antioxidants

Review Improvement of Testicular Steroidogenesis Using Flavonoids and Isoflavonoids for Prevention of Late-Onset Male Hypogonadism

Luc J. Martin 1,* and Mohamed Touaibia 2

1 Biology Department, Université de Moncton, Moncton, NB E1A 3E9, Canada 2 Chemistry and Biochemistry Department, Université de Moncton, Moncton, NB E1A 3E9, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-506-858-4938



Received: 19 February 2020; Accepted: 10 March 2020; Published: 13 March 2020


Abstract: Androgen production, being important for male fertility, is mainly accomplished by the Leydig cells from the interstitial compartment of the testis. Testosterone plays a critical role in testis development, normal masculinization, and the maintenance of spermatogenesis. Within seminiferous tubules, appropriate Sertoli cell function is highly dependent on testicular androgen levels and is essential to initiate and maintain spermatogenesis. During aging, testosterone production by the testicular Leydig cells declines from the 30s in humans at a rate of 1% per year. This review outlines the recent findings regarding the use of flavonoids and isoflavonoids to improve testosterone production, contributing to normal spermatogenesis and preventing age-related degenerative diseases associated with testosterone deficiency. With the cumulation of information on the actions of different flavonoids and isoflavonoids on steroidogenesis in Leydig cells, we can now draw conclusions regarding the structure-activity relationship on androgen production. Indeed, flavonoids having a 5,7-dihydroxychromen-4-one backbone tend to increase the expression of the steroidogenic acute regulatory protein (StAR), being critical for the entry of cholesterol into the mitochondria, leading to increased testosterone production from testis Leydig cells. Therefore, flavonoids and isoflavonoids such as chrysin, apigenin, luteolin, quercetin, and daidzein may be effective in delaying the initiation of late-onset hypogonadism associated with aging in males.

Keywords: testis; testosterone; androgen; Leydig cells; polyphenols; flavonoids

1. Introduction Testicular Leydig cells are the main site of androgen synthesis in mammals. Androgens are responsible for the development and maintenance of male sex organs and secondary sex characteristics. Specifically, embryonic testosterone is involved in the development of Wolffian ducts, leading to the formation of the epididymis, vas deferens, and seminal vesicles. In addition, testosterone is indirectly involved in the development of the genital tubercle to form the penis, scrotum, and prostate by its conversion to 5 α-dihydrotestosterone (DHT), the most potent androgen of the body. At puberty, testosterone plays a major role in masculinization of the brain, development of male sexual behavior, initiation and support of spermatogenesis, bone growth, maturation of the external genitalia, hair growth, and regulation of gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) secretion. Within the seminiferous tubules, Sertoli cells are stimulated by androgen through the expression of the androgen-binding protein (ABP, SHBG). In turn, activated Sertoli cells support germ cell division and normal spermatogenesis to maintain fertility. With aging, testosterone production declines, beginning in the 30s in men, at a rate of 1% per year [1]. Different conditions, such as exposure

Antioxidants 2020, 9, 237; doi:10.3390/antiox9030237 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 237 2 of 17 Antioxidants 2020, 9, 237 2 of 17 to endocrineyear [1]. Different disruptors, conditions, obesity, such diabetes as exposure and cancer to endocrine treatments, disruptors, exacerbate obesity, this diabetes decrease and in cancer serum testosteronetreatments, levels, exacerbate leading this to decrease the development in serum testosterone of late-onset levels, hypogonadism leading to [the2]. Age-relateddevelopment functional of late‐ alterationsonset hypogonadism include decreased [2]. Age response‐related functional of testicular alterations Leydig include cells to decreased the pituitary response LH [3 ].of Intesticular addition, middle-agedLeydig cells men to the have pituitary an increased LH [3]. prevalence In addition, of middle obesity‐aged and men insulin have resistance an increased associated prevalence with of low obesity and insulin resistance associated with low serum total testosterone and SHBG levels [4]. These serum total testosterone and SHBG levels [4]. These men are mainly responsible for the increase in men are mainly responsible for the increase in testosterone‐replacement therapies over the last testosterone-replacement therapies over the last decade [5]. Men with androgen deficiency may present decade [5]. Men with androgen deficiency may present with decreased libido, erectile dysfunction, with decreased libido, erectile dysfunction, decreased body hair, increased fatigue, decreased muscle decreased body hair, increased fatigue, decreased muscle mass and strength, increased body fat, low mass and strength, increased body fat, low bone mineral density, irritability, inability to concentrate, bone mineral density, irritability, inability to concentrate, and poor quality of life [6,7]. Therefore, andtherapies poor quality aiming of to life increase [6,7]. Therefore, serum levels therapies of testosterone aiming to may increase contribute serum to levels relieving of testosterone part of these may contributesymptoms to and relieving may involve part of nutritional these symptoms supplementation and may with involve flavonoids. nutritional In this supplementation review, a structure with‐ flavonoids.activity relationship In this review, for aplant structure-activity polyphenols with relationship flavonoid for backbone plant polyphenols on testosterone with flavonoid production backbone will onbe testosterone investigated production to draw conclusions will be investigated from cumulated to draw literature conclusions thus from far. cumulated literature thus far.

2. Steroidogenesis2. Steroidogenesis WithinWithin the the testis, testis, androgen androgen production, production, of which of which testosterone testosterone is the is mostthe most abundant, abundant, occurs occurs almost exclusivelyalmost exclusively in Leydig in cells. Leydig Androstenedione cells. Androstenedione and dehydroepiandrosterone and dehydroepiandrosterone (DHEA) (DHEA) are also are produced also byproduced the testis. by However, the testis. theirHowever, levels their of androgenicitylevels of androgenicity are lower are than lower those than ofthose testosterone. of testosterone. Adult LeydigAdult cells Leydig do express cells do the express aromatase the aromatase enzyme (CYP19A1) enzyme (CYP19A1) which is responsible which is responsible for the conversion for the of androgenconversion into of estrogen androgen [8]. into However, estrogen this [8]. rate However, of conversion this rate of testosterone of conversion into of estradiol testosterone is minimal into comparedestradiol to is the minimal secretion compared levels ofto androgen,the secretion and levels estrogen of androgen, may instead and estrogen play a bu mayffering instead action play on a the steroidogenesisbuffering action by on Leydig the steroidogenesis cells [9]. by Leydig cells [9]. TestosteroneTestosterone synthesis synthesis from from cholesterol cholesterol involves involves the sequential the sequential action action of several of several steroidogenic steroidogenic enzymes suchenzymes as the cholesterol such as the side-chain cholesterol cleavage side‐ enzymechain cleavage (CYP11A1), enzyme cytochrome (CYP11A1), P450 17cytochromeα-hydroxylase P450/20-lyase 17α‐ (CYP17A1),hydroxylase/20 3β-hydroxysteroid‐lyase (CYP17A1), dehydrogenase 3β‐hydroxysteroid (HSD3B1 dehydrogenase in rodents and (HSD3B1 HSD3B2 in in rodents humans), and and 17βHSD3B2-hydroxysteroid in humans), dehydrogenase and 17β‐hydroxysteroid type 3 (HSD17B3), dehydrogenase catalyzing type a 3 cascade (HSD17B3), of hydroxylation, catalyzing a cascadecleavage, dehydrogenation,of hydroxylation, and cleavage, isomerization dehydrogenation, reactions. and Testosterone isomerization synthesis reactions. from Testosterone cholesterol synthesis and its from cholesterol and its conversion to other estrogenic and androgenic active metabolites is presented conversion to other estrogenic and androgenic active metabolites is presented in Figure1. in Figure 1.

