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Immunological response to semen in the female genital tract
Abstract
When spermatozoa, seminal plasma and semen extender reach the uterus and interact with local leukocytes and endometrial cells, several immune mechanisms are initiated which have immediate, mid-term and long-term effects on ovulation, sperm cell selection, fertilization and pregnancy success by assuring the acceptance of fetal tissues. This report gives an overview on relevant key immune mechanisms following roughly the time axis after insemination. Detailed knowledge regarding these mechanisms will aid maximizing reproductive efficiency in livestock production. In the future, the many species involved will require a more comparative approach, since evidence is growing that endometrial physiology and the response to varying amounts and compositions of seminal plasma, various semen extenders, and variable numbers of spermatozoa also provoke different immune responses.
... Immunological abnormality can result from a defect in the maternal immune response or a defect in the husband's semen or spermatozoa in stimulation of the female reproductive tract (FRT) immune response [2]. Findings reveal that semen and spermatozoa immunogenicity result in the development of regulatory and effector T and B lymphocyte directed to paternal antigens [3,4] such as human leukocyte antigen (HLA) molecules. A result of this stimulated immunity may be the production of alloantibody against paternal HLA molecules. ...
... Therefore, our current study was conducted to obtain more information about APCA. As mentioned in the introduction, insemination results in the induction of inflammation in the FRT that accompanies recruitment of immune cells such as neutrophils, macrophages and lymphocytes to the FRT [3,4]. This response constitutes a pre-embryo implantation immune response that has a major role in the improvement of embryo implantation and successful pregnancy [4,15]. ...
... As mentioned in the introduction, insemination results in the induction of inflammation in the FRT that accompanies recruitment of immune cells such as neutrophils, macrophages and lymphocytes to the FRT [3,4]. This response constitutes a pre-embryo implantation immune response that has a major role in the improvement of embryo implantation and successful pregnancy [4,15]. We hypothesize that APCA may be produced in the context of this activated immunity and is directed to HLA molecules on spermatozoa. ...
Background: Anti-paternal cytotoxic antibody (APCA) is a pregnancy alloantibody and is directed to paternal HLA molecules. A recent study showed that this antibody is produced in the presence of the husband’s spermatozoa. Several studies have shown the absence or reduction of this antibody in the serum of recurrent spontaneous abortion (RSA) women. Note that the production of APCA has not been determined in the presence of spermatozoa in RSA women. A reason for the reduction or absence of APCA in the serum of RSA women may be the inability of the husband’s spermatozoa to induce APCA production by lymphocytes. Therefore, this study aims to investigate APCA production by the wife’s peripheral blood mononuclear cells (PBMCs) in the presence of the husband’s spermatozoa in RSA couples. Methods: Ten RSA couples and ten fertile couples were included in this study. The wife’s PBMCs (peripheral blood mononuclear cells) were cocultured with her husband’s spermatozoa and the supernatant assessed for the presence of IgG by the ELISA method and APCA by complement-dependent cytotoxicity (CDC) assay. Results: Results showed that the production of IgG (median = 740 ng/mL in the fertile and 64 ng/mL in the RSA group) and APCA (median = 78% in the fertile and 48.5% in the RSA group) was significantly lower in the RSA group as compared to the fertile group. Conclusions: We concluded that a possible cause for APCA reduction in RSA women may be the inability of the husband’s spermatozoa in induction of antibody production by the wife’s PBMC. Nevertheless, large studies are needed to confirm the results of the present study.
... Neutrophils summoned by endometrial cells and leukocytes are probably the primary mechanism of sperm selection in the uterus that causes their chemotaxis response toward dead, abnormal, and capacitated sperm (Ajdary et al., 2021, Pérez-Cerezales et al., 2018. Sperm-related signals that induce immunological reactions in the endometrium seem to be another way to negatively select and remove sperms with a lower potential for oocyte fertilizing (Pérez-Cerezales et al., 2018, Schuberth et al., 2008. ...
... In the oviduct, sperm selection depends on the cell's ability to bind to the epithelium. Sperm adhere to the epithelial cells by mediating factors expressed on the cell surface; for example, the binding of lectin-like protein on sperm to carbohydrate ligands on oviduct cells (Schuberth et al., 2008, Talevi and Gualtieri, 2010, Pérez-Cerezales et al., 2018. ADAMS are another group of proteins that might involve in the adhesion of sperm to a ligand, known as CD9, on the epithelial cells in the oviduct and select competent sperm based on the protein-to-protein interface. ...
... This adhesion does not form tightly and lets sperm move forward through the oviduct wall (Chen et al., 1999, Pérez-Cerezales et al., 2018, Suarez, 2004. Therefore, oviducts select sperm based on the superficial proteins required for attachment to the epithelial cell (Pérez-Cerezales et al., 2018, Schuberth et al., 2008. ...
... Semen deposition triggers the invasion of neutrophilic granulocytes, which phagocytize sperm in the reproductive tract during the first hours after insemination [7,37]. While sperms of most mammalian species survive in the oviduct for a maximum of five days after mating [38,39], our agents in the less sperm-friendly lower part of the female genital tract have a lifetime drawn from a normal distribution around 24 h (Table 1). ...
... This concerns for instance a more sophisticated simulation of immune responses. The female immune response develops gradually within the first hours after mating via infiltration of vagina, cervix, and uterus with leukocytes [7,37]. Consequently, a progressively increasing reduction of lifetime with time was chosen in the present model as a first simple approximation. ...
... Consequently, a progressively increasing reduction of lifetime with time was chosen in the present model as a first simple approximation. However, observations that preferentially viable sperm bind to porcine neutrophils in vitro [37] suggest the existence of so far unknown selective processes beyond the removal of damaged sperm. The complex processes occurring to sperm in the oviduct are a further topic to extend/complete the simulation of sperm migration to the oocyte. ...
Sperm migration in the female genital tract controls sperm selection and, therefore, reproductive success as male gametes are conditioned for fertilization while their number is dramatically reduced. Mechanisms underlying sperm migration are mostly unknown, since in vivo investigations are mostly unfeasible for ethical or practical reasons. By presenting a spatio-temporal model of the mammalian female genital tract combined with agent-based description of sperm motion and interaction as well as parameterizing it with bovine data, we offer an alternative possibility for studying sperm migration in silico. The model incorporates genital tract geometry as well as biophysical principles of sperm motion observed in vitro such as positive rheotaxis and thigmotaxis. This model for sperm migration from vagina to oviducts was successfully tested against in vivo data from literature. We found that physical sperm characteristics such as velocity and directional stability as well as sperm-fluid interactions and wall alignment are critical for success, i.e. sperms reaching the oviducts. Therefore, we propose that these identified sperm parameters should be considered in detail for conditioning sperm in artificial selection procedures since the natural processes are normally bypassed in reproductive in vitro technologies. The tremendous impact of mucus flow to support sperm accumulation in the oviduct highlights the importance of a species-specific optimum time window for artificial insemination regarding ovulation. Predictions from our extendable in silico experimental system will improve assisted reproduction in humans, endangered species, and livestock.
... Semen deposition triggers the invasion of neutrophilic granulocytes, which phagocytize sperm in the reproductive tract during the first hours after insemination [7,37]. While sperms of most mammalian species survive in the oviduct for a maximum of five days after mating [38,39], our agents in the less sperm-friendly lower part of the female genital tract have a lifetime drawn from a normal distribution around 24 h (Table 1). ...
... This concerns for instance a more sophisticated simulation of immune responses. The female immune response develops gradually within the first hours after mating via infiltration of vagina, cervix, and uterus with leukocytes [7,37]. Consequently, a progressively increasing reduction of lifetime with time was chosen in the present model as a first simple approximation. ...
... Consequently, a progressively increasing reduction of lifetime with time was chosen in the present model as a first simple approximation. However, observations that preferentially viable sperm bind to porcine neutrophils in vitro [37] suggest the existence of so far unknown selective processes beyond the removal of damaged sperm. The complex processes occurring to sperm in the oviduct are a further topic to extend/complete the simulation of sperm migration to the oocyte. ...
Sperm migration in the female genital tract controls sperm selection and, therefore, reproductive success as male gametes are conditioned for fertilization while their number is dramatically reduced. Mechanisms underlying sperm migration are mostly unknown, since in vivo investigations are mostly unfeasible for ethical or practical reasons. By presenting a spatio-temporal model of the mammalian female genital tract combined with agent-based description of sperm motion and interaction as well as parameterizing it with bovine data, we offer an alternative possibility for studying sperm migration in silico. The model incorporates genital tract geometry as well as biophysical principles of sperm motion observed in vitro such as positive rheotaxis and thigmotaxis. This model for sperm migration from vagina to oviducts was successfully tested against in vivo data from literature. We found that physical sperm characteristics such as velocity and directional stability as well as sperm-fluid interactions and wall alignment are critical for success, i.e. sperms reaching the oviducts. Therefore, we propose that these identified sperm parameters should be considered in detail for conditioning sperm in artificial selection procedures since the natural processes are normally bypassed in reproductive in vitro technologies. The tremendous impact of mucus flow to support sperm accumulation in the oviduct highlights the importance of a species-specific optimum time window for artificial insemination regarding ovulation. Predictions from our extendable in silico experimental system will improve assisted reproduction in humans, endangered species, and livestock.
... However, most of the inseminated spermatozoa do not reach the UTJs. Within 30 minutes of insemination about 20-25% of the spermatozoa are egressed by vaginal retrograde flow [4,5] while those spermatozoa remaining in utero are phagocytosed by polymorphonuclear leukocytes (PMNs) migrating from the endometrial lamina propria [1,[6][7][8][9]. Interestingly, such an inflammatory scenario is not seen in the pre-ovulatory UTJs [10,11], and it is normally transient (over a matter of hours) in the uterus [11]. ...
... Entry of semen into the sow genitalia leads to a brief uterine inflammation. It also seems to change the nature of the innate and acquired local immunity in the female reproductive tract after entry [1,7,9,12]. Such an effect ought to be initiated by signaling originating from the spermatozoa as well as from the complex SP and its battery of proteins, peptides and enzymes [13][14][15][16][17], cytokines/chemokines [18][19][20], sex hormones [21,22], extracellular vesicles (i.e., exosomes), lipoproteins and non-coding RNAs [23][24][25]. ...
... Earlier studies stated that pig oviductal sperm reservoirs (UTJ/isthmus) do not show signs of infiltration by immune cells nor display other obvious immune responses during the pre-ovulatory period [10,11]. In utero, by contrast, large numbers of cervically inseminated spermatozoa are lost due to inflammation-related leukocyte phagocytosis and CD4-CD8 lymphocyte attacks [1,[6][7][8][9]43]. Therefore, the female immediately reacts to the entry of semen with a transient, hour-lasting inflammation, where polymorphonuclear leukocytes are seen traversing the genital epithelium at the cervix and uterus to engulf spermatozoa as well as eliminating possible pathogens that accompany the non-sterile semen [1,9,44]. ...
