predominant expression of ACE2 in spermatogonia, Leydig and Sertoli cells [59] , but ACE2 has been found to be localized only in Leydig cells at the protein level [60, 61] . Although Ace2-null mice are fertile [62] , men with severely impaired spermatogenesis have lower levels of ACE2 than fertile men, suggesting that this enzyme may modulate sperm formation [32] . ACE2 has also been reported to play key roles in the regulation of testosterone production and in the local vascular regulatory system, in which it balances interstitial fluid volume by modulating the conversion of Ang II to Ang I [52] . Ang (1-7) has also been identified and characterized in mouse [61, 63] , rat [63] and human testes [32] , in the cytoplasm of Leydig cells, and in Sertoli cells and primary spermatocytes at lower levels [32] . This heptapeptide is involved in the regulation of spermatogenesis, since lower levels of Ang-(1-7) have been found in men with severe spermatogenesis impairment than in fertile men [32] . Similar to this peptide, MasR has been described to be present in the cytoplasm of mouse [60, 63] , human Leydig cells and inside the human seminiferous tubules in all layers of the normal seminiferous epithelium, being equally distributed between interstitial and tubular compartments [32] . However, its mRNA has been detected in both Leydig and Sertoli cells, with its expression being more pronounced in the latter, but not in developing germ cells [60] . Concerning MasR function, MasR-deficient mice have constitutive alterations in the activity of genes encoding steroidogenic enzymes within the testes [60, 64] . This finding and the observation that MasR and Ang-(1-7) are present in Leydig cells, suggest that both MasR and Ang-(1-7) could play a key role in modulating the production of testosterone [32] . Notably, it has been reported that MasR-deficient mice show marked reductions in testis weight, a significant increase of apoptotic cells during meiosis, the presence of giant cells and vacuoles in the seminiferous epithelium, and striking reductions in daily sperm production due to disturbed spermatogenesis [63, 65] , although the total numbers of Sertoli and Leydig cells are comparable in both wild-type and knockout animals [65] . Moreover, MasR levels are notably decreased or absent under conditions of severe spermatogenesis alteration in humans, providing insights into the role of the MasR in the regulation of spermatogenesis [32] .

Although there are no data in the literature that specifically show the presence of Ang (1-9) in the testes, the presence of Ang I and Ang (1-7), as well as the enzymes ACE, ACE2 and NEP, could indicate that this peptide is present in this tissue [6, 7] . On the other hand, NEP has been detected in human testes [66] and in the membranes of rat Sertoli cells [67] , and its activity is low in testicular homogenate [66] but high in Sertoli cells [67] . It has been suggested that this metalloendopeptidase may be related to sperm maturation and proacrosin activation [66] . Nevertheless, NEP-deficient mice have normal testicular function [68] . Similarly, an isoform of this enzyme, NEP2, has been observed in mouse [69] and human testes [70] . Studies have also demonstrated the presence of NEP2 mRNA in the seminiferous tubules, specifically in developing germ cells and mainly in spermatids [71] . However, the concrete function of NEP2 in the testes remains unknown.

Taken together, these data clearly indicate that this axis plays key roles in regulating steroidogenesis and spermatogenesis (Table 1) .

This pathway begins with the action of APN or APB. The metallopeptidase APN has been localized in both human [72] and mouse testes, showing high gene expression in Sertoli and Leydig cells [73] . APN seems to play a role in the inhibition of testosterone synthesis [74] . In fact, mice lacking APN present defects in spermatogenesis and infertility, as they have altered Sertoli cell function [73] . In addition, APB has been detected in mouse testes, and it also participates in inhibition of the synthesis of this hormone [74] (Table 1) .

Although the specific function of Ang IV in the testes remains unknown, Ang IV could also be involved in inhibition of testicular testosterone production [58, 74] . Likewise, although the presence of AT4R/IRAP in the testes has not yet been described, the presence of the other components of this axis could indicate that this receptor is present in this tissue. Despite the scarcity of evidence regarding the presence and functions of the main members of Ang IV/AT4R-IRAP axis in the testes, the available data show how this axis is also related to inhibition of testicular testosterone production and, therefore, in the regulation of spermatogenesis.

