The oviduct: from sperm selection to the epigenetic landscape of the embryo

The oviduct: from sperm selection to the epigenetic landscape of the embryo Abstract The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed. Introduction The oviduct is a convoluted tube bridging from the ovary to the uterus in the mammalian female. It is composed of three main longitudinal regions designated from the ovary to the uterus: the infundibulum (fimbriae in humans), in which most cells are ciliated epithelial cells; the ampulla, which also contains large numbers of ciliated epithelial cells and is the site of fertilization; and the isthmus, which contains a large number of secretory epithelial cells. The reproductive functions of the ovary and uterus have been well established, yet the role played by the oviduct in reproduction is far less understood. The oviduct transports the gametes and embryo, and provides biochemical and biophysical support for preimplantation development. In addition, there is direct and indirect evidence for two types of interactions, physical and molecular, among both gametes, embryos and oviduct. The incorrect functioning of the oviduct can lead to infertility or to pathophysiologies such as ectopic pregnancy due to impaired embryonic transport [1, 2]. The main difference between natural conception and in vitro fertilization (IVF) is that in the latter there is limited gamete selection, fertilization occurs under artificial conditions, and development is not subjected to the dynamics of the in vivo environment. When the oviduct is bypassed in IVF, there is no communication between embryo and oviduct, naturally a prerequisite for normal development and transport to the uterus. It is usually assumed that the in vitro culture medium is the main factor affecting embryo quality in vitro. However, we must not forget other factors including sperm selection. This is because of all the spermatozoa in an ejaculate, very few are selected by the female genital tract to fertilize the oocyte. The hardening of the zona pellucida (ZP) and other modifications that occur in vivo are other important factors for fertilization that are missing in vitro. Furthermore, the epigenetic marks of the embryo are reprogrammed during early development, and in vitro culture can have a profound effect on this. For example, it has been shown that the oxygen tension in vitro greatly impacts global DNA methylation of the embryo [3]. In effect, several studies have shown that an in vitro embryo environment may have long-term effects on subsequent offspring, possibly via epigenetic mechanisms at play during development [4, 5]. Due to the relative success of IVF, the role of the oviduct has been underrated. However, the oviduct not only serves as a passageway for gametes and embryo to enter the uterus. It also acts as a cofactor to facilitate sperm migration towards the egg, keep sperm alive for hours or days, select spermatozoa, and culture the embryo in a spatiotemporally dynamic system that will reprogram its genome and epigenome, thereby improving quality and ensuring implantation and subsequent normal development. In this review, we focus on the contents of oviductal fluid including the oviductosomes, and describe the role of the oviduct in sperm selection and how sperm regulates an immune response within it. Also, we discuss the role of the oviduct in early embryo development and how it may shape the epigenetic landscape of the embryo. A better understanding of the biology of the oviduct during sperm migration and preimplantation will help further the development of reproductive technologies. Oviductal fluid and extracellular vesicles (oviductosomes) The secretory cells of the oviduct epithelium secrete proteins and other factors that, together with plasma-derived constituents, form oviduct fluid (OF) [6, 7]. Oviduct fluid composition is complex including simple and complex carbohydrates, ions, lipids, phospholipids, and proteins [8]. Some of these constituents are metabolic substrates, such as lactate, pyruvate, amino acids and glucose, the concentrations of which differ from those present in the uterine fluid and serum [6, 9, 10]. Oviduct fluid protein concentrations are low compared to those of blood serum and also vary between species. Average protein concentrations in nonpregnant females have been estimated at 13.6–30.2 mg mL−1 in sheep [11, 12], 0.17 mg mL−1 in horses [13], and 20–80 mg mL−1 in humans [14]. Although some proteins such as albumin are in abundance, other proteins appear in lower amounts, with highly sensitive equipment or complex experimental processes needed to identify them. Early attempts to identify these low-level OF components were based on microarray analyses of epithelial cells to detect potentially secreted proteins. More recently, studies have shown that OF proteins can be directly detected by mass spectrometry. These studies have revealed the highly complex composition of this biological fluid and that it is modified by several factors. Dynamics of the protein composition of oviduct fluid Oviduct fluid composition differs according to estrus cycle stage as suggested by transcriptome studies in cows [15, 16], pigs [17], and humans [18, 19]. Proteomic analysis of oviduct epithelial cells suggests a similar protein composition during the follicular and luteal stages of the reproductive cycle in the pig [20]. However, quantitative differences in the OF proteome have been reported in sheep and cow during the estrous cycle [21]. In the pig, these differences amounted to a 79% increase in the relative abundance of proteins in estrus compared with a 21% increase in the luteal stage [22]. Oviduct cells react to oocyte and sperm presence by modifying their gene expression patterns as observed in pigs [23], cow embryos [24], and mouse seminal fluid [25]. Hence, changes in the oviduct microenvironment are induced by the presence of gametes, semen, or embryos. For instance, the reduced expression of cytokines Csf2, Lif, and Il6 in the oviduct has been reported in mated female mice not exposed to seminal plasma, and cytokines Efg and Trail were correlated with the presence of zygotes in the oviduct [25]. Furthermore, the presence of X or Y spermatozoa in the pig oviduct gives rise to changes in oviduct cell gene expression [23]. Two studies have also detected changes in the secreted OF proteome in response to the presence of gametes [26, 27]. More recently, differences have been identified in the cow oviduct transcriptome depending on ovulatory follicle size [28] and on the oviduct examined in the same individual—ipsilateral or contralateral—after ovulation [29]. This result was also observed using a proteomic approach [21]. Moreover, other studies have shown varying gene expression between the specific ampulla or isthmus regions of the oviduct where fertilization and early embryo development take place, respectively [30]. Protein composition of oviduct fluid In the mammalian species examined to date, OF composition is analogous suggesting that the main biological processes in the oviduct are conserved [16, 17, 20] (Figure 1). This idea is biologically supported by the success of embryo development in the oviducts of heterologous species [31]. Thus, some 2400 orthologous genes expressed in the pig and cow oviduct have been identified [17] and more than 1090 orthologous genes are common to the oviduct transcriptome of humans, pigs, and cows [16, 18, 32]. In the pig oviduct, nearly 5000 genes are expressed, of which 11% and 7% correspond to membrane and secreted proteins, respectively [17]. These proteins are theoretically more relevant because they directly contact the gametes and embryos. Through microarray analysis, the presence of 314 proteins secreted by oviduct epithelia was suggested. In a recent gel liquid chromatography MS/MS study performed in adult cyclic ewes, more than 600 proteins were identified in the OF [22]. Such differences in the gene expression of secreted proteins encountered between microarray and proteomic analyses may be the consequence of several factors, the most likely being related to the OF origin. Oviduct fluid composition is influenced by blood plasma transudate but is regulated by the ciliated and secretory epithelial cells which form a cell monolayer lining the lumen. Consequently, only some of the proteins detected in OF are transcribed in the oviduct. Albumin is a typical example. This protein is the most abundant in OF, but its transcript is not present in oviduct cells since the gene encoding this protein is only present in the liver. Figure 1. View largeDownload slide Venn diagram showing overlapping and nonoverlapping oviduct proteins in the pig [19], sheep [23], cow [21], and horse [15]. Figure 1. View largeDownload slide Venn diagram showing overlapping and nonoverlapping oviduct proteins in the pig [19], sheep [23], cow [21], and horse [15]. Data emerging so far from the detailed analysis of OF proteins have been a priori quite surprising because a considerable number of detected proteins are not typically secreted. For example, the presence of a large number of proteins that normally occur in the cytoplasm, nucleus, or mitochondria has been reported [22, 32]. In ewes, cytoplasmic proteins represent more than 40% of the proteins identified [22]. The most accepted hypothesis is that this protein presence is the result of epithelial cell shedding during renewal of the epithelium. The different proteins found in OF play different biological roles. Some contribute to oxidation or immune protection, fertilization, embryo development, or are proteases and their inhibitors. Expression of the anti-inflammatory cytokines TGFB1 and IL10 has been observed in the epithelial cells of the cow and pig oviduct [33, 34]. These molecules could be involved in maintaining tolerance to non–self-cells in the oviduct. This is discussed later in this review. Other proteins identified in OF play a relevant role in fertilization. Two abundant proteins are albumin and Oviductal glycoprotein 1 (OVGP1). Albumin has an important function in sperm capacitation via the removal of cholesterol from the plasma membrane [35]. OVGP1 also seems to play a key role in sperm physiology as it has been reported to induce the phosphorylation of sperm proteins during their capacitation [36, 37]. Other reports also exist of the interaction of OVGP1 with the ZP causing increased resistance of the ZP to protease treatment, or “ZP hardening.” This hardening of the ZP mediated by OVGP1 and OF has been related to polyspermy control before fertilization as an important event in some species such as the pig [38, 39]. Also importantly, knock-out (KO) mice for OVGP1 maintain their fertility, suggesting that this protein is not essential for fertilization [40]. Furthermore, OVGP1 is a pseudogene in the rat. However, it could be that other proteins of the same chitinase family act as paralogs and take over OVGP1 function. A similar functional model has been described for acrosin KO mice [41]. It has been recently confirmed that OVGP1 is secreted by in vitro cultured bovine oviduct epithelial monolayer under a system designed for modeling the oviduct suggesting a relevant role of this protein for the function of the oviduct [42]. The oviduct also plays an important role in protecting gametes and the preimplantation embryo. It is well known that free radicals have a deleterious effect on gametes and embryos [43] and the presence of antioxidant proteins involved in cell protection has been shown in the cow [21, 44] and sheep [22]. In addition, proteases and their inhibitors have been found in OF. However, it was not until a few years ago that the key role of OF in fertility was unveiled. Thus, following the development of genetically modified mice, the need for the fine modulation of proteases and their inhibitors for the survival of oocytes and embryos was reported [45, 46]. Immune cells, extracellular vesicles, and oviductosomes Several other components have been, detected in OF. Some are easy to identify like the cells of the immune system. Polymorphonuclear cells have been found in bovine OF with implications for a role in defense and possibly in the phagocytosis of spermatozoa [47]. Electron microscopy has revealed the presence of very small components surrounded by membranes. These structures are described as extracellular vesicles (EVs), a general term encompassing several different vesicle types, released by somatic cells present in body fluids, and containing bioactive molecules (i.e., proteins and RNAs, mRNAs, miRNAs) [48, 49] and lipids [50]. The terminology generally used for EV is size and origin associated such that exosomes (30–200 nm) arising from endosomes and microvesicles (MV) (100–1000 nm) are EVs that bud from the plasma membrane. These different EVs can be separated by ultracentrifugation [51]. Extracellular vesicles were first described in the mouse oviduct and then characterized by the presence of the sperm adhesion molecule 1 protein (SPAM1) [52]. Both EV types show significant heterogeneity in size and also in distribution according to the OF source, i.e., ampulla vs isthmus [53]. In addition, recent studies have revealed further EV heterogeneity indicating different subtypes of EV of different composition [51]. It would be interesting to identify, using current isolation procedures, the different EV subpopulations in OF collected from different oviduct regions and during different phases of the estrous cycle and assess their biological implications. Histological examination of the epithelium reveals two main cell types, ciliated and nonciliated, which probably produce different EV. This issue warrants further investigation. A recent study has identified 319 proteins present in EV harvested from cattle [44], 153 of these proteins have also been identified in the OF but other 166 proteins were not detected previously in the OF [21]. Almiñana et al. [44] found 175 proteins in EV obtained from both OF in vivo or the supernatants of cultured epithelial cells. However, it should be mentioned otherwise that their protein composition was specific to their in vivo or in vitro origin. Thus, some proteins were identified only in the in vivo setting (97) and others (47) only in in vitro culture. Further work is needed to examine the characteristics of other EV components (RNAm, miRNA, and lipids). Role of the oviduct in sperm selection and fertilization Sperm heterogeneity The ejaculate is a heterogeneous mix of different subpopulations of spermatozoa [54]. In addition to the abnormal or immotile fraction, within the normal motile subpopulation, a number of fractions can be differentiated through their specific properties. This means that spermatozoa can be grouped according to motility patterns [55]. The presence/absence of specific motility patterns and subpopulations has been correlated with fertility and the success of assisted reproductive technologies (ARTs) [56]. As a consequence, interest is growing in characterizing sperm subpopulations as an analytical tool for sperm quality in a number of mammalian species including cow [57], sheep [58], horse [59], and human [60], among others. Interestingly, sperm heterogeneity has also been shown at the epigenetic level through the finding that the same human ejaculate shows different sperm fractions bearing different