Reprod Dom Anim 49 (Suppl. 3), 27–36 (2014); doi: 10.1111/rda.12383 ISSN 0936–6768

Review Article Seminal Fluid and Immune Adaptation for Pregnancy – Comparative Biology in Mammalian Species† JE Schjenken and SA Robertson School of Paediatrics and Reproductive Health, Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia

Contents Seminal fluid delivered to the female reproductive tract at coitus not only promotes the survival and fertilizing capacity of spermatozoa, but also contains potent signalling agents that influence female reproductive physiology to improve the chances of conception and reproductive success. Male to female seminal fluid signalling occurs in rodents, domestic and livestock animals, and all other mammals examined to date. Seminal plasma is instrumental in eliciting the female response, by provision of cytokines and prostaglandins synthesized in the male accessory glands. These agents bind to receptors on target cells in the cervix and uterus, activating changes in gene expression leading to functional adaptations in the female tissues. Sperm also interact with female tract cells, although the molecular basis of this interaction is not yet defined. The consequences are increased sperm survival and fertilization rates, conditioning of the female immune response to tolerate semen and the conceptus, and molecular and cellular changes in the endometrium that facilitate embryo development and implantation. Studies in porcine, equine, bovine, ovine and canine species all show evidence of male–female signalling function for seminal fluid. There are variations between species that relate to their different reproductive strategies and behaviours, particularly the site of seminal fluid deposition and female reproductive tract anatomy. Although the details of the molecular mechanisms require more study, the available data are consistent with both the sperm and plasma fractions of seminal fluid acting in a synergistic fashion to activate inflammation-like responses and downstream female tract changes in each of these species. Insight into the biological function and molecular basis of seminal fluid signalling in the female will inform new interventions and management practices to support optimal reproductive outcomes in domestic, livestock and endangered animal species.

Introduction In addition to sperm, seminal fluid contains a complex mix of bioactive proteins and other agents produced by male accessory sex organs. These soluble factors have important effects after intromission to promote the survival of sperm and their functional capability, by protecting sperm from oxidative stress and providing metabolic support (Poiani 2006). It is less well appreciated that seminal fluid also affects reproductive events independently of sperm, by stimulating the female tissues to increase the chances of conception and progression to pregnancy. There are now a substantial †

The authors acknowledge the support of the NHMRC of Australia. © 2014 Blackwell Verlag GmbH

number of reports from a wide range of mammalian species which show that seminal fluid exerts a considerable influence on female reproductive tract physiology (Robertson 2005; Sharkey et al. 2012b) (Fig. 1). Advances in this field build on decades of research in invertebrates, where effects on female reproductive organs, immune system and behavioural responses are well known to promote fertilization and transmission of the male germ line (Avila et al. 2011). Extensive studies in Drosophila show that seminal fluid proteins, including accessory gland proteins can increase egg laying, ovulation, the production of antimicrobial proteins, remodelling of the female reproductive tract and reduce female receptivity to remating (Chapman and Davies 2004). In this short review, we summarize our current understanding of the physiological significance of seminal fluid signalling in a range of mammalian species, which despite their diverse reproductive tract anatomy and physiology, all share the attribute of female tract responsiveness to seminal fluid after coitus, with biological consequences that impact reproductive success. A better knowledge of this relatively unexplored aspect of reproductive biology will bring new insight into the regulation of reproduction in a range of socially, environmentally and economically important animal species.

Seminal Fluid and the Immune Adaptation to Tolerate Pregnancy The most obvious effect of seminal fluid in the female is the impact on the immune response, particularly the immune changes that are necessary to support the genetically different foetus. For successful pregnancy, a special state of immune ‘tolerance’ must exist from the very earliest time the embryo first contacts the maternal tissues, which for most species, occurs within days of conception. The conceptus is foreign or ‘non-self’ – it expresses antigens, including some encoded by major histocompatibility (MHC) genes, derived from paternal chromosomes. In healthy pregnancy, the conceptus does not experience immunological attack at implantation. A combination of strategies prevents foetal rejection – some aspects of the immune response are circumvented, while others are engaged and activated to skew the immune system towards tolerance. Seminal fluid seems to have a specific role in providing biological signals, specifically paternal antigens and cytokines, which drive the events leading to immune tolerance.