Mating or cervical deposition of spermatozoa or seminal plasma (SP) modifies the expression of genes affecting local immune defense processes at the oviductal sperm reservoir in animals with internal fertilization, frequently by down-regulation. Such responses may occur alongside sperm transport to or even beyond the reservoir. Here, immune-related gene expression was explored with cDNA microarrays on porcine cervix-to-infundibulum tissues, pre-/peri-ovulation. Samples were collected 24 h post-mating or cervical deposition of sperm-peak spermatozoa or SP (from the sperm-peak fraction or the whole ejaculate). All treatments of this interventional study affected gene expression. The concerted action of spermatozoa and SP down-regulated chemokine and cytokine (P00031), interferon-gamma signaling (P00035), and JAK/STAT (P00038) pathways in segments up to the sperm reservoir (utero-tubal junction (UTJ)/isthmus). Spermatozoa in the vanguard sperm-peak fraction (P1-AI), uniquely displayed an up-regulatory effect on these pathways in the ampulla and infundibulum. Sperm-free SP, on the other hand, did not lead to major effects on gene expression, despite the clinical notion that SP mitigates reactivity by the female immune system after mating or artificial insemination.
... In vitro incubation sperm from highly fertile bulls with seminal plasma from lowly fertile bulls decreases the oocyte penetration ability (Henault and Killian, 1996). Furthermore, differences in composition of seminal plasma between species and among males within the same species (Alghamdi et al., 2009;Robertson, 2005;Schuberth et al., 2008) result in variable fertility indexes (Maxwell et al., 2007;Moura et al., 2006;Robertson, 2005), as well as different uterine immune responses (Katila, 2001;Kotilainen et al., 1994;Matthijs et al., 2003;Pitnick et al., 2009) and freezability (Jobim et al., 2011;Zahn et al., 2005). ...
... The uterus has features of a mucosa-associated lymphoid tissue that varies with the estrous phase in mares. A neutrophilic based population is observed just before ovulation, and its inflammatory activity is triggered by insemination to protect the uterine environment against sperm, bacteria and other substances acting as foreign bodies (Katila, 2001;Pitnick et al., 2009;Schuberth et al., 2008). However, the endometrial physiology and its response to semen vary with the composition, amount of sperm, seminal plasma components or added substances such as extenders (Gorgens et al., 2005;Palm et al., 2006). ...
... The reduced sperm-PMN relation observed in fresh and diluted semen samples has been attributed to the presence of seminal plasma that suppresses the opsonisation, complement activation, chemiotaxis and, thus, blocks sperm phagocytosis (Alghamdi et al., 2004;Eisenbach, 2003;Katila, 1996;2001;Troedsson et al., 2001Troedsson et al., , 2005Schuberth et al., 2008). In contrast, seminal plasma promotes PMN binding and phagocytosis of non-viable spermatozoa (Troedsson et al., 2005). ...
Fertility results alter artificial insemination with frozen semen
in donkeys are very poor. Endometrial cytology and biopsies
on oestrus and post-insemination (PI) stages in four female
Catalonian donkeys were taken. Polymorphonuclear cells
(PMN) counting on cytology was done according Reilas
(2001). Biopsies were evaluated taking into account the
Kenney and Doig (1986) classification for mare endometrium.
A large amount of polymorphonuclear (PMN) population on
the PI cytology samples wer found showing an exacerbating
acute inflammatory response to frozen semen. In mares there is
a physiological inflammatory response to mating but it had not
been studied in donkeys. The response in jennies was similar to
that seen in mares that has developed persistent mating-
induced endometritis. Besides, all biopsy samples (oestrus and
PI) were classified as IIA (slight endometritis), highlighting the
presence of eosinophils in the stratum compactum. It differs
from mare where the presence of eosinophils is related to
anaphylaxis or strange body reaction and it seems to be a
characteristic on donkey’s healthy endometrium. Moreover,
occasionally PMN in the luminal epithelium and stratum
compactum in PI biopsies were found confirming the acute
endometritis developed by frozen semen. In conclusion,
donkey uterus is more sensible than mare uterus to frozen
semen. A hardest inflammatory response is induced, making
until now almost impossible the success of fertilization by
artificial insemination.
... Of the PGs present in SF, PGE2 has been identified as one of the predominant type detected [80,81]. PGE2 is a strong chemotactic agent for neutrophils [82]. ...
... Since the ovulatory process has been likened to an inflammatory reaction which includes immune cell infiltration of the tissue of the graafian follicles [21], it has been speculated that SF could mediate leukocyte trafficking from the uterus to the ovary and that migrating leukocytes can serve as vector in augmenting ovulation [82]. In mammals, studies have shown that SF advance the anticipated time of ovulation by significant number of hours [91]. ...
... Several in vitro and ex vivo studies have shown that TNF-α can induce ovulation or trigger events leading to ovulation [93] . These mediators may reach the ovarian stroma and preovulatory follicles via the lymphatic ducts and a counter-current transfer system from the uterine vein to the utero-ovarian artery and bind to receptors expressed on the surface of the ovarian cells [82]. While the effects of SF on ovulation has not been documented in humans, it is plausible that it act in similar manner to enhance the ovulatory process. ...
Inflammation is a multifaceted process that involves a host of resident and recruited immune cell types working together to promote the elimination of insult or injury and initiate tissue repair. In the female reproductive tract (FMRT), inflammation-mediated alteration in epithelial, vascular and immune functions are important components of complex physiological processes as well as many local and systemic pathologies. It is well established that intra-coital and post-coital function of seminal fluid (SF) goes beyond nutritive support for the
spermatozoa cells. There is emerging evidence to support a role for SF, in particular, the inflammatory bioactive lipids, and prostaglandins present in vast quantities in SF in localized immune modulation and regulation of pathways that can exacerbate inflammation in the FMRT. In sexually active women SF-mediated inflammation have been implicated in physiologic conditions such as ovulation, implantation and parturition while also enhancing tumorigenesis, and susceptibility to infection such as HIV. This review highlight the molecular mechanism by which SF regulates inflammatory pathways in the FMRT and how alterations in these pathways contribute to physiology and pathology of the female reproductive function. In addition, based on findings from TaqMan® 96-Well Plate Arrays, on neoplastic cervical epithelial cells treated with SF, we discuss some of our new findings on the role of SF as a potent driver of inflammatory and tumorigenic pathways in the cervix. SF was found to regulate components of eicosanoid signaling, kallikrien-kinin-bradykinin receptor signaling, toll-like
receptor-2 (TLR2) signaling and chemokine/cytokine signaling in neoplastic cervical epithelial cells. Although the detailed molecular mechanisms require additional studies, the available data suggests that SF can regulate a wide range of inflammatory pathways to augment pathologic conditions within the FMRT.
... When the female is in the oestrus stage a massive migration of leukocytes (mainly poly-morphonuclear neutrophils- PMNs) into the sub-epihelial stroma takes place (reviewed by Taylor et al., 2009 ). Contact of the semen constituents with the uterus and cervical tissues induces a series of immunological reactions and mechanisms (Schuberth et al., 2008). After natural mating or AI the PMN influx into the uterine lumen and activated PMNs bind to spermatozoa and phagocytose them. ...
... After natural mating or AI the PMN influx into the uterine lumen and activated PMNs bind to spermatozoa and phagocytose them. Given that in some aspects semen is a foreign material for the female organism , it seems logical to interpret many of the immune responses as actions to eliminate such material (Schuberth et al., 2008 ). Inflammation seems to be a normal process to remove spermatozoa and bacteria, producing an ideal environment for embryo implantation (Troedsson, 1997; Rozeboom et al., 1998). ...
... In species such horse, pig and cattle the onset of PMN chemotaxis by sperm is rapid and the duration of PMN infiltration relatively short. It has been hypothesized that PMN takes part in sperm cell selection, removing superfluous, non-motile or damaged spermatozoa (Tomlinson et al., 1992 ). Whether sperm cell phagocytosis is a selective or random process is still questionable (Schuberth et al., 2008). As already mentioned, this ensures effective removal of sperm and bacteria and the subsequent return of the endome-trium to a normal state, ready to receive the embryo (reviewed by Katila, 2012). ...
Assistant reproduction technologies are in constant evolution, among them the artificial insemi-nation (AI). AI has been successfully used in pigs for decades, especially to improve boar efficiency and productivity. Lately, swine AI has taken on a new lease of life since efficient AI is essential for solving future challenges in the porcine industry and to enhance productivity. The present paper summarizes several factors concerning AI, starting with an overview of some physiological aspects including the female reproductive tract and sperm transport, as well as sperm losses during insemination and uterus sperm selection. Strategies developed to reduce the number of sperm during the AI process, are also reviewed, along with their combination with new reproductive technologies for application in pig production in the near future.
... However, it is widely assumed that SP has a key role not only in the transport and survival of spermatozoa [1] but also in subsequent reproductive events [2][3][4]. For instance, in pigs, SP accelerates ovulation [5][6][7], promotes the synthesis of progesterone by supporting the development of corpora lutea [8], and improves reproductive outcomes [9][10][11][12]. The positive effects of SP on pregnancy outcomes have also been reported in other animal species [3,[13][14][15] and humans [16,17]. ...
... These findings confirmed previous studies indicating that the effect of SP administration at estrus is not only immediate [20,21] but also remains during the preimplantation time [22,24] and modifies the timing of embryo development [22]. Although the advanced embryo development detected in the SP group might be related to the influence that SP has on the time of ovulation [5][6][7], recent studies suggest that the timing of embryo development is controlled and modified at the molecular level by a possible direct effect of SP [22,23]. First, SP infused during estrus modifies the endometrial transcriptome on day 6 of gestation, upregulating the genes and pathways that are closely related to embryo development and maternal immune system tolerance [22]; second, such SP infusions also upregulate the expression of genes and pathways in day 6 blastocysts that are linked to processes that control embryonic development, implantation, or the progression of pregnancy [23]. ...
Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor β1 (TGF-β1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-β1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. On day 6 (day 0—onset of estrus), all “donors” were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant “donors” and cyclic “recipients,” incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-β1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.
... Following mating, spermatozoa from the reproductive tract must be removed without development of humoral or cellular immune responses to sperm cells. Indeed, deposition of sperm into the reproductive tract leads to an inflammatory response characterized by influx of phagocytic cells (neutrophils, macrophages, and dendritic cells) and T lymphocytes as well as increased local production of cytokines such as granulocyte-macrophage colony stimulating factor 2 (CSF2), IL-6 and monocyte chemotactic protein-1 (Schuberth et al. 2008). The most well studied of these cytokines with respect to their pro-developmental actions on the embryo in humans, mice, and pigs is CSF2. ...
... The immunization to these male antigens has potential consequences for fertility and pregnancy outcome (Vaiman et al. 1978;Tripathi et al. 1999). Deposition of semen in the female reproductive tract leads to an inflammatory response to semen, and contributes to the removal of sperm by the innate immune response (Schuberth et al. 2008) and thereby prevents the adaptive immune response against allogeneic antigens of sperm cells. ...
