The Birds, the Bees, and Innate Immunity.

The cytoplasm is usually a DNA-free zone, but during fertilization, sperm DNA enters oocyte cytoplasm and could potentially trigger a response. Abe et al. (2017) identify NLRP14 as a germ-cell-specific negative regulator of DNA sensing that may be of particular importance during fertilization.

Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization.

The altered immunological status of children conceived by assisted reproductive technology.

BACKGROUND: With the increased use of assisted reproductive technology (ART), assessing the potential health risks of children conceived on ART important to public health. Most research in this area has focused on the effects of ART on perinatal, metabolic, and oncological risks in children. Although an increased risk of immune-related diseases has been reported in children born after ART, there are no studies on the immunological status of these children. This study aimed to evaluate the impact of different embryo transfer methods and fertilization strategies on the immune status of the offspring. METHODS: A total of 69 children born to women treated with ART and a matched control group of 17 naturally conceived (NC) children, all aged from 3 to 6 years, were recruited in the reproductive hospital affiliated to Shandong University. The frequency of immune cells in the peripheral blood was assayed using flow cytometry; plasma cytokine levels were determined by multiplex cytokine immunoassay with human cytokine magnetic beads. RESULTS: Compared to children born after natural conception, children born after ART had elevated interferon-γ (IFN-γ) levels, regardless of embryo transfer and fertilization strategies. Children in the fresh-embryo transfer group had significantly higher IL-4 levels and a lower ratio of IFN-γ to IL-4 than those in the NC group ((P = 0.004, 10.41 ± 5.76 pg/mL vs 18.40 ± 7.01 pg/mL, P = 0.023, 1.00 ± 0.48 vs 0.67 ± 0.32, respectively). Similar results were shown in either the in vitro fertilization (IVF) group or the intra-cytoplasmic sperm injection (ICSI) group (P < 0.05 and P = 0.08 for IVF; P < 0.05 and P < 0.05 for ICSI, respectively). These alterations in IL-4 concentrations and the ratio of IFN-γ to IL-4 were statistically significantly correlated with supra-physical E2 (estradiol) levels on the day of hCG administration (R = 0.502, P = 0.017; R = - 0.537, P = 0.010, respectively). Consistently, the frozen embryo transfer did not result in alterations of these immune indicators in the offspring. Overall, there were no significant differences between the ART group and NC group in the frequencies of T cells, B cells, natural killer (NK) cells, CD4+T cells, CD8+T cells, T helper (TH)1 cells, TH17 cells, and regulatory T (Treg) cells and cytokine levels of IL-10 and IL-17a (all P > 0.05). CONCLUSIONS: Immunological alterations existed in children born after the use of ART. The elevated E2 levels before embryo implantation contributed to the increased IL-4 levels in children conceived by fresh embryo transfer. The assessment of immunological alteration is of importance to children conceived by ART for early monitoring and intervention.

Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process.

In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female partner, leading to the tolerogenic response enabling the embryo development and implantation as well as further progress of healthy pregnancy. Seminal plasma glycoproteins are rich in the unique immunomodulatory glycoepitopes that may serve as ligands for endogenous lectins that decorate the surface of immune cells. Such interaction may be involved in modulation of the maternal immune response. Among immunomodulatory glycans, Lewis type antigens have been of interest for at least two decades, while the importance of T/Tn antigens and related structures is still far from understanding. In the current work, we applied two plant lectins capable of distinguishing glycoepitopes with terminal GalNAc and Gal to identify glycoproteins that are their efficient carriers. By means of lectin blotting and lectin affinity chromatography followed by LC-MS, we identified lactotransferrin, prolactin inducible protein as well as fibronectin and semenogelins 1 and 2 as lectin-reactive. Net-O-glycosylation analysis results indicated that the latter three may actually carry T and/or Tn antigens, while in the case of prolactin inducible protein and lactotransferrin LacdiNAc and lactosamine glycoepitopes were more probable. STRING bioinformatics analysis linked the identified glycoproteins in the close network, indicating their involvement in immune (partially innate) processes. Overall, our research revealed potential seminal plasma ligands for endogenous Gal/GalNAc specific lectins with a possible role in modulation of maternal immune response during fertilization.