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JE Schjenken and SA Robertson immune tolerance Treg cells macrophages dendritic cells granulocytes

INFLAMMATORY CYTOKINES

epithelial cells

SEMINAL FLUID embryo development

endometrium

female tract to promote establishment and progression of pregnancy.

phagocytic clearance

uterine lumen

tissue remodeling

Fig. 1. Current understanding of the actions of seminal fluid in the female reproductive tract of species where seminal fluid is deposited in the uterus. Studies in mice and pigs show that after coitus, active moieties in seminal plasma and associated with sperm interact with epithelial cells lining the endometrium, to induce synthesis of proinflammatory cytokines and chemokines. In turn, these cause the recruitment and activation of inflammatory cells in the uterine endometrium, including macrophages, dendritic cells and granulocytes. The macrophages and dendritic cells have roles in inducing endometrial receptivity and in activating regulatory T cells (Treg cells) to mediate maternal immune tolerance of the embryo at implantation. Neutrophils traversing the endometrial epithelium into the luminal cavity act to clear seminal debris and maintain uterine sterility. Epithelial cytokines activated by seminal plasma are also secreted into the luminal fluid where they exert tropic actions on the developing preimplantation embryo [Updated from (Robertson 2005)]

The immediate response to insemination in mammals is a rapid and dramatic influx of inflammatory cells into the site of semen deposition (Robertson 2005). This leucocytic response is evident within an hour of intromission, with firstly neutrophils being released into the luminal space and then other leucocytes – macrophages, dendritic cells and T cells – invading the underlying reproductive tissues. The leucocytes in part respond directly to chemotactic agents in seminal plasma, but the response is amplified as a consequence of seminal fluid-induced production of cytokines and chemokines by epithelial cells lining the reproductive tract surface. The epithelial cell-derived cytokines and chemokines in turn cause leucocytes to extravasate from the blood stream and enter the reproductive tissues. This inflammation-like reaction to semen was first observed in rabbits (McDonald et al. 1952; Taylor 1982), but has since been described in several other species (Robertson 2005). Studies in rodents provide a comprehensive understanding of the molecular and cellular regulation of the inflammatory cascade that has informed experiments in other animals. The inflammatory response to seminal fluid has a central role in the clearance of excess sperm and seminal debris, and promotes recovery of the sterile status of the tract after introduction of micro-organisms at mating. Once this homoeostatic function is accomplished, there is a second and no less important phase in the immune adaptation, which proactively influences the

Seminal Fluid and Regulatory T cells To allow implantation, the female immune response to the conceptus is not switched off or suppressed, but instead immune cells are intimately involved in regulating invasion of conceptus cells at implantation (Trowsdale and Betz 2006). A key element of this active immune tolerance is engagement of special T lymphocytes known as regulatory T cells (Treg cells). Studies in mouse models show that Treg cells are induced to proliferate and then are recruited into the uterus before embryo implantation. Once in place, they permit the immune system to allow the intimate associations between maternal cells and the conceptus-derived placental trophoblasts that are required for adequate placental development and foetal growth. Treg cells operate as potent suppressors of inflammation and cell-mediated immunity (Rudensky 2011). They are critical for the prevention of immune rejection (Sakaguchi 2000; Shevach 2002) via a variety of mechanisms, generally involving suppression of cytokine production and effector function in T cells, B cells, NK cells, dendritic cell and macrophages. Genetic mismatch, particularly in the MHC genes, serves to increase the numbers of Treg cells in the implantation site, and this stimulates the likelihood of healthy pregnancy and benefits foetal growth. In mice, MHC disparate matings result in bigger litters, with heavier foetuses. This is likely to reflect the facilitatory effects of uterine NK cells and Treg cells on transformation of the uterine vasculature, which results in larger diameter blood vessels and increased vascular supply to the placenta (Madeja et al. 2011). A crucial role for seminal fluid in preparation for embryo implantation is through the provision of antigens and cytokines that stimulate expansion of the endometrial Treg-cell population. To ensure enough Treg cells are present in the implantation site, their activation and proliferation during the time between conception and implantation is critical. In mice, the female T-cell response to paternal MHC antigens is initiated immediately after immune cells first contact those antigens transmitted within seminal fluid at coitus, such that the T-cell pool generated during the preimplantation period is expanded by the time of implantation. Soluble MHC is present in seminal plasma, and cell-associated MHC is associated with sperm, seminal leucocytes and/or desquamated genital tract epithelial cells. These and other minor histocompatibility antigens are the same as those later expressed by the conceptus and gestational tissues if pregnancy ensues.

