The controversial roles of resveratrol in mammalian reproduction

: Resveratrol is one of the most investigated natural polyphenolic compounds and is contained in more than 70 types of plants and in red wine. The widespread interest in this polyphenol derives from its antioxidant, anti-inflammatory and anti-aging properties. Several studies have established that resveratrol regulates animal reproduction. However, the mechanisms of action and the potential therapeutic effects are still unclear. This review aims to clarify the role of resveratrol in the male and female reproductive functions, with a focus on animals of veterinary interest. In the female, resveratrol has been considered a phytoestrogen due to its capacity to modulate ovarian function and steroidogenesis via sirtuins, SIRT1, in particular. Resveratrol has also been used to enhance aged oocyte quality, and as a gametes cryo-protectant with mainly antioxidant, and anti-apoptotic effects. In the male, resveratrol enhanced testes function and spermatogenesis through activation of AMPK pathway. Furthermore, resveratrol has been supplemented to semen extenders improving the preservation of sperm quality. In conclusion, resveratrol has potentially beneficial effects for ameliorating ovarian and testes function. However, due to unclear data, further studies are necessary to consolidate these findings. blocked by estrogen antagonists. In these cell lines, resveratrol probably of action. The results of this study may be useful for developing potential therapies as a male contraceptive agent, where suppression of androgen action is needed [86].


Introduction
Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenol that belongs to the dietary stilbenes, a class of natural compounds that display significant biological activities of medicinal interest. This compound is one of the best known and most investigated polyphenols found in nature, is produced by more than 70 different types of plants, and is contained in red wine and in several botanical extracts [1]. The importance of resveratrol in medicine has been known since the 1940s; it was, in fact, initially extracted from plant roots and successfully used in traditional Japanese and Chinese medicine [2,3]. Resveratrol derives from phenylalanine through the activation of the enzyme stilbene synthase, and exists in two isomeric forms, trans-and cis-resveratrol [4] ( Figure 1). Trans-resveratrol is the most common form in plants and the most widely investigated, therefore, in the present review, we will mainly deal with this isoform. acts independently to the binding to ER, but, in any case, it shows an effect comparable to that of other tested phytoestrogens [27].
In 2002, Henry et al. [17] examined the effect of resveratrol administered to female rats in vivo. Although resveratrol did not show high affinity for ER, it was still able to determine effects on hypothalamic-pituitarygonadal axis regulatory genes, affecting the estrous cycles and inducing gonad hypertrophy in intact animals.
Instead, resveratrol did not replace the effect induced by 17-beta estradiol in rat gonadectomized females [17].
In Chinese Hamster Ovary cells (CHO-K1) it was found that resveratrol binds the two ER receptors (ER alpha and beta) with a similar affinity, but with an affinity approximately 7000 times lower than estradiol [15]. This is in contrast with findings obtained for other phytoestrogens, which bind the beta form of ER with higher affinity than the alpha form [28]. Interestingly, also DES, which has a structural analogy with resveratrol [16], shows a greater affinity for the alpha form of the ER [28].
Although there are conflicting data regarding resveratrol as an agonist of ER, the data testify to a potential role of this compound in enhancing the estrogenic effects of hormones and therefore as a modulator of the reproductive function.

Mechanisms of action: sirtuins
Growing evidences indicate the role of resveratrol in ovarian function and steroidogenesis modulation mediated by sirtuins [11,13] ( Figure 2).
Sirtuins are proteins of the nicotinamide adeninedinucleotide-dependent deacetylases family (or silent information regulator 2 family -SIRT family), that are well-known for their role in many cellular processes [29] such as apoptosis [30], cell reprogramming [31], and DNA repair [32]. Sirtuins are also involved in cancer progression [33], ovarian aging [34,35], redox homeostasis [36], glucose and lipid metabolism [37]. Since modification of the NAD+/NADH ratio controls the activity of SIRTs, all members of this family have a pivotal role in sensing the oxidative stress and energetic condition of the cell [38]. To date, seven members of the sirtuin family have been identified in mammals (SIRT1-7), each member playing a role in ovarian function (for an extensive review, see [38,39]. In fact, damage impairing SIRTs activity leads to fertility deficits [38,40,41]. Resveratrol is the most potent natural ligand of silent information regulator 2 type 1 (SIRT1). After a stressful event, SIRT1 is activated and binds different molecular targets, including NF-kB, p53, FOXO1, FOXO3, and 4, PGC-1alpha, liver X receptor, NBS1 and hypoxia-inducible factor 2a [42][43][44]. By activating these molecules, via SIRT1, resveratrol has a pivotal role in regulating energy homeostasis, gene silencing, genomic stability, and cell survival [42]. Resveratrol may also protect against ovarian aging through SIRT1-related cellular mechanisms, exerting an anti-oxidative effect that guards oocytes from age-dependent deficits [38].
In rat granulosa cells, resveratrol induced a transcript up-regulation of SIRT1, LH receptor, StAR, and P450 Thus, there is enough evidence that resveratrol may have beneficial effect in the reproductive function via sirtuins and specifically via SIRT1 even if the possibility that resveratrol acts through other pathways cannot be excluded ( Figure 2).