Figure 1. Common pathways for steroid synthesis within Leydig cells. Abbreviations for enzymes: Cyp11a1,Figure 1. P450 Common side-chain pathways cleavage; for steroid Hsd3b1, synthesis 3β-hydroxysteroid within Leydig cells. dehydrogenase; Abbreviations Cyp17a1,for enzymes: P450 17αCyp11a1,-hydroxylase P450/ 20-lyase;side‐chain Hsd17b3, cleavage; 17 Hsd3b1,β-hydroxysteroid 3β‐hydroxysteroid dehydrogenase; dehydrogenase; Srd5a1, 5 αCyp17a1,-reductase; P450 Cyp19a1, 17α‐ P450 aromatase. Abbreviations for steroids: DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone. Antioxidants 2020, 9, 237 3 of 17

In Leydig cells, as in other steroidogenic tissues, cholesterol is the primary substrate for steroid synthesis. Cholesterol may be synthesized from acetyl-coenzyme A or derived from plasma reserves through the receptor-mediated endocytosis of low-density lipoprotein (LDL) particles. In each case, cholesterol is stored in esterified form in cytoplasmic lipid droplets. The contribution of the two sources of cholesterol for steroidogenesis varies according to cell type, species, physiological status, and availability. Under normal physiological conditions, Leydig cells depend mainly on the endogenous production of cholesterol for testosterone synthesis. However, increased cholesterol demand can be fulfilled by increased extracellular cholesterol uptake from LDL and low-density lipoprotein (HDL) using the LDL receptor and Scavenger receptor class B, type I (SR-BI) surface receptors [10]. Translocation of cytoplasmic cholesterol to the inner mitochondrial membrane is initiated by the recruitment and activation of members of a carrier protein complex, including the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, at the outer mitochondrial membrane [11,12]. Once inside the mitochondria, cholesterol is converted to pregnenolone by the CYP11A1 enzyme through side-chain (C21-C27) cleavage (Figure1). This reaction also involves the electron carrier ferredoxin and NADPH: ferredoxin reductase as the electron donor [13]. To break the side chain of cholesterol, CYP11A1 hydroxylates the C22 and C20 of cholesterol to produce 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol, respectively. Afterward, CYP11A1 cuts the side chain of 20,22-dihydroxycholesterol to release pregnenolone (of 21 carbons) and a 6-carbon aldehyde [13]. The high affinity of CYP11A1 for 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol, along with very high rates of conversion, prevents the accumulation of these intermediates within the mitochondria. Once synthesized, pregnenolone dissociates from the active site of CYP11A1 to exit the mitochondria by diffusion and reaches the smooth endoplasmic reticulum (SER) where androgen synthesis is completed. Three enzymatic reactions involving HSD3B, CYP17A1, and HSD17B3 contribute to complete androgen synthesis. The HSD3B enzyme has dehydrogenase and isomerase activities as it is involved in the oxidation of the hydroxyl group on C3 to a ketone moiety and in the formation of a double bond at C5 of pregnenolone to form progesterone [14]. The CYP17A1 enzyme is then able to catalyze the hydroxylation of progesterone on C17 to generate the 17α-hydroxyprogesterone, followed by the cleavage of the C17-C20 bond to form androstenedione. Thereafter, the enzyme HSD17B3 catalyzes the conversion of androstenedione into testosterone using NADPH as a cofactor. Once produced, testosterone will be used to promote the initiation and maintenance of spermatogenesis within the testis as well as the development of male secondary sex characteristics.

Regulation of Steroidogenesis The expression of genes encoding the steroidogenic enzymes within Leydig cells is primarily regulated by the LH, which activates the cyclic adenosine monophosphate (cAMP)/Protein kinase A (PKA) signalling pathway. Binding of the LH to its G-protein-coupled membrane receptor (LHCGR) leads to the activation of adenylate cyclase, conversion of ATP to cAMP, and subsequent activation of PKA. Substrates for PKA involve the StAR, responsible for the transport of cholesterol inside mitochondria, and transcription factors critical for steroidogenic gene expressions. In addition to the cAMP/PKA pathway, other signalling pathways such as the mitogen-activated protein kinase (MAPK), Janus kinase/signal transducer and activator of transcription proteins (JAK/STAT), calcium/calmodulin-dependent protein kinase (CAMK), and protein kinase C (PKC) have been shown to influence steroidogenesis from testicular Leydig cells. Indeed, activation of the epidermal growth factor receptor (EGFR) contributes to the regulation of steroidogenesis by the modulation of MAPK signalling and its downstream extracellular-signal-regulated kinases 1 and 2 (ERK1/2) [15,16]. The JAK/STAT pathway has been shown to be critical for the modulation of steroidogenesis from Leydig cells by adipose-derived hormones such as leptin and resistin [17,18]. Increased intracellular Ca2+ levels result in increased concentrations of Ca2+ complexed to calmodulin, leading to the activation of the Ca2+/calmodulin kinase kinase (CAMKK) and its downstream target, Ca2+/calmodulin kinase I Antioxidants 2020, 9, 237 4 of 17

(CAMKI). CAMKI is expressed in adult Leydig cells and has been shown to be activated downstream of cAMP and to be essential for the StAR gene expression [19]. Others have shown that the co-activations of PKC and PKA play an important role in upregulation of StAR and steroidogenesis in Leydig cells [20,21]. In addition to the modulation of intracellular-signal transduction pathways, paracrine factors may also be shared through cell junctions and participate in the regulation of steroidogenesis within Leydig cells. Moreover, nutrients such as polyphenolic compounds found in plants may promote testosterone production through different regulatory mechanisms. Among these compounds, certain flavonoids have been shown to influence testosterone production and such action may depend on specific structural features.

3. Flavonoids Flavonoids are characterized by the C6-C3-C6 group in which two benzene rings are connected by a three-carbon segment. The structure of the different flavonoid types varies at the level of the oxygenated heterocycle formed by the chain of three carbons between the two benzene rings and hydroxyl (-OH) moieties of the A ring (see Figure2 for a description of the structures of flavonoids found in plants). The structure of the C3 region varies by its level of oxidation which is highest in flavonols. Catechins and dihydrochalcones are the most reduced flavonoids. On the other hand, the structure of the flavonoids corresponding to the minimum oxidation state—that is, to a complete opening and hydration of the heterocycle between the two flavonoid rings (A and B)—is not known inAntioxidants nature. 2020, 9, 237 5 of 17

Figure 2. Classification of major groups of flavonoids (a) and isoflavonoids (b). Figure 2. Classification of major groups of flavonoids (a) and isoflavonoids (b).

3.1. Flavonoids and Steroidogenesis Since their discovery, flavonoids have been associated with numerous health benefits including cancer prevention, reduced risk of cardiovascular and neurodegenerative diseases, and delayed‐ aging‐associated symptoms (reviewed in [23]). Having a chemical structure similar to cholesterol and other steroids, flavonoids may influence the production of androgens in Leydig cells. Therefore, more than 500 publications have reported effects of different flavonoids on testosterone production since the early 1960s. However, it is only recently that the molecular mechanisms of flavonoids affecting steroid synthesis have been partially elucidated.