Seminal Fluid and Embryotrophic Cytokines A second key role for seminal fluid signalling is to influence the cytokine and growth factor environment which controls the development of the pre-implantation embryo. As it traverses the female reproductive tract, the embryo is exposed to cytokines and growth factors secreted into the luminal compartment from oviduct © 2014 Blackwell Verlag GmbH

Seminal Plasma Signalling in the Female Tract

and uterine epithelial cells in precise spatial and temporal patterns. Embryos express cytokine receptors from conception until implantation, and several cytokines exert different effects on blastocyst cell number and viability, gene expression and developmental competence (Robertson et al. 1994; Sharkey et al. 1995). The identity and biological effects of several growth factors and cytokines targeting the pre-implantation embryo have been reviewed previously (Kane et al. 1997; Hardy and Spanos 2002). Factors including granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and leukemia inhibitory factor (LIF) promote blastocyst development, while others including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), and interferon-c (IFNc) exert potent inhibitory effects. The secretion profile of these cytokines is regulated at the mRNA level principally by ovarian steroid hormones and, in species where seminal fluid reaches the higher tract, by the signalling factors present in seminal fluid. The importance of seminal fluid signalling as a regulator of female tract cytokines is indicated by experiments in mice and other rodents. When the effect of seminal plasma is evaluated by comparing expression of oviduct cytokines in females mated to intact males and females mated with males from which the seminal vesicles are surgically removed to ablate the seminal stimulus, there are substantial effects on oviduct cytokine expression. In the absence of seminal plasma stimulation, oviduct expression of LIF, GM-CSF and interleukin-6 (IL6) was significantly decreased, while the inhibitory cytokine TRAIL was elevated (Bromfield et al. 2014). This was associated with a substantial reduction in the proportion of females achieving pregnancy.

Male to Female Signalling Factors in Seminal Fluid Several studies have aimed to identify the active factors present in seminal fluid. Early experiments in mice using surgical ablation to remove the male accessory glands demonstrated that many active factors in seminal fluid are derived from the seminal plasma fraction (Robertson et al. 1996). These seminal plasma factors include cytokines, sex hormones and prostaglandins (Aumuller and Riva 1992; Maegawa et al. 2002). Using protein chromatography, the cytokine transforming growth factor-b (TGFb) was identified as the principal active factor in mouse seminal plasma that induces the uterine leucocytic response (Tremellen et al. 1998). TGFb has also been identified as an important seminal fluid signalling factor in women (Sharkey et al. 2012a), pigs (O’Leary et al. 2011) and sheep (Scott et al. 2006b). In the mouse, the TGFb1 concentration is 70 ng/ml (Robertson et al. 2002), in humans the average TGFb concentration ranges from 219 ng/ml for TGFb1, 5 ng/ml for TGFb2 and 172 ng/ml for TGFb3 (Sharkey et al. 2012a), while in pigs, the TGFb1 and TGFb2 concentrations are approximately 150 ng/ml each (O’Leary et al. 2011). Studies to evaluate TGFb in seminal plasma of other species are yet to be undertaken, but are clearly important © 2014 Blackwell Verlag GmbH

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in understanding the importance of this active factor in seminal fluid signalling in other mammals. In addition to TGFb, prostaglandins have also been identified as putative signalling agents present in seminal plasma. In particular, prostaglandin E (PGE) is abundant in human seminal plasma, notably in the 19-hydroxy (19-OH) form (Templeton et al. 1978). In vitro experiments using human cervical explants have shown that 19-OH PGE induces the expression of IL8 and suppresses the anti-inflammatory molecule, secretory leucocyte protease inhibitor (Denison et al. 1999). While PGE is undetectable in rodent and porcine seminal fluid, prostaglandins are present in horse seminal plasma supporting the concept that this factor may play a role in seminal fluid signalling in this species (Claus et al. 1992). There are other active factors identified as potentially playing a role in seminal fluid signalling. Experiments in human tissues show effects of IL8 (Gutsche et al. 2003) and soluble human leukocyte antigen-g (HLA-G) (Larsen et al. 2011), while cysteine-rich secretory protein-3 (CRISP3) (Doty et al. 2011) and porcine sperm adhesion (PSP)-I and PSP-II (Rodriguez-Martinez et al. 2010) have been implicated as signalling agents in equine and porcine species, respectively. In addition to these factors, bacterial products including lipopolysaccharide (Schaefer et al. 2004) and potentially other ligands for Toll-like receptors (TLRs) also contribute to the capacity of seminal fluid to interact with the female reproductive tract. Not all agents act to promote gene expression changes in female tract cells; the type 1 cytokine IFNc is a potent inhibitor of seminal fluid signalling mediated by TGFb (Glynn et al. 2004). Not all seminal fluid signalling can be accounted for by soluble factors in seminal plasma. Studies in several species implicate sperm as also contributing, although the molecular basis of any direct sperm-mediated signalling remains unclear. This array of seminal fluid signalling components presumably acts in concert to regulate the female immune response to coitus. It seems likely that different species will utilize a range of factors in different proportions, depending on the species and the anatomical site of semen deposition. Substantial research will be required to identify the full range of active factors present in seminal fluid of different species, to define how the balance of different agents is regulated in the male, and to understand how alterations to the balance of signalling agents affect the female tract response after coitus.