Literally, reproductive immunology was born in bovine on-farm reproduction where seminal experiments intended for developing methods for embryo transfer in cattle were performed. Actually, these experiments led to two of major concepts and fundamental principles of reproductive immunology using the bovine species as a model for biomedical research, namely the concept of acquired immunological tolerance and the paradox of the semiallogeneic bovine foetus whereby such organism can develop within an immunologically competent host. Peter Medawar, a scientist who together with Frank Macfarlande Burnet shared the 1960 Nobel Prize in physiology or medicine for discovery of acquired immunological tolerance, while studying dizygotic cattle twins, thereby giving birth to reproductive immunology. Also, these findings significantly influenced development of organ transplants and showed that using farm animals as models for studying transplantation immunology had general relevance for mammalian biology and health including those of humans. However, the interest for further research of the fascinating maternal immune influences on pregnancy and perinatal outcomes and of the prevention and treatment of immunologically mediated reproductive disorders in viviparous mammals of veterinary relevance by veterinary immunologists and reproductive clinicians have been very scarce regarding the application of nonspecific immunomodulatory agents for prevention and treatment of subfertility and infertility in pigs and cattle, but still broadening knowledge in this area and hold great potential for improving such therapy in the future. The aim of the current overview is to provide up-to-date information and explaining/translating relevant immunology phenomena into veterinary practice for specialists and scientists/clinicians in reproduction of animals.
... After entering the vagina, semen and spermatozoa induce innate and adaptive immune responses in the FRT. The result of this activated immunity is memory regulatory lymphocytes and memory effector lymphocytes formation, which is directed to paternal antigens (7)(8)(9)(10). Therefore, any alteration in seminal and spermatozoa antigenicity can result in disturbed immune response and consequently pregnancy aberration (7)(8)(9)(10). ...
... The result of this activated immunity is memory regulatory lymphocytes and memory effector lymphocytes formation, which is directed to paternal antigens (7)(8)(9)(10). Therefore, any alteration in seminal and spermatozoa antigenicity can result in disturbed immune response and consequently pregnancy aberration (7)(8)(9)(10). As an allogeneic cell, spermatozoon can induce strong immune responses. ...
Background & objective:
Unexplained recurrent spontaneous abortion (URSA) is defined as an unknown cause of occurrence of three or more clinically detectable pregnancy losses before 20 weeks of gestation, but it occurs presumably as a result of the immune system dysfunctions. We supposed that the disruption of semen or spermatozoa might be responsible for the dysfunction of the immune system in women with URSA. Semen and spermatozoa (as antigens) induce female reproductive tract (FRT) immunity. This stimulated immunity is necessary for pregnancy occurrence. The disruption of semen or spermatozoa can be a result of altering a variety of surface molecules on spermatozoa, especially polymorphic human leukocyte antigen (HLA) molecules or antigens. Despite the importance of HLA antigens in reproduction, to the best of our knowledge, no one has studied the relation of HLA expression between spermatozoa and URSA. Therefore, this paper aims to assess this relation.
Methods:
Semen samples were collected from 15 URSA couples and 20 normal couples. After purification of normal spermatozoa, the HLA class I and II expressions were evaluated by flow cytometry methods.
Results:
Results showed that the expression of both HLA class I and II by spermatozoa, in URSA couples, was significantly less than the control couples.
Conclusion:
The decreased expression of polymorphic HLA class Ⅰ and Ⅱ by spermatozoa can be related to URSA occurrence.
... Nevertheless, some mares (and more frequently in jennies), are unable to eliminate inflammation, they consequently develop persistent breeding-induced endometritis, which is incompatible with pregnancy. Some extender components such as glycerol or egg yolk proteins play a major role in this reaction by inducing a greater migration of polymorphonuclear neutrophils (PMNN) in the endometrium [33,41]. ...
... Moreover, the use of frozen semen induces a stronger inflammatory reaction in the uterus than fresh semen in mares [20,38]. The absence of seminal plasma, certain cryoprotectants [29] such as glycerol, and egg yolk proteins favour this reaction by inducing greater PMNN migration to the endometrium [33,41]. ...
Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio®, lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio® and lactose-egg yolk based extender, respectively) –Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing.
... A further study suggested that SP induces an immediate and local cellular response in the uterus with a specific role of the utero-tubal junction [12]. Locally induced mediators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNFα) [13], may reach the pre-ovulatory follicle, bind to receptors expressed on the surface of ovarian cells and modify follicular responses to gonadotropins (LH). Most notably, proinflammatory cytokine TNFα promotes ovulations in the rat ovary [14]. ...
... Spermatozoa modulate mRNA concentrations of cytokines acting as pro-and anti-inflammatory mediators in porcine species at 3 h after insemination [36] and influence patterns of cytokine release in human cervical epithelial cells [41]. Together, the arrays of active seminal constituents, spermatozoa and semen extender initiate several immune mechanisms with immediate, mid-term and long-term effects on ovulation, sperm selection, fertilization and pregnancy outcome [13], which in its complexity is yet only partially understood. ...
Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations (r >0.08, p<0.05) of maturation promoting factors among each other and with cumulus cells on the side of the treated horn. In conclusion, SP orchestrates the gene network regulating the bidirectional communication between oocytes and surrounding somatic cells. The modulation of the immune-cytokine network of the female reproductive system could contribute to the previously reported SP-induced acceleration of ovulation in the porcine species.
... 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. ...
... 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][140][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]. ...
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 pre-implantation 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.
... Seminal plasma is known as both a transport and survival medium for mammalian sperm during and after ejaculation. It has been recognized for its active roles in targeting female reproductive tissues post-coitus and the potential for inducing modulations in the uterine environment (Robertson, 2005;Schuberth et al., 2008). It is thought that an inflammatory response following coitus in the female reproductive tract helps clear microorganisms and sperm cells from the tract (Hansen et al., 1987;Alghamdi et al., 2009). ...
Our objective was to determine if the addition of a concentrated human recombinant transforming growth factor beta-1 (TGF) to bovine semen at the time of AI would result in increased risk of pregnancy in beef and dairy cows. Suckled beef cows (n = 1,132) in 11 herds across 2 states and lactating dairy cows (n = 2,208) in one organic-certified herd were enrolled. Beef cows received fixed-time AI (FTAI) following a 7 d CO-Synch + controlled internal drug release estrous synchronization protocol. Dairy cows were inseminated following observation of natural estrus expression. Cows received either no treatment as a control (CON) or 10 ng of TGF in 10 μL added through the cut-end of a thawed straw of semen immediately prior to AI. At the time of FTAI of beef cows, the mean ± SD age was 5.0 ± 2.4 yr, BCS was 5.3 ± 0.7, and days postpartum was 78.2 ± 15.5 d. The overall pregnancy risk (PR) in beef cows was 55.2% to AI and 90.5% season-long. PR in beef cows was not affected (P = 0.27) by the addition of TGF (53.1% vs. 58.1%). Furthermore, there was no difference (P = 0.88) for season-long PR in beef cows that received TGF (91.2% vs. 91.5%). At the time of insemination of dairy cows, the mean ± SD lactation was 3.0 ± 1.3 lactations, BCS was 2.9 ± 0.3, days in milk was 115.6 ± 56.6 d, and cows had received 2.4 ± 1.5 inseminations/cow. The overall pregnancy risk to AI in dairy cows was 23.1%. PR to AI for dairy cows was not affected (P = 0.32) by addition of TGF (22.0% vs. 23.8%). In conclusion, PR to AI was not affected by addition of TGF to thawed semen immediately prior to AI in beef or dairy cows.
... Indeed, artificial insemination in cattle, typically conducted via the recto-vaginal method, involves navigating a catheter through the vulva, vagina, cervix, and ultimately into the uterus for semen deposition. This process poses a risk of introducing bacteria and tissue debris into the uterus at any point during catheter advancement [22,23]. To mitigate this risk, catheter sheaths originally designed for contamination-free embryo transfer can be repurposed for artificial insemination procedures [24]. ...
... The insemination or introduction of semen derived factors into the uterus triggers a myriad of immune responses which assist the spermatozoa in fertilization and aid implantation [132,133]. Endometrial inflammation is driven by seminal factors, such as prostaglandins, TGFβ and interleukins, which can stimulate the production of endometrial chemokines/cytokines and recruit immune cells that activate inflammatory pathways necessary for decidual tissue remodeling [133][134][135]. Further maternal immune responses are directed by paternal antigens in the seminal plasma, which are essential for immune tolerance of the developing embryo [136,137]. ...
As adults, our health can be influenced by a range of lifestyle and environmental factors, increasing the risk for developing a series of non-communicable diseases such as type 2 diabetes, heart disease and obesity. Over the past few decades, our understanding of how our adult health can be shaped by events occurring before birth has developed into a well-supported concept, the Developmental Origins of Health and Disease (DOHaD). Supported by epidemiological data and experimental studies, specific mechanisms have been defined linking environmental perturbations, disrupted fetal and neonatal development and adult ill-health. Originally, such studies focused on the significance of poor maternal health during pregnancy. However, the role of the father in directing the development and well-being of his offspring has come into recent focus. Whereas these studies identify the individual role of each parent in shaping the long-term health of their offspring, few studies have explored the combined influences of both parents on offspring well-being. Such understanding is necessary as parental influences on offspring development extend beyond the direct genetic contributions from the sperm and oocyte. This article reviews our current understanding of the parental contribution to offspring health, exploring some of the mechanisms linking parental well-being with gamete quality, embryo development and offspring health.
... In a study conducted in mouse, Hourcade et al. [9] illustrated that the spermatozoa in the uterus show a higher level of fragmented DNA compared to spermatozoa retrieved from the epididymis, going against the selective function of the cervical mucus. It has been suggested that the fragmentation of the DNA might be provoked by the immune responses occurring in the cervix and uterus in response to the sperm migration [10] or because of the presence of nucleases in the seminal fluid affecting the spermatozoa in the uterus [11,12]. Hourcade et al. [9] also demonstrated that there is a strong positive selection in the uterotubal junction for spermatozoa carrying low fragmented DNA. ...
... Additionally, the female reproductive tract is able to detect embryo presence prior to implantation and to accordingly modify its immune response [13,14] towards successful implantation [15]. Cytokines are major immune regulators linked to optimal endometrium receptivity and embryo growth [16][17][18]. The SP, spermatozoa and developing zygotes all contribute to modulating cytokine signaling in the female reproductive tract [4,[19][20][21][22]. ...
In the context of porcine embryo transfer (ET) technology, understanding the tightly regulated local uterine immune environment is crucial to achieve an adequate interaction between the transferred embryos and the receiving endometrium. However, information is limited on the uterine immune status of cyclic-recipient sows when receiving embryos during ET. The present study postulated that the anti- and proinflammatory cytokine profile 6 days after the onset of estrus differs between endometria from uninseminated cyclic sows and blastocyst-bearing sows. On Day 6 of the cycle, endometrial explants were collected from sows inseminated or not inseminated during the postweaning estrus and cultured for 22 h. The culture medium was then analyzed for the contents of a total of 16 cytokines using Luminex MAP® technology. The results showed important differences in the endometrial production of most cytokines between the sow categories, with a predominant anti-inflammatory environment displayed by the blastocyst-bearing endometria. These findings suggest that sperm, seminal plasma (SP) and/or early embryos modify the uterine environment by inducing an immune-tolerant cytokine profile already visible at Day 6. Whether the SP or some of its active components may help to develop strategies to maximize the reproductive performance of recipients after ET needs further investigation.
... This finding evidenced that the incidence of sows with small ovarian follicles at treatment time would be a major cause impairing the effectiveness of the GnRH agonist to properly synchronize ovulation in weaned sows. The poor responsiveness by sows with small follicles to the GnRH agonist treatment could be caused by the lack of LH receptors in small follicles, since granulosa cells acquire LH receptors when the follicles reach 0.5-0.6 cm in diameter [33]. The finding that, when treated with GnRH agonist, sows with small follicles showed a higher incidence of anestrus without ovulation than corresponding controls was also interesting. ...