3.1.1. Flavones Differently from falvans, flavones are characterized by the presence of the C2‐C3 double bond which is conjugated with the C4 carbonyl (Figure 2). In one of our studies, we found that 10 μM of chrysin (1), apigenin (2), luteolin (3), and baicalein (4) (Figure 3) stimulated cAMP‐dependent StAR, Cyp11a1, and Fdx1 (Ferredoxin 1) expression in MA‐10 Leydig cells [24]. The presence of the two hydroxyls in positions 5 and 7 (cycle A) and one or two hydroxyls in positions 3′ and 4′ (cycle C) are crucial for the biological effect of these molecules. Compared to chrysin (1), baicalein (4) has an extra hydroxyl in position 6 of the A cycle. Only luteolin (3) was able to increase the cAMP‐dependent accumulation of progesterone from MA‐10 Leydig cells, possibly through its positive regulation of StAR expression. Luteolin (3) also activated StAR expression and resulted in increased progesterone and testosterone syntheses in LC540 tumor Leydig cells [25]. Apigenin (2) and chrysin (1) have been shown to increase cAMP‐ dependent androgen production from testicular Leydig cells by increasing the StAR gene expression [26,27]. Antioxidants 2020, 9, 237 5 of 17

Based on the 2-Phenyl-3,4-dihydro-2H-1-benzopyran skeleton, there are four major groups of flavonoids (Figure2a): flavans, flavones, flavonols, and anthocyanidins [ 22]. Isoflavonoids are divided into two main families (Figure2b): isoflavans and isoflavones, both based on the 3-Phenyl-3,4-dihydro-2H-1-benzopyran skeleton [22]. The flavones are characterized by a planar structure attributed to the double bond in the central aromatic ring. Examples of flavones include apigenin and luteolin, found in parsley, thyme, and celery. The isoflavones are similar in structure, except that the B ring is associated with the carbon 3 rather than carbon 2 of the C ring. Examples of isoflavones are genistein and daidzein, found in soybeans. Flavonols is an important group of flavonoids characterized by the presence of a hydroxyl group at the position-3 of the C ring. Quercetin, myricetin and kaempferol, found in onions, apples, broccoli, cherries, tea, and berries, are members of this group. Flavanones such as naringenin and hesperedin, characterized by the absence of a double bond within the C ring, are mainly found in citrus fruits and plums. The flavonoids belonging to the catechin group, characterized by the absence of a double bond within the C ring and the presence of a hydroxyl group at the position-3 of the C ring, are mainly found in tea, apples, and red wine. Anthocyanidins such as cyanidin and delphinidin, characterized by the presence of two double bonds and a positive charge on the C-ring, are found in strawberries and other berries, such as blueberries and blackberries, in wine, and tea.

3.1. Flavonoids and Steroidogenesis Since their discovery, flavonoids have been associated with numerous health benefits including cancer prevention, reduced risk of cardiovascular and neurodegenerative diseases, and delayed-aging-associated symptoms (reviewed in [23]). Having a chemical structure similar to cholesterol and other steroids, flavonoids may influence the production of androgens in Leydig cells. Therefore, more than 500 publications have reported effects of different flavonoids on testosterone production since the early 1960s. However, it is only recently that the molecular mechanisms of flavonoids affecting steroid synthesis have been partially elucidated.

3.1.1. Flavones Differently from falvans, flavones are characterized by the presence of the C2-C3 double bond which is conjugated with the C4 carbonyl (Figure2). In one of our studies, we found that 10 µM of chrysin (1), apigenin (2), luteolin (3), and baicalein (4) (Figure3) stimulated cAMP-dependent StAR, Cyp11a1, and Fdx1 (Ferredoxin 1) expression in MA-10 Leydig cells [24]. The presence of the two hydroxyls in positions 5 and 7 (cycle A) and one or two hydroxyls in positions 30 and 40 (cycle C) are crucial for the biological effect of these molecules. Compared to chrysin (1), baicalein (4) has an extra hydroxylAntioxidants in 2020 position, 9, 237 6 of the A cycle. 6 of 17

OH O OH O OH O OH O HO

HO O HO O HO O HO O

Chrysin (1) Apigenin (2) OH Luteolin (3) OH Baicalein (4) OH Figure 3. Structures of chrysin (1), apigenin (2), luteolin ((3),), and baicalein ((44).).

OnlyAs for luteolin chrysin ( 3()1 was), apigenin able to ( increase2) was able the cAMP-dependentto decrease the levels accumulation of DAX1 of(dosage progesterone‐sensitive from sex MA-10reversal, Leydig adrenal cells, hypoplasia possibly critical through region, its positive on the X regulation chromosome, of StAR geneexpression. 1), an important Luteolin repressor (3) also of activatedStAR transcriptionStAR expression [27]. In and addition, resulted apigenin in increased (2) also progesterone inhibits COX2 and testosterone(cyclooxygenase syntheses‐2) expression, in LC540 tumorwhich Leydigmay contribute cells [25]. to Apigenin increased (2 )StAR and chrysinexpression. (1) have Increased been shown Cox2 expression to increase is cAMP-dependent associated with androgendecreased production expression fromof StAR testicular and reduced Leydig testosterone cells by increasing production the StAR in Leydiggene cells expression from aging [26,27 males]. [28]. However, others have shown that apigenin (2) inhibited the production of 5α‐androstane‐3α, 17β‐diol (DIOL), the main androgen of rat immature Leydig cells [29]. The activity of rat Hsd3b, Cyp17a1, and Hsd17b3 was inhibited by apigenin (2) with IC50 values in the 10 μM range [29]. In addition, human HSD3B2 and HSD17B3 were more than five times more sensitive to the inhibitory action of apigenin (2). Using H295R human adrenal cells, treatments with 10 μM apigenin for 24h resulted in a decrease of androstenedione and testosterone production [30,31]. In this cell model, apigenin (2) decreased the expressions of HSD3B2 and CYP17A1 [31]. Overall, apigenin (2) seems to increase steroidogenesis in Leydig cells mainly by increasing PKA‐dependent StAR protein expression. However, such effect may not translate into increased testosterone production as we have also reported that apigenin (1) inhibits Cyp11a1 expression in LC540 tumor Leydig cells [25]. Luteolin (3), a flavone, has been of growing interest in our laboratory for its potential in improving androgen production. Indeed, we have shown that luteolin (3) can increase Leydig cell steroidogenesis by upregulating StAR expression [24,25]. We also reported that luteolin (3) was able to activate Fdx1 (Ferredoxin 1) transcription in MA‐10 Leydig cells [24]. Fdx1 is important to supporting the Cyp11a1 steroidogenic enzyme activity by electron transfer. Similar to apigenin (2) and chrysin (1), luteolin (3) is also able to inhibit Cox2 expression, thus promoting StAR transcription [32]. Others have also reported that luteolin (3) is able to increase cAMP‐dependent steroidogenesis by improving mitochondrial import of cholesterol by increasing StAR protein levels in MA‐10 Leydig cells [33]. Furthermore, luteolin (3) increased StAR transcription by inhibiting the expression of Dax‐ 1 as reported for other flavonoids [33]. Chrysin (1), 5,7‐dihydroxyflavone, is present in high levels in honey, propolis, chamomile, mushrooms, fruit bark, and many plant extracts. Chrysin (1) increased cAMP‐dependent StAR protein levels and steroidogenesis in mouse MA‐10 Leydig cells, possibly by decreasing the expression of Dax1 [26]. Interestingly, chrysin (1) seems to increase the sensitivity of Leydig cells to cAMP‐dependent stimulation of StAR expression. Chrysin (1) is also able to inhibit the activity of the transcription factor NF‐κB, leading to reduced COX2 promoter activity [34]. Lower levels of Cox2 in Leydig cells contributes to increased StAR expression. In addition, others have shown that chrysin (1) is a potent inhibitor of the enzyme aromatase, which converts testosterone into estradiol [35]. This inhibitory effect of human aromatase activity has been assigned to different flavonoids in addition to chrysin (1) and apigenin (2) [36]. Therefore, chrysin (1) increases testosterone serum levels by more than 35% in adult male rats [37]. However, chrysin (1) supplementation did not change urine testosterone levels in humans after 21 days of treatment [38]. This may be attributed to the relatively low dose of chrysin (1) used in this research compared to studies with rodents. However, treatments of male mice for 10 days with up to 20 mg/kg of chrysin (1) had no effect on serum testosterone levels, while preventing the inhibition of testosterone production by exposure to the mycotoxin zearalenone [39]. Therefore, it has been suggested that chrysin (1) could be used to delay age‐related decline in StAR expression and testosterone production from Leydig cells [26,39,40]. Importantly, chrysin (1) failed to induce a significant increase in steroid production when MA‐10 Leydig cells were co‐ incubated with 22(R)hydroxycholesterol [26], suggesting this flavonoid only improves the entry of Antioxidants 2020, 9, 237 6 of 17

