Regulation of Male Fertility by the Renin-Angiotensin System
Abstract
This study was supported by grants from the Basque Government (GIC12/173) and University of the Basque Country (UPV/EHU) (EHUA14/17).
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International Journal of Molecular Sciences Review Regulation of Male Fertility by the ReninAngiotensin System Marta Gianzo 1and Nerea Subirán1,2,3,* 1 Department of Physiology, Faculty of Medicine and Nursery, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain; [email protected] 2Innovation in Assisted Reproduction Group, Biocruces-Bizkaia Health Research Institute, 48903 Barakaldo, Spain 3Research and Development Department, MEPRO Medical Reproductive Solutions, 20009 San Sebastian, Spain *Correspondence: ner[email protected] Received: 30 September 2020; Accepted: 19 October 2020; Published: 26 October 2020 Abstract: 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. Keywords: Renin-angiotensin system; male infertility; angiotensin; renin; testis; spermatozoa 1. Introduction 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 – 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]. Int. J. Mol. Sci. 2020,21, 7943; doi:10.3390/ijms21217943 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020,21, 7943 2 of 18 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]. 2. The Renin-Angiotensin System (RAS) 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].
Int. J. Mol. Sci. 2020,21, 7943 3 of 18 Int.J.Mol.Sci.2020,21,xFORPEERREVIEW3of18 Figure1.CurrentviewoftheRAS.Canonicalorsystemicpathwayisshowninblue,whiletheno‐ canonicalaxesareshowninred.Peptideabbreviations:AGT,angiotensinogen;AngI,angiotensinI; AngII,angiotensinII;AngIII,angiotensinIII;AngIV,angiotensin(3–8)orIV;Ang(1–9),angiotensin (1–9);Ang(1–7),angiotensin(1–7).Receptorabbreviations:AT1R,AngIIreceptortype1;AT2R,Ang IItype2receptor;AT4R/IRAP,AngIVreceptor;MasR,Angreceptor(1–7)orMasreceptor;PRR, (pro)reninreceptor.Enzymeabbreviations:ACE,angiotensin‐convertingenzyme;ACE2,angiotensin‐ convertingenzyme2;NEP,neutralendopeptidase;APA,aminopeptidaseA;APN,aminopeptidase N;APB,aminopeptidaseB. AlongwiththelocalsynthesisanduptakeofRASelementsbytissues,thereisincreasing evidencethatbothsynthesisanduptakealsooccuratthecellularlevel,whichsuggestsanew mechanismofactionforaphysiologicalsystemcalledtheintracellularRAS[14].Thisnew intracellularsystemischaracterizedbythepresenceofdifferentRAScomponentsinsidethecelland bythesynthesisofAngIIatanintracellularsite.Theconceptofthissystemisbasedon1)observation oftheexistenceofdiverseisoformsofAGTandrenin(asaresultofglycosylationandalternative splicing,respectively)anddifferentformsofACE(intracellularandsecreted),2)theexistenceof alternativeenzymesforthesynthesisofAngII(suchascathepsinandchymase),and3)intracellular detectionofthesecomponentsunderparticularcellularconditions.Inaddition,thesecomponents mustbeabletomediatebiologicaleffectsfromanintracellularlocationtobefunctionallyrelevant [15].Infact,changesincellstructureandgenetranscriptionhavebeenreportedtoinducethe mobilizationofintracellularcalciumdepositsandstimulatethegrowthofdifferentcelltypeshave beenreported[13,15].Inspiteofthat,thefunctionalrolesoftheintracellularRASinphysiologyand pathophysiologyhavenotyetbeenfullyelucidated. 2.1.AxesthatComposetheRAS Nowadays,RAShasbeendescribedasacomplexsystemcomposedbythecanonicalaxisaswell asthenon‐canonicalaxes(Figure1),whosecomponentsarewidelyexpressedinallcelltypesand organsinhumans.Therefore,RASplaysimportantrolesinnumerousphysiologicalevents,suchas renal,neuronal,cardiac,pancreatic,vascular,adrenal,pituitary,cognitive,ageing‐related, inflammatoryandreproductiveprocesses[16].Notably,oneofthemostrelevantfunctionsofthe RASisitsparticipationinvariousprocessesrelatedtomalefertility[7,8,17,18]. Figure 1. Current view of the RAS. Canonical or systemic pathway is shown in blue, while the no-canonical axes are shown in red. Peptide abbreviations: AGT, angiotensinogen; Ang I, angiotensin I; Ang II, angiotensin II; Ang III, angiotensin III; Ang IV, angiotensin (3–8) or IV; Ang (1–9), angiotensin (1–9); Ang (1–7), angiotensin (1–7). Receptor abbreviations: AT1R, Ang II receptor type 1; AT2R, Ang II type 2 receptor; AT4R/IRAP, Ang IV receptor; MasR, Ang receptor (1–7) or Mas receptor; PRR, (pro)renin receptor. Enzyme abbreviations: ACE, angiotensin-converting enzyme; ACE2, angiotensin-converting enzyme 2; NEP, neutral endopeptidase; APA, aminopeptidase A; APN, aminopeptidase N; APB, aminopeptidase B. 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. Axes that Compose the RAS 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].