methylated DNA regions [61]. Further, within the same ejaculate, spermatozoa coexist showing different degrees of DNA fragmentation [62]. Collectively, these lines of evidence point to the presence of a number of subpopulations of different quality in the same sample. Therefore, it is widely accepted that not all spermatozoa in an ejaculate are equally good at fertilization and that a high-quality fraction is selected within the female genital tract [63]. This may be especially critical for ARTs and in particular for the technique of intracytoplasmic sperm injection used in fertility treatments, because of the risk of fertilizing the oocyte with spermatozoa with errors that could affect the offspring in the long run [64]. Indeed, in human clinical practice, this risk may be higher if we consider the higher incidence of DNA fragmentation in the spermatozoa of infertile men [65–67]. Accordingly, sperm selection prior to ART is becoming an important research field in which the main challenges are to discover the sperm subpopulation selected within the female genital tract and the mechanisms involved in their selection in vivo. Only after acquiring this knowledge will we be able to design efficient in vitro methods for selecting high-quality spermatozoa for use in ARTs [68]. Selection mechanisms operating within the oviduct Mammalian spermatozoa have to overcome a number of obstacles in the female genital tract before reaching the fertilization site at the ampulla. Vaginal pH, resistance to sperm migration of the cervical mucus, the narrow utero-tubal junction, the meandering oviduct lumen, the response of the immune system, etc., are all physical/anatomical conditions of the female genital tract that configure a stringent selection mechanism for spermatozoa [68, 69]. Effectively, in all mammalian species examined to date it has been shown that of the many millions of spermatozoa ejaculated, only tens to hundreds reach the ampulla [70, 71], or site of fertilization. Presumably, this select group of spermatozoa have a greater fertilization capacity and better characteristics for supporting embryo development [68]. However, little is known about this sperm subpopulation, its relative effectiveness, and its selection criteria in vivo [68]. Migration of spermatozoa through the lower female genital tract already poses a first selective barrier, whereby viscosity, pH, immune response, etc., select those spermatozoa that are able to advance in this aggressive environment (Figure 2). However, it seems that the fine selection process occurs at the oviduct. Hourcade et al. in 2010 [72] noted in mice that spermatozoa with high-quality DNA are selected only within the oviduct, and that spermatozoa retrieved from the uterus actually show more DNA fragmentation than those harvested from the vagina (Figure 2). This might indicate that transit from the vagina through the cervix and uterus leads to extensive damage to the sperm population as a consequence of the aggressive conditions of pH, immune response, etc., encountered. Thus, we should perhaps view sperm selection in the oviduct as a way of sorting only those spermatozoa with a high-quality genetic content that have survived this initial transit. Figure 2. View largeDownload slide Model of sperm guidance and selection within the oviduct. The fraction of spermatozoa reaching the uterus contains high levels of fragmented DNA. Subsequently, within the oviduct spermatozoa with high DNA integrity are selected [19]. Near the utero–tubal junction, the spermatozoa attach to the epithelium in a region known as the sperm reservoir where they acquire the final maturation stage that capacitates them to fertilize the egg. Capacitated spermatozoa at the sperm reservoir (1) acquire a motility pattern known as hyperactive motility (2) that facilitates their detachment from the epithelium. Released spermatozoa are then guided by thermotaxis to the proximity of the egg at the ampulla. Finally, a gradient of chemoattractants generated by the COC guides the spermatozoa towards the egg for its fertilization. Figure 2. View largeDownload slide Model of sperm guidance and selection within the oviduct. The fraction of spermatozoa reaching the uterus contains high levels of fragmented DNA. Subsequently, within the oviduct spermatozoa with high DNA integrity are selected [19]. Near the utero–tubal junction, the spermatozoa attach to the epithelium in a region known as the sperm reservoir where they acquire the final maturation stage that capacitates them to fertilize the egg. Capacitated spermatozoa at the sperm reservoir (1) acquire a motility pattern known as hyperactive motility (2) that facilitates their detachment from the epithelium. Released spermatozoa are then guided by thermotaxis to the proximity of the egg at the ampulla. Finally, a gradient of chemoattractants generated by the COC guides the spermatozoa towards the egg for its fertilization. After spermatozoa have overcome the barriers of the vagina and cervix, they are transported by pro-ovarian contractions of the myometrium [69] to the uterotubal junction. At this site, the first specific selection process seems to take place. It has been reported that spermatozoa cannot pass through the junction if they do not possess certain proteins in the sperm head plasma membrane (Figure 2). This was discovered when null mutant mice for genes encoding a disintegrin and metalloproteinase 2 (ADAM2) [73], calmegin [74, 75], or testis-specific angiotensin converting enzyme [76, 77] were found to be infertile because their spermatozoa were neither able to pass through the uterotubal junction nor bind to the ZP. Although the mechanisms for this selection are unknown, it has been speculated that the functions of these proteins, especially ADAMS, might be to enable the spermatozoa to gain a foothold on the wall lining the junction and move forward onto the oviduct by lightly sticking to the epithelium [78]. Thus, ADAMS could function for binding the CD9 protein that is exposed on the oviductal epithelial cells, and in this way it could represent a specific mechanism of sperm selection through protein–protein interaction [79]. The spermatozoa entering the oviduct then attach to the epithelium of the isthmus in a fairly defined location depending on the species [69, 80–82] (Figure 2). This location is known as the sperm reservoir, and it has been proposed as the place where spermatozoa acquire the final maturation status known as capacitation [69] (see below). When spermatozoa are capacitated, they detach from the epithelium by acquiring hyperactive motility and swim up the oviduct towards the ampulla where fertilization occurs [69, 82, 83]. This adhesion is mediated via lectin-like molecules on the sperm surface that are able to bind carbohydrates exposed on the oviduct cell apical membrane [84]. Thus, the spermatozoon's specific protein profile enables it to pass through the uterotubal junction, and specific carbohydrate moieties responsible for its attachment to the oviduct epithelium configure a molecular passport of sorts ensuring the selection of the adequate spermatozoa [85]. Once spermatozoa detach from the sperm reservoir, they still need to swim through the oviduct to reach the ampulla where the egg awaits. The oviduct is narrow, convoluted, and relatively long (3–5 cm in humans). Thus, it has been suggested that spermatozoa must have some kind of navigation system to help them swim in the right direction towards the ampulla where fertilization occurs [86, 87]. So far, three such guidance mechanisms have been proposed on the basis of in vitro studies: thermotaxis—swimming up a temperature gradient (shown in rabbits, humans [88], and mice [89]), rheotaxis—swimming against a fluid flow (shown in mice and humans) [90], and chemotaxis—swimming up a concentration gradient of chemoattractant (shown in humans [91], rabbits [92], and mice [93]). Whereas rheotaxis seems to be a passive mechanism as a consequence of the hydrodynamics of motile spermatozoa [94], both thermotaxis and chemotaxis are active mechanisms that have been shown to be functional only in a specific subpopulation representing ∼10% of the total spermatozoa. In both cases, these subpopulations have to undergo capacitation in order to respond to migration stimuli [87, 95]. How capacitated spermatozoa acquire the ability to respond to both thermotaxis and chemotaxis is an unknown matter. However, a possibility is that the reorganization of the plasma membrane occurring during capacitation [96] determines the correct configuration/location of the thermosensors and chemosensors as well as their relation to the coupled transduction signaling in order to respond to the stimuli. According to this hypothesis, thermotaxis would function as the primary selective mechanism by guiding only capacitated spermatozoa released from the sperm reservoir through the relatively long distance separating the isthmus from the ampulla [97] (Figure 2). Then, once in the proximity of the oocyte, only those responding by chemotaxis would make contact with the cumulus oocyte complex (COC) and thus achieve fertilization [97] (Figure 2). Chemotaxis would not only guide the spermatozoa within the ampulla towards the COC but also through the layer of cumulus cells towards the egg, where a gradient of chemoattractant is established [98]. In addition, an increasing concentration of the chemoattractant within this layer would also potentiate the generation of hyperactive events in the spermatozoa as well as provoke the release of the acrosomal content to facilitate the penetration through the cumulus oophorus until reaching the surface of the egg [99]. Thus, the responsiveness of the spermatozoa to the chemoattractant could configure a stringent selective feature in the last stages prior fertilization. Sperm subpopulation selected within the oviduct Currently, the characteristic of the specific sperm subpopulation selected in vivo responsible for fertilizing the egg are unknown, but we do know that not all the spermatozoa in an ejaculate are able to fertilize the egg in vitro or even get close to it in vivo. This means IVF requires the placement of millions of spermatozoa directly on the egg [100], while, in vivo, only a very restricted number of spermatozoa make contact with the egg at the ampulla [95]. This indicates that either there are sperm selection mechanisms within the oviduct or that spermatozoa managing to reach the ampulla acquire the capacity to fertilize the egg during their transport within the oviduct. It is known that final maturation of the spermatozoon occurs within the female genital tract through its interaction with uterine and oviduct secretions [96]. This maturation process is known as capacitation and involves complex changes in the biochemical composition of the spermatozoon that enable it to fertilize the oocyte. During capacitation, membrane fluidity is enhanced through the loss of plasma membrane cholesterol [101], and changes have been observed in intracellular ion concentrations [102] along with hyperpolarization of the sperm plasma membrane [103], increased protein kinase A activity [104], and protein tyrosine phosphorylation [105]. It was generally assumed that all these changes prepared the spermatozoa for the acrosome reaction and fusion with the oocyte membrane [106]. However, in mice it has been recently shown that the acrosome reacts during sperm migration within the oviduct [107] and also that mice oocytes can be fertilized by spermatozoa that have completed their acrosome reaction before entering the cumulus oophorus [108]. These observations thus question the actual functioning of the acrosome and this issue has generated controversy among reproductive biologists. It is known that the capacitated sperm subpopulation shows a special motility pattern known as hyperactivation. Under low-viscosity conditions, this pattern involves vigorous movements produced by asymmetrical and high-amplitude waves in the flagella, resulting in erratic swimming trajectories [109]. This pattern allows the sperm to progress in viscoelastic conditions such as those encountered in the female genital tract [99, 110]. Thus, the acquisition of this motility type has been linked to fertility and to the ability of the spermatozoa to respond to migration stimuli such as thermotaxis [111] and chemotaxis [112]. It has been estimated that under in vitro conditions, only a small fraction of spermatozoa, about 10%, is capacitated [113]. However, this proportion of capacitated sperm within the female tract in vivo is unknown and neither do we know whether it affects all spermatozoa in the same way at the same time. For instance, it has been suggested that capacitation might occur sequentially in different sperm subpopulations during their storage in the sperm reservoir. This would ensure continuous waves of capacitated spermatozoa that would be continuously detached from the epithelium where they are stored and then swim towards the egg [113]. In this way, synchrony between oocyte and spermatozoa for fertilization would be ensured for a single copula. As aforementioned, only capacitated spermatozoa are able to migrate within a temperature gradient by thermotaxis and respond by chemotaxis to swim towards the oocyte. Thereby, thermotaxis and chemotaxis can be seen as mechanisms for selecting capacitated spermatozoa. Indeed, this premise opens the way for a promising field of research on the selection of spermatozoa using these migration properties. In future, it would be interesting to check the quality of spermatozoa responding by both thermotaxis and chemotaxis and examine their efficiency in ARTs. In addition, this subpopulation needs to be characterized to find specific markers that could be suitable for male fertility diagnosis or for sperm selection using techniques such as magnetic-activated cell sorting [114]. Sperm regulation of the female immune response at conception To ensure pregnancy success from the moment in which the sperm reaches the female reproductive tract, careful regulation of the maternal immune system is needed to allow selective immune privilege for male gametes and the forthcoming fetus, while maintaining a reasonable immune response to pathogens. This phenomenon is known as the “immunological paradox of pregnancy” and has an immediate effect on ovulation, sperm selection and fertilization, but also a long-term effect in assuring the acceptance of fetal tissues [115]. Insemination constitutes the beginning of communication between the female immune system and paternal antigens. Although the volume and fractions of the ejaculate differ between species, contact with maternal tissues induces a postmating inflammatory response in most mammals [116–119] (Figure 3). Uterine and cervical cells then synthesize proinflammatory cytokines [118, 120] that induce an influx of leukocytes into the uterine lumen [121]. In particular, neutrophils attract monocytes and dendritic cells, and are able to recruit, activate, and program antigen presenting cells. Finally, seminal plasma will determine if these cells activate or suppress other immune mechanisms such as T-cell activation [122]. This inflammatory response needs to be resolved before the embryo implants for pregnancy to succeed. To this end, increasing progesterone levels inhibit the synthesis of certain