The GnRH agonist buserelin (GnRH), used to synchronize ovulation in weaned sows, attains only 70–80% effectivity, owing to several reasons of ovarian origin. This study evaluated in particular whether mean ovarian follicle size at treatment and the season of weaning are among those influencing GnRH responsiveness. The experiment was carried out in a temperate-region farm with 352 sows of 1–6 parities weaned either in winter–spring (WS, 174 sows) or in summer–autumn (SA, 178 sows). The sows were randomized into two groups: GnRH (10 µg of buserelin acetate at 86 h after weaning, 172 sows) and control (180 sows). The ovaries were transrectally scanned from weaning to ovulation and the sows clustered according to their mean follicular size at treatment time: small (<0.5 cm in diameter), medium (0.5 to 0.64 cm) and large (0.65 to 1.09 cm). In total, 88.33% of the GnRH-treated sows ovulated, with 82% of them within the expected time window (120–132 h after weaning). In contrast, 95.45% of the unresponsive sows had small follicles at the time of treatment and were mostly weaned in SA (20.45%) than in WS (4.76%). In conclusion, the conspicuous presence of sows having small ovarian follicles at treatment time compromises the efficiency of the GnRH agonist buserelin to synchronize ovulation in weaned sows, which occurs more frequently in summer–autumn weaning.
... Consequently, for a number of years, seminal plasma was regarded primarily as a transport vehicle for sperm (Garner and Hafez, 1993). However, more recently, studies have demonstrated that the rich mixture of compounds in seminal plasma have profound effects on boar sperm; the sow's reproductive tract; and interactions between the two (Waberski, 1997;Robertson, 2007;Schuberth et al., 2008;Rodriquez-Martinez et al., 2009). As a result, seminal plasma, especially its protein component, is now believed to play an active role in fertilization and the successful establishment of pregnancy. ...
... Nevertheless, as commercial sex workers, HESN women are more exposed to semen from multiple partners. Semen can induce a local mucosal inflammatory response [68], thus could also potentially mediate NK cell activation. ...
Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4⁺ T cells, we observed that NKp30⁺ and Siglec-7⁺ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.
... To determine the role of glycans in immune evasion we incubated the HF and LF bull spermatozoa with the PMNs isolated from female buffaloes presumably simulating in vivo conditions. The insemination or intromission of any liquid in the uterus has been found to trigger multiple immune mechanisms that assist in sperm selection and fertilization of the egg by the spermatozoa (98). It also initiates en-masse recruitment of the leukocytes in the uterine lumen through the sub-epithelial stroma, known as the leukocyte reaction. ...
The glycans on the plasma membrane of cells manifest as the glycocalyx, which serves as an information-rich frontier that is directly in contact with its immediate milieu. The glycoconjugates (GCs) that adorn most of the mammalian cells are also abundant in gametes, especially the spermatozoa where they perform unique reproduction-specific functions e.g., inter-cellular recognition and communication. This study aimed to implicate the sperm glycosylation pattern as one of the factors responsible for low conception rates observed in buffalo bulls. We hypothesized that a differential abundance of glycans exists on the spermatozoa from bulls of contrasting fertilizing abilities endowing them with differential immune evasion abilities. Therefore, we investigated the role of glycan abundance in the phagocytosis and NETosis rates exhibited by female neutrophils (PMNs) upon exposure to such spermatozoa. Our results indicated that the spermatozoa from high fertile (HF) bulls possessed a higher abundance of O-linked glycans e.g., galactosyl (β-1,3)N-acetylgalactosamine and N-linked glycans like [GlcNAc]1-3, N-acetylglucosamine than the low fertile (LF) bull spermatozoa. This differential glycomic endowment appeared to affect the spermiophagy and NETosis rates exhibited by the female neutrophil cells (PMNs). The mean percentage of phagocytizing PMNs was significantly different (P < 0.0001) for HF and LF bulls, 28.44 and 59.59%, respectively. Furthermore, any introduced perturbations in the inherent sperm glycan arrangements promoted phagocytosis by PMNs. For example, after in vitro capacitation the mean phagocytosis rate (MPR) rate in spermatozoa from HF bulls significantly increased to 66.49% (P < 0.01). Likewise, the MPR increased to 70.63% (p < 0.01) after O-glycosidase & α2-3,6,8,9 Neuraminidase A treatment of spermatozoa from HF bulls. Moreover, the percentage of PMNs forming neutrophil extracellular traps (NETs) was significantly higher, 41.47% when exposed to spermatozoa from LF bulls vis-à-vis the spermatozoa from HF bulls, 15.46% (P < 0.0001). This is a pioneer report specifically demonstrating the role of O-linked glycans in the immune responses mounted against spermatozoa. Nevertheless, further studies are warranted to provide the measures to diagnose the sub-fertile phenotype thus preventing the losses incurred by incorrect selection of morphologically normal sperm in the AI/IVF reproduction techniques.
... These changes in embryonic development might be associated with variations in ovulation time in response to SP treatment. The SP alters the endocrine-immune-cytokine system in preovulatory follicles (Einer-Jensen and Hunter, 2005;O'Leary et al., 2006) by decreasing the LH peak ovulation interval and, therefore, hastening the time of ovulation (Schuberth et al., 2008). Accordingly, embryos collected from sows treated with SP should have reached a more advanced developmental stage than those collected from non-treated sows. ...
Seminal plasma (SP) supports not only sperm function but also the ability of spermatozoa to withstand biotechnological procedures as artificial insemination, freezing or sex sorting. Moreover, evidence has been provided that SP contains identifiable molecules which can act as fertility biomarkers, and even improve the output of assisted reproductive technologies by acting as modulators of endometrial and embryonic changes of gene expression, thus affecting embryo development and fertility beyond the sperm horizon. In this overview, we discuss current knowledge of the composition of SP, mainly proteins and cytokines, and their influence on semen basic procedures, such as liquid storage or cryopreservation. The role of SP as modulator of endometrial and embryonic molecular changes that lead to successful pregnancy will also be discussed.
... Inactivation and/or removal of these factors may affect in vivo capacitation (Calvete et al. 1997); they are also necessary for events leading to successful fertilization such as sperm-zona pellucida interactions and sperm-oocyte fusion, as reviewed in Rodriguez- Martinez et al. (2009). Additionally, SP has been shown to modulate the immune response in the uteri of pigs, humans and other mammals (Kelly and Critchley 1997;O'Leary et al. 2004;Rodriguez-Martinez et al. 2010;Rozeboom et al. 1999;Schuberth et al. 2008;Veselsky et al. 2000) by modifying gene expression affecting local processes of immune defense at the oviductal sperm reservoir (Alvarez-Rodriguez et al. 2019;Sharkey et al. 2012); also reviewed in Schjenken and Robertson (2014). Properties of seminal plasma such as the ability to maintain uncapacitated sperm state and to immune-suppress the female reproductive tract are widely exploited in pig semen handling/ processing, storage/extension/preservation and AI (Rodriguez- Martinez et al. 2009;Rodríguez-Martínez and Peña Vega 2013). ...
Mammalian fertilization remains a poorly understood event with the vast majority of studies done in the mouse model. The purpose of this review is to revise the current knowledge about semen deposition, sperm transport, sperm capacitation, gamete interactions and early embryonic development with a focus on the porcine model as a relevant, alternative model organism to humans. The review provides a thorough overview of post-ejaculation events inside the sow’s reproductive tract including comparisons with humans and implications for human fertilization and assisted reproductive therapy (ART). Porcine methodology for sperm handling, preservation, in vitro capacitation, oocyte in vitro maturation, in vitro fertilization and intra-cytoplasmic sperm injection that are routinely used in pig research laboratories can be successfully translated into ART to treat human infertility. Last, but not least, new knowledge about mitochondrial inheritance in the pig can provide an insight into human mitochondrial diseases and new knowledge on polyspermy defense mechanisms could contribute to the development of new male contraceptives.
... The observed differences in embryo development could be directly related to changes in the time of ovulation in response to SP exposure. Previous studies have reported that SP modifies the endocrine-immune-cytokine network in preovulatory follicles (25,26) leading to an acceleration of ovulation through a decrease in the interval LH peak-ovulation time-point (27). Consequently, embryos derived from heterologous SP-treated sows should be in a more advanced stage of development. ...
Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract.
Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos.
Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation.
Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others.
Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.
... Following mating, spermatozoa from the reproductive tract must be removed without development of humoral or cellular immune responses to sperm cells. Indeed, deposition of sperm into the reproductive tract leads to an inflammatory response characterized by influx of phagocytic cells (neutrophils, macrophages, and dendritic cells) and T lymphocytes as well as increased local production of cytokines such as granulocyte-macrophage colony stimulating factor 2 (CSF2), IL-6 and monocyte chemotactic protein-1 (Schuberth et al. 2008). The most well studied of these cytokines with respect to their pro-developmental actions on the embryo in humans, mice, and pigs is CSF2. ...
Background
The specific role of non-invasive functional testing in a risk stratification of patients with incomplete revascularization after primary percutaneous coronary intervention (pPCI) still needs to be evaluated. The aim of our study was to assess negative prognostic value of stress echocardiography (SECHO) after successful pPCI and incomplete revascularization of non-culprit lesions.
Methods
Our study consisted of 202 patients (mean age 59±10 years, male 142) successfully treated with pPCI, who performed SECHO according to Bruce protocol in order to assess residual ischemia in coronary artery with non-culprit lesion. Duke treadmill score, functional capacity (Metabolic Equivalents - METs), achieved target heart rate (THR), heart rate recovery (HRR), wall motion score index (WMSI) and ejection fraction were interrogated in all patients. Slow HRR was defined as ≤18 beats/min. Median follow-up of the patients was 70 months (IQR 55–83 months) for the occurrence of cardiovascular death and non-fatal myocardial infarction. We also assessed the independent predictors for the occurrence of the adverse events.
Results
Out of 202 patients, 42 (20.8%) had positive SECHO test, 4 patients (1.98%) had died due to non-cardiac causes and 7 patients (3.5%) were lost to follow-up. From the remaining 149 patients with negative SECHO, 13 (8.7%) had an adverse event (7 cardiovascular deaths and 6 non-fatal MI). Negative predictive value of SECHO test was 91.3%. Univariate predictors of adverse events were slow HRR (HR 4.343 [95% CI 1.473–14.011], p=0.008), and not achieved THR (HR 0.322 [95% CI 0.105–0.985], p=0.047). By multivariate analysis, only slow HRR remained independent predictor of adverse events (HR 3.324 [95% CI 1.013–10.906], p=0.048).
Conclusion
SECHO test has excellent negative prognostic value in patients with incomplete revascularization of non-culprit lesions after successful pPCI. Still, particular care should be taken to the patients with slow HRR and negative SECHO due to increased risk for the occurrence of adverse events.
Acknowledgement/Funding
Ministry of Education and Science of the Republic of Serbia (Grant No III41022)
... Semen contains a variety of immune activating factors which have a major role in the induction of an immune response in the FRT. One of these is HLA molecules which are present in soluble form in seminal plasma and also in membrane form on the surface of cells (such as epithelia and leukocytes) existing in semen (Bromfield, 2014;Katila, 2012;Robertson & Sharkey, 2016;Schjenken & Robertson, 2015;Schuberth et al., 2008). ...