Evidence for Seminal Fluid Signalling in Various Mammalian Species Seminal fluid signalling in mice The most extensive studies to define the role of seminal fluid in the female immune response to pregnancy are in mice. In mice, the immune response to seminal fluid is initiated when seminal proteins interact with oestrogenprimed cervical and uterine epithelial cells to activate synthesis of pro-inflammatory cytokines including GMCSF, IL6, macrophage chemotactic protein-1 (MCP-1), IL8 (KC) and an array of other chemokines (Sanford

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et al. 1992; Robertson et al. 1996, 1998). The presence of these inflammatory cytokines elicits recruitment from the blood of inflammatory leucocytes including macrophages, dendritic cells and granulocytes, resulting in the accumulation of these cells in the subepithelial stromal tissue. During this initial acute phase, large populations of neutrophils are also observed to migrate through the epithelial surface and efflux into the luminal cavity (De et al. 1991; McMaster et al. 1992; Robertson et al. 1996). This is a transient response and is resolved prior to embryo implantation, in parallel with the decline in inflammatory cytokine release as rising progesterone levels inhibit synthesis. Detailed studies in different surgical and genetic models in mice have revealed that the female immune response to seminal fluid contributes to the establishment of pregnancy via various pathways. In addition to clearance from the uterine cavity of micro-organisms introduced at mating (Robertson 2005) and induction of Treg cells to confer tolerance at implantation (Guerin et al. 2011), seminal fluid induces the oviduct and uterus to provide trophic factors that promote the development of the pre-implantation embryo (Robertson et al. 2011), and induces expression of embryo attachment ligands and angiogenic factors that promote embryo implantation (Jasper et al. 2011). Another, less well-explored pathway through which seminal fluid may support progression to pregnancy is through effects on the ovary. Experiments in mice show that macrophage populations in corpora lutea are augmented by exposure of the female tract to seminal plasma constituents (Gangnuss et al. 2004). These cells are now understood to have central functions in the development of the luteal vasculature and the progesterone secretion required for endometrial receptivity (Care et al. 2013). The pathway for establishment of tolerance towards paternal antigens is well defined in mice. Using T-cell transgenic mice that have large populations of clonal T cells reactive with male antigens, it can be clearly demonstrated that priming of the female immune response commences when the female is exposed to male transplantation antigens in the seminal fluid (Moldenhauer et al. 2009). This is a crucial first step in eliciting the expansion of antigen-specific populations of Treg cells that prevent immune destruction of the embryo (Robertson et al. 2009). In addition to providing antigen and immune regulatory cytokines to stimulate Treg-cell proliferation, seminal fluid promotes recruitment of Treg cells into the implantation site through stimulating expression of the Treg-cell chemokine, CCL19 (Guerin et al. 2011). Within 2 days of conception, an expanded population of Treg cells can be detected within the uterine lymph nodes (Aluvihare et al. 2004; Zhao et al. 2007; Robertson et al. 2009). By day 3.5 post-coitum, when embryos implant in mice, Treg cells also accumulate in the uterus and are increased >two-fold compared with oestrous numbers (Robertson et al. 2009; Guerin et al. 2011). In mice, this increase in Treg cells clearly depends on seminal plasma, as when seminal vesicles are excised from males before mating, there is no expansion in Tregcell numbers (Robertson et al. 2009; Guerin et al. 2011). TGFb is the prime candidate for this seminal plasma