Effects of resveratrol on oocyte vitrification
Resveratrol interferes with the endocrine and paracrine communications taking place between the cumulus oophorous and the oocyte. In domestic species, it is well documented that resveratrol enhances the quality of  the developmental competence of vitrified oocytes, only when added to the IVM medium, but not when resveratrol was added as a pre-treatment of the vitrification process.
Phytomelatonin is a well-known product used in phytomedicine for its antioxidant properties [55]. In this context, Lee et al. [56] investigated the synergistic properties of melatonin and resveratrol to ameliorate porcine IVM of oocytes. These authors found that the association of the two compounds in the medium of cumulus-oocyte complexes undergoing IVM supported a synergistic increase in oocyte nuclear maturation and total cell numbers of parthenogenetic activated blastocysts, and improved the development of somatic cell nuclear transfer embryos [56].
In 2015, Ma et al. [57] reported that SIRT1 expression is notably reduced in in vitro-aged oocytes of sows. These aged oocytes are characterized by aberrations such as spindle and chromosome defects, anatomical misdistribution of cortical granules and mitochondria. Resveratrol treatment during pig oocyte maturation reduced (probably via SIRT1) these aging-caused defects [57]. While SIRT1 impairs mitochondria number and functionin oocytes; the supplementation with resveratrolincrease mitochondria in the developing oocytes, improvingtheir own competence [57]. A treatment with resveratrol (2 µM for 14 days) in oocyte-granulosa cell complexes, collected from early antral follicles of gilt ovaries, revealed that it enhanced SIRT1 expression and ATP content in oocytes, improved the blastocyst formation, and ameliorated the growing ability of oocytes [58].
Cow: In cows, resveratrol supplemented to the in vitro cultured (IVC) medium and/or vitrification solution (VS), at 0.5 µM concentration to protect embryos by the negative effect of cryopreservation, partially restored their quality [59]. In fact, resveratrol addition to IVC medium or VS partially compensated the gene expression increase for FOXO3A and PNPLA2, but not for BCL2L1 and BAX, restoring GSH content in bovine embryos [59]. Similar results, such as decreasing ROS levels, either directly or indirectly by increasing GSH levels in oocytes, were obtained in bovine adding different antioxidants, including resveratrol, to the medium during IVM [60]. The same moderate amounts of resveratrol added to the culture medium (0.5 µM) achieved beneficial effects on embryo as suggested by the higher development and hatching rates recorded after 48h post-warming culture [61].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 September 2020 doi:10.20944/preprints202009.0003.v1 Since during cryopreservation, functional aberrations in oocytes may intervene due to lipid content variation and formation of reactive oxygen species, Sprícigo et al. [62] assessed the effect of L-carnitine and/or resveratrol addition to maturation medium before calf oocyte vitrification. L-Carnitine is known both for its modulating activity on lipid metabolism and for its antioxidant action [62]. L-Carnitine and resveratrol supplementation before vitrification decreased spindle damage, and resveratrol addition-modulated apoptosis [62]. The addition of L-carnitine or resveratrol before vitrification positively affected the expression of gene of vitrified/warmed oocytes [62].
Sugiyama et al. [63] collected oocytes and granulosa cell complexes from early antral follicles of aged cows (>10 age years) and examined the effects of resveratrol on mitochondrial generation, degradation, and quality in oocytes grown in vitro [63]. Interestingly, resveratrol affected both oocytes and granulosa cells, improving the quality of growing oocytes, through up-regulation of mitochondrial biogenesis and degradation of growing oocytes and by modulating genes in granulose cells whose expression level are associated to developmental competence of oocytes and embryos [63]. To evaluate SIRT1-mediated resveratrol effects on mitochondrial function of oocytes and on mechanisms responsible for blockage of polyspermic fertilization, In 2014 Takeo et al. found that 20 µM resveratrol improved, via SIRT1 up-regulation, the quality of oocytes cultured in vitro, by ameliorating mitochondrial quantity and quality, ATP content, and their fertilization reducing polyspermic fertilization [64].
Taken together, these findings suggest that resveratrol might be effective in improving oocyte maturation and, thereby, increasing their quality for in vitro fertilization.