As for chrysin (1), apigenin (2) was able to decrease the levels of DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on the X chromosome, gene 1), an important repressor of StAR transcription [27]. In addition, apigenin (2) also inhibits COX2 (cyclooxygenase-2) expression, which may contribute to increased StAR expression. Increased Cox2 expression is associated with decreased expression of StAR and reduced testosterone production in Leydig cells from aging males [28]. However, others have shown that apigenin (2) inhibited the production of 5α-androstane-3α, 17β-diol (DIOL), the main androgen of rat immature Leydig cells [29]. The activity of rat Hsd3b, Cyp17a1, and Hsd17b3 was inhibited by apigenin (2) with IC50 values in the 10 µM range [29]. In addition, human HSD3B2 and HSD17B3 were more than five times more sensitive to the inhibitory action of apigenin (2). Using H295R human adrenal cells, treatments with 10 µM apigenin for 24h resulted in a decrease of androstenedione and testosterone production [30,31]. In this cell model, apigenin (2) decreased the expressions of HSD3B2 and CYP17A1 [31]. Overall, apigenin (2) seems to increase steroidogenesis in Leydig cells mainly by increasing PKA-dependent StAR protein expression. However, such effect may not translate into increased testosterone production as we have also reported that apigenin (1) inhibits Cyp11a1 expression in LC540 tumor Leydig cells [25]. Luteolin (3), a flavone, has been of growing interest in our laboratory for its potential in improving androgen production. Indeed, we have shown that luteolin (3) can increase Leydig cell steroidogenesis by upregulating StAR expression [24,25]. We also reported that luteolin (3) was able to activate Fdx1 (Ferredoxin 1) transcription in MA-10 Leydig cells [24]. Fdx1 is important to supporting the Cyp11a1 steroidogenic enzyme activity by electron transfer. Similar to apigenin (2) and chrysin (1), luteolin (3) is also able to inhibit Cox2 expression, thus promoting StAR transcription [32]. Others have also reported that luteolin (3) is able to increase cAMP-dependent steroidogenesis by improving mitochondrial import of cholesterol by increasing StAR protein levels in MA-10 Leydig cells [33]. Furthermore, luteolin (3) increased StAR transcription by inhibiting the expression of Dax-1 as reported for other flavonoids [33]. Chrysin (1), 5,7-dihydroxyflavone, is present in high levels in honey, propolis, chamomile, mushrooms, fruit bark, and many plant extracts. Chrysin (1) increased cAMP-dependent StAR protein levels and steroidogenesis in mouse MA-10 Leydig cells, possibly by decreasing the expression of Dax1 [26]. Interestingly, chrysin (1) seems to increase the sensitivity of Leydig cells to cAMP-dependent stimulation of StAR expression. Chrysin (1) is also able to inhibit the activity of the transcription factor NF-κB, leading to reduced COX2 promoter activity [34]. Lower levels of Cox2 in Leydig cells contributes to increased StAR expression. In addition, others have shown that chrysin (1) is a potent inhibitor of the enzyme aromatase, which converts testosterone into estradiol [35]. This inhibitory effect of human aromatase activity has been assigned to different flavonoids in addition to chrysin (1) and apigenin (2)[36]. Therefore, chrysin (1) increases testosterone serum levels by more than 35% in adult male rats [37]. However, chrysin (1) supplementation did not change urine testosterone levels in humans after 21 days of treatment [38]. This may be attributed to the relatively low dose of chrysin (1) used in this research compared to studies with rodents. However, treatments of male mice for 10 days with up to 20 mg/kg of chrysin (1) had no effect on serum testosterone levels, while preventing the inhibition of testosterone production by exposure to the mycotoxin zearalenone [39]. Therefore, it has been suggested that chrysin (1) could be used to delay age-related decline in StAR expression and testosterone production from Leydig cells [26,39,40]. Importantly, chrysin (1) failed to induce a significant increase in steroid production when MA-10 Leydig cells were co-incubated with 22(R)hydroxycholesterol [26], suggesting this flavonoid only improves the entry of cholesterol into the mitochondria by regulating StAR protein levels and has no effects on steroidogenic enzyme activity. Baicalein (4) is an important flavonoid found in several plants such as roots of Scutellaria baicalensis Georgi [41] and Oroxylum indicum [42]. As for other flavones, we have shown that low concentrations of baicalein (4) increased cAMP-dependent StAR promoter activation in MA-10 Leydig cells [24]. In addition, Cyp11a1 and Fdx1 promoter activity was also enhanced by co-treatments with cAMP as reported for apigenin (2), luteolin (3), and chrysin (1). However, others recently reported that Antioxidants 2020, 9, 237 7 of 17

cholesterol into the mitochondria by regulating StAR protein levels and has no effects on steroidogenic enzyme activity. Baicalein (4) is an important flavonoid found in several plants such as roots of Scutellaria baicalensis Georgi [41] and Oroxylum indicum [42]. As for other flavones, we have shown that low concentrations of baicalein (4) increased cAMP‐dependent StAR promoter activation in MA‐10 AntioxidantsLeydig cells2020 ,[24].9, 237 In addition, Cyp11a1 and Fdx1 promoter activity was also enhanced by7 of co 17‐ treatments with cAMP as reported for apigenin (2), luteolin (3), and chrysin (1). However, others recently reported that baicalein (4) administration for four weeks reduced serum testosterone and baicalein (4) administration for four weeks reduced serum testosterone and FSH and LH levels FSH and LH levels following treatments of a rat model for polycystic ovary syndrome (PCOS) [43]. following treatments of a rat model for polycystic ovary syndrome (PCOS) [43]. In their study, In their study, baicalein (4) instead decreased the expression of StAR, Hsd3b, Cyp11a1, and Cyp19a1 baicalein (4) instead decreased the expression of StAR, Hsd3b, Cyp11a1, and Cyp19a1 in ovarian tissues. in ovarian tissues. Such discrepancy in responses to baicalein (4) may be attributed to the pathological Such discrepancy in responses to baicalein (4) may be attributed to the pathological condition of PCOS condition of PCOS and different responses to baicalein (4) according to the sex of animals. Overall, and different responses to baicalein (4) according to the sex of animals. Overall, flavones seem to have flavones seem to have a stimulatory effect on the StAR gene expression in Leydig cells. However, a stimulatory effect on the StAR gene expression in Leydig cells. However, such action may not always such action may not always translate into increased testosterone production. translate into increased testosterone production.

3.1.2.3.1.2. IsoflavonesIsoflavones InIn mice,mice, estrogenestrogen receptorreceptor (ER)-(ER)‐β β isis detecteddetected inin everyevery cellcell typetype ofof thethe testis,testis, whereaswhereas ER-ER‐α α isis mainlymainly expressedexpressed byby LeydigLeydig cellscells [44[44].]. ExpressingExpressing thethe enzymeenzyme aromatase,aromatase, SertoliSertoli cellscells synthesizesynthesize estrogenestrogen from from androgenandrogen [ [45],45], whichwhich hashas anan inhibitoryinhibitory eeffectffect onon steroidogenesis from Leydig cells [[46].46]. However,However, since since adult adult Leydig Leydig cells cells produce produce higher higher levels of levels estrogen of [estrogen47], the physiological [47], the physiological significance ofsignificance estrogen-mediated of estrogen regulatory‐mediated interactions regulatory between interactions Sertoli cellsbetween and Leydig Sertoli cells cells remains and Leydig to be better cells defined.remains Havingto be better phytoestrogenic defined. Having effects, phytoestrogenic isoflavones such effects, as daidzein isoflavones (5) and such genistein as daidzein (6) (Figure (5) and4) maygenistein disrupt (6) the(Figure paracrine 4) may signalling disrupt the by paracrine estrogen betweensignalling Sertoli by estrogen and Leydig between cells. Sertoli The 5-hydroxyland Leydig substitutioncells. The 5‐hydroxyl (cycle A) substitution is crucial since (cycle it is A) the is onlycrucial di ffsinceerence it is between the only these difference two molecules. between these Indeed, two daidzeinmolecules. (5 )Indeed, impairs Leydigdaidzein cell (5 testosterone) impairs Leydig production cell testosterone by inhibiting production the expression by levelsinhibiting of StAR, the Cyp11a1,expression and levels Hsd3b1 of StAR, in neonatal Cyp11a1, mouse and Hsd3b1 testes [in48 neonatal]. Genistein mouse (6) inhibitedtestes [48]. progesterone Genistein (6) secretion inhibited throughprogesterone the down-regulation secretion through of theStAR downexpression‐regulation in MA-10 of StAR Leydig expression cells [in49 MA]. Others‐10 Leydig have cells reported [49]. thatOthers genistein have reported (6) exposure that genistein was also (6 able) exposure to inhibit was testosterone also able to inhibit production testosterone from fetal production mouse testes from byfetal decreasing mouse testes StAR, by Cyp11a1, decreasing Hsd3b1, StAR, and Cyp11a1, Cyp17a1 Hsd3b1, expression and levels Cyp17a1 [50]. Genistein expression also levels inhibited [50]. HSD3BGenistein and also HSD17B3 inhibited enzyme HSD3B activities and HSD17B3 from rat enzyme and human activities testes from [51 ].rat therefore, and human high testes levels [51]. of isoflavonestherefore, high may levels have detrimentalof isoflavones eff ectsmay on have testicular detrimental steroidogenesis effects on testicular during the steroidogenesis early neonatal period.during the early neonatal period. OH OH O OH O