JE Schjenken and SA Robertson

factor, as TGFb is clearly implicated in Treg-cell generation from precursor T cells (Chen et al. 2003), and furthermore, exogenous TGFb increased vaginal Treg-cell numbers and prevented spontaneous foetal loss in abortion-prone mice when delivered at coitus (Clark et al. 2008). Interestingly, sperm may also provide Treg-inducing and Treg-stabilizing factors, as females mated with vasectomized males show reduced expression of the signature Treg-cell transcription factor forkhead Box P-3 (Foxp3) (Guerin et al. 2011). The importance of the seminal plasma fraction in pregnancy success by pathways independent of Treg cells can be examined by studying syngeneic pregnancies, where there is no MHC disparity between sire and dam. Even in this case, where Treg cells are not required for pregnancy success, the success and quality of the outcome are compromised if females are not exposed to seminal plasma (Bromfield et al. 2014). Experiments in which the seminal vesicle, prostate or coagulating glands are surgically removed from mice, rats and hamsters prior to mating each show that seminal vesicle fluid is the most vital non-sperm component of the ejaculate, and support the interpretation that seminal plasma is essential for optimal sperm survival and fertilization potential (Pang et al. 1979; Queen et al. 1981; Peitz and Olds Clarke 1986; O et al. 1988). In mice, embryo transfer protocols generally employ recipients exposed to seminal plasma by mating to vasectomized males, but foetal loss and abnormality is considerably greater when embryos are transferred after pseudopregnancy achieved without exposure to male fluids (Watson et al. 1983). Confirmation that the effects of seminal plasma are mediated, at least partly, through promoting embryotrophic cytokines in the female tract is provided by studies showing that when recipient females are mated with seminal vesicle-deficient males, transferred embryos give rise to foetuses with impaired growth trajectories and placental development (Bromfield et al. 2014). In other rodent species, implantation rates and foetal growth are similarly impaired unless females are inseminated prior to embryo transfer (Carp et al. 1984). Seminal fluid signalling in human There are a limited number of studies on the role of seminal fluid in the peri-conceptional period in humans. The cervix is the site for seminal fluid deposition following coitus in women and is a major effector site for immune responses in the female genital tract (Pudney et al. 2005). Early studies documented that artificial insemination (AI) of human sperm into the cervix results in an influx of leucocytes, predominantly neutrophils into the surrounding tissue (Pandya and Cohen 1985; Thompson et al. 1992). The seminal plasma fraction is demonstrated to be important in this process, as in vitro and in vivo experiments show that addition of seminal plasma results in the induction of GM-CSF, IL6, IL8, MCP-1, Macrophage inflammatory protein-3a (Mip-3a) and IL1a (Sharkey et al. 2007, 2012b). In addition to the increase in cytokines and chemokines observed in vivo, leucocyte recruitment of predominantly macrophages and dendritic cells and also memory T cells was demonstrated (Sharkey et al. 2012b). © 2014 Blackwell Verlag GmbH

Seminal Plasma Signalling in the Female Tract

Interestingly, there is some evidence consistent with a role for seminal plasma in induction of Treg cells in women, although the definitive experiments have not yet been conducted. In in vitro experiments, seminal plasma induces the differentiation of tolerogenic dendritic cells, potentially through effects mediated by E-series prostaglandins (Remes Lenicov et al. 2012). TGFb within seminal plasma appears to play a major role in this process; however, TGFb alone does not completely explain the signalling properties of seminal fluid (Sharkey et al. 2012a). Seminal plasma also contains substantial concentrations of other immune-deviating cytokines and molecules involved in Treg-cell production from na€ıve T cells, including HLA-G5, TGFb and PGE2 (Kelly and Critchley 1997; Hutter and Dohr 1998; Robertson et al. 2002). Further studies are required to identify the full range of active factors present in seminal fluid, to increase our understanding of seminal fluid signalling in humans. Clinical studies are consistent with an effect of seminal fluid exposure on the outcomes of pregnancy. Immunological conditions of pregnancy such as pre-eclampsia are more common in the first conception, after a short period of cohabitation, after a long period of cohabitation when barrier contraceptives are used or when pregnancy is conceived with a new male partner in multiparous mothers (Klonoff-Cohen et al. 1989; Robillard et al. 1993, 1994; Trupin et al. 1996; Dekker et al. 1998; Saftlas et al. 2014). Data from IVF and related reproductive technologies support a benefit of seminal fluid signalling at conception, with the absence of seminal plasma in the in vitro setting potentially contributing to higher rates of implantation failure and a decrease in embryo quality. IVF success is limited for some subsets of patients with potential consequences for embryos, seen through subtle changes in birthweight and health outcomes of IVF children (Schieve et al. 2002; Maher et al. 2003; Ceelen et al. 2007, 2008). Taking these findings into account, strategies have been devised to utilize factors induced by seminal plasma to improve IVF outcomes, with addition of GM-CSF to IVF culture media significantly improving the survival of transferred embryos to week 12 and live birth (Ziebe et al. 2013). Seminal fluid signalling in pigs In pigs, seminal fluid induces a well-described inflammatory cascade regulated by similar processes to mouse and humans. Like mice, pigs are intrauterine inseminators resulting in direct contact between seminal fluid and epithelial cells lining the endometrium. The most immediate response to seminal fluid is rapid recruitment of neutrophils into the uterine lumen (Lovell and Getty 1968; Claus 1990; Rozeboom et al. 1998), followed by neutrophil binding to viable, but not non-viable spermatozoa (Taylor et al. 2008). The influx of neutrophils is regulated at least partly by seminal plasma proteins PSP-I and PSP-II, which are major proteins in boar seminal plasma (Garcia et al. 2008). In vivo studies show that a heterodimer of PSP-I and PSP-II acts as a postmating pro-inflammatory mediator targeting neutrophils in pigs (Rodriguez-Martinez et al. 2010). Exposing sperm to this complex preserves sperm viability, motility © 2014 Blackwell Verlag GmbH