Background:
After coitus and insemination, an inflammatory response is evident in the female reproductive tract (FRT). Semen contains a variety of immune-activating components that have a major role in the induction of an immune response in the FRT. One of the most important is (human leukocyte antigen) HLA molecules which are present in soluble form in seminal plasma and in membrane form on the surface of cells (such as epithelial and leukocytes) existing in semen. Nevertheless, there is considerable debate over the expression of HLA antigens by human spermatozoa. Considering the critical role of HLA molecules in reproduction and the induction of an immune response, it is very important to clearly define HLA expression by spermatozoa and the role of these molecules in sperm morphology, motility, and strength to fertilize an egg. Therefore, the objective of this study was to determine HLA expression by ejaculated spermatozoa. The results of this study will facilitate the design of future studies.
Method:
Semen samples were collected from 50 healthy men with normal semen status by masturbation after 2-3 days of sexual abstinence. After purification of normal spermatozoa, HLA class I & II expression was evaluated by quantitative real-time PCR and flow cytometry methods.
Results:
The results showed the expression of both HLA class I & class II by spermatozoa. The results also showed that the expression of HLA class Ⅱ was significantly more than HLA class Ⅰ.
Conclusion:
Spermatozoa express both HLA class I & class II molecules.
... An inflammatory response is induced in the reproductive tract by deposition of semen during natural mating (see reviews by Robertson, 2005;Schuberth et al., 2008;Katila, 2012;Bromfield, 2016). The inflammatory response to mating is less well described in cattle than in some other species but includes accumulation of neutrophils (Howe and Black, 1963;Mattner, 1968) and, based on responses to seminal plasma, changes in gene expression in the endometrium (Ibrahim et al., 2019). ...
... An inflammatory response is induced in the reproductive tract by deposition of semen during natural mating (see reviews by Robertson, 2005;Schuberth et al., 2008;Katila, 2012;Bromfield, 2016). The inflammatory response to mating is less well described in cattle than in some other species but includes accumulation of neutrophils (Howe and Black, 1963;Mattner, 1968) and, based on responses to seminal plasma, changes in gene expression in the endometrium (Ibrahim et al., 2019). ...
An inflammatory response is induced in the reproductive tract by deposition of semen during natural mating. This response might facilitate establishment and maintenance of pregnancy and alter the phenotype of the offspring by modifying the microenvironment of the reproductive tract. Here, we hypothesized that intrauterine infusion of 0.5 mL of seminal plasma at the time of artificial insemination (AI) in first-service lactating Holstein cows will improve pregnancy success after insemination. Cows were inseminated (511 primiparous cows inseminated with X-sorted semen, 554 multiparous cows inseminated with X-sorted semen, and 627 multiparous cows inseminated with conventional semen) using the Double-Ovsynch protocol. Cows were randomly assigned to receive intrauterine infusion of either 0.5 mL of seminal plasma or saline immediately after AI. There was no overall effect of seminal plasma infusion on the percentage of inseminated cows diagnosed pregnant at d 32 or 60 after AI, pregnancy loss, or percent of inseminated cows calving. If cows were inseminated with conventional semen, seminal plasma reduced pregnancies at d 32 and tended to reduce calvings. There was no effect of seminal plasma if cows were inseminated with X-sorted semen. Seminal plasma infusion increased the birth weight of heifer calves born using X-sorted semen but not conventional semen. These results do not support a beneficial effect of seminal plasma on pregnancy success after AI, but exposure to seminal plasma may program fetal development to affect phenotype at birth.
... As a result, epithelial cells turn out to be unprotected from pathogens that also target the cervical regions (Ghosh, 2014;Anderson et al., 2014;Wira et al., 2015) producing an E2 and E2 receptor (ESR1) mediated window of vulnerability to infection during ovulation (Salinas-Munoz et al., 2018). In contrast, studies performed during insemination demostrate a sperm dependent wave of neutrophils after insemination (Matthijs et al., 2003;Schuberth et al., 2008;Sharkey et al., 2007, Sharkey et al., 2012. This apparently https://doi.org/10.1016/j.jri.2019.02.002 contradictory finding highlights a gap in our knowledge about how cervical mucosa responds to sperm and the neutrophils driving forces during insemination. ...
Female reproductive mucosa must allow allogenic sperm survival whereas at the same time, avoid pathogen infection. To preserve sperm from neutrophil attack, neutrophils disappear from the vagina during the ovulatory phase (high estradiol); although the mechanisms that regulate neutrophil influx to the vagina during insemination remain controversial.
We investigated the sex hormone regulation of the neutrophil migration through the cervix during insemination and revealed that ovulatory estradiol dose fades the CXCL1 epithelial expression in the ectocervix and fornix; hence, retarding neutrophil migration and retaining them in the epithelium. These mechanisms spare sperm from neutrophil attack to preserve reproduction, but might compromise immunity. However, luteal progesterone dose promotes the CXCL1 gradient expression to restore neutrophil migration, to eliminate sperm and prevent sperm associated pathogen dissemination. Surprisingly, these mechanisms are hormone dependent and independent of the insemination. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil transepithelial migration in the fornix and ectocervix.
... The immune system of the female genital tract epithelium has evolved by making contact with sperm, with male's ejaculate substances and with potentially infectious agents [12,62,[100][101][102][103]. For that, differences in specificity or magnitude of local and systemic immune responses are expected. ...
Immune defense is a key feature in the life history of organisms, expensive to maintain, highly regulated by individuals and exposed to physiological and evolutionary trade-offs. In chelicerates, relatively scarce are the studies that relate postcopulatory mechanisms and immune response parameters. This work makes an approximation to the female’s immunological consequences produced after the placement of a foreign body in the genitalia of three scorpions species, two species that normally receive genital plugs during mating (Urophonius brachycentrus and U. achalensis) and one that does not (Zabius fuscus). Here we performed the first morphological description of the natural plugs of the two Urophonius species. We described complex three zoned structure anchored to the female genital atrium and based on this information we placed implants in the genitalia (for eliciting the local immune response) of virgin females of the three species and measured the immune encapsulation response to this foreign body. We found a greater and heterogeneous response in different zones of the implants in the plug producing species. To corroborate the specificity of this immune response, we compared the local genital reaction with the triggered response at a systemic level by inserting implants into the female body cavity of U. brachycentrus and Zabius fuscus. We found that the systemic response did not differ between species and that only in the plug producing species the local response in the genitalia was higher than the systemic one. We also compared the total hemocyte load before and after the genital implantation to see if this parameter was compromised by the immunological challenge. We confirmed that in Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. Besides, we find correlations between the body weight and the immunological parameters, as well as between different immunological parameters with each other. Complementarily, we characterized the hemocytes of the three scorpion species for the first time. This comparative study can help to provide a wider framework of the immunological characteristics of the species, their differences and their relationship with the particular postcopulatory mechanism such as the genital plugs.
... The immunological responses to sperm and seminal plasma in the female tract are of considerable interest as these processes influence sperm capacitation, transport, selection, fertilisation as well as early embryo development (Schuberth et al., 2008). The local immune responses of the epithelial lining, regulated by its secretions, constitute the main part of the mucosal innate immunity inside the uterus and oviduct which is largely mediated by cytokines, chemokines, and prostaglandins (Bulek et al., 2010). ...
The ability to predict the fertility of bulls before semen is released into the field has been a long-term objective of the animal breeding industry. However, the recent shift in the dairy industry towards the intensive use of young genomically selected bulls has increased its urgency. Such bulls, which are often in the highest demand, are frequently only used intensively for one season and consequently there is limited time to track their field fertility. A more pressing issue is that they produce fewer sperm per ejaculate than mature bulls and therefore there is a need to reduce the sperm number per straw to the minimum required without a concomitant reduction in fertility. However, as individual bulls vary in the minimum number of sperm required to achieve their maximum fertility, this cannot be currently achieved without extensive field-testing. Although an in vitro semen quality test, or combination of tests, which can accurately and consistently determine a bull's fertility and the optimum sperm number required represent the 'holy grail' in terms of semen assessment, this has not been achieved to date. Understanding the underlying causes of variation in bull fertility is a key prerequisite to achieving this goal. In this review, we consider the reliability of sire conception rate estimates and then consider where along the pregnancy establishment axis the variation in reproductive loss between bulls occurs. We discuss the aetiology of these deficiencies in sperm function and propose avenues for future investigation.
... In a study conducted in mouse, Hourcade et al., [9] illustrated that the spermatozoa in the uterus show a higher level of fragmented DNA compared to spermatozoa retrieved from the epididymis, going against the selective function of the cervical mucus. It has been suggested that the fragmentation of the DNA might be provoked by the immune responses occurring in the cervix and uterus in response to the sperm migration [10] or because of the presence of nucleases in the seminal fluid affecting the spermatozoa in the uterus [11,12]. Hourcade et al., [9] also demonstrated that there is a strong positive selection in the uterotubal junction for spermatozoa carrying low fragmented 70 DNA. ...
... Immunologic studies of sperm transport have also shown that once a cohort of spermatozoa reaches the uterus, they induce the "postmating inflammatory response." This involves an influx of neutrophils into the uterine lumen [86,87] which, in the pig at least, selectively reject or permit the further progress of the spermatozoa. The major histocompatibility system (MHC) has been widely researched for its relevance to reproduction, and it is likely that mechanisms exist to identify and select the best match between maternal and paternal MHC characteristics [88,89]. ...
... Of the 1.5 to 3 billion inseminated sperm, approximately 0.1 to 0.3 million sperm make up the sperm reservoir at the utero-tubal junction, and some 2000 sperm will enter the oviduct; similar quantities of sperm were measured within the sperm reservoir and the oviduct when low dose intra-uterine insemination is performed (Sumransap et al, 2007;Tummaruk et al, 2007). These consistent numbers suggest that sperm residing at the utero-tubal junction and the oviduct have actively escaped cervix and uterine interactions, as opposed to being passively transported, and that sperm interact with or adsorb secreted factors from the cervix or the uterus and likewise with the mucous/epithelial cells of the uterus and/or with infiltrating leukocytes entering the uterine fluid (Schuberth et al, 2008;Taylor et al, 2008, 2009a, Taylor et al, 2009b, Taylor et al, 2008c). The various interactions that occur between sperm and the environment of the female reproductive tract prepare the sperm surface for fertilization and/or to reduce the quantity of sperm that reach the oviduct (reviewed byRath et al, 2016). ...
The sperm cell has a unique, polarized and segregated surface that is modified extensively by the changing environments in both the male and the female reproductive tracts. The sperm cannot refresh its surface, as protein translation and membrane recycling by intracellular vesicular transport have ceased upon its maturation. So, how is the sperm surface modified in the reproductive tracts and how do these processes affect fertilization? This review traces these modifications as boar sperm travels from their liberation from the Sertoli cell into the lumen of seminiferous tubules of the testis to the site of fertilization in the ampulla of the oviduct in the sow, via an artificial insemination route. The effect of sperm dilution for artificial insemination as well as more extensive sperm processing for in vitro fertilization, cryopreservation, or sex sorting, are also discussed with respect to how these procedures affect sperm surface organization and fertilization capacity. This article is protected by copyright. All rights reserved.