chemokines, but the transition seems to be also driven by seminal plasma components [118, 123]. Figure 3. View largeDownload slide Regulation of the maternal immune system at conception. After insemination, cytokines present in seminal plasma induce an inflammatory response in the female reproductive tract. The release of proinflammatory cytokines by epithelial cells attracts leukocytes and dendritic cells that recruit and modulate T-regulatory cells (Treg), inducing immune tolerance to paternal and fetal antigens. This cascade of events ensures that successful pregnancy is established. Figure 3. View largeDownload slide Regulation of the maternal immune system at conception. After insemination, cytokines present in seminal plasma induce an inflammatory response in the female reproductive tract. The release of proinflammatory cytokines by epithelial cells attracts leukocytes and dendritic cells that recruit and modulate T-regulatory cells (Treg), inducing immune tolerance to paternal and fetal antigens. This cascade of events ensures that successful pregnancy is established. Studies in mice, gilts, and humans have shown that the female immune response is mainly mediated by seminal plasma, while spermatozoa play a secondary role [118, 123–125]. Seminal plasma originates in the male sex accessory glands and contains a range of molecules that provide the sperm with metabolic support and protection from the acidic environment of the vagina [126]. Specific factors present in seminal plasma interact with uterine cells to induce the expression of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, and many other chemokines [127]. However, the main trigger for an immune response seems to be transforming growth factor-β (TGF-β). This cytokine is present in its precursor form in seminal fluid, and is activated in the female reproductive tract [128]. It can subsequently either directly activate multiple immune cell types [129] or indirectly induce the expression of other cytokines such as prostaglandin E2 (PGE2) and IL-8 [128, 130]. It has been shown that both TGF-β and PGE2 inhibit the function of natural killer cells and neutrophils [126]. Another key event triggered by exposure to seminal plasma is T-cell priming to respond correctly to paternal antigens, activating immune tolerance in the female reproductive system [131]. Therefore, seminal plasma would play a dual role in inducing and resolving inflammation. The role of the female reproductive tract in sperm selection remains controversial. Although they represent less than 15% of the total cell fraction, immature germ cells, epithelial cells, and leukocytes are also present in semen [132]. In contrast to spermatozoa, these cells express paternal major histocompatibility complex (MHC) molecules that could trigger a response by the maternal immune system [133]. It has also been hypothesized that aged, dead, or capacitated spermatozoa induce the chemotaxis of neutrophils mediated by endometrial cells or leukocyte signaling and that this takes part in sperm cell selection [117, 119, 134, 135]. However, selective sperm phagocytosis is still questionable as studies in the pig have indicated that intact viable sperm cells are more likely to bind to neutrophils in vitro, though in these studies contact was transient and the phagocytic role of neutrophils was inhibited by seminal plasma in vivo [136–138]. Similar interactions between neutrophils and spermatozoa have been described in other species such as horses, ruminants, and humans, but the underlying molecular mechanism is still unclear because the presence of sperm surface molecules that could be recognized by neutrophils has not been demonstrated; thus, interactions could be mediated just by random attachment [139–141]. Other hypotheses are that sperm bindings could induce signaling pathways promoting subsequent inflammatory responses, or that there could be a negative selection process against spermatozoa which are not able to attach to epithelial cells and not able to fertilize the oocyte [122]. Before embryo hatching takes place, it is thought that the ZP acts as a physical barrier against the maternal immune system [142] and protects the early embryo from the oviductal environment [143]. In particular, carbohydrate sequences expressed on the ZP could play a key role in protecting the embryo [144]. Following this, to allow the development of fetal trophoblast cells posthatching, maternal immune mechanisms need to be controlled, and the proinflammatory environment needs to be switched to an anti-inflammatory state (Figure 3). Increasing maternal progesterone concentrations together with levels of TGF-β and PGE2 in the seminal plasma play a key role. TGF-β induces the generation of antigen-specific regulatory T (Treg) cells in peripheral tissues. After paternal antigen-driven activation, they proliferate in paraaortic lymph nodes draining the female reproductive tract to later mediate in the processes of T-cell proliferation and awareness of paternal and fetal antigens [129, 145]. Furthermore, they are able to regulate T-cell production of IL-10, which induces the generation of anti-inflammatory cytokines and inhibits the production of antibodies [146]. These mechanisms are also enhanced by progesterone, which elicits the production of PGE2 by epithelial cells [147] and promotes the secretion of more IL-10 [148]. In this way, immune cells can regulate their proliferation and activation and even promote their own death. Treg cells are essential during this process, and their impairment has been associated with reproductive disorders such as miscarriage, pre-eclampsia, intrauterine growth restriction, and preterm birth [149–151]. Although maternal recognition of pregnancy takes place in the uterus, studies have shown that early embryos can induce signaling pathways that are recognized in the oviduct. Early studies in horses, rats, and hamsters revealed that fertilized embryos are transported to the uterus faster than nonfertilized oocytes because of certain signaling molecules they produce such as PGE2 [152–154]. Some years later, the impact of the embryo on the oviduct was confirmed by gene expression analysis in the mouse, pig, and cow [155–158]. Interestingly, one of the genes upregulated in the oviduct by the presence of four-cell stage pig embryos was TGF-β binding protein II [156]. Moreover, in pigs and cows, embryo presence induced the downregulation of genes in the oviduct related to the immune system, particularly those related to inflammation, the complement system and the MHC [157, 158]. In conclusion, successful pregnancy establishment requires the precise regulation of the female immune system. Insemination induces a fast inflammatory reaction followed by proliferation and activation of immune cells that are able to regulate their own response to paternal and fetal antigens. However, while studies in recent years have provided a broad knowledge of the mechanisms involved, there are still details that need to be filled in. This is important to maximize reproductive efficiency in livestock, as varying sperm concentrations or semen extenders elicit different immune responses. Moreover, deficient immune tolerance to male antigens in the female reproductive tract is still an important cause of infertility in humans. Thus, by elucidating the mechanisms involved in these immune responses, effective therapies for infertile couples could be developed [126]. Role of the oviduct in early embryo development In mammalian species, the oviduct is the place where life begins as this is where fertilization occurs and where the first 3–4 days of embryo development take place [159]. As detailed in Section “Oviductal fluid and extracellular vesicles (oviductosomes)”, the oviduct epithelium is responsible for secreting the different components of OF [6, 7]. Thus, the regulation of this epithelium and its secretions are important to generate an optimal environment for early embryo development. Transcriptomic approaches have identified different functional groups of genes involved in regulating the oviduct over the estrous cycle [15]. Recently, in bovine oviduct epithelial cells (BOEC), Cerny et al. [16] identified large numbers of genes differentially expressed in the follicular and luteal stage of estrus, many of which were exclusive to either the ampulla or isthmus. Transcriptional differences between the isthmus and ampulla regions of the oviduct at the time the embryo is exposed to these environments have been reported by our group [160]. In line with these observations, Seytanoglu et al. [20] reported significant differences in the proteomic profiles of the oviduct according to estrus cycle stage (follicular vs luteal phase) in cattle. The oviduct and the early embryo in vivo It is well known that maternal recognition of pregnancy in mammals occurs in the uterus [161]. However, limited data are available on embryo signaling in the oviduct. In the 1960s, it was found in mares that nonfertilized oocytes remained in the oviduct [153], while fertilized embryos that produced PGE2 were transported to the uterus [152]. Further, it was shown in hamsters that embryos were transported to the uterus 1 day earlier than nonfertilized oocytes [154]. In rats, when one-cell embryos were transferred to the oviducts of recipients, 3 days later they had reached the morula stage and remained in the oviduct, whereas when four-cell embryos were transferred, 3 days later a significant proportion of those embryos at the blastocyst stage were located in the uterus [162]. At the transcriptome level, Lee et al. [155] showed that the presence of embryos in the oviduct upregulated the expression of specific genes in mice including thymosin beta 4, ribosomal protein L41, and nonmuscle myosin light chain 3. In pigs, it was found that most of the newly expressed genes were detected at the four-cell stage and beyond, and three of them were identified as porcine TGF-α, porcine TGF-β-binding protein II, and porcine atrial natriuretic factor receptor-like [156]. In another study in pigs, Almiñana et al. [163] reported that the presence of embryos downregulated the expression of genes related to the immune system. The lack of similar data in mono-ovulatory species may be partly due to the fact that, in litter-bearing species, the effect of any putative embryo signal(s) may be magnified [157, 164]. In a recent study by our group, it was necessary to transfer multiple embryos (up to 50) into the oviduct of heifers to detect differences in transcriptomes, while when a single embryo was present in the oviduct (pregnant vs cyclic heifers) no differences were found, suggesting a local effect of the embryo [157]. In addition, Smits et al. [164] reported the local impact of the embryo on the transcriptome of the equine oviduct epithelium. In these studies in cattle, horses, and pigs, the presence of an embryo induced subtle changes in the oviduct expression of genes related to immune functions. This reduced reactivity of the immune system is perhaps not that surprising given the semiallogenic nature of the embryo/fetus. Without the appropriate control of the maternal immune system, the embryo will be rejected [165]. However, the semiallogenic fetus, which expresses paternal antigens, is able to avoid immunological rejection [166]. In addition, results of immunological studies have indicated that circulating P4 blocks the capacity of antigen-presenting cells to present the embryo antigen to Th lymphocytes and creates maternal immunological tolerance [167]. More recently, it was shown that the presence of an embryo in the ipsilateral oviduct of pregnant mares induced higher abundances of 11 and lower abundances of 2 proteins compared with the contralateral side, and higher abundances of 19 proteins compared with the ipsilateral side of nonpregnant mares. This supports the hypothesis that the equine embryo interacts with the oviduct affecting secretion pattern of proteins involved in pregnancy-related pathways [13]. The oviduct and the early embryo in vitro The goal of in vitro embryo production is to simulate in vivo conditions as closely as possible to obtain high-quality embryos capable of continued development and implantation, and finally leading to viable births [168]. Nonetheless, embryos produced in vitro are inferior in quality in terms of morphology, cryotolerance, transcript expression profiles, and pregnancy rates after transfer compared to those derived in vivo (for a review see [169]). In this regard, the role of the female reproductive tract (oviduct and uterine horns) has been underestimated. The reason for this is that it is possible to produce competent embryos in vitro. Indeed, after their transfer to the uterus, thousands of live calves, lambs, kids, and babies have been born. Nevertheless, the culture of in vitro-produced bovine zygotes in vivo in the ewe oviduct improves the quality of the resulting blastocysts so that they resemble the quality of totally in vivo produced embryos [170, 171]. Conversely, the culture of in vivo-produced bovine zygotes in vitro gives rise to low-quality blastocysts [170]. Gad et al. [172] explored the consequences of the culture conditions before and during embryonic genome activation (EGA) on bovine embryonic developmental rates via global gene expression patterns using the homologous cow oviduct. Embryo development was similar irrespective of where culture took place. However, the blastocyst transcriptome was clearly influenced by abnormal culture conditions, confirming once more the significant effect of culture conditions during EGA. It should be underscored that isolated oviduct ex vivo culture systems have been successfully used for the in vitro culture of mouse, rat, hamster, pig, and cow embryos up to the morula/blastocyst stage (for a review see [31]). In an effort to mimic in vivo conditions and improve embryo quality, several embryo culture systems have been developed. The in vitro culture of BOEC is considered a suitable model to produce embryos of better quality and also to explore oviduct–embryo interactions [173]. These cells can be cultured as monolayers or cell suspensions, or under polarizing conditions. The drawback of monolayers is that they dedifferentiate loosing important morphological characteristics [174] such as cell height, cilia, secretory granules, and bulbous protrusions [175, 176]. In a study by Cordova et al. [177], the use of BOEC for embryo culture in vitro at the early stages of embryo development (up to day 4) was found to improve development and quality in terms of specific gene transcripts. This period of culture coincides with the in vivo situation when the embryo is still in the oviduct. Recently, we reported that an extended culture BOEC monolayer can be successfully used for coculture, with no differences in embryo development (35%) when compared either with coculture with fresh recovered cells or normal culture in synthetic oviductal fluid [178]. The benefit of this system over classic coculture systems is that it provides homogenous results. Moreover, it has been shown that BOEC are capable of adapting their transcriptome profile in response to signals produced by the embryo [24], making it a useful in vitro model to examine embryo–maternal interactions during the early stages of embryo development, when the embryo is still in the