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and mitochondrial activity compared with vehicletreated sperm. This preservation may be due to the PSP-I/PSP-II heterodimer adhering to the acrosomal area of sperm (Caballero et al. 2006). In addition to neutrophils, seminal fluid elicits accumulation of macrophages, dendritic cells, granulocytes and lymphocytes within the endometrial stroma (Bischof et al. 1994, 1995; Engelhardt et al. 1997). Experiments with vasectomized boars show that seminal plasma constituents contribute to this phase of the response (Bischof et al. 1994). Studies in gilts have shown that specific factors present in seminal plasma interact with uterine cells to induce expression of GMCSF, IL6, MCP-1 and IL10 and that neuroendocrine stimulation resulting from the physical act of mating is not necessary for this response. GM-CSF, IL6 and MCP-1 have chemotactic properties and are implicated in regulating macrophage and dendritic cell recruitment into the endometrial stroma. Interestingly, exposure to sperm in addition to seminal plasma alters the seminal plasma response (O’Leary et al. 2004; Taylor et al. 2009). While the luminal neutrophil response is resolved within 24 h (Rozeboom et al. 1998), the inflammatory cells infiltrating the endometrium appear to persist and undergo differentiation in the tissue for several days, expanding the endometrial MHC class II+ dendritic cell pool for the duration of the pre-implantation period (Bischof et al. 1994; O’Leary et al. 2004). This transition in leucocyte phenotype is likely to be driven by the more rapid termination of inflammatory cytokine synthesis that occurs in gilts exposed to seminal plasma (O’Leary et al. 2004). Studies in pigs have been instrumental for exploring the effects of seminal fluid on ovarian function. In oestrous pigs, there is a reduction in the interval between the LH surge and ovulation in response to natural mating (Signoret et al. 1972) or seminal plasma administered transcervically (Waberski et al. 1997). The effects appear to be mediated by local transport of seminal constituents or semen-induced effector molecules originating in proximal parts of the tract, because the effect is seen only in ovaries ipsilateral to uterine horns receiving the seminal stimulus. Seminal plasma also promotes the development of the corpus luteum and steroidogenic capacity (O’Leary et al. 2006), with infiltrating macrophages implicated as mediating cells in this process. Seminal fluid signalling in rabbit The first reported evidence of an inflammatory reaction to semen was observed in rabbits in 1952 (McDonald et al. 1952), when pseudopregnant female rabbits or oestrous rabbits were inseminated with semen and the resulting leucocyte influx was examined. The study demonstrated that a leucocytic influx in response to seminal fluid was evident under both hormonal conditions, but that a stronger response occurred in the uterus of pseudopregnant rabbits. Interestingly, administration of sterile epididymal sperm failed to induce a significant reaction, leading the authors to suggest that bacterial contamination was the reason for the leucocytic influx (McDonald et al. 1952). Subsequent studies showed

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that both seminal plasma and sperm may contribute to the initiation of the leucocytic response in rabbits (Phillips and Mahler 1977a,b). In the presence of seminal plasma, rabbit sperm show similar viability and potentially increased motility, but fertility was not improved (Castellini et al. 2000). Rabbits have also been utilized to provide evidence for rapid transport of seminal fluid components to distal parts of the female reproductive tract. Using bucks immunized with fd-tet, male-derived immunoglobulin could be detected in the uterus within 20 min of intromission (Jappel 1992). Seminal fluid signalling in horse and donkey In equine species, seminal fluid accesses the uterus after coitus and the endometrial response is an active area of investigation because of its relevance to a condition known as mating-induced endometritis, a major issue in these species, particularly horses (Watson 2000; Katila 2012). Endometritis is generally considered a pathological condition, but recent studies have advanced the notion that an appropriate form of endometrial inflammation is a physiologically normal process which has evolved to benefit the establishment of pregnancy, as observed for human and mice. Early studies into endometritis in the horse have focused on the role of sperm in initiating the inflammation-like response and have identified that sperm alone is chemotactic for equine neutrophils. Neutrophils are rapidly recruited into the uterus following exposure to sperm where they peak between 4 and 8 h and are mostly removed by 48 h (Katila 1995). After recruitment into the uterus, neutrophils bind to and then phagocytize sperm (Troedsson et al. 2001; Alghamdi et al. 2004). The presence of seminal plasma inhibits this process in viable but not non-viable sperm (Troedsson et al. 2005) potentially through formation of neutrophil extracellular traps (NETs), which reduce sperm ability to bind to epithelial cells (Alghamdi and Foster 2005). Furthermore, a role for CRISP3 in seminal plasma has been indicated in mediating the interaction between sperm and neutrophils (Doty et al. 2011). Examination of T-cell populations by immunohistochemistry has shown that an increase in CD4+ but not CD8+ T cells in the uterine body can be observed at 6 and 48 h following insemination (Tunon et al. 2000). In addition, as observed in human and mouse, uterine seminal plasma treatment induces the expression of IL1b, IL6, TNF and cyclooxygenase-2 (COX2) genes in the endometrial tissue, suggesting that the actions of seminal plasma in the horse are similar to those observed in mouse and human. Fertility studies examining the impact of seminal plasma in the mare reveal contrasting results. An effect of seminal plasma can be observed when inflammation is induced in the uterus by the prior administration of dead spermatozoa. In this case, delivery of sperm in the presence of seminal plasma results in a 77% conception rate in mares, compared with only 4.5% conception in mares inseminated with sperm in the absence of seminal plasma (Troedsson et al. 2002). In contrast, when there is no prior induction of uterine inflammation, conception rates do not differ (Rigby et al. 2001; Portus et al. 2005). This has led to suggestions that seminal plasma is