HO O HO O

Daidzein (5) Genistein (6) . Figure 4. Structures of daidzein (5) and genistein (6). Figure 4. Structures of daidzein (5) and genistein (6). Although numerous studies have suggested that a phytoestrogenic action of isoflavones contributedAlthough to decreased numerous testosterone studies have levels suggested in men, it that was concludeda phytoestrogenic from a meta-analysis action of isoflavones including 32contributed reports that to neither decreased soy testosterone foods nor isoflavone levels in supplementation men, it was concluded had a significant from a meta eff‐ectanalysis on bioavailable including testosterone32 reports levelsthat neither [52]. In soy addition, foods treatments nor isoflavone of MA-10 supplementation Leydig cells with had 0.1-100 a significantµM genistein effect (6) foron 48hbioavailable had no e fftestosteroneect on cell viability, levels [52]. basal In progesteroneaddition, treatments synthesis, of MA and‐10 the Leydig expression cells ofwith steroidogenic 0.1‐100 μM genesgenistein such (6) as forStAR 48h, hadTspo no, Cyp11a1, effect on andcell viability,Hsd3b1, whilebasal progesterone decreasing hCG-stimulated synthesis, and the progesterone expression productionof steroidogenic when genes combined such with as StAR the, phtalate Tspo, Cyp11a1, MEHP [and53]. Hsd3b1 However,, while perinatal decreasing exposure hCG of‐stimulated male rats toprogesterone soy isoflavones production induced when proliferation combined of with Leydig the phtalate cells and MEHP increased [53]. the However, levels of perinatal StAR, CYP11A1, exposure HSD3B,of male andrats CYP17A1,to soy isoflavones compensating induced for proliferation the decrease of in Leydig HSD17B3 cells steroidogenic-enzyme and increased the levels activity of StAR, [54]. Moreover,CYP11A1, exposure HSD3B, and to genistein CYP17A1, (6) compensating during perinatal for development the decrease inin maleHSD17B3 rodents steroidogenic resulted in‐ reducedenzyme anogenitalactivity [54]. distance, Moreover, lower exposure testosterone to genistein serum levels, (6) during and decreased perinatal testicular development mass compared in male torodents male ratsresulted under in a reduced normal dietanogenital [55–57]. distance, In addition, lower low testosterone doses of 10 serum nM genistein levels, and (6) inhibits decreased testosterone testicular secretion by fetal Leydig cells through interaction with the estrogen receptor α and reduction of StAR and steroidogenic-enzyme gene expression during early fetal development (E12.5), a critical period for male programming [50]. Therefore, fetal exposure to phytoestrogen may disturb the development and function of the male reproductive system. Antioxidants 2020, 9, 237 8 of 17 mass compared to male rats under a normal diet [55–57]. In addition, low doses of 10 nM genistein (6) inhibits testosterone secretion by fetal Leydig cells through interaction with the estrogen receptor α and reduction of StAR and steroidogenic‐enzyme gene expression during early fetal development (E12.5), a critical period for male programming [50]. Therefore, fetal exposure to phytoestrogen may disturb the development and function of the male reproductive system. Genistein (6) and daidzein (5) have been reported to inhibit the activity of 5α‐reductase in vitro [58] and to reduce the plasma levels of 5α‐dihydrotestosterone (DHT) in male rats [59]. Dihydrotestosterone is the main prostatic androgen responsible for the development of prostate cancer [60]. Interestingly, lifetime exposure to flavonoids such as daidzein (5) and genistein (6) increased serum and testicular testosterone levels in rats [61].

3.1.3. Flavonols Flavonols, especially quercetin (7), have been well documented for their positive effects on testicular function and steroidogenesis. Indeed, treatments of MA‐10 Leydig cells with 10 μM of quercetin (7), myricetin (8), or pentaacetylquercetin (9) (Figure 5) resulted in increased cAMP‐ dependent expressions of StAR, Cyp11a1, and Fdx1, contributing to increased steroidogenesis and accumulation of progesterone [62]. However, there were no changes in serum testosterone levels following quercetin (7) supplementation of healthy men for eight weeks [63]. Such discrepancy in the effects of quercetin on testosterone production may be attributed to differences between species. Quercetin (7), which does not have an extra hydroxyl in the position 5′ of the B‐cycle as in myricetin (8), has been shown to improve steroidogenesis and testosterone levels in male mice Antioxidants 2020, 9, 237 8 of 17 exposed to bisphenol A, widely used in the production of plastics [64]. However, the molecular mechanism responsible for such improvement may be attributable to the antioxidative properties of quercetinGenistein (7). (6Supporting) and daidzein its (5 )positive have been effect reported on tosteroidogenesis, inhibit the activity quercetin of 5α-reductase improvesin vitro Creb1[58] transcriptionaland to reduce the activity, plasma as levels well of as 5α Cyp11a1-dihydrotestosterone and Fdx1 promoter (DHT) in activity male rats [62]. [59 Creb1]. Dihydrotestosterone is an important activatoris the main of prostaticsteroidogenic androgen‐gene responsibleexpression, forincluding the development StAR, in Leydig of prostate cells [65–67]. cancer [ 60Others]. Interestingly, have also reportedlifetime exposure that quercetin to flavonoids (7) increased such as daidzeinStAR mRNA (5) and levels, genistein StAR (6 )promoter increased activity, serum and and testicular steroid hormonetestosterone production levels in from rats [ 61MA]. ‐10 Leydig cells [49]. Quercetin (7) may increase StAR gene expression in response to cAMP stimulation by reducing Dax1 protein levels in Leydig cells [33]. The expression of3.1.3. StAR Flavonols and steroidogenesis are also increased by blocking Cox2 signalling in response to quercetin (7), asFlavonols, well as chrysin especially (1), apigenin quercetin (2), (7 and), have luteolin been (3 well) treatments documented of Leydig for their cells positive[33]. Quercetin effects ( on7) alsotesticular contributes function to andthe improvement steroidogenesis. of testosterone Indeed, treatments production of MA-10in rats Leydigexposed cells to either with cadmium 10 µM of quercetinchloride or (7 ),the myricetin herbicide (8 ),atrazine, or pentaacetylquercetin where the enzyme (9) (Figure activities5) resulted of Hsd3b in and increased Hsd17b3 cAMP-dependent are recovered [68,69].expressions Indeed, of StAR we ,haveCyp11a1, shownand thatFdx1 quercetin, contributing (7) can to increasedactivate the steroidogenesis expression of and Hsd3b accumulation in rat LC540 of progesteronetumor Leydig [62 cells]. However, [25]. An thereacetylated were noform changes of quercetin in serum (7 testosterone), pentaacetylquercetin levels following (9), quercetinsuccessfully (7) increasedsupplementation cAMP‐ ofdependent healthy men accumulation for eight weeks of progesterone [63]. Such discrepancy from MA in‐10 the Leydig effects ofcells, quercetin possibly on throughtestosterone the productionactivation of may StAR be attributedand Cyp11a1 to di transcriptionsfferences between [62]. species. Such chemical modification may improve the bioavailability of quercetin (7) in vivo. O OH O O OH O O O OH OH O O HO O OH HO O O O Quercetin (7) OH Myricetin (8) OH Pentaacetylquercetin (9) O OH OH O O O