Prorenin is known to be produced and secreted by the testes [75] and it has been found a correlation between sperm density and the prorenin level in semen [76] . Similarly, it has been shown that Leydig cells are capable of producing renin [6, 34, 40] and that renin levels are directly related to testicular testosterone concentrations [38] . Thus, it could be hypothesized that this axis is positively involved in steroidogenesis (Table 1) . Nonetheless, as previously described, the (pro)renin/PRR axis was described after the existence of the PRR was discovered [12] . No evidence has yet been reported showing that this receptor is present in the testes; therefore, further studies should be performed in order to demonstrate the presence of PRR and consequently the existence of this axis as well as to understand the functional role of prorenin and/or renin independent of the effects of the classical RAS cascade.

In order to acquire fertilization ability, spermatozoa must undergo a series of processes, such as motility acquisition; capacitation; the acrosomal reaction; and oocyte recognition, fusion and activation [77] . These processes must be perfectly regulated to ensure proper development. It is known that tight control is exerted by the joint actions of complex biological systems, including the RAS [21, 40, 77] . Accumulated evidence regarding the presence, distributions and specific functions of different members of this peptide system in sperm cells suggests that the RAS regulates male reproductive function by acting directly on the fertility potential of spermatozoa [8] . In addition, as has been observed for testicular function, the different RAS axes could exert opposite roles, which would result in fine modulation of reproductive function (Table 2 ). [27, 78] ; although they have not yet been specifically detected in sperm cells. sACE has also been observed in seminal plasma at higher concentrations than in blood plasma and it is positively correlated with sperm concentration and motility [40, 76] , although oligospermic patients have sACE levels similar to those of normozoospermic men [79] . Even though AGT has not been described in spermatozoa, it plays a key role in the function of these cells, since Agt-deficient male mice show reduced fertility, as their sperm cells are less likely to fertilize oocytes than those of wild-type mice [80] .

On the other hand, other RAS members, such as tACE, Ang II, and Ang II receptors, have been specifically detected in germinal cells. Specifically, tACE is present in spermatids and mature sperm of different species [81] [82] [83] , being located mainly in the acrosomal region, the equatorial segment, the postacrosomal region and the intermediate piece of sperm cells [84, 85] . Although tACE enzymatic activity is low in immature rats, it increases with sexual maturity, suggesting that its presence is dependent on sexual maturation [51, [86] [87] [88] [89] [90] . Moreover, different studies have demonstrated the involvement of tACE in sperm motility [90] [91] [92] [93] , capacitation [87, 89] , the acrosome reaction [89] and sperm-oocyte fusion [88] . Interestingly, the levels of tACE on the sperm surface differ during different phases of the fertilization process because tACE is released during capacitation [87, 89] and the acrosome reaction [89] . It has been supposed that this release may have a physiological role in the regulation of sperm physiology as well as in sperm fertilization ability [88] . In this sense, the lack of sACE in male mice does not affect fertility, while animals lacking both ACE isoforms show reduced fertility due to failure of spermatozoa transport through the oviduct and gamete fusion [94, 95] . On the other hand, tACE has been proposed to be an essential protein in the sperm-oocyte fusion process [96] , although specific tACE3-IZUMO1 binding is not required for oocyte fertilization [52] . In addition, patients enrolled in in vitro fertilization (IVF) programmes found that aberrant, reduced or absent expression of the tACE protein on sperm underlies failed fertilization [51] , and it is also involved in embryo development, since higher percentages of tACE-positive cells and fewer enzyme molecules per spermatozoon in semen samples are positively correlated with better embryo quality and development [93] .

Ang II has been widely studied in sperm cells and both Ang II receptors, AT1R and AT2R, have been identified in sperm cells. Ang II is evidently a key modulator of sperm function given that this peptide is involved in sperm motility, capacitation, and the acrosomal reaction [88, 89, [97] [98] [99] . AT1R has been detected in developing spermatids and mature spermatozoa in humans and other mammalian species [33, [98] [99] [100] [101] and is located in the tails of human sperm [100] . Targeted deletion of the AT1BR gene (Agtr1b) in mice results in high AT1BR transcriptional activity in mature and immature sperm cells [6] . Treatment with antagonists of ATIR receptor (losartan) inhibits sperm motility and Ang I and II increases not only the percentage of motile spermatozoa but also the linear velocities of the spermatozoa, which proves that AT1R is involved in sperm motility [97] . AT2R has been described in mouse [98] and human spermatozoa [33] , and it may play also an important role in sperm motility, as it has been observed that AT2R is related to human sperm concentration and motility, and the percentage of AT2R-positive spermatozoa is lower in asthenozoospermic patients (sperm samples with suboptimal motility) than in normozoospermic males [33] .