JE Schjenken and SA Robertson

required for protection of sperm against phagocytosis by neutrophils in an inflamed uterus, but is less important for conception in a normal quiescent uterine environment (Katila 2005). Studies evaluating the effectiveness of AI in the donkey have demonstrated that commonly used equine cryoprotectants are not suitable, possibly due to toxic effects on the female reproductive tract (Vidament et al. 2009; Rota et al. 2012). In an effort to improve AI rates in donkeys, a number of semen extenders have been compared with seminal plasma in glycerol, or ethylene glycol cryoprotected sperm (Rota et al. 2012). While the use of semen extenders improved total and progressive motility of sperm, there was an improved pregnancy rate when seminal plasma was present. Seminal plasma also increased the female neutrophil response; however, the neutrophil-mediated clearance of sperm was reduced due to suppressed sperm–neutrophil attachment (Rota et al. 2012; Miro et al. 2013). Together, these observations are consistent with a regulatory role for seminal plasma during the peri-conception period in equine species. Seminal fluid signalling in cows In the bovine, little is known about the interactions between seminal fluid and the female reproductive tract. Bovine semen, unlike equine semen, is naturally deposited in the vagina and sperm migrate through the cervix into the uterus leaving the bulk of seminal plasma behind in the lower tract (Alghamdi et al. 2009). However, ejaculated sperm are coated in major seminal plasma proteins which may allow seminal plasma to indirectly function in the bovine uterus (Gwathmey et al. 2006; Alghamdi et al. 2010). Despite this, the potential effects of seminal plasma in the uterus are pertinent, given the common practice of intrauterine AI to yield a higher reproductive efficiency. In studies utilizing intrauterine AI, there is evidence for neutrophil migration but not sperm binding following delivery of sperm alone (Alghamdi et al. 2009). The addition of bovine seminal plasma to diluted or washed sperm has been demonstrated to increase sperm viability, motility and protection (Baas et al. 1983; Garner et al. 2001; Gwathmey et al. 2006). Further, in the presence of seminal plasma, bovine neutrophils have more cell membrane damage, produce less reactive oxygen species following mitogenic stimulation and have a lower transmigration in response to IL8, compared with control (Aloe et al. 2012). However, an increase in sperm–neutrophil binding and NET formation is also be observed in the presence of seminal plasma (Alghamdi et al. 2009). Overall, these studies suggest that bovine seminal plasma has a regulatory effect on inflammatory processes in the uterus following insemination and that neutrophil–sperm interactions may be a physiologically relevant pathway in bovine fertility. Seminal fluid signalling in sheep As observed in other species, mating in sheep induces a transient inflammatory response that draws macrophages and neutrophils into the cervical and uterine tissues © 2014 Blackwell Verlag GmbH