Ovulatory follicular fluid induces sperm phagocytosis by neutrophils, but oviductal fluid around oestrus suppresses its inflammatory effect in the buffalo oviduct in vitro.

We have recently shown that the conditioned media from bovine oviductal epithelial cell culture suppress sperm phagocytosis by neutrophils, suggesting that the oviduct around oestrus supplies the anti-inflammatory microenvironment. To investigate the immune response of neutrophils toward the sperm at ovulation in the buffalo oviduct, we examined (a) a detailed distribution of neutrophils in the oviduct in buffaloes, (b) the effect of ovulatory follicular fluid (FF) and oviductal fluid (OF) on sperm phagocytosis by neutrophils, and (c) the interaction of the ovulatory FF with OF on sperm phagocytosis by neutrophils in vitro. Buffalo oviducts were collected from healthy reproductive tracts at a local slaughterhouse. A detailed observation by histological examination and transmission electron microscopy revealed that neutrophils exist in the oviduct epithelium and lumen throughout the oestrous cycle in buffaloes. The number of neutrophils at the oestrus stage was higher in ampulla compared with those in isthmus, whereas they remained relatively constant at the dioestrus stage. Two hours of preincubation of neutrophils with FF enhanced sperm phagocytosis through the formation of neutrophil extracellular traps (NETs) together with H2 O2 production, whereas OF around oestrus (eOF) suppressed sperm phagocytosis, NETs formation, and H2 O2 production and relieved the above FF-induced inflammatory response. Our findings show that neutrophils exist in the healthy cyclic oviduct across bovine species, and the OF supplies a strong anti-inflammatory environment that could minimize the inflammatory effect of the FF that flows into the oviduct lumen after ovulation and supports the occurrence of fertilization.

Paternal priming of maternal tissues to optimise pregnancy success.

The question of 'how does the allogeneic fetus survive gestation in the face of the maternal immune system?' has yet to be definitively answered. Several acceptable mechanisms exist to facilitate survival of the semi-allogeneic fetus in various species; paramount is the immunological separation of maternal and fetal tissues during gestation. However, keen observation of the maternal immune system during pregnancy has noted maternal immune tolerance to paternal-specific antigens. A mechanism by which the maternal immune system tolerates specific paternal antigens expressed on the fetus would be far more beneficial than the previously proposed immune indolence that would leave the mother susceptible to infection. In species like human or rodent, implantation occurs days after fertilisation and, as such, the mechanisms to establish antigen-specific tolerance must be initiated very early during pregnancy. We and others propose that these mechanisms are initiated at the time of insemination when paternal antigens are first introduced to the maternal immune system. Indeed, a new paradigm demonstrating the importance of paternal-maternal communication at the time of insemination is becoming evident as it relates to maternal tolerance to fetal antigen and ultimately pregnancy success.

The autoimmune regulator prevents premature reproductive senescence in female mice.

Loss-of-function mutations in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyglandular syndrome type 1 (APS-1), which commonly manifests as infertility in women. AIRE is a transcriptional regulator that promotes expression of tissue-restricted antigens in the thymus, including antigens specific to the ovary. Thymic expression of ovarian genes under AIRE's control may be critical for preventing ovarian autoimmune disease. Because mice lacking Aire are an important APS-1 model, we examined the reproductive properties of female Aire-deficient (Aire(-/-)) mice. Female Aire(-/-) mice on the BALB/c background were examined for reproductive parameters, including fertility, litter sizes, and ovarian follicular reserves. Although delayed puberty was observed in Aire(-/-) mice, all mice entered puberty and exhibited mating behavior. Only 50% of Aire(-/-) females gave an initial litter, and only 16% were able to produce two litters. Ovarian histopathologic examination revealed that 83% of previously bred females lost all ovarian follicular reserves. Among virgin females, follicular depletion was observed in 25% by 8 wk, and by 20 wk, 50%-60% of mice lost all follicles. This was associated with elevated serum follicle-stimulating hormone level and ovarian infiltration of proliferating CD3+ T lymphocytes. Ovulation rates of 6-wk-old Aire(-/-) mice were reduced by 22%, but this difference was not statistically significant. Finally, transplantation experiments revealed that follicular loss depended on factors extrinsic to the ovary. These results suggest that immune-mediated ovarian follicular depletion is a mechanism of infertility in Aire(-/-) mice. The results have important implications in the pathogenesis of ovarian autoimmune disease in women.