oviduct. Short-term (24 h) BOEC cell suspension cultures serve to maintain morphological characteristics as well as gene markers present in the cells in vivo such as OVGP1, estrogen, and P4 receptors [174]. In addition, according to preliminary results from our laboratory, BOEC suspension cultures resemble in vivo controls more than monolayers in terms of morphology and the oviduct epithelial cell markers OVGP1, GPX4, and FOXJ1 [179]. Polarized cell culture maintains the polarized asymmetrical structure of oviduct epithelial cells, and it seems that this system preserves detailed morphological features of the porcine oviduct as well as oviduct-specific markers [180]. As aforementioned, an important component of the oviduct environment is the OF. When porcine oocytes were treated with OF before fertilization, a significant increase in cleavage rates and blastocyst yields was observed, suggesting embryo protection by OF against adverse effects on mitochondrial DNA transcription or replication and apoptosis [181]. When cattle oocytes were exposed to OF before fertilization, no effect was observed on embryo development and the morphology of the resulting blastocysts, though differences in RNA expression appeared in specific transcripts of the embryos derived from oocytes treated with OF [182]. Recently, we observed that low concentrations of OF (<5%) in embryo culture medium in the absence of serum had a positive effect on development and quality in terms of cryotolerance, cell number, and the expression of quality-related genes [53]. The EVs found in OF are important for intercellular communication and play a key role in regulating physiological and pathological processes. It has been shown that EV can horizontally transfer mRNAs to other cells, which can then be translated into functional proteins at the new location [183]. Burns et al. [184] noted differences in molecular contents according to pregnancy status in the ewe. This suggests a differential source of MV (endometrial epithelia or conceptus trophectoderm) supporting the notion that MV in uterine fluid play a biological role in conceptus–endometrial interactions which may be important for establishing and maintaining pregnancy. Further, these authors moreover showed that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication between those tissues during the establishment of pregnancy in sheep (Burns et al. [185]). Recently, we presented firm evidence that EV derived from BOEC conditioned media and OF improve blastocyst quality and induce cryoprotection in in vitro cultures to the same extent as classic coculture with fresh BOEC monolayers and control media supplemented with fetal calf serum [178]. Thus, EV presence in OF and their effects on early embryonic development may be truly essential, and culture systems containing EV may provide new insight into early embryo–maternal communication thus improving embryo quality in current in vitro production systems. How the oviduct shapes the epigenetic landscape of the embryo Based on the different mechanisms and cell–cell and cell–fluid interactions previously described, the oviduct may be viewed as a highly complex ecosystem where every cell and molecule are interconnected and finely tuned. In the oviduct, proteins inside the cells and OF are responsible for a number of functions, all directed towards the same common goal: to provide the most suitable conditions for the optimal development of the offspring. However, not only proteins are crucial. Also, additional molecules such as glycosaminoglycans, proteoglycans, carbohydrates, ions, hormones, growth factors, and cytokines etc. [8] play a role in the equilibrium of the system and in the mechanisms needed for successful fertilization and the first steps of embryo development. In addition, subtle changes in the physiological characteristics of OF, including O2 concentration (affecting reactive oxygen species [ROS] levels), CO2, pH, temperature (affecting enzymatic activity), osmotic pressure, water, and electrolyte concentration (e.g., Ca2+ affecting exocytotic processes involving proteins secreted by the epithelial cells) or even viscosity (e.g., affecting sperm motility and oocyte or early embryo transport), can be determining factors for both the correct development of the embryo and the reliable transmission of the genetic information within embryonic cells and from the perspective of shaping the epigenetic environment. The zygote and the early embryo are, indeed, highly dynamic in terms of metabolic and cell division activities. While in the oviduct, they take from the surrounding environment all the nutrients and factors they cannot find in their yolk reserves to continue their development. In addition, they are likely receptive to all the signals and messages received from the oviduct cells arriving via different ways of communication, including exosomes and their miRNAs within. In this context, continuous DNA replication is one of the predominant features of embryonic cells. Although producing precise copies of the genome sequence is the goal pursued by this constant DNA replication, after each cell cleavage, the methylation status of the replicated DNA and most epigenetic marks at the histone level undergo dramatic change. This leads to the almost full reprogramming of the cells that will enable their pluripotency to form any tissue of the body. As stated by Ross and Cánovas, during the life cycle, there are moments in which the epigenetic information needs to be reset for the initiation of a new organism [186]. These moments occur during the formation of the primordial germ cells in the embryo and during the development of the early embryo itself, shortly after fertilization in the oviduct. Since epigenetic remodeling begins at the ooplasm, most evidence holds maternal RNA responsible for this remodeling. However, a number of studies have also indicated a key role of environmental factors for the correct establishment of epigenetic marks supporting the hypothesis of the developmental origins of health and disease (DOHaD) [187]. Although most studies to date have focused on the uterine environment and the impacts of a lack or excess of nutrients for the mother during pregnancy [188, 189], it is clear that the major epigenetic reprogramming event in development takes place in the oviduct and not in the uterus. Consequently, it would be of great interest to know the extent to which changes in the oviduct environment can affect the epigenetic marks of the embryo. Data from children conceived through assisted reproduction reveal a major incidence of some imprinting disorders such as Beckwith–Wiedemann syndrome, Angelman syndrome, and Silver–Russell syndrome compared with naturally conceived babies [190]. In addition, imprinted genes, together with active retrotransposons, are precisely the only genomic regions that, in theory, escape the erasing of DNA methylation marks during the first days of development in the oviduct. Thus, the immediate question that arises is whether embryos whose development was initiated out of the oviduct environment carry a wrong methylation pattern in their imprinting genes which, under physiological circumstances, would have been avoided. Or if we turn the question around: Has the oviduct the capacity to shield the offspring from imprinting disorders? This question remains unanswered prompting the need for a consistent body of research in this field. It has been well documented in the mouse [191], human [192, 193], and monkey [194] that dramatic reshaping of the DNA methylation landscape during the early stages of development (from zygote to blastocyst) involves a drop of around 60–80% of global DNA methylation in the oocyte and spermatozoa, to 20–40% in the blastocyst. In other species such as the pig, global DNA methylation at the blastocyst stage seems to be as low as 12–15% [195]. Although the time lines in each species vary and differences have been reported in the stage at which the maximum drop in methylation is observed, what is clear is that the conceptus spends a long fraction of this period in the oviduct. Consequently, any minor change in the oviduct ecosystem might have consequences on the global DNA demethylation occurring at this point in the conceptus genome, as well as in the remethylation initiated almost simultaneously [194]. Moving forward in the life of the conceptus, if the methylation state of the DNA at a precise gene body or specific genome feature with regulatory capacity changes or is not correct, the corresponding protein encoded by this gene will not be expressed or will be produced in aberrant amounts or with abnormal sequences, with direct consequences on embryo health. Returning to the oviduct environment and the factors affecting it, it would not be difficult to predict a number of correlations between those factors and errors in the epigenetic marks of the embryonic cells. To start with, changes in water and electrolyte transport will affect the osmotic balance and nutrient transport through biological membranes. Accordingly, it has been shown that a lack of folate or methyl group nutrients in the maternal diet could be responsible for DNA hypomethylation [196]. Although such studies looked at DNA from human leukocytes and results were related to cancer [197, 198], it would not be surprising to obtain similar findings in embryonic cells collected from the oviduct-stage conceptus, given the parallels between cancer and embryo metabolism. Another important factor, temperature, has been directly correlated with distribution patterns of DNA methylation across the whole genome in mammals, reptiles and fish [199]. This factor, besides influencing sex determination in fish and plants [200], also affects factors such as growth rate, e.g., in salmon [201] or insulin, sensitivity in mice through changes in DNA methylation and histone deacetylation [202]. In parallel, temperature gradients in the oviduct are thought to influence in events shortly before and after fertilization [203]. Taken together, the data suggest that any modification in oviduct temperature could lead to aberrations during genome methylation reprogramming with the subsequent consequences on the embryonic phenotype. As a third example, we could mention the impacts of abnormal ROS levels in the oviduct lumen. Ten-eleven translocation enzymes (TET1, TET2, and TET3) are partially responsible for the global demethylation process affecting DNA, from the zygote to the blastocyst stage. The mechanism of action of these enzymes consists of the conversion of oxidize 5 methyl cytosine (5 mC) to 5 hydroxy mC and further to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). It is therefore tempting to speculate that alterations in the ROS levels of the oviduct could affect the active DNA demethylation process occurring at this time in the embryo. Finally, two recent studies have shown direct correlation between the presence of oviduct factors during embryo culture and DNA methylation marks in the pig and cow. In the first of these studies [195], pig blastocysts produced in vitro with or without oviduct and uterine fluids as additives in the culture medium were compared with embryos produced in vivo. Whole-genome DNA methylation datasets from individual blastocysts showed global methylation patterns that were closer to those of the in vivo-produced blastocysts when cultured in the presence of these reproductive fluids. In addition, the in vitro-produced blastocysts had higher cell numbers, an improved hatching ability, and also their gene expression patterns were closer to those of in vivo-produced blastocysts. Furthermore, in embryos produced in the absence of reproductive fluids methylation changes were observed in genes whose methylation could be critical, such as insulin like growth factor 2 receptor IGF2R and Neuronatin NNAT. In the second study [204], the culture of bovine embryos with OF induced DNA methylation changes in specific genomic regions in resulting blastocysts. The authors examined the methylation state of four developmentally important genes (mitochondrial transcription termination factor 2 (MTERF2), ATP binding cassette subfamily A member 7 (ABCA7), Olfactomedin 1 (OLFM1), GDP-mannose 4,5 dehydratase (GMDS)) and Long Interspersed Element-1 (LINE-1) retrotransposons in 7–8-day blastocysts. The data indicated reduced methylation levels of MTERF2 when OF was added to the culture medium from the zygote to 16-cell stages, and the elevated expression of MTERF2 mRNA. Moreover, LINE-1 showed higher CpG methylation levels and decreased expression in the embryos produced in the presence of OF compared to its absence. These two studies can be considered consistent proof of the oviduct's effect on the epigenetic landscape of the embryo. As such, they represent the first steps towards the development of more physiological culture media for assisted reproduction both in animals and humans. Conclusions Although mammalian oviducts have long been considered mere conduits for gametes and embryos, recent studies document that the oviduct and its secretions regulate and/or provide a dynamic microenvironment for (1) transport of both male and female gametes to the site of fertilization, (2) final gamete maturation, (3) sperm selection, (4) prevention of polyspermy, (5) fertilization, (6) early cleavage and embryonic development, (7) embryo genome activation, (8) embryo maternal communication, and (9) transport of the embryo to the uterus. In this review, we have focus in the role of the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. There are many studies probing the downside of the ARTs that link to some of the critical functionalities of the oviduct affecting embryo development. The challenge is to continue to develop and optimize in vitro systems to maximize embryo production and quality by emulating the conditions and processes occurring in vivo within the oviduct. Thus, it is possible that by a more efficient selection of the spermatozoa the initial quality of the embryo would be improved. Also by including molecules secreted by the oviduct to the media used for the incubation/culture of both gametes and embryo the adverse periconceptional environment in in vitro-derived embryos could be reduced, and in this way we could increase the efficiency of the current systems for in vitro embryo production in the livestock market as well as in human clinics. Conflict of Interest: The authors have declared that no conflict of interest exists. Footnotes † Grant Support: This work has been funded by the State Secretariat for Research, Development and Innovation of the Spanish Ministry of Economy, Industry and Competitiveness through the projects AGL2015-66145R, AGL2015-70140-R, AGL2015-70159-P, AGL2015-66341, and by the Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia through the projects 19357/PI/14 and 20040/GERM/16. References 1. Wang H, Guo Y, Wang D, Kingsley PJ, Marnett LJ, Das SK, DuBois RN, Dey SK. Aberrant cannabinoid signaling impairs oviductal transport of embryos. Nat Med  2004; 10: 1074– 1080. Google Scholar CrossRef Search ADS PubMed  2. Lopez-Cardona AP, Perez-Cerezales S, Fernandez-Gonzalez R, Laguna-Barraza R, Pericuesta E, Agirregoitia N, Gutierrez-Adan A, Agirregoitia E. CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways. 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Abstract