... 1796, Outubro, 2014. lúmen uterino em resposta ao antígeno (SCHUBERTH et al., 2008SILVA, F.M., BLUME, H. e OLIVEIRA, R.A. Endometrite persistente pós-cobertura. PUBVET, Londrina, V. 8, N. 20, Ed. 269, Art. ...
... Following mating, spermatozoa from the reproductive tract must be removed without development of humoral or cellular immune responses to sperm cells. Indeed, deposition of sperm into the reproductive tract leads to an inflammatory response characterized by influx of phagocytic cells (neutrophils, macrophages, and dendritic cells) and T lymphocytes as well as increased local production of cytokines such as granulocyte-macrophage colony stimulating factor 2 (CSF2), IL-6 and monocyte chemotactic protein-1 (Schuberth et al. 2008). The most well studied of these cytokines with respect to their pro-developmental actions on the embryo in humans, mice, and pigs is CSF2. ...
The advantages of using minicomputers for research into the physical and mechanical properties of man-made fibres are considered. Their use in data recording systems is particularly mentioned. Details are given of other applications such as the computation of statistics and the evaluation of rheological and viscoelastic properties. It is stated that low-cost minicomputers in conjunction with peripheral units provide great advantages when processing experimental results.
... This exemplifies the principle that females can target paternal antigens and use the immune system to facilitate selection (Dorus et al., 2012). Immunological studies of sperm transport have also shown that once a cohort of spermatozoa reaches the uterus, they induce the 'post-mating inflammatory response' which Sperm selection in vivo prompts an influx of neutrophils into the uterine lumen (Schuberth et al., 2008). This provides another level of sperm selection mediated by direct cell-cell interactions. ...
The capacity for sperm storage within the female reproductive tract occurs widely across all groups of vertebrate species and is exceptionally well developed in some reptiles (maximum duration seven years) and fishes (maximum duration >1 year). Although there are many reports on both the occurrence of female sperm storage in diverse species and its adaptive benefits, few studies have been directed toward explaining the mechanisms involved. In this article we review recent findings in birds and mammals in an effort to develop hypotheses that could be translated into research applications in animal breeding technologies. There are pockets of evidence to suggest that the local epithelial cells, sometimes arranged as sperm storage tubules, can respond to spermatozoa by producing heat shock proteins as well as providing an environment rich in antioxidants. Moreover, the local immune system seems to tolerate the arrival of spermatozoa, while retaining the ability to combat the arrival of infectious microorganisms Expected final online publication date for the Annual Review of Animal Biosciences Volume 4 is February 15, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Immunologic studies of sperm transport have also shown that once a cohort of spermatozoa reaches the uterus, they induce the "postmating inflammatory response." This involves an influx of neutrophils into the uterine lumen [86,87] which, in the pig at least, selectively reject or permit the further progress of the spermatozoa. The major histocompatibility system (MHC) has been widely researched for its relevance to reproduction, and it is likely that mechanisms exist to identify and select the best match between maternal and paternal MHC characteristics [88,89]. ...
Research over the past 3 decades has caused a major shift in the way that the oviduct, or fallopian tube, is perceived. Previously, it was regarded as little more than the anatomic site for fertilization, where spermatozoa and oocytes meet as they travel in opposite directions. However, this view has been radically altered by the realization that both spermatozoa and oocytes elicit changes in the biochemical composition of oviductal fluid through the induction of novel gene expression. Moreover, it has also been shown that only a privileged sperm population, selected on the basis of multiple criteria, is permitted to enter the oviduct, where they are subjected to even more selection processes that control their motility and capacitation status, thus either inhibiting or facilitating their progress toward the oocyte. Even more recently, it has become apparent that the oviduct has some ability to differentiate the genetic signatures of X- and Y-bearing spermatozoa. Although how exactly this is achieved is unknown, it prompts us to speculate that the oviduct may also be capable of distinguishing other genetically encoded properties of individual spermatozoa and that there must ultimately be a huge payoff in terms of selective animal breeding.
Copyright © 2015 Elsevier Inc. All rights reserved.
Background
Macrophages can phagocytose sperm, especially damaged spermatozoa, in the female genital tract. The semenogelin I-derived peptide SgI-52 in seminal plasma exhibits seminal plasma motility inhibitor (SPMI) activity and can inhibit sperm motility. This raises the question of the role played by SPMIs in macrophage-mediated phagocytosis of sperm. We speculated that SgI-52 promotes sperm clearance by macrophages. Therefore, we investigated the phagocytosis of sperm in different states using this peptide.
Methods
SgI-52 was fluorescently labeled, and its binding site for sperm was observed. The ability of macrophages to phagocytose sperm was observed using fluorescence confocal microscopy. Spermatozoa from different sources were co-cultured with SgI-52 in BWW medium for 4 and 22 h to compare the differences in their phagocytosis by macrophages. Sperm motility, induced acrosome reaction, mitochondrial membrane potential, and ATP content were examined after incubation with SgI-52.
Results
SgI-52 could bind to spermatozoa in different states, mainly to the tail, and then spread to the acrosome. This effect was more pronounced in demembranated spermatozoa. SgI-52 promoted phagocytosis of spermatozoa by macrophages, decreased the mitochondrial membrane potential, and increased the average ATP content of spermatozoa (P < 0.05).
Conclusions
We found for the first time that SgI-52 can bind to spermatozoa in different states and promote their phagocytosis by macrophages. Therefore, we speculate that SgI-52 is involved in the screening of sperm in the female reproductive tract and has potential value in improving assisted reproductive technology.
Artificial insemination (AI) with incorrectly selected spermatozoa accounts for a significant loss to the farmers and the dairy industry. Therefore, accurate prediction of male fertility is of major economic value, especially in a farm set-up. To maximize the efficiency of modern livestock systems, novel ways to ascertain the fertilizing ability of the spermatozoa are required. Numerous factors are known to govern the male fertility; however, the sugars (glycans) on the sperm surface are emerging as a novel and key factor regulating various aspects of the sperm fertilizing ability. A highly specialized epithelial cell, the mammalian spermatozoon is embellished with a rich array of functionally diverse and structurally complex glycans that manifest as a thick glycocalyx. These glycans which are often conjugated either to lipids or proteins exist as glycoconjugates (GCs) apart from their independent existence on the sperm surface. The glycans in the glycocalyx of mammalian spermatozoa are multifunctional molecules which perform reproduction-specific tasks such as cervical mucus penetration, communication with various cells in the female reproductive tract (FRT), and recognition of the oocyte, etc. The testicular spermatozoa only have a rudimentary glycocalyx which, however, is substantially transformed in thickness, composition, and structure during the epididymal sperm-surface remodeling (SSR) events. The sugars in the sperm glycocalyx have been proposed to exist in a specific three-dimensional conformation termed as Sperm Associated Glyco-Topography (SpAGT). This topography of glycans is recognized and presumably interpreted by the patterns recognition receptors (PRRs) on the female immune cells, e.g. neutrophils. The glycans in the sperm glycocalyx are known to interact with various inhibitory receptors on these cells which facilitate sperm survival in the FRT. Nevertheless, to decode the information about survival in the sperm glycome, the identity, valence, linkage, attachment sites, and structures of the individual sugar units in the sperm glycocalyx need to be determined.KeywordsBuffaloArtificial inseminationSpermatozoaGlycocalyxFertility
Background
A complex of effector and predominant regulatory immune responses are induced in the female reproductive tract (FRT) due to insemination that is necessary to achieve pregnancy. The expression of immune regulatory molecules by spermatozoa indicates the significance of the interaction between spermatozoa and immune cells recruited to the FRT in the preparation of appropriate immunity for pregnancy occurrence. One of the immune regulatory molecules is CD5 whose expression by spermatozoa has not yet been investigated. Therefore, the aim of this study is to investigate the expression of CD5 on the surface of human spermatozoa. Semen samples were collected from 30 healthy men with normal semen status. CD5 expression on purified spermatozoa was evaluated by flow cytometry methods.
Results
The results showed the mean ± SD percentage of CD5 positive spermatozoa was 49.41 ± 8.73.
Conclusion
CD5 is expressed on spermatozoa.
This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodine (PI) stain, acrosome integrity by fluorescein isothiocyanate and peanut agglutinine (FITC-PNA) and PI stain, plasma membrane functionality by hypo-osmotic swelling (HOS) test, and DNA denaturation by acridine orange (AO) test. There were no significant differences in the percentages of total motility, acrosome integrity, and DNA fragmentation among the extenders after thawing. Kinematics parameters showed significantly higher values in BotuCrio than in lactose extenders (P < .05). BotuCrio and lactose colostrum extender yielded significantly better rates for HOS-test, linearity, straightness, and wobble than egg-yolk extender (P < .05). However, in relation to sperm viability, lactose egg yolk extender showed significantly better results in comparison to the others seminal experimental media (P < .05). In conclusion, the incorporation of mare colostrum into cryopreservation media protected the sperm against cold-shock; therefore, it may be a good cryoprotectant agent alternative in extenders for freezing stallion semen.
The uterine microenvironment during the first 7 days after ovulation accommodates and facilitates sperm transit to the oviduct and constitutes the sole source of nutrients required for development of preimplantation embryos. Knowledge of the composition of uterine fluid is largely incomplete. Using untargeted mass spectrometry, we characterized the uterine metabolome during the first 7 days of the estrous cycle. Bovine uteri were collected on day 0 (N=4), 3 (N=4), 5 (N=3) and 7 (N=4) relative to ovulation and flushed with Dulbecco’s phosphate‐buffered saline. A total of 1,993 molecular features were detected of which 184 peaks with putative identification represent 147 unique metabolites, including amino acids, benzoic acids, lipid molecules, carbohydrates, purines, pyrimidines, vitamins, and other intermediate and secondary metabolites. Results revealed changes in the uterine metabolome as the cow transitions from ovulation to day 7 of the estrous cycle. The majority of metabolites reached maximum intensity on either day 5 or day 7 relative to ovulation. Moreover, several metabolites found in uterine fluid have signaling capabilities and some have been shown to affect preimplantation embryonic development. In conclusion, the metabolome of the bovine uterus changes during early stages of the estrous cycle and is likely to participate in the regulation of preimplantation embryonic development. Data reported here will serve as basis for future studies aiming to evaluate maternal regulation of preimplantation embryonic development and optimal conditions for culture of embryos.
This article is protected by copyright. All rights reserved.
Donkey frozen semen shows good spermatozoa (spz) survival results, however fertility results are very bad in jennies. Donkey frozen semen is able to penetrate oocytes in vitro and to fertilize mares. Jennies express a high endometrial inflammatory response 6h after AI with frozen semen. High amount of polymorphonuclear neutrophils (PMNs) in cytological smears and biopsies, and intense cyclooxygenase-2 (COX-2) immunohistochemical labelling were detected. In vivo and in vitro experiences showed that seminal plasma (SP), eliminated during semen freezing process, has an important role in modulating this response. SP reduces the PMN-spz binding in vitro. Moreover, SP decreases COX-2 expression and eosinophils and neutrophils migration in vivo. In vitro, PMN-spz incubation showed that SP is able to activate PMNs and preserves sperm survival and motility. After 3-4h of incubation, the main part of spz attached to PMNs were alive, and several attached spz set free had good motility parameters when assessed by CASA system. Fractioning SP attending proteins molecular weight (MW), we observed that low MW fractions (3-10 kDa) induced a rapid spz death, however fractions between 30-100 kDa showed an interesting maintenance of survival and motility as well as a control of reactive oxygen species (ROS) production. Moreover, donkey SP has melatonin and donkey spz have melatonin receptors. Spz produce ROS but PMNs produce much more. Therefore, in understanding the role of SP fractions on sperm survival and the antioxidant effect of SP, melatonin and other antioxidant SP compounds are very important to increase donkey frozen semen AI results.