In conclusion, this canonical axis plays a key role in the proper functioning of sperm cells since it is involved in physiological processes such as sperm motility, capacitation, the acrosome reaction, and sperm-egg recognition and fusion (Table 2) (Table 2 ). Even though Ang (1-7) has not been localized in mature spermatozoa [32, 102] , incubation of seminal samples from asthenozoospermic patients with this peptide increases the proportion of progressive motile spermatozoa through MasR signalling [102] . In addition, in the sperm of asthenozoospermic patients, Ang-(1-7) may exert some effects independent of MasR, perhaps through binding to receptors such as the MRGPRD receptor (a GPCR related to MasR) or AT2R [102] . With reference to the specific receptor, it has been observed that MasR is present in human mature spermatozoa at the mRNA and protein levels [32, 102] . MasR is located in the human sperm head, with the highest levels at the acrosomal region, but also in the tail. In addition, this receptor is functional, and its activation participates in regulating sperm motility [102] .

The presence of another enzyme in this non-canonical RAS axis, NEP, has also been broadly proved. In fact, this endopeptidase has been located at higher concentrations and with stronger enzymatic activity in human seminal fluid than in other body tissues [28] . It has also been detected at the gene and protein levels in sperm cells; it is located in the neck in a small proportion of human spermatozoa [103] . Congruent with its location, this enzyme participates in the regulation of sperm motility, since its inhibition improves the motility of capacitated spermatozoa, increasing the percentage of sperm with progressive motility [103] . Moreover, NEP activity is altered in asthenozoospermic patients compared to normozoospermic men [29] . Likewise, the NEP-homologous enzyme NEP2 [69] has been described to be present around the equatorial segment in human spermatozoa, and its selective inhibition with phosphoramidon increases sperm progressive motility [71] . However, the effects observed after inhibition of both neprilysins are mediated, at least in part, by tachykinins [71] . In addition, animal studies have shown that NEP2-deficient mice exhibit less efficient egg fertilization than wild-type mice and that large proportions of oocytes do not develop normally after fertilization with NEP2-null mouse spermatozoa [68] .

The all components of the Ang IV/AT4R-IRAP axis have not yet been detected in spermatozoa. In spite of that, presence of APN, the enzyme responsible for the formation of the Ang IV peptide, could indicate that both this peptide and its receptor are present in these cells. APN is present in human seminal fluid [28] and exhibits significantly higher activity and concentrations in seminal fluid than in other body tissues [28] . In sperm, this enzyme has also been detected at the gene and protein levels; it is located at the plasma membrane of the equatorial/postacrosomal region of the sperm head, in the neck and uniformly distributed along the tail [103] . In mussel spermatozoa, this enzyme induces the acrosome reaction [104, 105] . Moreover, APN has been negatively related to sperm motility [31, 103, 106, 107] , and its activity is altered in asthenozoospermic patients compared to normozoospermic men [29] (Table 2) . Moreover, the alteration in APN activity induces cellular toxicity in sperm cells, which causes persistent damage [29, 106] and produces several adverse consequences and affecting early embryonic development in mice [106, 107] . These findings are consistent with the fact that the cumulative probabilities of having developed blastocysts and viable embryos increase by 1.5-fold when semen samples with relatively low APN levels are used during the intracytoplasmic sperm injection (ICSI) technique (Gianzo et al., unpublished data) . Strong evidences, therefore, indicate that APN play a key role in sperm fertility ability.