Figure 5. StructuresStructures of quercetin ( 7), myricetin ( 8), and pentaacetylquercetin (9). Quercetin (7), which does not have an extra hydroxyl in the position 5 of the B-cycle as in Icariin (11) is a prenylated flavonol glycoside (Figure 6) derived from 0kaempferol (10) and myricetin (8), has been shown to improve steroidogenesis and testosterone levels in male mice exposed isolated from several species of plant belonging to the genus Epimedium, such as the horny goat weed. to bisphenol A, widely used in the production of plastics [64]. However, the molecular mechanism responsible for such improvement may be attributable to the antioxidative properties of quercetin (7). Supporting its positive effect on steroidogenesis, quercetin improves Creb1 transcriptional activity, as well as Cyp11a1 and Fdx1 promoter activity [62]. Creb1 is an important activator of steroidogenic-gene expression, including StAR, in Leydig cells [65–67]. Others have also reported that quercetin (7) increased StAR mRNA levels, StAR promoter activity, and steroid hormone production from MA-10 Leydig cells [49]. Quercetin (7) may increase StAR gene expression in response to cAMP stimulation by reducing Dax1 protein levels in Leydig cells [33]. The expression of StAR and steroidogenesis are also increased by blocking Cox2 signalling in response to quercetin (7), as well as chrysin (1), apigenin (2), and luteolin (3) treatments of Leydig cells [33]. Quercetin (7) also contributes to the improvement of testosterone production in rats exposed to either cadmium chloride or the herbicide atrazine, where the enzyme activities of Hsd3b and Hsd17b3 are recovered [68,69]. Indeed, we have shown that quercetin (7) can activate the expression of Hsd3b in rat LC540 tumor Leydig cells [25]. An acetylated form of quercetin (7), pentaacetylquercetin (9), successfully increased cAMP-dependent accumulation of progesterone from MA-10 Leydig cells, possibly through the activation of StAR and Cyp11a1 transcriptions [62]. Such chemical modification may improve the bioavailability of quercetin (7) in vivo. Icariin (11) is a prenylated flavonol glycoside (Figure6) derived from kaempferol ( 10) and isolated from several species of plant belonging to the genus Epimedium, such as the horny goat weed. Icariin (11) has been shown to reverse the adverse effects of di(2-ethylhexyl) phtalate (DEHP) on primary Leydig cell proliferation and testosterone synthesis [70]. Specifically, icariin (11) allowed to recover from the inhibitory effects of DEHP on the expression of steroidogenic enzymes (Cyp11a1, Hsd3b1, and Hsd17b3) Antioxidants 2020, 9, 237 9 of 17

Icariin (11) has been shown to reverse the adverse effects of di(2‐ethylhexyl) phtalate (DEHP) on primary Leydig cell proliferation and testosterone synthesis [70]. Specifically, icariin (11) allowed to recover from the inhibitory effects of DEHP on the expression of steroidogenic enzymes (Cyp11a1, Hsd3b1, and Hsd17b3) and the transcription factor Nr5a1 (Sf‐1), known to regulate steroidogenic gene expression. In addition, icariin (11) also contributed to the increase of the expressions of peripheral‐type benzodiazepine receptor (PBR) and steroidogenic acute regulatory protein (StAR), leading to increased testosterone production in adult‐male rat testes [71]. However, others have reported that icariin (11) induces apoptosis in mouse Leydig tumor cells (mLTC1) [72], suggesting an anticancer potential. Recently, rutin (12) (Figure 6), a glycosylated quercetin (7) found in citrus fruits, was shown to reverse the decreased levels of serum testosterone, LH, and FSH, as well as the impaired sperm quality, induced by carbon tetrachloride in male rats [73]. Carbon tetrachloride is an important environmental contaminant inducing male hypogonadism. Rutin (12) also partially reversed Antioxidants 2020, 9, 237 9 of 17 cadmium‐induced decline in plasma testosterone levels in male rats by increasing Hsd3b and Hsd17b3 enzyme activity [74,75]. and theTaxifolin transcription (13) (Figure factor 6), Nr5a1 also (Sf-1),known known as dihydroquercetin, to regulate steroidogenic belongs to gene the expression.subclass of fla Invanonols addition, icariinand is found (11) also in red contributed onions. Taxifolin to the increase(13) has been of the shown expressions to inhibit of androgen peripheral-type production benzodiazepine in immature receptorrat Leydig (PBR) cells and by inhibiting steroidogenic the acuteactivities regulatory of Hsd3b protein and Cyp17a1 (StAR), leading enzymes to [76]. increased However, testosterone human productionHSD3B2 and in CYP17A1 adult-male were rat testes less sensitive [71]. However, to taxifolin others (13 have) compared reported to that rat icariin enzymes. (11) inducesOverall, apoptosisflavonols such in mouse as quercetin Leydig tumor(7) are cellspromising (mLTC1) in increasing [72], suggesting steroid an anticancer potential.

OH OH O O HO HO O HO O O OH OH O OH O OH O Icariin (11) Kaempferol (10) OH O HO HO O HO OH HO HO OH O O OH HO O OH O OH HO OH OH Rutin (12) HO O O OH O Taxifolin (13) HO

Figure 6. Structures of kaempferol (10), icariin (11), rutin ((12),), and taxifolintaxifolin ((13).).