After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization.
However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved
in controlling sperm transport and fertilization include the female reproductive tract environment, cell‑cell interactions, gene
expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA
status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport
within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the
progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology
influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do
not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as
seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and
shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte.
Asian Journal of Andrology (2016) 18, 1–7; doi: 10.4103/1008-682X.186880; published online: 13 September 2016
Introduction: This project aims to investigate the nature of cryo-induced DNA damage in the spermatozoa of the model species Xenopus tropicalis. We have turned to Xenopus as a tool to achieve this because the genome is available and we aim to identify specific genomic regions that are most affected during sperm cryopreservation.
Materials and methods: The sperm chromatin dispersion (SCD) test was used to show DNA damage and the potent DNA repair inhibitor 3-aminobenzamide (3-AB) was used to reveal the effects of sperm DNA damage in whole embryos produced by IVF. Morphological assessment and in situ hybridization was used to analyse the phenotypes. Biochemical techniques were used to validate genes, particularly gastrula markers which were identified as potential hotspots for DNA damage.
Results and discussion: Single and double stranded DNA breakage was induced by sperm cryopreservation. In fresh sperm samples 6.06% showed fragmentation compared to 17.65% in cryopreserved sperm samples. Furthermore, the dynamics of DNA damage revealed the chromatin is less stable following the freezethaw process. This is demonstrated in vivo by treating embryos derived from cryopreserved sperm with 3-AB. Over 65% of these embryos exhibit developmental abnormalities, mostly grastrula defects, compared to 17% abnormal embryos without 3-AB. Based on the observed phenotypes fibroblast growth factor 8 (Fgf8), a gene necessary for proper gastrulation, was identified as one of the genes affected most by cryopreservation. By better understanding cryo-induced damage we will be able to develop the practical applications of genetic resource banking and improve its generic success across a wider range of species.
Tribbles (TRIB) proteins, a family of evolutionary conserved psuedokinase proteins, modulate various signalling pathways within the cell. The regulatory roles of tribbles make them an important part of a number of biological processes ranging from cell proliferation to metabolism, immunity, inflammation and carcinogenesis. Innate immune system plays a pivotal role during the regulation of reproductive process that allows successful creation of an offspring. Its involvement initiates from fertilization of the oocyte by spermatozoon and lasts throughout early embryonic development, pregnancy and labour. Therefore, there is a close cooperation between the reproductive system and the innate immune system. Evidence from our lab has demonstrated that improper activation of the innate immune system can reduce embryo implantation, thus leading to infertility. Therefore, control mechanisms regulating the innate immune system function can be critical for successful reproductive events.
Recent insemination techniques bypass the interactions between sperm and the uterine wall because the semen is deposited deep into the tip of uterine horn or directly into the oviduct. Such techniques allow high dilution of the ejaculates. After normal mating, semen entering the uterus communicates with the uterine milieu. Intact sperm of high mitochondrial membrane potential bind to uterine epithelial cells, whereas most of the unbound sperm in the uterine lumen have damaged membranes. Lectins are the most likely factors to mediate these sperm-uterine interactions. The lectin wheat germ agglutinin is known to induce the strongest binding of sperm, whereas binding is impaired when sialic acid receptors are blocked by wheat germ agglutinin. This suggests that sialic acid is involved in porcine sperm-endometrium interactions, and it is hypothesized that the use of a semen extender supplemented with sialidase would allow insemination with reduced sperm numbers. A lack of contact of sperm and seminal plasma with the uterine wall, as a result of deep insemination, may adversely affect (1) events during ovulation, (2) induction of immunologic tolerance against paternal antigens, (3) preparation of the endometrium for implantation and placentation, and (4) immunologic support required for the fetus during pregnancy. Seminal plasma is known to signal post-insemination changes in the uterine endometrium involving the redistribution of leukocytes. This may involve migration of leukocytes from the uterine wall to the ovary, as seminal plasma particularly increases the appearance of the major histocompatibility complex class II-positive cells. Uterine epithelial cells respond to sperm binding by the production of pro- or anti-inflammatory cytokines. These cytokines may include synchronizing substances, transferred through a counter-current pathway to the ipsilateral ovary, thereby accelerating the final maturation of preovulatory follicles and advancing time of ovulation. In several species, an ovulation-inducing factor exists in seminal plasma, first identified as ß-nerve growth factor in camelid semen, indicating another pathway that influences the hypothalamic-pituitary-gonadal axis. In summary, low-dose inseminations may not necessarily require semen deposition deep into the uterine horn, as binding inhibitors can circumvent the binding of sperm to the uterine wall. However, subsequent immune-relevant events that control ovulation and prepare the uterine milieu for the developing embryo should be taken into account.
The onset and duration of the inflammatory response after insemination (AI) were studied in eight mares. The mares were inseminated during estrus with fresh semen containing 600 million spermatozoa. Uterine fluid was absorbed into a tampon 0.5, 1, 2, 4, 8, 12, 24, and 48 h after AI. Only one sample per mare was taken during a given estrous period. All mares were examined by rectal palpation and ultrasonography before AI and before sampling. Bacteria were cultured from semen and uterine fluid by means of a 48-h incubation on blood agar. Spermatozoa and neutrophils were counted from the fluid by a Bürker chamber. Spermatozoa—the majority with heads and tails detached—were present in high but steadily decreasing numbers from 0.5 to 4 h and had completely disappeared at 48 h after AI. Corynebacteria, which were cultured from all ejaculates, were cultured in all mares at 1 h and in most mares at 0.5 and 2 h after AI. The first neutrophils appeared as early as 0.5 h after AI. The numbers of neutrophils increased steadily, reaching their highest levels at 8 h after AI. High levels persisted until 24 h and had disappeared at 48 h after AI. The values at 4, 8, 12, and 24 h were significantly different from those at other sampling points. Clinical signs of inflammation (discharge, increase in endometrial edema) were evident from 4 to 24 h after AI.
Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the
source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared
neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography
procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated
with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human
cathepsin G was dose dependent with optimal concentration at 0.5–1 μg/ml. Cathepsin G is chemotactic rather than chemokinetic
for monocytes, as demonstrated by checkerboard analysis. Cathepsin G–induced monocyte chemotaxis is partially pertussis toxin
sensitive implying the involvement of a G protein–coupled receptor. Enzymatic activity of cathepsin G is associated with
its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic
activity of cathepsin G can also be completely blocked by α1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G
is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these
in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses
of 0.05 to 5 μg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous
injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition
of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of
neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.
In patients with accessory gland infections or subjects who have sperm antibodies in their semen, the presence of macrophages with phagocytic activity on ejaculated spermatozoa is significant. Light microscopy cannot certify phagocytosis because it does not give a three-dimensional view of the cells and can lead one to mistake superficial adherence of the spermatozoa to the macrophage for phagocytic activity. For that reason, scanning electron microscopy was used in this study. The samples, fixed with 2.5% glutaraldehyde in phosphate-buffered saline, were processed for observation with light microscopy (Giemsa or Papanicolaou stain) or with scanning electron microscopy (cell selection, critical point drying and paladium-platinum sputtering). With scanning electron microscopy, inactive macrophages had large membrane folds and a globular structure similar to those seen in ascites, whereas when active, they decreased in volume and developed a surface with granules or blebs. Inactive macrophages were rarely seen. A few minutes after mixing the different fractions of the ejaculate, phagocytosis reached such a level of activity that the spermatozoa partly covered the macrophages. Thus, we observed that the spermatozoa were caught by the head first in some instances but by the main-piece fragment of the tail first in other instances; very rarely were they taken by the midportion, between the head and tail. The presence in the ejaculate of macrophages with phagocytic activity on living, motile spermatozoa thus indicates that the encounter between the macrophages and spermatozoa was a result of the assemblage of components that make up the ejaculate. In this way the contributions of the prostatic gland and seminal vesicles play an important part in the spermiophagy of spermatozoa.
Seminal plasma was obtained from bulls of known fertility and was assessed for its effect on serum-induced phagocytosis of bull spermatozoa. A non-dialysable component was found to inhibit neutrophil phagocytic uptake of spermatozoa. The component was not destroyed by heating (56 degrees C for 30 min) or removed by ether. Use of a bactericidal assay confirmed the inhibition and suggested that inhibition does not permanently impair neutrophil function. Immunoperoxidase staining demonstrated the presence of bovine IgM, IgG1 and IgG2 on spermatozoa incubated in serum. Affinity of spermatozoa for the immunoglobulins was reduced when seminal plasma was added to the serum. These results suggest that bull seminal plasma can regulate phagocytic ingestion of spermatozoa. While the mechanism of this regulation remains obscure, it may be important in providing protection to spermatozoa immediately after ejaculation.
This presentation reviews current information on the events that lead to rupture of an ovarian follicle. It contains a summary of the morphological changes that occur at the apex of a follicle wall during ovulation. Existing information shows that the tenacious connective tissue layers of the tunica albuginea and theca externa must be weakened before the follicle wall can dissociate and break open under the force of a modest intrafollicular pressure. These changes are probably dependent on transformation of quiescent thecal fibroblasts into proliferating cells in a manner that is characteristic of tissue responses to inflammatory reactions. The metabolic factors that initiate transformation of the fibroblasts are uncertain, but they are probably generated by gonadotropin-induced changes in the theca interna and granulosa of a follicle as these layers begin to luteinize during the ovulatory process.
The shortening of the time interval between the onset of oestrus and ovulation in sows by the transcervical administration of seminal plasma was investigated in 23 German Landrace gilts, using the technique of single horn infusions (Mariensee model) in combination with the transcutaneous sonographic monitoring of ovaries. Preparative surgery comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. The left ovary was loosely tied to the ventral abdominal wall for better sonographic distinction. The animals were used in two to four consecutive cycles. After detection of oestrus by the teaser boar, the patent (right) horns were filled by transcervical infusion of 100 ml of a variety of test solutions. Ovulation was probed by transcutaneous sonography at intervals of 4 h thereafter. Native seminal plasma provoked ovulation in the ipsilateral ovary of the treated horn 10.7 h earlier than in the contralateral ovary. This effect was reduced to 7.3 h after charcoal treatment of seminal plasma; addition of 10 micrograms oestradiol restored the effect in full, while 10 micrograms of oestradiol in PBS shortened the time interval to only 3.3 h versus the control ovary. Little effect was seen with oestrone sulfate, none with prostaglandins in PBS or with PBS alone. The preliminary characterization of the nonsteroidal component of seminal plasma advancing ipsilateral ovulation after transcervical infusion suggests a proteinaceous nature. The activity resides in the 1-10 kDa fraction separated by ultrafiltration and is lost after treatment with pronase.