Progesterone's effects on the psychology of disease avoidance: support for the compensatory behavioral prophylaxis hypothesis.

In the human menstrual cycle, luteal phase immunomodulation prevents the maternal immune system from attacking the half-foreign blastocyst should conception occur, thereby facilitating implantation and development. However, tolerance of the conceptus comes at the cost of increased vulnerability to infection. The compensatory behavioral prophylaxis hypothesis (Fessler, 2001; Fessler & Navarrete, 2003) holds that evolved psychological mechanisms enhance avoidance of potential contaminants during periods of reproductive immunomodulation so as to decrease the likelihood of infection. Because such immunomodulation is triggered by progesterone, this hormone is predicted to correspondingly enhance behavioral prophylaxis and the motivational states underlying it. We investigated specific domains of disease avoidance psychology in a nonclinical sample of women (n=120) by measuring salivary progesterone in naturally cycling women. We find that progesterone correlates directly with the degree to which women report emotions, thoughts, and behaviors consonant with enhanced prophylaxis.

Immunological response of fallopian tube epithelial cells to spermatozoa through modulating cytokines and chemokines.

BACKGROUND: Spermatozoa interactions with fallopian tubes may influence fertilization. The purpose was to investigate cytokines, chemokines and growth factors expression from human fallopian tube epithelial cells (OE-E6/E7) exposed to spermatozoa. METHODS: Fresh semen samples were obtained from 10 healthy normozoospermic men. Sperms were prepared and co-cultured with OE-E6/E7. The cell line without spermatozoa was considered as the control group. Afterwards, Expression of 84 cytokines from OE-E6/E7 cell line in the presence and absence of spermatozoa were measured using PCR-array. Quantitative PCR was performed on seven genes to confirm the results of PCR-array analysis. Differentially expressed genes were subjected to www.geneontology.org and www.pantherdb.org to perform GO enrichment and panther pathway analysis. The concentration of IL-8, IL-10, IL-1B and BMP-4 in culture medium were analyzed by ELISA. RESULTS: Sperm interaction with the epithelial cells resulted in a significant increase in expression of TGF-ß2, BMP-4, IL-10, IL-9, and CD40LG markers. Moreover, expression of IL-16, IL-17F, SPP-1, CXCL-13, MSTN, IL-1A, IL-1B, IL-8, BMP-7, CSF-2, CSF-3, VEGF-A, OSM, LTA, TNF, TNFRSF11B, TNFSF11, CCL-11, CCL-20, CCL-24, CCL-3, CCL-8, CX3CL1 and CXCL-9 were considerably reduced in presence of spermatozoa. Panther pathway analysis discovered 3 pathways for upregulated genes including gonadotropin-releasing hormone receptor, TGF-beta and interleukin signaling pathways. Furthermore, 9 pathways were detected for down-regulated genes. Inflammation signaling pathway which is mediated by chemokine and cytokine contains the most number of genes. CONCLUSION: This study indicates that sperm modifies expression of cytokines, chemokines and growth factors from OE-E6/E7. Moreover, altered genes expression are toward higher survival chance of the spermatozoa.

The molecular basis of allorecognition in ascidians.

The process of allorecognition consists of an ability to discriminate self from non-self. This discrimination is used either to identify non-self cells and reject them ("non-self histocompatibility") or to identify self cells and reject them (as in the avoidance of self-fertilization by hermaphrodites ("self incompatibility"). The molecular basis governing these two distinct systems has been studied recently in hermaphroditic ascidian urochordates. Harada et al. postulated two highly polymorphic self-incompatibility loci, Themis (A and B), that are transcribed from both strands, forward to yield sperm (s-) trans-membrane antigen, and reverse to yield the egg vitelline coat (v-) receptor. De Tomaso et al. characterized a candidate histocompatibility locus, encoding a highly variable immunoglobulin. Nyholm et al. isolated its candidate allorecognition receptor, fester. Only a minute similarity was found in the structure of the genes involved. It appears that ascidian harbor two very separate types of labeling and recognition genetic systems: one for self and the other for non-self.