Abstract The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed. Introduction The oviduct is a convoluted tube bridging from the ovary to the uterus in the mammalian female. It is composed of three main longitudinal regions designated from the ovary to the uterus: the infundibulum (fimbriae in humans), in which most cells are ciliated epithelial cells; the ampulla, which also contains large numbers of ciliated epithelial cells and is the site of fertilization; and the isthmus, which contains a large number of secretory epithelial cells. The reproductive functions of the ovary and uterus have been well established, yet the role played by the oviduct in reproduction is far less understood. The oviduct transports the gametes and embryo, and provides biochemical and biophysical support for preimplantation development. In addition, there is direct and indirect evidence for two types of interactions, physical and molecular, among both gametes, embryos and oviduct. The incorrect functioning of the oviduct can lead to infertility or to pathophysiologies such as ectopic pregnancy due to impaired embryonic transport [1, 2]. The main difference between natural conception and in vitro fertilization (IVF) is that in the latter there is limited gamete selection, fertilization occurs under artificial conditions, and development is not subjected to the dynamics of the in vivo environment. When the oviduct is bypassed in IVF, there is no communication between embryo and oviduct, naturally a prerequisite for normal development and transport to the uterus. It is usually assumed that the in vitro culture medium is the main factor affecting embryo quality in vitro. However, we must not forget other factors including sperm selection. This is because of all the spermatozoa in an ejaculate, very few are selected by the female genital tract to fertilize the oocyte. The hardening of the zona pellucida (ZP) and other modifications that occur in vivo are other important factors for fertilization that are missing in vitro. Furthermore, the epigenetic marks of the embryo are reprogrammed during early development, and in vitro culture can have a profound effect on this. For example, it has been shown that the oxygen tension in vitro greatly impacts global DNA methylation of the embryo [3]. In effect, several studies have shown that an in vitro embryo environment may have long-term effects on subsequent offspring, possibly via epigenetic mechanisms at play during development [4, 5]. Due to the relative success of IVF, the role of the oviduct has been underrated. However, the oviduct not only serves as a passageway for gametes and embryo to enter the uterus. It also acts as a cofactor to facilitate sperm migration towards the egg, keep sperm alive for hours or days, select spermatozoa, and culture the embryo in a spatiotemporally dynamic system that will reprogram its genome and epigenome, thereby improving quality and ensuring implantation and subsequent normal development. In this review, we focus on the contents of oviductal fluid including the oviductosomes, and describe the role of the oviduct in sperm selection and how sperm regulates an immune response within it. Also, we discuss the role of the oviduct in early embryo development and how it may shape the epigenetic landscape of the embryo. A better understanding of the biology of the oviduct during sperm migration and preimplantation will help further the development of reproductive technologies. Oviductal fluid and extracellular vesicles (oviductosomes) The secretory cells of the oviduct epithelium secrete proteins and other factors that, together with plasma-derived constituents, form oviduct fluid (OF) [6, 7]. Oviduct fluid composition is complex including simple and complex carbohydrates, ions, lipids, phospholipids, and proteins [8]. Some of these constituents are metabolic substrates, such as lactate, pyruvate, amino acids and glucose, the concentrations of which differ from those present in the uterine fluid and serum [6, 9, 10]. Oviduct fluid protein concentrations are low compared to those of blood serum and also vary between species. Average protein concentrations in nonpregnant females have been estimated at 13.6–30.2 mg mL−1 in sheep [11, 12], 0.17 mg mL−1 in horses [13], and 20–80 mg mL−1 in humans [14]. Although some proteins such as albumin are in abundance, other proteins appear in lower amounts, with highly sensitive equipment or complex experimental processes needed to identify them. Early attempts to identify these low-level OF components were based on microarray analyses of epithelial cells to detect potentially secreted proteins. More recently, studies have shown that OF proteins can be directly detected by mass spectrometry. These studies have revealed the highly complex composition of this biological fluid and that it is modified by several factors. Dynamics of the protein composition of oviduct fluid Oviduct fluid composition differs according to estrus cycle stage as suggested by transcriptome studies in cows [15, 16], pigs [17], and humans [18, 19]. Proteomic analysis of oviduct epithelial cells suggests a similar protein composition during the follicular and luteal stages of the reproductive cycle in the pig [20]. However, quantitative differences in the OF proteome have been reported in sheep and cow during the estrous cycle [21]. In the pig, these differences amounted to a 79% increase in the relative abundance of proteins in estrus compared with a 21% increase in the luteal stage [22]. Oviduct cells react to oocyte and sperm presence by modifying their gene expression patterns as observed in pigs [23], cow embryos [24], and mouse seminal fluid [25]. Hence, changes in the oviduct microenvironment are induced by the presence of gametes, semen, or embryos. For instance, the reduced expression of cytokines Csf2, Lif, and Il6 in the oviduct has been reported in mated female mice not exposed to seminal plasma, and cytokines Efg and Trail were correlated with the presence of zygotes in the oviduct [25]. Furthermore, the presence of X or Y spermatozoa in the pig oviduct gives rise to changes in oviduct cell gene expression [23]. Two studies have also detected changes in the secreted OF proteome in response to the presence of gametes [26, 27]. More recently, differences have been identified in the cow oviduct transcriptome depending on ovulatory follicle size [28] and on the oviduct examined in the same individual—ipsilateral or contralateral—after ovulation [29]. This result was also observed using a proteomic approach [21]. Moreover, other studies have shown varying gene expression between the specific ampulla or isthmus regions of the oviduct where fertilization and early embryo development take place, respectively [30]. Protein composition of oviduct fluid In the mammalian species examined to date, OF composition is analogous suggesting that the main biological processes in the oviduct are conserved [16, 17, 20] (Figure 1). This idea is biologically supported by the success of embryo development in the oviducts of heterologous species [31]. Thus, some 2400 orthologous genes expressed in the pig and cow oviduct have been identified [17] and more than 1090 orthologous genes are common to the oviduct transcriptome of humans, pigs, and cows [16, 18, 32]. In the pig oviduct, nearly 5000 genes are expressed, of which 11% and 7% correspond to membrane and secreted proteins, respectively [17]. These proteins are theoretically more relevant because they directly contact the gametes and embryos. Through microarray analysis, the presence of 314 proteins secreted by oviduct epithelia was suggested. In a recent gel liquid chromatography MS/MS study performed in adult cyclic ewes, more than 600 proteins were identified in the OF [22]. Such differences in the gene expression of secreted proteins encountered between microarray and proteomic analyses may be the consequence of several factors, the most likely being related to the OF origin. Oviduct fluid composition is influenced by blood plasma transudate but is regulated by the ciliated and secretory epithelial cells which form a cell monolayer lining the lumen. Consequently, only some of the proteins detected in OF are transcribed in the oviduct. Albumin is a typical example. This protein is the most abundant in OF, but its transcript is not present in oviduct cells since the gene encoding this protein is only present in the liver. Figure 1. View largeDownload slide Venn diagram showing overlapping and nonoverlapping oviduct proteins in the pig [19], sheep [23], cow [21], and horse [15]. Figure 1. View largeDownload slide Venn diagram showing overlapping and nonoverlapping oviduct proteins in the pig [19], sheep [23], cow [21], and horse [15]. Data emerging so far from the detailed analysis of OF proteins have been a priori quite surprising because a considerable number of detected proteins are not typically secreted. For example, the presence of a large number of proteins that normally occur in the cytoplasm, nucleus, or mitochondria has been reported [22, 32]. In ewes, cytoplasmic proteins represent more than 40% of the proteins identified [22]. The most accepted hypothesis is that this protein presence is the result of epithelial cell shedding during renewal of the epithelium. The different proteins found in OF play different biological roles. Some contribute to oxidation or immune protection, fertilization, embryo development, or are proteases and their inhibitors. Expression of the anti-inflammatory cytokines TGFB1 and IL10 has been observed in the epithelial cells of the cow and pig oviduct [33, 34]. These molecules could be involved in maintaining tolerance to non–self-cells in the oviduct. This is discussed later in this review. Other proteins identified in OF play a relevant role in fertilization. Two abundant proteins are albumin and Oviductal glycoprotein 1 (OVGP1). Albumin has an important function in sperm capacitation via the removal of cholesterol from the plasma membrane [35]. OVGP1 also seems to play a key role in sperm physiology as it has been reported to induce the phosphorylation of sperm proteins during their capacitation [36, 37]. Other reports also exist of the interaction of OVGP1 with the ZP causing increased resistance of the ZP to protease treatment, or “ZP hardening.” This hardening of the ZP mediated by OVGP1 and OF has been related to polyspermy control before fertilization as an important event in some species such as the pig [38, 39]. Also importantly, knock-out (KO) mice for OVGP1 maintain their fertility, suggesting that this protein is not essential for fertilization [40]. Furthermore, OVGP1 is a pseudogene in the rat. However, it could be that other proteins of the same chitinase family act as paralogs and take over OVGP1 function. A similar functional model has been described for acrosin KO mice [41]. It has been recently confirmed that OVGP1 is secreted by in vitro cultured bovine oviduct epithelial monolayer under a system designed for modeling the oviduct suggesting a relevant role of this protein for the function of the oviduct [42]. The oviduct also plays an important role in protecting gametes and the preimplantation embryo. It is well known that free radicals have a deleterious effect on gametes and embryos [43] and the presence of antioxidant proteins involved in cell protection has been shown in the cow [21, 44] and sheep [22]. In addition, proteases and their inhibitors have been found in OF. However, it was not until a few years ago that the key role of OF in fertility was unveiled. Thus, following the development of genetically modified mice, the need for the fine modulation of proteases and their inhibitors for the survival of oocytes and embryos was reported [45, 46]. Immune cells, extracellular vesicles, and oviductosomes Several other components have been, detected in OF. Some are easy to identify like the cells of the immune system. Polymorphonuclear cells have been found in bovine OF with implications for a role in defense and possibly in the phagocytosis of spermatozoa [47]. Electron microscopy has revealed the presence of very small components surrounded by membranes. These structures are described as extracellular vesicles (EVs), a general term encompassing several different vesicle types, released by somatic cells present in body fluids, and containing bioactive molecules (i.e., proteins and RNAs, mRNAs, miRNAs) [48, 49] and lipids [50]. The terminology generally used for EV is size and origin associated such that exosomes (30–200 nm) arising from endosomes and microvesicles (MV) (100–1000 nm) are EVs that bud from the plasma membrane. These different EVs can be separated by ultracentrifugation [51]. Extracellular vesicles were first described in the mouse oviduct and then characterized by the presence of the sperm adhesion molecule 1 protein (SPAM1) [52]. Both EV types show significant heterogeneity in size and also in distribution according to the OF source, i.e., ampulla vs isthmus [53]. In addition, recent studies have revealed further EV heterogeneity indicating different subtypes of EV of different composition [51]. It would be interesting to identify, using current isolation procedures, the different EV subpopulations in OF collected from different oviduct regions and during different phases of the estrous cycle and assess their biological implications. Histological examination of the epithelium reveals two main cell types, ciliated and nonciliated, which probably produce different EV. This issue warrants further investigation. A recent study has identified 319 proteins present in EV harvested from cattle [44], 153 of these proteins have also been identified in the OF but other 166 proteins were not detected previously in the OF [21]. Almiñana et al. [44] found 175 proteins in EV obtained from both OF in vivo or the supernatants of cultured epithelial cells. However, it should be mentioned otherwise that their protein composition was specific to their in vivo or in vitro origin. Thus, some proteins were identified only in the in vivo setting (97) and others (47) only in in vitro culture. Further work is needed to examine the characteristics of other EV components (RNAm, miRNA, and lipids). Role of the oviduct in sperm selection and fertilization Sperm heterogeneity The ejaculate is a heterogeneous mix of different subpopulations of spermatozoa [54]. In addition to the abnormal or immotile fraction, within the normal motile subpopulation, a number of fractions can be differentiated through their specific properties. This means that spermatozoa can be grouped according to motility patterns [55]. The presence/absence of specific motility patterns and subpopulations has been correlated with fertility and the success of assisted reproductive technologies (ARTs) [56]. As a consequence, interest is growing in characterizing sperm subpopulations as an analytical tool for sperm quality in a number of mammalian species including cow [57], sheep [58], horse [59], and human [60], among others. Interestingly, sperm heterogeneity has also been shown at the epigenetic level through the finding that the same human ejaculate shows different sperm fractions bearing different methylated DNA regions [61]. Further, within the same ejaculate, spermatozoa coexist showing different degrees of DNA fragmentation [62]. Collectively, these lines of evidence point to the presence of a number of subpopulations of different quality in the same sample. Therefore, it is widely accepted that not all spermatozoa in an ejaculate are equally good at fertilization and that a high-quality fraction is selected within the female genital tract [63]. This may be especially critical for ARTs and in particular for the technique of intracytoplasmic sperm injection used in fertility treatments, because of the risk of fertilizing the oocyte with spermatozoa with errors that could affect the offspring in the long run [64]. Indeed, in human clinical practice, this risk may be higher if we consider the higher incidence of DNA fragmentation in the spermatozoa of infertile men [65–67]. Accordingly, sperm selection prior to ART is becoming an important research field in which the main challenges are to discover the sperm subpopulation selected within the female genital tract and the mechanisms involved in their selection in vivo. Only after acquiring this knowledge will we be able to design efficient in vitro methods for selecting high-quality spermatozoa for use in ARTs [68]. Selection mechanisms operating within the oviduct Mammalian spermatozoa have to overcome a number of obstacles in the female genital tract before reaching the fertilization site at the ampulla. Vaginal pH, resistance to sperm migration of the cervical mucus, the narrow utero-tubal junction, the meandering oviduct lumen, the response of the immune system, etc., are all physical/anatomical conditions of the female genital tract that configure a stringent selection mechanism for spermatozoa [68, 69]. Effectively, in all mammalian species examined to date it has been shown that of the many millions of spermatozoa ejaculated, only tens to hundreds reach the ampulla [70, 71], or site of fertilization. Presumably, this select group of spermatozoa have a greater fertilization capacity and better characteristics for supporting embryo development [68]. However, little is known about this sperm subpopulation, its relative effectiveness, and its selection criteria in vivo [68]. Migration of spermatozoa through the lower female genital tract already poses a first selective barrier, whereby viscosity, pH, immune response, etc., select those spermatozoa that are able to advance in this aggressive environment (Figure 2). However, it seems that the fine selection process occurs at the oviduct. Hourcade et al. in 2010 [72] noted in mice that spermatozoa with high-quality DNA are selected only within the oviduct, and that spermatozoa retrieved from the uterus actually show more DNA fragmentation than those harvested from the vagina (Figure 2). This might indicate that transit from the vagina through the cervix and uterus leads to extensive damage to the sperm population as a consequence of the aggressive conditions of pH, immune response, etc., encountered. Thus, we should perhaps view sperm selection in the oviduct as a way of sorting only those spermatozoa with a high-quality genetic content that have survived this initial transit. Figure 2. View largeDownload slide Model of sperm guidance and selection within the oviduct. The fraction of spermatozoa reaching the uterus contains high levels of fragmented DNA. Subsequently, within the oviduct spermatozoa with high DNA integrity are selected [19]. Near the utero–tubal junction, the spermatozoa attach to the epithelium in a region known as the sperm reservoir where they acquire the final maturation stage that capacitates them to fertilize the egg. Capacitated spermatozoa at the sperm reservoir (1) acquire a motility pattern known as hyperactive motility (2) that facilitates their detachment from the epithelium. Released spermatozoa are then guided by thermotaxis to the proximity of the egg at the ampulla. Finally, a gradient of chemoattractants generated by the COC guides the spermatozoa towards the egg for its fertilization. Figure 2. View largeDownload slide Model of sperm guidance and selection within the oviduct. The fraction of spermatozoa reaching the uterus contains high levels of fragmented DNA. Subsequently, within the oviduct spermatozoa with high DNA integrity are selected [19]. Near the utero–tubal junction, the spermatozoa attach to the epithelium in a region known as the sperm reservoir where they acquire the final maturation stage that capacitates them to fertilize the egg. Capacitated spermatozoa at the sperm reservoir (1) acquire a motility pattern known as hyperactive motility (2) that facilitates their detachment from the epithelium. Released spermatozoa are then guided by thermotaxis to the proximity of the egg at the ampulla. Finally, a gradient of chemoattractants generated by the COC guides the spermatozoa towards the egg for its fertilization. After spermatozoa have overcome the barriers of the vagina and cervix, they are transported by pro-ovarian contractions of the myometrium [69] to the uterotubal junction. At this site, the first specific selection process seems to take place. It has been reported that spermatozoa cannot pass through the junction if they do not possess certain proteins in the sperm head plasma membrane (Figure 2). This was discovered when null mutant mice for genes encoding a disintegrin and metalloproteinase 2 (ADAM2) [73], calmegin [74, 75], or testis-specific angiotensin converting enzyme [76, 77] were found to be infertile because their spermatozoa were neither able to pass through the uterotubal junction nor bind to the ZP. Although the mechanisms for this selection are unknown, it has been speculated that the functions of these proteins, especially ADAMS, might be to enable the spermatozoa to gain a foothold on the wall lining the junction and move forward onto the oviduct by lightly sticking to the epithelium [78]. Thus, ADAMS could function for binding the CD9 protein that is exposed on the oviductal epithelial cells, and in this way it could represent a specific mechanism of sperm selection through protein–protein interaction [79]. The spermatozoa entering the oviduct then attach to the epithelium of the isthmus in a fairly defined location depending on the species [69, 80–82] (Figure 2). This location is known as the sperm reservoir, and it has been proposed as the place where spermatozoa acquire the final maturation status known as capacitation [69] (see below). When spermatozoa are capacitated, they detach from the epithelium by acquiring hyperactive motility and swim up the oviduct towards the ampulla where fertilization occurs [69, 82, 83]. This adhesion is mediated via lectin-like molecules on the sperm surface that are able to bind carbohydrates exposed on the oviduct cell apical membrane [84]. Thus, the spermatozoon's specific protein profile enables it to pass through the uterotubal junction, and specific carbohydrate moieties responsible for its attachment to the oviduct epithelium configure a molecular passport of sorts ensuring the selection of the adequate spermatozoa [85]. Once spermatozoa detach from the sperm reservoir, they still need to swim through the oviduct to reach the ampulla where the egg awaits. The oviduct is narrow, convoluted, and relatively long (3–5 cm in humans). Thus, it has been suggested that spermatozoa must have some kind of navigation system to help them swim in the right direction towards the ampulla where fertilization occurs [86, 87]. So far, three such guidance mechanisms have been proposed on the basis of in vitro studies: thermotaxis—swimming up a temperature gradient (shown in rabbits, humans [88], and mice [89]), rheotaxis—swimming against a fluid flow (shown in mice and humans) [90], and chemotaxis—swimming up a concentration gradient of chemoattractant (shown in humans [91], rabbits [92], and mice [93]). Whereas rheotaxis seems to be a passive mechanism as a consequence of the hydrodynamics of motile spermatozoa [94], both thermotaxis and chemotaxis are active mechanisms that have been shown to be functional only in a specific subpopulation representing ∼10% of the total spermatozoa. In both cases, these subpopulations have to undergo capacitation in order to respond to migration stimuli [87, 95]. How capacitated spermatozoa acquire the ability to respond to both thermotaxis and chemotaxis is an unknown matter. However, a possibility is that the reorganization of the plasma membrane occurring during capacitation [96] determines the correct configuration/location of the thermosensors and chemosensors as well as their relation to the coupled transduction signaling in order to respond to the stimuli. According to this hypothesis, thermotaxis would function as the primary selective mechanism by guiding only capacitated spermatozoa released from the sperm reservoir through the relatively long distance separating the isthmus from the ampulla [97] (Figure 2). Then, once in the proximity of the oocyte, only those responding by chemotaxis would make contact with the cumulus oocyte complex (COC) and thus achieve fertilization [97] (Figure 2). Chemotaxis would not only guide the spermatozoa within the ampulla towards the COC but also through the layer of cumulus cells towards the egg, where a gradient of chemoattractant is established [98]. In addition, an increasing concentration of the chemoattractant within this layer would also potentiate the generation of hyperactive events in the spermatozoa as well as provoke the release of the acrosomal content to facilitate the penetration through the cumulus oophorus until reaching the surface of the egg [99]. Thus, the responsiveness of the spermatozoa to the chemoattractant could configure a stringent selective feature in the last stages prior fertilization. Sperm subpopulation selected within the oviduct Currently, the characteristic of the specific sperm subpopulation selected in vivo responsible for fertilizing the egg are unknown, but we do know that not all the spermatozoa in an ejaculate are able to fertilize the egg in vitro or even get close to it in vivo. This means IVF requires the placement of millions of spermatozoa directly on the egg [100], while, in vivo, only a very restricted number of spermatozoa make contact with the egg at the ampulla [95]. This indicates that either there are sperm selection mechanisms within the oviduct or that spermatozoa managing to reach the ampulla acquire the capacity to fertilize the egg during their transport within the oviduct. It is known that final maturation of the spermatozoon occurs within the female genital tract through its interaction with uterine and oviduct secretions [96]. This maturation process is known as capacitation and involves complex changes in the biochemical composition of the spermatozoon that enable it to fertilize the oocyte. During capacitation, membrane fluidity is enhanced through the loss of plasma membrane cholesterol [101], and changes have been observed in intracellular ion concentrations [102] along with hyperpolarization of the sperm plasma membrane [103], increased protein kinase A activity [104], and protein tyrosine phosphorylation [105]. It was generally assumed that all these changes prepared the spermatozoa for the acrosome reaction and fusion with the oocyte membrane [106]. However, in mice it has been recently shown that the acrosome reacts during sperm migration within the oviduct [107] and also that mice oocytes can be fertilized by spermatozoa that have completed their acrosome reaction before entering the cumulus oophorus [108]. These observations thus question the actual functioning of the acrosome and this issue has generated controversy among reproductive biologists. It is known that the capacitated sperm subpopulation shows a special motility pattern known as hyperactivation. Under low-viscosity conditions, this pattern involves vigorous movements produced by asymmetrical and high-amplitude waves in the flagella, resulting in erratic swimming trajectories [109]. This pattern allows the sperm to progress in viscoelastic conditions such as those encountered in the female genital tract [99, 110]. Thus, the acquisition of this motility type has been linked to fertility and to the ability of the spermatozoa to respond to migration stimuli such as thermotaxis [111] and chemotaxis [112]. It has been estimated that under in vitro conditions, only a small fraction of spermatozoa, about 10%, is capacitated [113]. However, this proportion of capacitated sperm within the female tract in vivo is unknown and neither do we know whether it affects all spermatozoa in the same way at the same time. For instance, it has been suggested that capacitation might occur sequentially in different sperm subpopulations during their storage in the sperm reservoir. This would ensure continuous waves of capacitated spermatozoa that would be continuously detached from the epithelium where they are stored and then swim towards the egg [113]. In this way, synchrony between oocyte and spermatozoa for fertilization would be ensured for a single copula. As aforementioned, only capacitated spermatozoa are able to migrate within a temperature gradient by thermotaxis and respond by chemotaxis to swim towards the oocyte. Thereby, thermotaxis and chemotaxis can be seen as mechanisms for selecting capacitated spermatozoa. Indeed, this premise opens the way for a promising field of research on the selection of spermatozoa using these migration properties. In future, it would be interesting to check the quality of spermatozoa responding by both thermotaxis and chemotaxis and examine their efficiency in ARTs. In addition, this subpopulation needs to be characterized to find specific markers that could be suitable for male fertility diagnosis or for sperm selection using techniques such as magnetic-activated cell sorting [114]. Sperm regulation of the female immune response at conception To ensure pregnancy success from the moment in which the sperm reaches the female reproductive tract, careful regulation of the maternal immune system is needed to allow selective immune privilege for male gametes and the forthcoming fetus, while maintaining a reasonable immune response to pathogens. This phenomenon is known as the “immunological paradox of pregnancy” and has an immediate effect on ovulation, sperm selection and fertilization, but also a long-term effect in assuring the acceptance of fetal tissues [115]. Insemination constitutes the beginning of communication between the female immune system and paternal antigens. Although the volume and fractions of the ejaculate differ between species, contact with maternal tissues induces a postmating inflammatory response in most mammals [116–119] (Figure 3). Uterine and cervical cells then synthesize proinflammatory cytokines [118, 120] that induce an influx of leukocytes into the uterine lumen [121]. In particular, neutrophils attract monocytes and dendritic cells, and are able to recruit, activate, and program antigen presenting cells. Finally, seminal plasma will determine if these cells activate or suppress other immune mechanisms such as T-cell activation [122]. This inflammatory response needs to be resolved before the embryo implants for pregnancy to succeed. To this end, increasing progesterone levels inhibit the synthesis of certain chemokines, but the transition seems to be also driven by seminal plasma components [118, 123]. Figure 3. View largeDownload slide Regulation