Mating in rodents evokes an inflammatory-like reaction within the uterine endometrium, characterized by extensive infiltration and activation of macrophages, dendritic cells, and granulocytes. This response is initiated when seminal vesicle gland-derived factors in the ejaculate stimulate uterine epithelial cells to release proinflammatory cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments in which seminal vesicle secretions were fractionated by Sephacryl S-400 chromatography and assayed in vitro for GM-CSF-stimulating activity revealed that the seminal moiety coeluted with transforming growth factor beta1 (TGFbeta1) in the 150-440-kDa range and was neutralized by anti-TGFbeta1 antibodies. Comparable amounts of recombinant TGFbeta1 stimulated GM-CSF release in cultures of uterine epithelial cells from estrous mice and, when instilled into the uterine lumen, caused an increase in GM-CSF content and an infiltration of leukocytes into the endometrium similar to the postmating response. These results show that seminal vesicular fluid contains TGFbeta1 at levels sufficient to be the primary causative agent in the postmating inflammatory cascade through induction of GM-CSF synthesis by uterine epithelial cells. Seminal TGFbeta1 is thus implicated as a key factor in initiation of the remodeling events and immunological changes that occur in the uterus during the preimplantation period of pregnancy.
The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.
Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.
Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN.
Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.
Regulatory T cells (Tregs) are crucial for maintaining immune tolerance to self-antigens but can also dampen T cell immunity to tumor-associated antigens (TAAs). They are thought to be one of the main obstacles tempering successful immunotherapy. In this article, the molecular and cellular phenotype of Tregs in the tumor microenvironment, the trafficking profile, and their multiple modes of suppressive mechanisms are discussed. The impact of epigenetics and genetics in Treg development and function, as well as Treg metabolism, is reviewed. Applicably, the strategies of therapeutically targeting Tregs in cancer and the potential effects of cancer chemotherapy and radiation therapy on Tregs are incorporated in the discussion.
Cervical smears were taken before, and at intervals after, artificial insemination and scored for the ratio of leukocytes to squamous cells. Most women showed a significant rise in this ratio after insemination with whole fresh or frozen/thawed semen, but this was not seen when fresh or frozen sperm-free seminal plasma was used. Some women had received spermatozoa naturally less than 24 hours before our tests, and most showed some leukocytosis before insemination; even these women showed further response to artificial insemination with spermatozoa. It is concluded that women, like rabbits, normally respond by leukocytosis to cervical deposition of spermatozoa. The primary function of the leukocytes may be sperm selection by phagocytosis or the "mopping up" of introduced bacteria. Some of the patients became pregnant at the monitored cycle; so it is very clear that this is a physiologic response of the cervix, not a pathologic leukocytic reaction.
The function of the extracellular matrix (ECM) can be broadly divided into two. First, it acts as a structural framework for tissue. The structural demands on endometrium change with pregnancy, and after parturition there is a need to return the ECM to its non-pregnant state.The hormonally controlled compositional and biomechanical changes in endometrial ECM during the reproductive cycle are some of the most rapid and extensive seen in adult physiology.
A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.
Human spermatozoa activated human polymorphonuclear leukocytes (PMN) in the presence of serum. Activation of PMN was studied by measuring the emission of chemiluminescence by the PMN. The amount of chemiluminescence emitted depended on the number of spermatozoa and the serum concentration, and the presence of antibody or complement. Spermatozoa were able to activate PMN in the presence of heat-inactivated serum (only antibody, no complement), serum obtained from a patient with agammaglobulinemia (without antibodies and only complement as opsonin), and in MgEGTA agammaglobulinemic serum (only the alternative pathway of complement intact). In the presence of heat-inactivated agammaglobulinemic serum no significant chemiluminescence was observed. It was concluded that spermatozoa activate the alternative pathway of complement. Dead spermatozoa were more able to activate PMN than viable spermatozoa.
The influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells and the cytokine production of spermatozoa were investigated in vitro. Seminal plasma and spermatozoa were collected from healthy volunteers and were screened by enzyme-linked immunosorbent assay for cytokines. Epithelial and stromal cells from fertile women were cultured on matrigel or polystyrol and incubated with pooled seminal plasma or with transforming growth factor b1 (TGF-b1), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), which were found to be significantly concen- trated in seminal plasma. Endometrial cytokine expression was analysed by RNase protection assay and supported by RT-PCR. Supernatants of highly purified spermatozoa did not contain detectable levels of IL-1b, IL-6 and VEGF. Screening of seminal plasma revealed concentrations >10-fold above the serum level for TGF-b1, IL-8 and VEGF. Incubation of epithelial cells with 0.1, 1 and 10% seminal plasma resulted in concentration-dependant stimulation of IL-1b, IL-6 and LIF mRNA expression. Maximum stimulation was found in epithelial cells from tissue samples taken in the mid secretory phase. Epithelial mRNA expression of IL-1b, IL-6 and LIF increased by stimulation with TGF-b1 and IL-8, but not with VEGF. In conclusion, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. This effect might at least in part be exerted by TGF-b1 and IL-8, abundantly present in seminal plasma. The in-vivo physiological relevance of these in-vitro studies remains to be determined.
The attachment of the blastocyst to the uterine luminal epithelium and the subsequent invasion by trophoblast cells through the stroma and deciduum occur in a highly regulated manner by remodeling of the extracellular matrix. We investigated the temporal and spatial expression of mRNAs for four matrix metalloproteinases (MMPs; MMP-2 [gelatinase A], MMP-3 [stromelysin 1], MMP-9 [gelatinase B], and MMP-13 [collagenase 3]) and tissue inhibitors of metalloproteinases (TIMPs; TIMP-1, TIMP-2, and TIMP-3) in the mouse uterus from days 1 to 8 of pregnancy. Northern blot analyses showed the transcripts for MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in whole uterine RNA on these days. However, MMP-13 mRNA was not detected in the uterus, and only weak signals for MMP-3 mRNA were detected in the myometrium. Striking expression was observed with MMP-2 mRNA in the subepithelial stroma on days 3–5. With the progression of decidualization on day 6, signals were primarily in the secondary decidual zone. On day 8, MMP-2 mRNA was localized at the site of placenta formation in the mesometrial pole. Signals for MMP-9 mRNA were first detected in a small population of stromal cells exclusively at the site of implantation on day 5 at the antimesometrial pole. However, the most pronounced expression was noted in trophoblast giant cells on day 8. TIMP-1 mRNA was present in the myometrium on day 1. On days 2–5, modest signals were detected in the stroma, and on days 6 and 8, they were in the secondary decidual zone. Localization of TIMP-2 mRNA was similar to that of TIMP-1 except it was restricted to the stroma on day 1. The regulation of TIMP-3 was more pronounced. While a gradual increase in signals was observed in stromal cells from days 1 to 4, strong signals were detected in antimesometrial stromal cells at the sites of blastocyst attachment on day 5. On days 6 and 7, even stronger signals were present in the primary decidual zone surrounding the embryo, and on day 8 signals were localized primarily in the mesometrial decidual bed. These results suggest that MMP-2 may participate in the early phase of decidualization and neovascularization required for placentation. The restricted MMP-9 expression in stromal cells on day 5 and in trophoblast giant cells on day 8, coupled with the expression of TIMP-3 in the stroma surrounding the embryo, suggests that a fine balance between MMP-9 and TIMP-3 may regulate trophoblast invasion in the uterus. Dev. Genet. 21:44–54, 1997. © 1997 Wiley-Liss, Inc.
After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co- incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.
A preliminary investigation was undertaken further to determine the function of the leukocytic cells found in semen. We performed semen analysis and quantified leukocyte subsets using immunocytochemical staining techniques in ejaculates of 351 patients. Leukocyte profiles were examined in relation to sperm morphological data for evidence of a sperm removal/selection process. Three types of seminal phagocytic cell were found to contain spermatozoa: small polymorphonuclear leukocytes (approximately 10-12 microns), monocytes of similar size and much larger (30-40 microns) macrophages capable of engulfing multiple sperm heads. The total leukocyte count (P less than 0.01), the numbers of phagocytic cells i.e. polymorphonuclear leukocytes (P less than 0.05), monocyte/macrophages (P less than 0.01) and HLA-DR positive cells (P less than 0.01), were significantly higher in those samples with greater than 50% ideal sperm forms. Significantly fewer of these same cell types were observed in samples with greater than 50% head defects. There was no difference in the number of tail or midpiece defects between leukocytospermic (greater than 10(6)/ml) and non-leukocytospermic semen samples. Oligozoospermic samples contained significantly fewer leukocytes (P less than 0.005), although above a concentration of 5 x 10(6)/ml, the sperm number was not correlated with leukocyte number. These data, along with repeated observation of spermatozoa or sperm fragments within phagocytic cells, support the hypothesis that leukocytes have a role in the removal of abnormal spermatozoa from the ejaculate.
Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.
The ultrastructure of the surface epithelia from the uterotubal junction (UTJ), and the adjacent tubal isthmic and endometrial regions, was studied in preovulatory oestrus gilts, either unmated or inseminated 12 h before with fresh boar semen. The simple columnar epithelium of the UTJ consisted of non-ciliated (secretory) and ciliated cells. Secretory vesicles occurred in the secretory cells, especially in inseminated gilts. Lymphocytes, monocytes and macrophages were found dispersed basally among the epithelial cells. Phagocytosis of epithelial cells undergoing apoptosis was seen throughout the UTJ at oestrus, increasing after insemination. Neutrophilic granulocytes were found in the lamina propria of the uterine component of the UTJ, but only occasionally in the epithelium. After insemination, neutrophils invaded the uterine epithelium, to actively participate in intraepithelial phagocytosis or move into the lumen, engulfing spermatozoa. Neutrophils were absent from the UTJ proper and the isthmic epithelium, irrespective of the presence of spermatozoa in the lumen. Those spermatozoa in the uterine lumen that escaped phagocytosis had severely damaged plasma membranes, whereas those in the UTJ proper--concentrated towards the deep furrows of the diverticulae--mostly showed normal sperm ultrastructure.
The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.
Cervical smears were taken before, and at intervals after, artificial insemination and scored for the ratio of leukocytes to squamous cells. Most women showed a significant rise in this ratio after insemination with whole fresh or frozen/thawed semen, but this was not seen when fresh or frozen sperm-free seminal plasma was used. Some women had received spermatozoa naturally less than 24 hours before our tests, and most showed some leukocytosis before insemination; even these women showed further response to artificial insemination with spermatozoa. It is concluded that women, like rabbits, normally respond by leukocytosis to cervical deposition of spermatozoa. The primary function of the leukocytes may be sperm selection by phagocytosis or the "mopping up" of introduced bacteria. Some of the patients became pregnant at the monitored cycle; so it is very clear that this is a physiologic response of the cervix, not a pathologic leukocytic reaction.
Human spermatozoa activated human polymorphonuclear leukocytes (PMN) in the presence of serum. Activation of PMN was studied by measuring the emission of chemiluminescence by the PMN. The amount of chemiluminescence emitted depended on the number of spermatozoa and the serum concentration, and the presence of antibody or complement. Spermatozoa were able to activate PMN in the presence of heat-inactivated serum (only antibody, no complement), serum obtained from a patient with agammaglobulinemia (without antibodies and only complement as opsonin), and in MgEGTA agammaglobulinemic serum (only the alternative pathway of complement intact). In the presence of heat-inactivated agammaglobulinemic serum no significant chemiluminescence was observed. It was concluded that spermatozoa activate the alternative pathway of complement. Dead spermatozoa were more able to activate PMN than viable spermatozoa.