Human sperm DNA damage has an effect on immunological interaction between spermatozoa and fallopian tube.

BACKGROUND: Toll-like receptors play a crucial role in the immunological interaction between the spermatozoa and fallopian tube and contribute to the ovulation, sperm capacitation, fertilization, and pregnancy. OBJECTIVES: To investigate the expression of toll-like receptors and their adaptor molecules and cytokines under the effect of spermatozoa with high DNA fragmentation (high DF) in human fallopian tube cell line (OE-E6/E7) and compare to those in normal spermatozoa. MATERIALS AND METHODS: Fresh semen samples were obtained from 10 unexplained infertile males with high DF (more than 20%) and from 10 healthy donors with a DF less than 3%. After sperm preparation, samples were co-cultured with OE-E6/E7. Toll-like receptors, myeloid differentiation factor 88 (MyD88), TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing IFN-b (TRIF), TRIF-related adapter molecule as well as IL-6, IL-8, IFN-ß, and TNFα mRNA expression were evaluated by quantitative real-time PCR. Protein levels of these cytokines and chemokines were measured using ELISA method. RESULTS: TLR 1-6 mRNA expression in OE-E6/E7 was significantly higher under the effect of spermatozoa with high DF compared to the spermatozoa with low DF. Furthermore, significantly increased mRNA expression of MyD88, TIRAP, and TRIF was observed in the high DF group compared to the low DF group, except TRIF-related adapter molecule. Moreover, the expression of IL-6 and IL-8 in the high DF group was significantly higher than low DF group, although there was no significant difference in IFN-ß and TNFα expression between the groups. DISCUSSION AND CONCLUSION: Damage-associated molecular patterns from DNA damage activate TLR signaling pathway in human fallopian tubes and result in the upregulation of inflammatory cytokines and chemokines. This situation may provide pathologic environment for capacitation, fertilization, embryo development, and implantation in female reproductive tract and can be one of the mechanisms of infertility in men with high DF.

Antibodies to two ZP3 B cell epitopes affect zona pellucida assembly.

Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335-342) and ZP3 (171-180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.

Obtaining and characterization of anti-testis monoclonal antibodies: Invaluable tools toward the identification of testis antigens involved in fertilization.

BACKGROUND: Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. OBJECTIVE: Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. METHODS: To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. RESULTS: By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. CONCLUSIONS: So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.

Apparent involvement of a beta1 type integrin in coral fertilization.

Integrins are involved in a wide variety of cell adhesion processes, and have roles in gamete binding and fusion in mammals. Integrins have been also discovered in the scleractinian coral Acropora millepora (Cnidaria: Anthozoa). As a first step toward understanding the molecular basis of fertilization in corals, we examined the effect of polyclonal antisera raised against recombinant coral integrins on gamete interactions in A. millepora. Antiserum raised against integrin betacn1 dramatically decreased the binding of Acropora sperm to eggs and significantly decreased fertilization rates relative to preimmune serum and seawater controls. However, the antiserum against AmIntegrin alpha1 did not affect significantly either sperm-egg binding or fertilization. One possible explanation for this is that AmIntegrin alpha1 may preferentially mediate interactions with RGD-containing ligands, whereas mammalian alpha6 integrin (which is most directly implicated in gamete interactions) preferentially interacts with laminin-related ligands. Our results suggest that beta1 type integrins are involved in the fertilization process in Acropora and that some functions of these molecules may have been conserved between corals and mammals.

Is HLA-E a possible genetic marker relevant for natural conception?

BACKGROUND: HLA-E products, class Ib human leukocyte antigens, act in the immunology of human reproduction as modulators of the maternal immune system during pregnancy. AIMS: To evaluate HLA-E role in the establishment of a viable pregnancy. MATERIALS & METHODS: HLA-E was genotyped by sequence-based typing (SBT) and analyzed for specific polymorphisms, comparing couples who underwent assisted reproduction treatment (ART) and fertile control couples. RESULTS: There was a significant difference in HLA-E allele and genotype distributions between ART couples and control couples. The allele HLA-E*01:03 was observed in 63.2% of ART men and in 35.1% of fertile men (P = 0.0032). CONCLUSION: These results suggest that HLA-E allelic variants may play a role in the modulation of immune responses in the context of the inability of natural conception and establishment of a viable pregnancy.