of the maternal immune system at conception. After insemination, cytokines present in seminal plasma induce an inflammatory response in the female reproductive tract. The release of proinflammatory cytokines by epithelial cells attracts leukocytes and dendritic cells that recruit and modulate T-regulatory cells (Treg), inducing immune tolerance to paternal and fetal antigens. This cascade of events ensures that successful pregnancy is established. Figure 3. View largeDownload slide Regulation of the maternal immune system at conception. After insemination, cytokines present in seminal plasma induce an inflammatory response in the female reproductive tract. The release of proinflammatory cytokines by epithelial cells attracts leukocytes and dendritic cells that recruit and modulate T-regulatory cells (Treg), inducing immune tolerance to paternal and fetal antigens. This cascade of events ensures that successful pregnancy is established. Studies in mice, gilts, and humans have shown that the female immune response is mainly mediated by seminal plasma, while spermatozoa play a secondary role [118, 123–125]. Seminal plasma originates in the male sex accessory glands and contains a range of molecules that provide the sperm with metabolic support and protection from the acidic environment of the vagina [126]. Specific factors present in seminal plasma interact with uterine cells to induce the expression of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, and many other chemokines [127]. However, the main trigger for an immune response seems to be transforming growth factor-β (TGF-β). This cytokine is present in its precursor form in seminal fluid, and is activated in the female reproductive tract [128]. It can subsequently either directly activate multiple immune cell types [129] or indirectly induce the expression of other cytokines such as prostaglandin E2 (PGE2) and IL-8 [128, 130]. It has been shown that both TGF-β and PGE2 inhibit the function of natural killer cells and neutrophils [126]. Another key event triggered by exposure to seminal plasma is T-cell priming to respond correctly to paternal antigens, activating immune tolerance in the female reproductive system [131]. Therefore, seminal plasma would play a dual role in inducing and resolving inflammation. The role of the female reproductive tract in sperm selection remains controversial. Although they represent less than 15% of the total cell fraction, immature germ cells, epithelial cells, and leukocytes are also present in semen [132]. In contrast to spermatozoa, these cells express paternal major histocompatibility complex (MHC) molecules that could trigger a response by the maternal immune system [133]. It has also been hypothesized that aged, dead, or capacitated spermatozoa induce the chemotaxis of neutrophils mediated by endometrial cells or leukocyte signaling and that this takes part in sperm cell selection [117, 119, 134, 135]. However, selective sperm phagocytosis is still questionable as studies in the pig have indicated that intact viable sperm cells are more likely to bind to neutrophils in vitro, though in these studies contact was transient and the phagocytic role of neutrophils was inhibited by seminal plasma in vivo [136–138]. Similar interactions between neutrophils and spermatozoa have been described in other species such as horses, ruminants, and humans, but the underlying molecular mechanism is still unclear because the presence of sperm surface molecules that could be recognized by neutrophils has not been demonstrated; thus, interactions could be mediated just by random attachment [139–141]. Other hypotheses are that sperm bindings could induce signaling pathways promoting subsequent inflammatory responses, or that there could be a negative selection process against spermatozoa which are not able to attach to epithelial cells and not able to fertilize the oocyte [122]. Before embryo hatching takes place, it is thought that the ZP acts as a physical barrier against the maternal immune system [142] and protects the early embryo from the oviductal environment [143]. In particular, carbohydrate sequences expressed on the ZP could play a key role in protecting the embryo [144]. Following this, to allow the development of fetal trophoblast cells posthatching, maternal immune mechanisms need to be controlled, and the proinflammatory environment needs to be switched to an anti-inflammatory state (Figure 3). Increasing maternal progesterone concentrations together with levels of TGF-β and PGE2 in the seminal plasma play a key role. TGF-β induces the generation of antigen-specific regulatory T (Treg) cells in peripheral tissues. After paternal antigen-driven activation, they proliferate in paraaortic lymph nodes draining the female reproductive tract to later mediate in the processes of T-cell proliferation and awareness of paternal and fetal antigens [129, 145]. Furthermore, they are able to regulate T-cell production of IL-10, which induces the generation of anti-inflammatory cytokines and inhibits the production of antibodies [146]. These mechanisms are also enhanced by progesterone, which elicits the production of PGE2 by epithelial cells [147] and promotes the secretion of more IL-10 [148]. In this way, immune cells can regulate their proliferation and activation and even promote their own death. Treg cells are essential during this process, and their impairment has been associated with reproductive disorders such as miscarriage, pre-eclampsia, intrauterine growth restriction, and preterm birth [149–151]. Although maternal recognition of pregnancy takes place in the uterus, studies have shown that early embryos can induce signaling pathways that are recognized in the oviduct. Early studies in horses, rats, and hamsters revealed that fertilized embryos are transported to the uterus faster than nonfertilized oocytes because of certain signaling molecules they produce such as PGE2 [152–154]. Some years later, the impact of the embryo on the oviduct was confirmed by gene expression analysis in the mouse, pig, and cow [155–158]. Interestingly, one of the genes upregulated in the oviduct by the presence of four-cell stage pig embryos was TGF-β binding protein II [156]. Moreover, in pigs and cows, embryo presence induced the downregulation of genes in the oviduct related to the immune system, particularly those related to inflammation, the complement system and the MHC [157, 158]. In conclusion, successful pregnancy establishment requires the precise regulation of the female immune system. Insemination induces a fast inflammatory reaction followed by proliferation and activation of immune cells that are able to regulate their own response to paternal and fetal antigens. However, while studies in recent years have provided a broad knowledge of the mechanisms involved, there are still details that need to be filled in. This is important to maximize reproductive efficiency in livestock, as varying sperm concentrations or semen extenders elicit different immune responses. Moreover, deficient immune tolerance to male antigens in the female reproductive tract is still an important cause of infertility in humans. Thus, by elucidating the mechanisms involved in these immune responses, effective therapies for infertile couples could be developed [126]. Role of the oviduct in early embryo development In mammalian species, the oviduct is the place where life begins as this is where fertilization occurs and where the first 3–4 days of embryo development take place [159]. As detailed in Section “Oviductal fluid and extracellular vesicles (oviductosomes)”, the oviduct epithelium is responsible for secreting the different components of OF [6, 7]. Thus, the regulation of this epithelium and its secretions are important to generate an optimal environment for early embryo development. Transcriptomic approaches have identified different functional groups of genes involved in regulating the oviduct over the estrous cycle [15]. Recently, in bovine oviduct epithelial cells (BOEC), Cerny et al. [16] identified large numbers of genes differentially expressed in the follicular and luteal stage of estrus, many of which were exclusive to either the ampulla or isthmus. Transcriptional differences between the isthmus and ampulla regions of the oviduct at the time the embryo is exposed to these environments have been reported by our group [160]. In line with these observations, Seytanoglu et al. [20] reported significant differences in the proteomic profiles of the oviduct according to estrus cycle stage (follicular vs luteal phase) in cattle. The oviduct and the early embryo in vivo It is well known that maternal recognition of pregnancy in mammals occurs in the uterus [161]. However, limited data are available on embryo signaling in the oviduct. In the 1960s, it was found in mares that nonfertilized oocytes remained in the oviduct [153], while fertilized embryos that produced PGE2 were transported to the uterus [152]. Further, it was shown in hamsters that embryos were transported to the uterus 1 day earlier than nonfertilized oocytes [154]. In rats, when one-cell embryos were transferred to the oviducts of recipients, 3 days later they had reached the morula stage and remained in the oviduct, whereas when four-cell embryos were transferred, 3 days later a significant proportion of those embryos at the blastocyst stage were located in the uterus [162]. At the transcriptome level, Lee et al. [155] showed that the presence of embryos in the oviduct upregulated the expression of specific genes in mice including thymosin beta 4, ribosomal protein L41, and nonmuscle myosin light chain 3. In pigs, it was found that most of the newly expressed genes were detected at the four-cell stage and beyond, and three of them were identified as porcine TGF-α, porcine TGF-β-binding protein II, and porcine atrial natriuretic factor receptor-like [156]. In another study in pigs, Almiñana et al. [163] reported that the presence of embryos downregulated the expression of genes related to the immune system. The lack of similar data in mono-ovulatory species may be partly due to the fact that, in litter-bearing species, the effect of any putative embryo signal(s) may be magnified [157, 164]. In a recent study by our group, it was necessary to transfer multiple embryos (up to 50) into the oviduct of heifers to detect differences in transcriptomes, while when a single embryo was present in the oviduct (pregnant vs cyclic heifers) no differences were found, suggesting a local effect of the embryo [157]. In addition, Smits et al. [164] reported the local impact of the embryo on the transcriptome of the equine oviduct epithelium. In these studies in cattle, horses, and pigs, the presence of an embryo induced subtle changes in the oviduct expression of genes related to immune functions. This reduced reactivity of the immune system is perhaps not that surprising given the semiallogenic nature of the embryo/fetus. Without the appropriate control of the maternal immune system, the embryo will be rejected [165]. However, the semiallogenic fetus, which expresses paternal antigens, is able to avoid immunological rejection [166]. In addition, results of immunological studies have indicated that circulating P4 blocks the capacity of antigen-presenting cells to present the embryo antigen to Th lymphocytes and creates maternal immunological tolerance [167]. More recently, it was shown that the presence of an embryo in the ipsilateral oviduct of pregnant mares induced higher abundances of 11 and lower abundances of 2 proteins compared with the contralateral side, and higher abundances of 19 proteins compared with the ipsilateral side of nonpregnant mares. This supports the hypothesis that the equine embryo interacts with the oviduct affecting secretion pattern of proteins involved in pregnancy-related pathways [13]. The oviduct and the early embryo in vitro The goal of in vitro embryo production is to simulate in vivo conditions as closely as possible to obtain high-quality embryos capable of continued development and implantation, and finally leading to viable births [168]. Nonetheless, embryos produced in vitro are inferior in quality in terms of morphology, cryotolerance, transcript expression profiles, and pregnancy rates after transfer compared to those derived in vivo (for a review see [169]). In this regard, the role of the female reproductive tract (oviduct and uterine horns) has been underestimated. The reason for this is that it is possible to produce competent embryos in vitro. Indeed, after their transfer to the uterus, thousands of live calves, lambs, kids, and babies have been born. Nevertheless, the culture of in vitro-produced bovine zygotes in vivo in the ewe oviduct improves the quality of the resulting blastocysts so that they resemble the quality of totally in vivo produced embryos [170, 171]. Conversely, the culture of in vivo-produced bovine zygotes in vitro gives rise to low-quality blastocysts [170]. Gad et al. [172] explored the consequences of the culture conditions before and during embryonic genome activation (EGA) on bovine embryonic developmental rates via global gene expression patterns using the homologous cow oviduct. Embryo development was similar irrespective of where culture took place. However, the blastocyst transcriptome was clearly influenced by abnormal culture conditions, confirming once more the significant effect of culture conditions during EGA. It should be underscored that isolated oviduct ex vivo culture systems have been successfully used for the in vitro culture of mouse, rat, hamster, pig, and cow embryos up to the morula/blastocyst stage (for a review see [31]). In an effort to mimic in vivo conditions and improve embryo quality, several embryo culture systems have been developed. The in vitro culture of BOEC is considered a suitable model to produce embryos of better quality and also to explore oviduct–embryo interactions [173]. These cells can be cultured as monolayers or cell suspensions, or under polarizing conditions. The drawback of monolayers is that they dedifferentiate loosing important morphological characteristics [174] such as cell height, cilia, secretory granules, and bulbous protrusions [175, 176]. In a study by Cordova et al. [177], the use of BOEC for embryo culture in vitro at the early stages of embryo development (up to day 4) was found to improve development and quality in terms of specific gene transcripts. This period of culture coincides with the in vivo situation when the embryo is still in the oviduct. Recently, we reported that an extended culture BOEC monolayer can be successfully used for coculture, with no differences in embryo development (35%) when compared either with coculture with fresh recovered cells or normal culture in synthetic oviductal fluid [178]. The benefit of this system over classic coculture systems is that it provides homogenous results. Moreover, it has been shown that BOEC are capable of adapting their transcriptome profile in response to signals produced by the embryo [24], making it a useful in vitro model to examine embryo–maternal interactions during the early stages of embryo development, when the embryo is still in the oviduct. Short-term (24 h) BOEC cell suspension cultures serve to maintain morphological characteristics as well as gene markers present