Int. J. Mol. Sci. 2020,21, 7943 4 of 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 (2–8) 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 AT2Rand 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 (3–8), 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]. 3. The RAS and Male Fertility 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 – 29 ], testes [ 9 , 30 ], and spermatozoa [ 8 , 31 – 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 [8,18,22,29,31–33].
Int. J. Mol. Sci. 2020,21, 7943 5 of 18 3.1. Regulation of Testicular Function by Local RAS 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. Int.J.Mol.Sci.2020,21,xFORPEERREVIEW5of18 processes,itiseasytounderstandthestrictendocrine,paracrineandautocrineregulationtowhich theymustbesubjectandhowtheirderegulationcancontributetomaleinfertility[4,5].Thefirstpoint ofhormonalregulationoftesticularfunctionisthehypothalamic‐pituitary‐testicularaxis[5].Several membersoftheRASarepresentinthetestes,suchasreninandAngII,andareregulatedbysex hormonesaswellasbygonadotropins[30,34](Figure2).Likewise,variousparacrineandautocrine mechanismsmodulatetesticularfunctionatthelocallevelbyactingondifferenttypesofcellspresent inthistissue,suchasSertoli,Leydigand/orspermatogeniccells[4].Atthispoint,thelocalRAS becomesinvolved;thisRASisisolatedfromtheplasmaRASbyatesticularbloodbarrierthatprotects malefertilityfromsubstancessuchasAT1RblockersandACEinhibitors[22].Withinthislocal system,numerousresearchershavefoundevidenceofthesynthesisandpresenceofcomponentsof differentaxesoftheRAS. Figure2.EndocrineregulationofthetesticularRAS.Atthetesticularlevel,aftergonadotropin stimulation,thelevelsofAngIIandreninincrease,whiletheexpressionofthegenesencodingboth AngIIreceptorsaswellasthelevelsofthereceptorsdecrease.Ontheotherhand,after hypophysectomy,thelevelsofrenindecrease,butthemRNAexpressionofAT1RandAT2R increases.Atthecellularlevel,inLeydigcells,exogenousadministrationofgonadotropinsincreases thelevelsofAngIandIIandrenin,whereasoestrogentreatmentorhypophysectomydecreasesrenin levels. 3.1.1.TheRenin/ACE/AngII/AT1R/AT2RAxis Allmembersofthisaxishavebeenfoundinthetestesofvariousmammals,includinghumans (Table1).Theinitialstudiescarriedoutinthisregardshowedthatthemajorelementsofthisaxis, AGT[34]andrenin[35–37],arepresentinLeydigcells.Atfirstitwasthoughtthattheseproteins originatedinthesystemicRAS,sincethetesticularlevelsofAGTandreninandsubsequentlyACE, AngIIanditsreceptorsincreaseatthebeginningofpubertywiththeappearanceofLHandFSHin thebloodstream[8,38,39].Later,itwasobservedthattheirtranscriptsaresynthesizedinLeydigcells [6,34,40],indicatingtheirlocalproductionandthelinkedregulationbetweenthesystemicandlocal RAS[22].Inaddition,ithasbeenfoundthatincreasesinreninlevelscauseincreasesintestosterone synthesis[38];however,furtherstudiesarenecessarytoelucidatewhethersuchincreasesin testosteroneareduetodirectactiononthePRRorsignallingthroughthecanonicalpathway. Figure 2. Endocrine regulation of the testicular 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 – 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
Int. J. Mol. Sci. 2020,21, 7943 6 of 18 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 – 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.