The role of antisperm antibodies during fertilization and for immunological infertility.

Antisperm antibodies (ASA) occur in men and in women. In the male they may cause an autoimmune disease, 'immune infertility'. In order to understand the functional relevance of ASA it is necessary to characterize the cognate antigens of ASA by different methods. In the literature, several immunologically characterized sperm proteins--as cognate antigens of naturally occurring ASA or of artificially produced antibodies--are quoted with respect to different sperm functions in the course of fertilization: cervix mucus penetration, zona binding, zona penetration, oolemma binding, and pronucleus formation. In a number of these functions, sperm proteins were already identified as cognate antigens of ASA. As practical consequences of the research on ASA-related sperm proteomics those ASA will be identified, which decrease male fertility by inhibiting sperm functions that are essential for fertilization.

Immunocontraceptive approaches in females.

In the female reproductive system, there are multiple points, where immunointerception is feasible. Among these, the most promising are those that interfere immunologically at the level of sperm-egg interaction or neutralize human chorionic gonadotropin (hCG), which is required for the establishment and maintenance of pregnancy. Immunization studies in various animal models including non-human primates with zona pellucida (ZP) glycoproteins have demonstrated curtailment of fertility. To circumvent the ovarian pathology often observed following immunization with ZP glycoproteins, synthetic peptides devoid of 'oophoritogenic' T cell epitopes as immunogens have been proposed. Though of utility for controlling wildlife populations, its application in humans will have to wait till the safety of ZP glycoprotein vaccine is established beyond doubt. Clinical trials in women have established that immunocontraceptive vaccine based on Beta-subunit of hCG inhibits fertility. To make it a practical proposition, novel strategies have to be adopted to increase its immunogenicity so that 'contraceptive' antibody titers are achieved in near to 100% recipients.

The role of cytokines in gestation.

Lymphohemopoietic cytokines are now recognized to be central participants in the cellular communication events underlying the complex and dynamic remodeling processes required to accommodate the semiallogeneic conceptus during mammalian reproduction. Cytokines are identified to be particular importance in mediating communications between the conceptus and maternal cells, particularly the uterine epithelium and infiltrating leukocytes, both prior to implantation and as the placenta develops. In this review we summarize recent experimental data concerning the synthesis of various cytokines in uterine and conceptus-derived tissues and highlight current hypotheses for their roles in establishing and maintaining successful pregnancy. It is concluded that complex and finely balanced cytokine networks underpin precise regulatory mechanisms controlling the rate and degree of conceptus development and invasion into maternal tissues.

Study on the role of a sperm membrane-associated antigen in canine fertilization.

The Mab 4B12 produced by us against capacitated boar spermatozoa was found to recognize a protein located in the acrosome portion of capacitated boar spermatozoa which is shared by different animal species, dogs included. It was shown that Mab 4B12 might affect fertilizing ability in vitro of boar spermatozoa. Using indirect immunofluorescence (IIF) test, we provide evidence here that Mab 4B12 stained the acrosome of the capacitated but not of the ejaculated and acrosome-reacted canine spermatozoa. The biological experiments using hemizona assay functional test in this study provide evidence supporting the involvement of Mab 4B12 corresponding antigen in the functional steps required for canine sperm-zona pellucida binding. These results together with the data on cell and tissue specificity of the 4B12 antigen suggest its contraceptive potential for canine fertilization.

Human spermatozoal FA-1 binds with ZP3 of porcine zona pellucida.

Experiments were carried out to study the binding of the major porcine zona pellucida glycoprotein ZP3 (Mr = 55,000) with human FA-1, a fertilization-related antigen isolated from human male germ cell plasma membranes and with GA-1, a germ cell antigen isolated from murine male germ cell plasma membranes. FA-1 showed a dose-dependent binding with ZP3 in an enzyme-linked immunosorbent assay (ELISA) and by the Western blot procedure. FA-1 also bioneutralized the sperm ligand activity of ZP3 in a boar sperm/porcine zona pellucida attachment assay. In contrast, GA-1 displayed no reactivity for ZP3 using any of these procedures. These results indicate that FA-1 but not GA-1 has zona pellucida binding activity in the porcine system.