Large amounts of renin and its precursor, prorenin, have been detected in human seminal fluid [76, 78] . However, the putative effects of these molecules on the physiology of spermatozoa have not yet been clarified. As previously indicated, the presence of PRR is essential for the existence of the (pro)renin/PRR axis [12] . In addition, we have recently described the presence of PRR in human spermatozoa at the gene and protein levels and this receptor seems to play a role in sperm motility (Gianzo et al., unpublished data) . PRR is located mainly in the proximal region over the acrosome and in the postacrosomal region of the head; to a lesser extent, it also extends along the sperm tail. Given these novel data, along with the high concentrations of renin and its precursor, prorenin, observed in human seminal fluid [76, 78] , it can be assumed that this non canonical axis could be important in the regulation of sperm functions ( Table 2 ).

The descriptions of different elements in the canonical and non-canonical enzymatic RAS pathways in the male reproductive system indicate that both pathways may be actively present, regulating male fertility by paracrine and autocrine mechanisms. In the testes, several RAS components are present in Leydig, Sertoli and spermatogenic cells at different maturation stages, indicating that these elements can modulate testosterone production and spermatogenesis. RAS functions at the seminal level are involved in the regulation of essential processes of male fertility, such as motility acquisition, the acrosomal reaction, oocyte fertilization, and early embryonic development. However, even more interestingly, certain elements of these RAS axes, such as tACE, AT2R and APN, can aid in the diagnosis and treatment of human male fertility by acting as potential biomarkers of high-quality embryos, providing added value in the pursuit of male fertility prognosis.

Even though many functions of the RAS in male fertility are clear, numerous other relevant aspects of the male reproductive system remain unknown, such as (1) whether all elements of the RAS are present in testes, sperm and seminal fluid and the functions that they exert in these tissues; (2) whether the various components play important roles in the regulation and/or formation of testes; (3) whether the various RAS elements found in the seminal fluid play roles relevant to the survival and/or transport of sperm through the female reproductive tract; and (4) whether the levels of these components could vary under different pathological conditions. A more complete knowledge of the overall function of the RAS in the male reproductive system could improve understanding of this system in sperm and in male fertility-related physiology and physiopathology. Likewise, elucidation of RAS function could support the development of new diagnostic tools or therapeutic strategies for male infertility and the establishment of useful biomarkers for the selection of optimal spermatozoa for use in assisted reproduction techniques. 

The authors declare no conflict of interest.

Genetics of male infertility: From research to clinic

New Generation of Diagnostic Tests for Infertility: Review of Specialized Semen Tests

Mouse Models in Male Fertility Research

Regulation of Spermatogenesis by paracrine/autocrine Testicular Factors

Endocrinology of the Testis and Male Reproduction

Physiology of Local Renin-Angiotensin Systems

The Renin-Angiotensin System and Male Reproduction: New Functions for Old Hormones

Isoforms of Angiotensin I-Converting Enzyme in the Development and Differentiation of Human Testis and Epididymis

International Union of Pharmacology. XXIII. The Angiotensin II Receptors

The Renin-Angiotensin System and Cancer: Old Dog, New Tricks

Pivotal Role of the renin/prorenin Receptor in Angiotensin II Production and Cellular Responses to Renin

Renin-Angiotensin System Revisited

Intracellular Renin and the Nature of Intracrine Enzymes

The Intracellular Renin-Angiotensin System: A New Paradigm

Mas Receptor Axis: Fetal Programing, Sex Differences, and Intracellular Pathways

Tissue Renin-Angiotensin Systems and Reproduction

Devil and Angel in the Renin-Angiotensin System: ACE-Angiotensin II-AT1 Receptor Axis Vs. ACE2-Angiotensin-(1-7)-Mas Receptor Axis

Angiotensin-(1-7) and Mas: New Players of the Renin-Angiotensin System

)) Receptor is the Enzyme Insulin-Regulated Aminopeptidase

Pro)Renin and Receptor: An Update

Angiotensin II Receptors: Localization of Type I and Type II in Rat Epididymides of Different Developmental Stages

Angiotensinogen Expression by Rat Epididymis: Evidence for an Intrinsic, Angiotensin-Generating System

Characterization of Contractile Response to Angiotensin in Epididymal Rat Vas Deferens

Identification, Distribution, and Expression of Angiotensin II Receptors in the Normal Human Prostate and Benign Prostatic Hyperplasia