3.1.4.Recently, Flavanones rutin (12) (Figure6), a glycosylated quercetin ( 7) found in citrus fruits, was shown to reverse the decreased levels of serum testosterone, LH, and FSH, as well as the impaired sperm quality, inducedNaringenin by carbon (14 tetrachloride, Figure 7), ina flavanone male rats [ 73found]. Carbon in grapefruits, tetrachloride has is been an important reported environmental to inhibit the contaminantactivity of Hsd17b3 inducing and male Hsd3b hypogonadism. in male rats Rutin after (12 daily) also partiallysubcutaneous reversed injections cadmium-induced of 15 mg/kg decline [77]. inHowever, plasma testosteronethese changes levels in enzyme in male activity rats by had increasing no consequences Hsd3b and on Hsd17b3 testosterone enzyme levels. activity Oppositely, [74,75]. oral administration of naringenin (14) to ma Taxifolin (13) (Figure6), also known as dihydroquercetin,le rats for 10 belongs weeks toresulted the subclass in increased of flavanonols serum testosterone levels [78]. Therefore, the route of delivery of flavonoids may influence the result and is found in red onions. Taxifolin (13) has been shown to inhibit androgen production in immature outcome in certain experimental designs. Naringenin (14) also prevented the decreases of serum rat Leydig cells by inhibiting the activities of Hsd3b and Cyp17a1 enzymes [76]. However, human testosterone and inhibin B in rats receiving chemotherapeutic drugs such as cisplatin and doxorubicin HSD3B2 and CYP17A1 were less sensitive to taxifolin (13) compared to rat enzymes. Overall, flavonols [79]. Hesperidin glycoside, found in citrus fruits such as lemon and oranges, has been reported to such as quercetin (7) are promising in increasing steroidogenic enzymes activities and may prevent reverse vanadium‐induced decline in testosterone serum levels in male rats [80]. Hesperidin (16), a age-related decline in testosterone production in men. glycosylated form of hesperetin, (Figure 7) was also reported to reverse the decline of testosterone 3.1.4. Flavanones Naringenin (14, Figure7), a flavanone found in grapefruits, has been reported to inhibit the activity of Hsd17b3 and Hsd3b in male rats after daily subcutaneous injections of 15 mg/kg [77]. However, these changes in enzyme activity had no consequences on testosterone levels. Oppositely, oral administration of naringenin (14) to male rats for 10 weeks resulted in increased serum testosterone levels [78]. Therefore, the route of delivery of flavonoids may influence the result outcome in certain experimental designs. Naringenin (14) also prevented the decreases of serum testosterone and inhibin B in rats receiving chemotherapeutic drugs such as cisplatin and doxorubicin [79]. Hesperidin glycoside, found in citrus fruits such as lemon and oranges, has been reported to reverse vanadium-induced decline in testosterone serum levels in male rats [80]. Hesperidin (16), a glycosylated form of hesperetin, (Figure7) was also reported to reverse the decline of testosterone production in diabetic rats [ 81]. Antioxidants 2020, 9, 237 10 of 17 production in diabetic rats [81]. Therefore, flavanones may have the potential to prevent testosterone decline in response to endocrine disruptors, and possibly to aging. OH HO Antioxidants 2020, 9, 237 HO 10 of 17 O OH OH Antioxidants 2020, 9, 237 OH O O 10 of 17 O Therefore, flavanones may have the potential to prevent testosterone declineO in response to endocrine HO O disruptors,production andin diabetic possibly rats toHO [81]. aging. Therefore,O flavanones mayHO hav HO O O OH OH OH O OH O HO OH O HO Hesperidin (16) Naringenin (14) Hesperetin (15) O OH OH Figure 7. OHStructures of naringenin (14O), hesperetin (15),O and hesperidin (16). O O HO O HO O HO 3.1.5. Catechins HO O O OH In male rats, catechin (17), epicatechin (18), and epigallocatechin gallate (EGCG, 19) (Figure 8) OH O OH O have been shown to increase plasma testosterone levels after only 8h of treatmentOH O [82]. Moreover, catechinsNaringenin were ( 14effective) in increasingHesperetin human (15) chorionic gonadotropinHesperidin (hCG)(16) ‐stimulated testosterone production from purified rat Leydig cells [82]. Specifically, epicatechin (18), which is a catechin (17) Figure 7. Structures of naringenin ( 14), hesperetin (15), and hesperidin (16). epimer, increased the activity of Hsd17b3. However, there is evidence that green tea polyphenols 3.1.5.have an Catechins inhibitory effect on testosterone production from rat Leydig cells by inhibiting the PKA/PKC 3.1.5. Catechins pathways, Cyp11a1, and Hsd17b3 [83]. In addition, chronic green tea consumption was also In male rats, catechin (17), epicatechin (18), and epigallocatechin gallate (EGCG, 19) (Figure8) associatedIn male with rats, decreased catechin (plasma17), epicatechin testosterone (18), levels and epigallocatechin due to enhanced gallate aromatase (EGCG, expression 19) (Figure [84]. 8) have been shown to increase plasma testosterone levels after only 8h of treatment [82]. Moreover, haveHowever, been oralshown administration to increase ofplasma catechin testosterone (17) resulted levels in increased after only plasma 8h of treatmenttestosterone [82]. levels Moreover, in male catechins were effective in increasing human chorionic gonadotropin (hCG)-stimulated testosterone catechinsrats by inhibiting were effective aromatase in increasing activity [85]. human Moreover, chorionic injections gonadotropin with catechins (hCG)‐stimulated or its derivatives testosterone also production from purified rat Leydig cells [82]. Specifically, epicatechin (18), which is a catechin (17) productionincreased testosterone from purified plasma rat Leydig concentrations cells [82]. [82].Specifically, Such discrepancy epicatechin in (18 the), whicheffects is of a catechincatechins ( 17on) epimer, increased the activity of Hsd17b3. However, there is evidence that green tea polyphenols epimer,testosterone increased levels theneeds activity further of investigation Hsd17b3. However, to better there define is theirevidence regulatory that green mechanisms. tea polyphenols have an inhibitory effect on testosterone production from rat Leydig cells by inhibiting the PKA/PKC haveEGCG an inhibitory (19), isolated effect on from testosterone green tea, production has been fromreported rat Leydig to inhibit cells estradiolby inhibiting and theprogesterone PKA/PKC pathways, Cyp11a1, and Hsd17b3 [83]. In addition, chronic green tea consumption was also associated productionpathways, Cyp11a1,by swine granulosaand Hsd17b3 cells [83].[86]. In addition,addition, EGCGchronic (19 green) inhibits tea testosteroneconsumption production was also with decreased plasma testosterone levels due to enhanced aromatase expression [84]. However, associatedfrom both basalwith anddecreased hCG‐stimulated plasma testosterone primary Leydig levels cells due without to enhanced affecting aromatase cell viability expression [83]. In [84]. this oral administration of catechin (17) resulted in increased plasma testosterone levels in male rats by However,study, they oral found administration that EGCG of (19 catechin) inhibited (17) resultedthe PKA/PKC in increased signalling plasma pathways testosterone and decreased levels in male the inhibiting aromatase activity [85]. Moreover, injections with catechins or its derivatives also increased enzymerats by inhibiting activity of aromatase Cyp11a1 and activity Hsd17b3. [85]. Moreover, Due to the injectionsimportant with variability catechins in the or itseffects derivatives of catechins also testosterone plasma concentrations [82]. Such discrepancy in the effects of catechins on testosterone increasedon androgen testosterone production, plasma more concentrations research should [82]. be Suchperformed discrepancy to better ine thdefine effects the ofpotential catechins these on levels needs further investigation to better define their regulatory mechanisms. testosteronemolecules have levels to maintainneeds further testosterone investigation levels toaccording better define to aging. their regulatory mechanisms. EGCG (19), isolated from green tea, has been reported to inhibit estradiol and progesterone OH production by swine granulosa cells [86]. In addition, EGCG (19) inhibits testosterone production OH from both basal and hCG‐ stimulatedOH primary Leydig cells withoutOH affecting cell viability [83]. In this study, they found that EGCG (19) inhibited the PKA/PKC signallingHO pathwaysO and decreased the HO O HO O OH enzyme activity of Cyp11a1 andOH Hsd17b3. Due to the importantOH variability in the effects of catechins on androgen production, more research should be performed to better define the potential these OH O molecules have to maintainOH testosterone levels according to aging. OH OH OH OH O OH Catechin (17) Epicatechin (18) OH OH EpigallocatechinOH gallate (19) OH OH HO O HO O HO O OH Figure 8. StructuresOH of catechin (17), epicatechin (18), andOH epigallocatechin gallate (19). Figure 8. Structures of catechin (17), epicatechin (18), and epigallocatechin gallate (19). OH O EGCG (19), isolatedOH from green tea, has been reported to inhibit estradiol and progesterone 3.1.6. Anthocyanidins OH OH OH productionOH by swine granulosa cells [86]. In addition, EGCG (19) inhibits testosteroneO production fromAnthocyanidins bothCatechin basal ( and17) hCG-stimulated are colored pigmentsEpicatechin primary highly Leydig (18 abundant) cells without in berries, affecting currants, cell viabilitygrapes, and [83]. tropical In this Epigallocatechin gallate (19) OH study,fruits. They they foundhave antioxidant that EGCG and (19 )antimicrobial inhibited the properties PKA/PKC [87]. signalling Although pathways these flavonoids and decreased have not the OH enzymebeen specifically activity of studied Cyp11a1 for and their Hsd17b3. potential Due to to regulate the important testosterone variability production, in the eff theyects of may catechins improve on androgen production, more research should be performed to better define the potential these molecules Figure 8. Structures of catechin (17), epicatechin (18), and epigallocatechin gallate (19). have to maintain testosterone levels according to aging. 3.1.6. Anthocyanidins Anthocyanidins are colored pigments highly abundant in berries, currants, grapes, and tropical fruits. They have antioxidant and antimicrobial properties [87]. Although these flavonoids have not been specifically studied for their potential to regulate testosterone production, they may improve Antioxidants 2020, 9, 237 11 of 17