Int. J. Mol. Sci. 2020,21, 7943 7 of 18 Table 1. Regulation of the testicular function by local RAS. Axis Component Function References Renin/ACE/Ang II/ AT1R/AT2R axis AGT Local production of Ang II Dzau et al. 1987, Speth et al. 1999 ACE Local production of Ang II - Ang II Negative regulation of testosterone production Regulation of testicular growth and/or differentiation Dufau et al. 1989, Khanum and Dufau 1988 Schunkert et al. 1993; Leung and Sernia 2003 Hirai et al. 1998 Ang III Negative regulation of testosterone production de la Chica-Rodriguez et al. 2008, Martinez-Martos et al. 2011 AT1R Negative regulation of spermatogenesis and testosterone production Vinson et al. 1995 Hirai et al. 1998 APA Negative regulation of testosterone production de la Chica-Rodriguez et al. 2008 ACE2/Ang (1–7)/ MasR axis ACE2 Positive regulation of spermatogenesis Positive regulation of testosterone production Reis et al. 2010 Pan et al. 2013 Ang-(1–7) Positive regulation of testosterone production Positive regulation of spermatogenesis Alenina et al. 2002, Leal et al. 2009, Xu et al. 2007 Reis et al. 2010 MasR Positive regulation of spermatogenesis Regulation of testosterone production Reis et al. 2010 NEP Regulation of sperm maturation and proacrosin activation Erdos et al., 1985 Ang IV AT4R-IRAP axis Ang IV Negative regulation of testosterone production de la Chica-Rodriguez et al. 2008, Martinez-Martos et al. 2011 APN Alteration of Sertoli cells function Negative regulation of spermatogenesis and testosterone production Osada et al. 2001 Martinez-Martos et al. 2011 APB Negative regulation of testosterone production Martinez-Martos et al. 2011 (Pro)renin/ PRR axis (Pro)renin Positive regulation of spermatogenesis Mukhopadhyay et al. 1995 Renin Positive regulation of testosterone production Parmentier et al. 1983 3.1.2. The ACE2/Ang (1–7)/MasR Axis 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
Int. J. Mol. Sci. 2020,21, 7943 8 of 18 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). 3.1.3. The Ang IV/AT4R-IRAP Axis 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.
Int. J. Mol. Sci. 2020,21, 7943 9 of 18 3.1.4. The (Pro)renin/PRR Axis 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. 3.2. Regulation of Sperm Phisiology by Local RAS 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). Table 2. Regulation of the sperm physiology by local RAS. Axis Component Function References Renin/ACE/Ang II/ AT1R/AT2R axis AGT Involved in sperm-oocyte fusion Tempfer et al. 2000 tACE Regulation of sperm motility, capacitation, acrosome reaction and sperm-oocyte fusion Involved in embryo quality and development Siems et al. 1991, Yamaguchi et al. 2006, Foresta et al. 1987, Kohn et al. 1995, Hagaman et al. 1998, Krege et al. 1995 , Foresta et al. 1991, Gianzo et al. 2018, Gianzo et al. 2018 ACE3 Involved in sperm-oocyte fusion Inoue et al. 2010 Ang II Regulation of sperm motility, capacitation and acrosome reaction Foresta et al. 1991, Kohn et al. 1995, Sabeur et al. 2000, Vinson et al. 1996, Wennemuth et al. 1999 AT1R Regulation of sperm motility Vinson et al. 1996 AT2R Regulation of sperm motility Gianzo et al., 2016 ACE2/Ang (1–7)/ MasR axis Ang-(1–7) Positive regulation of sperm motility Valdivia et al. 2020 MasR Regulation of sperm motility Valdivia et al. 2020 NEP Negative regulation of sperm motility Subiran et al. 2008 NEP2 Negative involved in oocyte fertilization and embryo development Regulation of sperm motility Carpentier et al. 2004 Pinto et al. 2010
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