Angiotensin II in Human Seminal Fluid

Peptidase Activities in Human Semen

Enkephalin-Degrading Enzymes in Normal and Subfertile Human Semen

Induction of Renin-Angiotensin System in Human Testis in Vivo

Control of APN/CD13 and NEP/CD10 on Sperm Motility

-7) and its Receptor Mas are Expressed in the Human Testis: Implications for Male Infertility

Angiotensin II Type 2 Receptor is Expressed in Human Sperm Cells and is Involved in Sperm Motility

A Comparative Study of the Distributions of Renin and Angiotensinogen Messenger Ribonucleic Acids in Rat and Mouse Tissues

Evidence for Intracellular Formation of Angiotensins: Coexistence of Renin and Angiotensin-Converting Enzyme in Leydig Cells of Rat Testis

Detection of Renin mRNA in Mouse Testis by Hybridization with Renin cDNA Probe

Analysis by Immunocytochemistry and in Situ Hybridization of Renin and its mRNA in Kidney, Testis, Adrenal, and Pituitary of the Rat

Pituitary-Dependent Renin-Like Immunoreactivity in the Rat Testis

Gonadotropin-Dependent Renin in the Rat Testes

Angiotensin II: A Reproductive Hormone Too?

Regulation of Renin Angiotensins by Gonadotropic Hormones in Cultured Murine Leydig Tumor Cells. Release of Angiotensin but Not Renin

Concentrations of Angiotensin-Converting Enzyme in Tissues of the Rat

Sperm-Specific Expression of Angiotensin-Converting Enzyme (ACE) is Mediated by a 91-Base-Pair Promoter Containing a CRE-Like Element

Two Putative Active Centers in Human Angiotensin I-Converting Enzyme Revealed by Molecular Cloning

Molecular Cloning of Human Testicular Angiotensin-Converting Enzyme: The Testis Isozyme is Identical to the C-Terminal Half of Endothelial Angiotensin-Converting Enzyme

Mouse Angiotensin-Converting Enzyme is a Protein Composed of Two Homologous Domains

Structure of the Angiotensin I-Converting Enzyme Gene. Two Alternate Promoters Correspond to Evolutionary Steps of a Duplicated Gene

Transcription of Testicular Angiotensin-Converting Enzyme (ACE) is Initiated within the 12th Intron of the Somatic ACE Gene

Transgenic Mice Demonstrate a Testis-Specific Promoter for Angiotensin-Converting Enzyme

Testicular Angiotensin I-Converting Enzyme (E.C. 3.4.15.1)

Human Sperm Devoid of Germinal Angiotensin-Converting Enzyme is Responsible for Total Fertilization Failure and Lower Fertilization Rates by Conventional in Vitro Fertilization

Angiotensin-Converting Enzymes Play a Dominant Role in Fertility

Angiotensin II Receptors and Inhibitory Actions in Leydig Cells

LH Action in the Leydig Cell: Modulation by Angiotensin II and Corticotropin Releasing Hormone, and Regulation of P450(17) Alpha mRNA

Pituitary-Dependent Expression of the Testicular Angiotensin II Receptor and its Subtypes in Rats

Angiotensin II Receptors in Testes

The Distribution of Angiotensin II Type 1 Receptors, and the Tissue Renin-Angiotensin Systems

In Vivo Administration of Doxazosin in Rats Highly Decreases Serum Circulating Levels of Testosterone through a Mechanism Involving the Testicular Renin-Angiotensin System

ScRNA-Seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, A Target for SARS-CoV-2 Infection in Spermatogonia

A Novel Angiotensin-Converting Enzyme-Related Carboxypeptidase (ACE2) Converts Angiotensin I to Angiotensin 1-9

Cell Type-Specific Expression of the Mas Proto-Oncogene in Testis

Angiotensin-Converting Enzyme 2 is an Essential Regulator of Heart Function

The Role of Angiotensin-(1-7) Receptor Mas in Spermatogenesis in Mice and Rats

Alterations in Gene Expression in the Testis of Angiotensin-(1-7)-Receptor Mas-Deficient Mice

Sustained Long-Term Potentiation and Anxiety in Mice Lacking the Mas Protooncogene

Neutral Metalloendopeptidase in Human Male Genital Tract. Comparison to Angiotensin I-Converting Enzyme

