Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane

Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose

The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs

Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose

The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs

The inhibitory effect of ethanol on antigen-induced degranulation is not affected by blocking alcohol dehydrogenase.

<p>IgE-sensitized cells were pretreated or not (Ctrl) for 15 min with ethanol (0.5%) and/or 4-MP (0.1 or 1.0 mM). The cells were non-activated or activated with antigen (100 ng/ml) in the presence of ethanol and/or 4-MP for 15 min and degranulation was determined. Data are means ± SEs (n = 6). Statistical significance of differences between antigen-activated control cells and ethanol-treated cells is also shown. 4-</p

Model of FcεRI-mediated activation in ethanol-pretreated mast cells.

<p>In nonactivated cells (A), the topography of FcεRI and other signaling molecules, such as SRC family kinase LYN, protein tyrosine phosphatase (PTP), and adaptor proteins (LAT, PAG, and NTAL), prevents signaling. An important role in this process is played by the plasma membrane cholesterol. Aggregation of the FcεRI-IgE complexes by multivalent antigen (B) induces topographical changes that lead to formation of the FcεRI signalosome and enhanced tyrosine phosphorylation of the FcεRI β and γ subunits by LYN and SYK kinases. This results in enhanced degranulation, calcium response, cytokine production and numerous other events. In the cells exposed to ethanol and/or with reduced amount of cholesterol (C), the topography of plasma membrane molecules is slightly modified, resulting in increased tyrosine phosphorylation of some signaling molecules even in nonactivated cells. Aggregation of the receptor in ethanol-treated cells leads to suboptimal topographical changes resulting in reduced tyrosine phosphorylation of the FcεRI β and γ subunits by LYN and SYK kinases and/or enhanced activity of the corresponding phosphatases (D). This leads to reduced degranulation, calcium response, cytokine production and other events. Ethanol could also bind directly to some cytoplasmic or plasma membrane proteins, such as ion channel proteins, and in this way inhibit the cell signaling.</p

Short-term exposure to ethanol inhibits antigen-induced degranulation and calcium response in BMMCs.

<p>IgE-sensitized cells were preincubated for 15 min with various concentrations of ethanol (0–1%), which was also present during antigen-mediated activation. (A, B) Degranulation (release of β-glucuronidase) was measured 5 min (A) or 15 min (B) after exposure of the cells to the indicated concentrations of antigen. (C, D) Calcium response after addition of antigen (arrow, Ag, 100 ng/ml) was measured in the presence of 1 mM extracellular calcium (C), or in its absence (D), followed by addition of 1 mM calcium (arrow, Ca²⁺ in D). Calcium levels in the absence of antigen activation but in the presence of 1% ethanol is also shown in C and D (empty circles). Data are means ± SEs (n = 6–8). Statistical significance of intergroup differences is shown in A and B. In C and D, statistical significance of differences between control cells (0% ethanol) and cells exposed to 0.2% ethanol (violet line), 0.5% ethanol (green line) or 1% ethanol (red line) calculated for the corresponding time intervals (coloured lines) are also indicated.</p

Pretreatment with ethanol inhibits tyrosine phosphorylation of FcεRI β and γ subunits and some other proteins involved in FcεRI signaling.

<p>(A) IgE-sensitized cells were preincubated for 15 min with BSS-BSA alone (Ctrl) or supplemented with ethanol (0.5%) and/or Mβ and then activated or not with antigen (100 ng/ml) in the presence or absence of the compounds. After 5 min the cells were solubilized in 0.2% Triton X-100 and FcεRI was immunoprecipitated (IP) from postnuclear supernatants. The immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with PY-20-HRP conjugate. For loading controls, the same membrane was stripped and re-blotted with FcεRI-β-chain-specific antibody. Representative immunoblots from three to five independent experiments are shown on the left. The immunoblots were analyzed by densitometry and the fold increase in tyrosine FcεRI-β and -γ chain phosphorylation, normalized to non-activated cells and the amount of FcεRI-β chain, is also shown on the right. (B) IgE-sensitized cells were incubated and activated as above. Five min after triggering the cells were solubilized, size fractionated, and tyrosine phosphorylated proteins were detected by immunoblotting with the phosphoprotein-specific antibodies. Antibodies for the corresponding proteins were used for detection of loading controls. Representative immunoblots from three to four independent experiments are shown. (C) The immunoblots were analyzed by densitometry. Fold increases of protein tyrosine phosphorylation, normalized to control (Ctrl) non-activated cells and the corresponding protein loads are shown. (D and E) IgE-sensitized cells were incubated with the drugs as in A and then activated with antigen (100 ng/ml) in the presence of the drugs for the indicated time intervals. The cells were solubilized, size fractionated, and LYN phosphorylated on Tyr 507 (D) or Tyr 416 (E) was detected by immunoblotting with the corresponding antibodies. After stripping, the membranes were developed for LYN used as a loading control. Fold increase in protein tyrosine phosphorylation, normalized to non-activated cells (Ctrl) and protein load, is also shown. Means ± SEs and the statistical significance of differences in A, C, E, and D were calculated from three to five independent experiments.</p

Protective effect of cholesterol against ethanol-mediated inhibition of ROS production in antigen-activated BMMCs.

<p>(A) IgE-sensitized cells were incubated for 15 min with the indicated concentrations of ethanol, which was also present during the activation. Then the cells were activated or not with antigen (250 ng/ml) and ROSs were determined using H₂DCFDA as a substrate. The values on y-axes indicate fluorescence intensities observed 10 min after triggering. (B) The cells were exposed to BSS-BSA supplemented or not with ethanol (0.5%), Mβ (2 mM) and/or sMβ (2 mM), and after 20 min activated or not with antigen (250 ng/ml). ROSs were determined as above. Data are means ± SEs (n = 6–8). The statistical significance of the intergroup differences is also shown.</p

Inhibitory effect of ethanol on mast cell-mediated PCA.

<p>PCA was performed as described in Materials and methods. Sensitizing IgE in PBS and PBS alone were injected into left and right ears, respectively. (A) Representative photographs of ears of the mice injected intraperitoneally with 0.5 ml (per mouse weighing 20 g) PBS alone (0%) or with 0.5 ml of PBS containing 5%, 10%, or 20% ethanol, followed by intravenous administration of Evans blue and antigen in PBS. (B) Quantitative data for ear-tissue extracted Evans blue from left (IgE) and right (PBS) ears in mice treated as above. Means ± SEs were calculated from 3–4 animals in each group. Statistically significant differences between control mice injected with PBS alone and mice injected with 10% or 20% ethanol in PBS are shown.</p