Impact of resveratrol on male reproductive function and spermatogenesis
Estrogens were identified in testes where they play a paracrine regulatory function [65][66][67], suggesting a possible role for resveratrol, given its structural similarity to estradiol, as previously reported in this review.
Several studies reported that resveratrol modulates the estrogen-response system acting as regulator of male reproductive function [68]. However, the role of resveratrol in male reproductive function is not clearly established yet, although considerable work has been done. Some studies indicate that resveratrol arguably improves sperm quality in humans [69,70] and domestic animals [71][72][73][74][75]. This seems to be possible thanks to its capacity to pass through the blood-testis barrier, imparting its protective effects in the testis [76].
Resveratrol administration was shown to (  Resveratrol administration in vivo was used to treat infertility. In men affected by dyszoospermia, resveratrol promoted spermatogenesis by ameliorating the effect induced by 2,5-hexanedione [68]. In this study, it was also established that the expression of c-kit, a specific marker protein of spermatogenic cell membranes, was regulated by resveratrol [68]. Resveratrol has been extensively used during cancer therapy since its beneficial effect in preserving male reproductive function has been demonstrated. In this scenario, resveratrol administration preserved the metabolic pathways involved in erectile function and provided functional was indeed correlated with a protective effect on radiation-induced spermatogenesis injury [82]. The results of this study demonstrated that resveratrol can act with other antioxidant molecules to enhance sperm maturation [82]. On the contrary, in the same study, no effect on the protection of Leydig cells as a source of testosterone was observed [82].
The positive effects of resveratrol havealso been shown in metabolic disorders such as diabetes. Abdeli et al. [83] demonstrated that resveratrol ameliorated Type 1 diabetes mellitus-induced abnormal sperm formation, oxidative stress, and DNA damage and had some effects on PARP signaling pathway in the rat testis [83].
Despite considerable data on the effects of resveratrol, the mechanisms underlying this phenomenon are still unclear. According to several studies, resveratrol directly acts on the expression of sirtuin-1 [42,45,84].
According to Seneret al.