Possible Involvement of Proteases in the Regulation of Spermatogenesis

Reduced Fertility in Male Mice Deficient in the Zinc Metallopeptidase NL1

Molecular Cloning and Biochemical Characterization of a New Mouse Testis Soluble-Zinc-Metallopeptidase of the Neprilysin Family

Molecular Cloning, Tissue Distribution, and Chromosomal Localization of MMEL2, a Gene Coding for a Novel Human Member of the Neutral Endopeptidase-24.11 Family

Autocrine Regulation of Human Sperm Motility by Tachykinins

Alanyl Aminopeptidase from Human Seminal Plasma: Purification, Characterization, and Immunohistochemical Localization in the Male Genital Tract

Male Reproductive Defects Caused by Puromycin-Sensitive Aminopeptidase Deficiency in Mice

Diet-Induced Hypercholesterolemia Impaired Testicular Steroidogenesis in Mice through the Renin-Angiotensin System

Prorenin Secretion from Human Testis: No Evidence for Secretion of Active Renin or Angiotensinogen

Human Seminal Fluid Contains Significant Quantities of Prorenin: Its Correlation with the Sperm Density

Control of Sperm Motility and Fertility: Diverse Factors and Common Mechanisms

Active and Inactive Renin in Human Seminal Plasma

Angiotensin I Converting Enzyme in the Ejaculate of Fertile and Infertile Men

Genetic Control of Fertility and Embryonic Waste in the Mouse: A Rolefor Angiotensinogen

Localization of the Antigotensin-Converting Enzyme Activity in Testis and Epididymis

Gametes Contain Angiotensin Converting Enzyme (Kininase II)

Isolation and Characterization of a Protein with Homology to Angiotensin Converting Enzyme from the Periacrosomal Plasma Membrane of Equine Spermatozoa

Reproduction and the Renin-Angiotensin System

Ultrastructural Localization of Angiotensin-Converting Enzyme in Ejaculated Human Spermatozoa

Angiotensin Converting Enzyme in Semen and its Possible Role in Capacitation

Angiotensin-Converting Enzyme Content of Human Spermatozoa and its Release during Capacitation

Evidence for the Involvement of Sperm Angiotensin Converting Enzyme in Fertilization

Release of Angiotensin-Converting Enzyme (ACE) from Human Spermatozoa during Capacitation and Acrosome Reaction

Activity of Testis Angiotensin Converting Enzyme (ACE) in Ejaculated Human Spermatozoa

Angiotensin-Converting Enzyme and Other Peptidolytic Enzymes in Human Semen and Relations to its Spermatologic Parameters

Aberrant Distribution of ADAM3 in Sperm from both Angiotensin-Converting Enzyme (Ace)-and Calmegin (Clgn)-Deficient Mice

Human Sperm Testicular Angiotensin-Converting Enzyme Helps Determine Human Embryo Quality

Male-Female Differences in Fertility and Blood Pressure in ACE-Deficient Mice

Angiotensin-Converting Enzyme and Male Fertility

Identification and Disruption of Sperm-Specific Angiotensin Converting Enzyme-3 (ACE3) in Mouse

Distribution and Function of Angiotensin II Receptors in Mouse Spermatozoa

Effects of Angiotensin II on the Acrosome Reaction in Equine Spermatozoa

Angiotensin II Receptors in Rat and Human Sperm

Angiotensin II Induces Acrosomal Exocytosis in Bovine Spermatozoa

Role of Angiotensin-(1-7) Via MAS Receptor in Human Sperm Motility and Acrosome Reaction

Expression of Enkephalin-Degrading Enzymes in Human Semen and Implications for Sperm Motility

Aminopeptidase-Like Protease Released from Oocytes Affects Oocyte Surfaces and Suppresses the Acrosome Reaction in Establishment of Polyspermy Block in Oocytes of the Mussel Mytilus Edulis

GPI-Anchored Aminopeptidase is Involved in the Acrosome Reaction in Sperm of the Mussel Mytilusedulis

Elevated Aminopeptidase N Affects Sperm Motility and Early Embryo Development

Effect of Aminopeptidase N on Functions and Fertility of Mouse Spermatozoa in Vitro