Seminal Plasma Signalling in the Female Tract

(Mattner 1968; Scott et al. 2006a). This is despite the fact that semen is deposited into the cranial vagina and around the cervical os of ewes, rather than directly into the uterus. As observed in humans and mice, ovine seminal plasma contains the active factor TGFb (Scott et al. 2006b). Studies of the uterine inflammatory response at oestrus have shown that both sperm and seminal plasma contribute to the macrophage and neutrophil infiltration as well as increased endometrial IL8 secretion. Exposure to whole semen or washed sperm but not seminal plasma alone induced secretion of GM-CSF. However, a limitation of this study was the possible contribution of bacterial contamination, as antibiotic addition partially reduced the neutrophil response to seminal fluid (Scott et al. 2009). Seminal fluid signalling in dogs There are a limited number of studies examining the role of seminal fluid in the immune response to coitus in bitches. Following coitus, canine sperm are deposited in the cranial vagina and distributed throughout the female reproductive tract via vaginal and uterine contractions. Due to the potential length between coitus and fertilization in canine (up to 9 days), studies have focused on sperm storage in the reproductive tract and it has been established that epithelial cells of the uterine gland and uterine tube junction tether sperm and act to provide a sperm reservoir (Rijsselaere et al. 2004; England et al. 2012, 2013). This interaction is thought to promote sperm survival and fertility and reduce polyspermia (Suarez 2008). In the canine, studies have shown that seminal plasma has beneficial effects on fertility. The addition of seminal plasma to intravaginally administered sperm was shown to increase the odds of conception when compared to sperm combined with Tyrode’s albumin lactate pyruvate media (a medium used for canine sperm during computer-assisted motility assessment) (Nothling et al. 2005). In the first study to document the canine response to seminal fluid, the role of seminal fluid in the canine uterus was examined following AI and it was demonstrated that seminal plasma induces vasodilation (England et al. 2012). Further, sperm attachment to uterine epithelium was shown to be hindered without the presence of seminal plasma, potentially due to sperm interaction with neutrophils. Seminal fluid signalling in other species In a number of other species, there is evidence for seminal fluid function during the peri-conception period of early pregnancy. In cats, assessment of Foxp3, cytotoxic T-lymphocyte-associated protein-4 (Ctla4) and CD25 mRNA levels supports the conclusion that Treg cells are higher in early pregnancy compared with late pregnancy. This study did not examine the role of seminal fluid, but one interpretation is that seminal fluid induces Treg cells following coitus as it does in other species (Lockett et al. 2010). Studies in camels have shown that factors in seminal plasma are required for the induction of ovulation (Li and Zhao 2004), consistent with reports in pigs that seminal fluid influences © 2014 Blackwell Verlag GmbH

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ovarian function including ovulation and corpus luteum development (O’Leary et al. 2006). Studies in birds provide evidence for immunological responses in the presence of components of seminal fluid. Sperm within the fowl oviduct are phagocytized by immune cells, likely to be macrophages (Koyanagi and Nishiyama 1981).

Conclusions In all species examined so far, seminal fluid appears to play an integral role in eliciting inflammatory changes in the female reproductive tract following coitus. Utilizing rodents and also pigs as experimental models has allowed considerable insight into the molecular and cellular basis for this response and its role in the physiology of embryo implantation and establishing successful pregnancy. There are remarkable similarities in the female response in mice and pigs, where the uterus is the site of seminal fluid deposition. In both species, factors in seminal plasma account for the majority of the endometrial changes with induction of proinflammatory cytokines and chemokines, which combined with endogenous chemokines in seminal plasma cause recruitment of macrophages and dendritic cells. Sperm appear to be particularly important for inducing the first phase of neutrophil recruitment into the luminal cavity of the tract, and additionally contribute by interacting with seminal plasma factors to further modulate cytokine expression and regulate leucocyte influx. NET formation induced by neutrophil– sperm interaction has the potential to further attenuate the immunological environment and may contribute to influencing the female response to sperm, perhaps ultimately impacting which sperm are available to fertilize oocytes. The cascade of changes in local leucocyte populations and cytokine synthesis persists through the pre-implantation period and has sets the course of the female immune response to implantation. In both species, exposure to seminal fluid alters the dynamics of pre-implantation embryo development with an increase in the number of fertilized oocytes attaining viable blastocyst stage. Both also provide evidence that seminal factors promote corpus luteum development and progesterone synthesis. The commonality between these species is consistent with an evolutionarily conserved pathway that has substantial influence on the reproductive process. Furthermore, there is emerging understanding of the similarities and differences in the seminal fluid response of women, where the cervix is the site of seminal fluid deposition. Here, it seems that the cervical response at least to some extent mirrors that seen in the uterus of intrauterine ejaculators. There is less knowledge on how seminal fluid functions in other mammalian species. In sheep and cows where seminal fluid is deposited in the cervix, there is preliminary evidence that signalling factors associated with sperm can influence immune events in the higher tract. Clearly, there is still much to learn about how and why seminal fluid signalling varies between species, the evolutionary value of this event in reproductive biology, and the potential benefit that seminal fluid may impart

34

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during the establishment of pregnancy in many animals. This is particularly relevant in species which are commonly bred using AI, where purified sperm is combined with artificial semen extenders for fertilization and the normal process of seminal fluid signalling is averted. Future studies should focus on defining the effects of seminal fluid exposure on conception and implantation, as well as foetal development and postnatal growth trajectory, viability and health of offspring. A better understanding of seminal fluid signalling in a range of animal species will no doubt lead to a better understanding of this important area of biology

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Author Contribution JES and SAR both critically reviewed the literature and drafted the manuscript.

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Submitted: 0 0000; Accepted: 21 Jun 2014 Author’s address (for correspondence): SA Robertson, School of Paediatrics and Reproductive Health, Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia. E-mail: sarah.robertson@ adelaide.edu.au

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