Use of resveratrol in sperm cryopreservation
Cryopreservation of sperm is commonly used for the management and long-term preservation of male fertility in humans and domestic animals [87,88]. In humans, sperm cryopreservation is a suitable tool to deal with males affected by azoospermia or susceptible to infertility following cancer treatment [87]. In domestic animals, cryopreservation is used to preserve semen of endangered species or to accelerate the rate of genetic improvement [88]. However, freeze-thawing processes induce oxidative stress in mammalian spermatozoa, decreasing sperm quality and motility, with subsequent reduction of capacity of the spermatozoa to fertilize the oocyte [76, 89,90]. The main source of oxidative stress has been related to the high concentration of polyunsaturated fatty acids located on sperm membranes, which determines the production of large amounts of ROS [91,92] during freeze-thawing procedures [93][94][95][96][97][98]. ROS are mainly represented by hydrogen peroxide (H2O2), superoxide anions (O2-), hydroxyl radicals (OH-). These molecules are generated during intermediate steps of oxygen reduction. It is well-known that ROS have a negative impact on cell morphology and genomic integrity determining damage of cellular proteins, DNA and plasma membrane lipids, due to their free radical nature [99]. Although a low and controlled concentration of ROS is required for sperm functionality, such as hyperactivation, capacitation, acrosome reaction and zona binding events [100][101][102], when ROS reach very high levels, oxidative stress occurs, leading to sperm damage [99].
In the ejaculate, the levels of ROS are controlled by a balance between their cellular production and the catabolism of antioxidative compounds. This equilibrium of ROS levels can be maintained because the seminal plasma of the ejaculate contains antioxidant molecules, membrane stabilizers, and sugars [103,104], among others. However, during the procedure of dilution and cooling, the semen is markedly exposed to oxidative stress since spermatozoa do not have adequate reserves of natural antioxidants functioning to reduce the negative impact of ROS, which induce lipid peroxidation (LPO) during the preparation of sperm for cryopreservation [105,106]. Most of the ROS production occurs in mitochondria during the mechanism of oxidative phosphorylation [70]. Cryopreservation has been correlated with sperm mitochondrial dysfunction Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 September 2020 doi:10.20944/preprints202009.0003.v1 that occurs because of temperature changes, ice formation, and osmotic stress [107]. Furthermore, ROS levels in the sperm significantly increased during the cryopreservation process [101].
External antioxidants have been supplemented to freezing extenders to ameliorate oxidative stress during the cryopreservation process [108]. This practice allows to preserve sperm quality that is affected by oxidation after the freeze-thaw process [108]. The supplementation of antioxidants to the extenders has led to an enhancement in values for the post-thaw sperm quality variables in several species including bull [109], stallion [110], red deer stag [111], dog [112], ram [113], buck goat [114,115], and boar [116,117] semen.
In this scenario, resveratrol has been extensively used as a suitable antioxidant supplement to semen extenders in human, mouse, ram, bull, and boar semen [70,81,[118][119][120][121]. In particular, in vivo and in vitro studies indicated that resveratrol improves sperm quality during the cryopreservation process [69,122], thanks to its protective function against lipid peroxidation (LPO) and DNA damage caused by ROS [70,123].
In humans, resveratrol has been reported to minimize post-thawing DNA damage to spermatozoa [120].
Similarly, in cattle, it has been observed that supplementation of resveratrol in semen extender improved postthaw bull sperm quality, in terms of sperm motility, mitochondrial activity, and DNA integrity [120]. The ability of resveratrol to act as antioxidant was also proved using induced oxidative stress in vitro, where it was reported that mouse [119], cattle [124], and human spermatozoa [70] can be protected by resveratrol.
Furthermore, in frozen-thawed ram sperm, the addition of resveratrol to the tris-egg yolk-glycerol extender was shown to reduce sperm mitochondrial membrane potential [125].There are many other studies that demonstrated how resveratrol may act as antioxidant. All these studies lead to common conclusions that can be summarized in the ability of resveratrol to: 1) reduce ROS production in the mitochondria, 2) scavenge superoxide radicals, including superoxide anion, hydroxyl radical, and metal-induced radicals, 3) inhibit lipid peroxidation, and 4) regulate the expression of antioxidant cofactors and enzymes [126][127][128]. However, even if there is a clear positive effect on sperm quality, there are no data that indicate that resveratrol may improve motility of freeze-thawed spermatozoa. Furthermore, as discussed until now, the use of resveratrol in sperm preservation was extensively related to its supplementation to extenders before cryopreservation. However, in a recent study, it was shown that resveratrol supplementation in washing and fertilization media improved fertilization capability of bovine sex-sorted spermatozoa with respect to not-treated ones, increasing blastocyst percentage and quality following IVF [129]. This occurred because the spermatozoa had a decreased oxidative stress, since mitochondrial function and acrosomal integrity were ameliorated [129].  AMPK is a key kinase involved in regulating the cellular redox state by switching the metabolic pathway under stressful conditions [64,65]. It was observed that resveratrol activated AMPK in somatic cells in vitro [132]. In human spermatozoa, it was demonstrated that AMP-activated protein kinases are mainly present in the whole flagellum and the post-equatorial region of the head [133]. Related to these findings, supplementation of resveratrol increased AMPK activity and was beneficial for protection against cryopreservation-induced oxidative stress of human spermatozoa by improving DNA integrity and transcripts, which was used as markers of sperm quality [133]. Similar results were obtained in boar [134] and goat [135] spermatozoa. In both studies, the addition of resveratrol activated AMPK phosphorylation, allowing the reduction of ROS production, while enhancing the sperm antioxidative defense system such as GSH level and activities of glutathione peroxidase (GPx), SOD, and catalase ( Figure 4). However, while it is well-established that resveratrol is capable of activating AMPK, the exact mechanism by which this occurs remains to be clarified [136]. This is because the activation of AMPK can take place through a variety of complex and apparently contradictory mechanisms, which include an increase in the AMP/ATP ratio [132], inhibition of mitochondrial ATP synthase [137,138], ROS (independent of the AMP/ATP ratio) [139,140], as well as upstream serine/threonine kinases, such as LKB1 (Peutz-Jeghers protein) [141,142], and calcium/calmodulin-dependent protein kinase kinase b (CaMKKb) [143,144].

Conclusions
Resveratrol is a natural polyphenol with antioxidant, anti-inflammatory, and anti-aging properties. In several studies, it has been shown that resveratrol modulates both female and male reproduction.
In the female, resveratrol is considered a phytoestrogen with a chemical structure similar to that of some estrogens. Interestingly, resveratrol is potentially usable alone or in combination with other hormones for its moderate estrogenic effect. Moreover, resveratrol exerts a steroidogenesis modulation in the ovary via sirtuins, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 September 2020 doi:10.20944/preprints202009.0003.v1 especially SIRT1. Finally, resveratrol is as a quality enhancer of aged oocytes and a gametes cryo-protectant, with mainly antioxidant, and anti-apoptotic effects.
In the male, resveratrol modulates the reproductive function by: 1) enhancing the production of testosterone, 2) triggering penile erection, and 3) improving spermatogenesis including sperm differentiation and number in the testes and ejaculate, respectively. The mechanisms of action seem to be exerted by activating AMPK pathway. Finally, resveratrol is a suitable antioxidant to supplement to semen extenders thanks to its beneficial effect in preserving sperm quality.