3.1.6. Anthocyanidins Anthocyanidins are colored pigments highly abundant in berries, currants, grapes, and tropical fruits. They have antioxidant and antimicrobial properties [87]. Although these flavonoids have not beenAntioxidants specifically 2020, 9, 237 studied for their potential to regulate testosterone production, they may improve11 of 17 steroidogenesis as they are known to inhibit COX2 activity and to modulate the activity of the steroidogenesis as they are known to inhibit COX2 activity and to modulate the activity of the MAPK MAPK pathway [88,89], both of which are known to influence StAR expression and activity in pathway [88,89], both of which are known to influence StAR expression and activity in Leydig cells. Leydig cells. Interestingly, lead (Pb) mediated decrease in progesterone production from R2C Interestingly, lead (Pb) mediated decrease in progesterone production from R2C Leydig cells was Leydig cells was prevented by cyanidin-3-glucoside (20, Figure9) by protecting mitochondrial prevented by cyanidin‐3‐glucoside (20, Figure 9) by protecting mitochondrial function and increasing function and increasing steroidogenic gene (StAR, Hsd3b, and Cyp11a1) expression [90]. In addition, steroidogenic gene (StAR, Hsd3b, and Cyp11a1) expression [90]. In addition, this anthocyanin also this anthocyanin also upregulated the activities of the MAPK and PKA signalling pathways [90], upregulated the activities of the MAPK and PKA signalling pathways [90], promoting steroid promoting steroid production. Cyanidin-3-glucoside (20) was also reported to improve the expression production. Cyanidin‐3‐glucoside (20) was also reported to improve the expression levels of levels of steroidogenic proteins (StAR, Hsd3b, and Cyp11a1) and of the LH receptor within the testes steroidogenic proteins (StAR, Hsd3b, and Cyp11a1) and of the LH receptor within the testes of mice of mice exposed to cadmium, an important neuroendocrine disruptor [91]. Thus, anthocyanidins may exposed to cadmium, an important neuroendocrine disruptor [91]. Thus, anthocyanidins may promote testosterone production by Leydig cells through their antioxidant properties. promote testosterone production by Leydig cells through their antioxidant properties. OH OH

HO O

OH HO O OH OH HO Cyanidin-3-glucoside (20) Figure 9. Structure of cyanidin-3-glucoside (20). Figure 9. Structure of cyanidin‐3‐glucoside (20). Considering the entire data reported on the regulation of steroidogenesis by flavonoids and Considering the entire data reported on the regulation of steroidogenesis by flavonoids and isoflavonoids, a structure-activity relationship may explain the differences in inhibition and activation isoflavonoids, a structure‐activity relationship may explain the differences in inhibition and of androgen production. Indeed, isoflavonoids with the phenol group in the position 3 of cycle C, activation of androgen production. Indeed, isoflavonoids with the phenol group in the position 3 of such as isoflavones (daidzein (5) and genistein (6)), have been suggested to preferentially inhibit cycle C, such as isoflavones (daidzein (5) and genistein (6)), have been suggested to preferentially HSD3B2 and HSD17B3 rather than aromatase enzyme activities [92]. This may explain the more potent inhibit HSD3B2 and HSD17B3 rather than aromatase enzyme activities [92]. This may explain the inhibitory action of isoflavones on testosterone production from H295R adrenal cells, compared to a more potent inhibitory action of isoflavones on testosterone production from H295R adrenal cells, flavonoid such as apigenin (2) which has its phenol group in the position 2 of cycle C [30]. compared to a flavonoid such as apigenin (2) which has its phenol group in the position 2 of cycle C Aging is associated with reduced StAR protein levels within adult Leydig cells, resulting in [30]. defective mitochondrial cholesterol import and lower testosterone production [93,94]. As previously Aging is associated with reduced StAR protein levels within adult Leydig cells, resulting in reported [95], age-related decline in testosterone production may be delayed by increasing StAR and/or defective mitochondrial cholesterol import and lower testosterone production [93,94]. As previously Cyp11a1 gene expressions using supplementation with flavonoids or chemical derivatives. In aging reported [95], age‐related decline in testosterone production may be delayed by increasing StAR males, Cox2 activity is increased in Leydig cells, resulting in decreased StAR gene expression and and/or Cyp11a1 gene expressions using supplementation with flavonoids or chemical derivatives. In testosterone production [28]. Interestingly, flavonoids such as chrysin (1), apigenin (2), luteolin (3), aging males, Cox2 activity is increased in Leydig cells, resulting in decreased StAR gene expression and quercetin (7) can promote StAR expression and steroidogenesis by inhibiting Cox2-dependent and testosterone production [28]. Interestingly, flavonoids such as chrysin (1), apigenin (2), luteolin signalling. Therefore, the intake of these flavonoids may delay age-related decline in testosterone (3), and quercetin (7) can promote StAR expression and steroidogenesis by inhibiting Cox2‐ production in males. dependent signalling. Therefore, the intake of these flavonoids may delay age‐related decline in 4.testosterone Conclusions production in males.

4. ConclusionImportantly, plasma concentrations of flavonoids for an adequate response of testicular Leydig cells are in the low micromolar range which can be attained with high quality nutrition mainly Importantly, plasma concentrations of flavonoids for an adequate response of testicular Leydig composed of fruits and vegetables. In mammals, appropriate Sertoli cell function is highly dependent cells are in the low micromolar range which can be attained with high quality nutrition mainly on testicular androgen levels to support normal spermatogenesis. According to the cumulated composed of fruits and vegetables. In mammals, appropriate Sertoli cell function is highly dependent literature and our data, flavonoids having a 5,7-dihydroxychromen-4-one backbone tend to increase on testicular androgen levels to support normal spermatogenesis. According to the cumulated StAR expression, contributing to increased testosterone production from testis Leydig cells (Figure 10). literature and our data, flavonoids having a 5,7‐dihydroxychromen‐4‐one backbone tend to increase StAR expression, contributing to increased testosterone production from testis Leydig cells (Figure 10). Although numerous polyphenols demonstrate either activation or inhibition effects on androgen biosynthesis, consideration should be taken for combined exposure to naturally occurring flavonoids and isoflavonoids and their potential additive or synergistic effects on steroidogenesis. Antioxidants 2020, 9, 237 12 of 17

Although numerous polyphenols demonstrate either activation or inhibition effects on androgen biosynthesis, consideration should be taken for combined exposure to naturally occurring flavonoids andAntioxidants isoflavonoids 2020, 9, 237 and their potential additive or synergistic effects on steroidogenesis. 12 of 17

FigureFigure 10.10. Summary of the mechanism of actionaction ofof flavonoidsflavonoids andand isoflavonoidsisoflavonoids onon testosteronetestosterone productionproduction from from testicular testicular Leydig Leydig cells. Flavonoidscells. Flavonoids and isoflavonoids and isoflavonoids mainly regulate mainly steroidogenesis regulate throughsteroidogenesis the modulation through oftheStAR modulationgene expression. of StAR Dotted gene linesexpression. represent Dotted the unknown lines represent regulatory the mechanismsunknown regulatory of flavonoids mechanisms and isoflavonoids. of flavonoids and isoflavonoids.

Author Contributions: Writing—original draft preparation, L.M. and M.T.; writing—review and editing, L.M. Author Contributions: Writing—original draft preparation, L.J.M. and M.T.; writing—review and editing, L.J.M. andand M.T.;M.T.; funding acquisition, L.M. L.J.M. and and M.T. M.T. All All authors authors have have read read and and agreed agreed to the to the published published version version of the of themanuscript. manuscript. Funding:Funding: This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), CanadaCanada (grant(grant numbers:numbers: 386557 toto L.J.M.L.J.M. andand 0456004560 toto M.T.).M.T.). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.

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