in the cells in vivo such as OVGP1, estrogen, and P4 receptors [174]. In addition, according to preliminary results from our laboratory, BOEC suspension cultures resemble in vivo controls more than monolayers in terms of morphology and the oviduct epithelial cell markers OVGP1, GPX4, and FOXJ1 [179]. Polarized cell culture maintains the polarized asymmetrical structure of oviduct epithelial cells, and it seems that this system preserves detailed morphological features of the porcine oviduct as well as oviduct-specific markers [180]. As aforementioned, an important component of the oviduct environment is the OF. When porcine oocytes were treated with OF before fertilization, a significant increase in cleavage rates and blastocyst yields was observed, suggesting embryo protection by OF against adverse effects on mitochondrial DNA transcription or replication and apoptosis [181]. When cattle oocytes were exposed to OF before fertilization, no effect was observed on embryo development and the morphology of the resulting blastocysts, though differences in RNA expression appeared in specific transcripts of the embryos derived from oocytes treated with OF [182]. Recently, we observed that low concentrations of OF (<5%) in embryo culture medium in the absence of serum had a positive effect on development and quality in terms of cryotolerance, cell number, and the expression of quality-related genes [53]. The EVs found in OF are important for intercellular communication and play a key role in regulating physiological and pathological processes. It has been shown that EV can horizontally transfer mRNAs to other cells, which can then be translated into functional proteins at the new location [183]. Burns et al. [184] noted differences in molecular contents according to pregnancy status in the ewe. This suggests a differential source of MV (endometrial epithelia or conceptus trophectoderm) supporting the notion that MV in uterine fluid play a biological role in conceptus–endometrial interactions which may be important for establishing and maintaining pregnancy. Further, these authors moreover showed that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication between those tissues during the establishment of pregnancy in sheep (Burns et al. [185]). Recently, we presented firm evidence that EV derived from BOEC conditioned media and OF improve blastocyst quality and induce cryoprotection in in vitro cultures to the same extent as classic coculture with fresh BOEC monolayers and control media supplemented with fetal calf serum [178]. Thus, EV presence in OF and their effects on early embryonic development may be truly essential, and culture systems containing EV may provide new insight into early embryo–maternal communication thus improving embryo quality in current in vitro production systems. How the oviduct shapes the epigenetic landscape of the embryo Based on the different mechanisms and cell–cell and cell–fluid interactions previously described, the oviduct may be viewed as a highly complex ecosystem where every cell and molecule are interconnected and finely tuned. In the oviduct, proteins inside the cells and OF are responsible for a number of functions, all directed towards the same common goal: to provide the most suitable conditions for the optimal development of the offspring. However, not only proteins are crucial. Also, additional molecules such as glycosaminoglycans, proteoglycans, carbohydrates, ions, hormones, growth factors, and cytokines etc. [8] play a role in the equilibrium of the system and in the mechanisms needed for successful fertilization and the first steps of embryo development. In addition, subtle changes in the physiological characteristics of OF, including O2 concentration (affecting reactive oxygen species [ROS] levels), CO2, pH, temperature (affecting enzymatic activity), osmotic pressure, water, and electrolyte concentration (e.g., Ca2+ affecting exocytotic processes involving proteins secreted by the epithelial cells) or even viscosity (e.g., affecting sperm motility and oocyte or early embryo transport), can be determining factors for both the correct development of the embryo and the reliable transmission of the genetic information within embryonic cells and from the perspective of shaping the epigenetic environment. The zygote and the early embryo are, indeed, highly dynamic in terms of metabolic and cell division activities. While in the oviduct, they take from the surrounding environment all the nutrients and factors they cannot find in their yolk reserves to continue their development. In addition, they are likely receptive to all the signals and messages received from the oviduct cells arriving via different ways of communication, including exosomes and their miRNAs within. In this context, continuous DNA replication is one of the predominant features of embryonic cells. Although producing precise copies of the genome sequence is the goal pursued by this constant DNA replication, after each cell cleavage, the methylation status of the replicated DNA and most epigenetic marks at the histone level undergo dramatic change. This leads to the almost full reprogramming of the cells that will enable their pluripotency to form any tissue of the body. As stated by Ross and Cánovas, during the life cycle, there are moments in which the epigenetic information needs to be reset for the initiation of a new organism [186]. These moments occur during the formation of the primordial germ cells in the embryo and during the development of the early embryo itself, shortly after fertilization in the oviduct. Since epigenetic remodeling begins at the ooplasm, most evidence holds maternal RNA responsible for this remodeling. However, a number of studies have also indicated a key role of environmental factors for the correct establishment of epigenetic marks supporting the hypothesis of the developmental origins of health and disease (DOHaD) [187]. Although most studies to date have focused on the uterine environment and the impacts of a lack or excess of nutrients for the mother during pregnancy [188, 189], it is clear that the major epigenetic reprogramming event in development takes place in the oviduct and not in the uterus. Consequently, it would be of great interest to know the extent to which changes in the oviduct environment can affect the epigenetic marks of the embryo. Data from children conceived through assisted reproduction reveal a major incidence of some imprinting disorders such as Beckwith–Wiedemann syndrome, Angelman syndrome, and Silver–Russell syndrome compared with naturally conceived babies [190]. In addition, imprinted genes, together with active retrotransposons, are precisely the only genomic regions that, in theory, escape the erasing of DNA methylation marks during the first days of development in the oviduct. Thus, the immediate question that arises is whether embryos whose development was initiated out of the oviduct environment carry a wrong methylation pattern in their imprinting genes which, under physiological circumstances, would have been avoided. Or if we turn the question around: Has the oviduct the capacity to shield the offspring from imprinting disorders? This question remains unanswered prompting the need for a consistent body of research in this field. It has been well documented in the mouse [191], human [192, 193], and monkey [194] that dramatic reshaping of the DNA methylation landscape during the early stages of development (from zygote to blastocyst) involves a drop of around 60–80% of global DNA methylation in the oocyte and spermatozoa, to 20–40% in the blastocyst. In other species such as the pig, global DNA methylation at the blastocyst stage seems to be as low as 12–15% [195]. Although the time lines in each species vary and differences have been reported in the stage at which the maximum drop in methylation is observed, what is clear is that the conceptus spends a long fraction of this period in the oviduct. Consequently, any minor change in the oviduct ecosystem might have consequences on the global DNA demethylation occurring at this point in the conceptus genome, as well as in the remethylation initiated almost simultaneously [194]. Moving forward in the life of the conceptus, if the methylation state of the DNA at a precise gene body or specific genome feature with regulatory capacity changes or is not correct, the corresponding protein encoded by this gene will not be expressed or will be produced in aberrant amounts or with abnormal sequences, with direct consequences on embryo health. Returning to the oviduct environment and the factors affecting it, it would not be difficult to predict a number of correlations between those factors and errors in the epigenetic marks of the embryonic cells. To start with, changes in water and electrolyte transport will affect the osmotic balance and nutrient transport through biological membranes. Accordingly, it has been shown that a lack of folate or methyl group nutrients in the maternal diet could be responsible for DNA hypomethylation [196]. Although such studies looked at DNA from human leukocytes and results were related to cancer [197, 198], it would not be surprising to obtain similar findings in embryonic cells collected from the oviduct-stage conceptus, given the parallels between cancer and embryo metabolism. Another important factor, temperature, has been directly correlated with distribution patterns of DNA methylation across the whole genome in mammals, reptiles and fish [199]. This factor, besides influencing sex determination in fish and plants [200], also affects factors such as growth rate, e.g., in salmon [201] or insulin, sensitivity in mice through changes in DNA methylation and histone deacetylation [202]. In parallel, temperature gradients in the oviduct are thought to influence in events shortly before and after fertilization [203]. Taken together, the data suggest that any modification in oviduct temperature could lead to aberrations during genome methylation reprogramming with the subsequent consequences on the embryonic phenotype. As a third example, we could mention the impacts of abnormal ROS levels in the oviduct lumen. Ten-eleven translocation enzymes (TET1, TET2, and TET3) are partially responsible for the global demethylation process affecting DNA, from the zygote to the blastocyst stage. The mechanism of action of these enzymes consists of the conversion of oxidize 5 methyl cytosine (5 mC) to 5 hydroxy mC and further to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). It is therefore tempting to speculate that alterations in the ROS levels of the oviduct could affect the active DNA demethylation process occurring at this time in the embryo. Finally, two recent studies have shown direct correlation between the presence of oviduct factors during embryo culture and DNA methylation marks in the pig and cow. In the first of these studies [195], pig blastocysts produced in vitro with or without oviduct and uterine fluids as additives in the culture medium were compared with embryos produced in vivo. Whole-genome DNA methylation datasets from individual blastocysts showed global methylation patterns that were closer to those of the in vivo-produced blastocysts when cultured in the presence of these reproductive fluids. In addition, the in vitro-produced blastocysts had higher cell numbers, an improved hatching ability, and also their gene expression patterns were closer to those of in vivo-produced blastocysts. Furthermore, in embryos produced in the absence of reproductive fluids methylation changes were observed in genes whose methylation could be critical, such as insulin like growth factor 2 receptor IGF2R and Neuronatin NNAT. In the second study [204], the culture of bovine embryos with OF induced DNA methylation changes in specific genomic regions in resulting blastocysts. The authors examined the methylation state of four developmentally important genes (mitochondrial transcription termination factor 2 (MTERF2), ATP binding cassette subfamily A member 7 (ABCA7), Olfactomedin 1 (OLFM1), GDP-mannose 4,5 dehydratase (GMDS)) and Long Interspersed Element-1 (LINE-1) retrotransposons in 7–8-day blastocysts. The data indicated reduced methylation levels of MTERF2 when OF was added to the culture medium from the zygote to 16-cell stages, and the elevated expression of MTERF2 mRNA. Moreover, LINE-1 showed higher CpG methylation levels and decreased expression in the embryos produced in the presence of OF compared to its absence. These two studies can be considered consistent proof of the oviduct's effect on the epigenetic landscape of the embryo. As such, they represent the first steps towards the development of more physiological culture media for assisted reproduction both in animals and humans. Conclusions Although mammalian oviducts have long been considered mere conduits for gametes and embryos, recent studies document that the oviduct and its secretions regulate and/or provide a dynamic microenvironment for (1) transport of both male and female gametes to the site of fertilization, (2) final gamete maturation, (3) sperm selection, (4) prevention of polyspermy, (5) fertilization, (6) early cleavage and embryonic development, (7) embryo genome activation, (8) embryo maternal communication, and (9) transport of the embryo to the uterus. In this review, we have focus in the role of the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. There are many studies probing the downside of the ARTs that link to some of the critical functionalities of the oviduct affecting embryo development. The challenge is to continue to develop and optimize in vitro systems to maximize embryo production and quality by emulating the conditions and processes occurring in vivo within the oviduct. Thus, it is possible that by a more efficient selection of the spermatozoa the initial quality of the embryo would be improved. Also by including molecules secreted by the oviduct to the media used for the incubation/culture of both gametes and embryo the adverse periconceptional environment in in vitro-derived embryos could be reduced, and in this way we could increase the efficiency of the current systems for in vitro embryo production in the livestock market as well as in human clinics. Conflict of Interest: The authors have declared that no conflict of interest exists. Footnotes † Grant Support: This work has been funded by the State Secretariat for Research, Development and Innovation of the Spanish Ministry of Economy, Industry and Competitiveness through the projects AGL2015-66145R, AGL2015-70140-R, AGL2015-70159-P, AGL2015-66341, and by the Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia through the projects 19357/PI/14 and 20040/GERM/16. References 1. Wang H, Guo Y, Wang D, Kingsley PJ, Marnett LJ, Das SK, DuBois RN, Dey SK. Aberrant cannabinoid signaling impairs oviductal transport of embryos. Nat Med  2004; 10: 1074– 1080. Google Scholar CrossRef Search ADS PubMed  2. Lopez-Cardona AP, Perez-Cerezales S, Fernandez-Gonzalez R, Laguna-Barraza R, Pericuesta E, Agirregoitia N, Gutierrez-Adan A, Agirregoitia E. CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways. 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Biology of ReproductionOxford University Press

Published: Mar 1, 2018

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