The molecular basis of mouse sperm-zona pellucida binding: a still unresolved issue in developmental biology

During murine fertilization, sperm bind to the specialized extracellular matrix of the egg, known as the zona pellucida (ZP). This matrix is composed of three major glycoproteins designated ZP1, ZP2, and ZP3. Three models for sperm-ZP binding are now under consideration. The domain-specific model posits that adhesion relies primarily on interactions between N-glycans located within the C-terminal domain of ZP3 and a lectin-like egg-binding protein in the sperm plasma membrane. However, this model does not explain recent results obtained in studies with ZP2(mut) mice. In the supramolecular structure model, sperm bind to a three-dimensional zona matrix that depends on the cleavage status of ZP2. This paradigm does not explain the potent inhibitory effect of specific carbohydrate sequences or a C-terminal glycopeptide (gp55) derived from ZP3. Recently, O-glycans linked at Thr(155) and Thr(162) of ZP3 were implicated as potential ligands that mediate initial sperm-ZP binding. This novel model will be reviewed. A major challenge is to develop an alternate model for sperm-ZP binding that fits as much of the data as possible. Such a model is presented in this review. This paradigm could explain how the inability to cleave ZP2(mut) in ZP2(mut) mice could result in continued sperm binding to two-cell stage embryos without the formation of a supramolecular binding complex. These novel insights should guide future experiments that will eventually determine the molecular basis underlying gamete binding in the mouse and other eutherian mammals.     

Inhibition of lipoxygenase-1 by tetrahydrocurcumin

Tetrahydrocurcumin (THC)—the reduced form of curcumin—is more hydrophilic than its parent compound. It possesses higher stability in aqueous medium compared to curcumin. Lipoxygenase (LOX) enzyme is implicated in inflammatory conditions. THC was investigated for the inhibition of soy LOX-1. THC-inhibited LOX-1 with an IC₅₀ value of 44.6 ± 0.6 μM. Kinetics of inhibition revealed mixed linear type with a K _i value of 46 μM. THC-inhibited LOX-1 by preventing the activation of LOX-1 as evidenced from spectroscopic studies. Molecular docking simulations suggested the binding of THC near the iron cofactor. This study helps in further understanding of the anti-inflammatory property of curcumin derivatives and provides valuable leads as an alternative of curcumin with better solubility and stability at physiological pH.     

Zinc binding in bovine milk

About 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100,000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations greater than or equal to 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and approximately 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and Pi were gradually rendered soluble as the pH of milk was lowered and at pH 4.6 greater than 95% of the Zn, Ca and Pi were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6.7. About 40% of the total Zn in skim milk was non-sedimentable at 0.2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1-50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or Pi was solubilized at EDTA concentrations up to 1.0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and Pi by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to 1.2 mg/l (approximately 32% of the original Zn). Thus, approximately 32% of the Zn in skim milk is directly bound to caseins, while approximately 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0.2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4.6) co-precipitated with Ca and Pi on raising the pH to 6.7 and heating for 2 h at 40 degrees C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6.6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.     

Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression

Polymorphisms in 5′-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine α_s1-casein encoding gene (CSN1S1), which is affected by an A→G exchange at −175 bp (CSN1S1^−175bp). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1^−175bp binding site. Further investigations comprised genotyping of the variable CSN1S1^−175bp activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1^−175bp*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1^−175bp*G showed higher content (%) as well as quantity (g/d) of α_s1-casein than CSN1S1^−175bp*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1^−175bp*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.     

Inhibition of the wine spoilage yeast Dekkera bruxellensis by bovine lactoferrin-derived peptides

The antimicrobial action of lactoferrin (LF)-derived peptides against Dekkera bruxellensis strains isolated from spoiled wines has been examined. The study included a fifteen-residue peptide (LfcinB(17-31)) derived from bovine lactoferricin B and a bovine LF pepsin hydrolysate (LFH). In vitro assays showed the inhibitory properties of LfcinB(17-31) on D. bruxellensis growth with IC(50) and MIC values in the micromolar range. Strains tested showed different sensitivity to the peptide. LfcinB(17-31) showed fungicidal properties towards all strains tested in laboratory growth medium. However, the extent of fungicidal activity was strain-dependent in must and wine, confirming the different antimicrobial action of peptides depending on both the food matrix and the target micro-organism. The binding of LfcinB(17-31) to D. bruxellensis cells was visualized by fluorescence microscopy and correlated with the fungicidal activity in the different matrixes. LfcinB(17-31) and LFH showed growth inhibitory properties in wine suggesting their potential use for spoilage control.     

Genetic variability in the humoral immune response to bovine herpesvirus-1 infection in dairy cattle and genetic correlations with performance traits

Bovine herpesvirus-1 (BoHV-1) is a viral pathogen of global significance that is known to instigate several diseases in cattle, the most notable of which include infectious bovine rhinotracheitis and bovine respiratory disease. The genetic variability in the humoral immune response to BoHV-1 has, to our knowledge, not ever been quantified. Therefore, the objectives of the present study were to estimate the genetic parameters for the humoral immune response to BoHV-1 in Irish female dairy cattle, as well as to investigate the genetic relationship between the humoral immune response to BoHV-1 with milk production performance, fertility performance, and animal mortality. Information on antibody response to BoHV-1 was available to the present study from 2 BoHV-1 sero-prevalence research studies conducted between the years 2010 to 2015, inclusive; after edits, BoHV-1 antibody test results were available on a total of 7,501 female cattle from 58 dairy herds. National records of milk production (i.e., 305-d milk yield, fat yield, protein yield, and somatic cell score; n = 1,211,905 milk-recorded cows), fertility performance (i.e., calving performance, pregnancy diagnosis, and insemination data; n = 2,365,657 cows) together with animal mortality data (i.e., birth, farm movement, death, slaughter, and export events; n = 12,853,257 animals) were also available. Animal linear mixed models were used to quantify variance components for BoHV-1 as well as to estimate genetic correlations among traits. The estimated genetic parameters for the humoral immune response to BoHV-1 in the present study (i.e., heritability range: 0.09 to 0.16) were similar to estimates previously reported for clinical signs of bovine respiratory disease in dairy and beef cattle (i.e., heritability range: 0.05 to 0.11). Results from the present study suggest that breeding for resistance to BoHV-1 infection could reduce the incidence of respiratory disease in cattle while having little or no effect on genetic selection for milk yield or milk constituents (i.e., genetic correlations ranged from ?0.13 to 0.17). Moreover, even though standard errors were large, results also suggest that breeding for resistance to BoHV-1 infection may indirectly improve fertility performance while also reducing the incidence of mortality in older animals (i.e., animals >182 d of age). Results can be used to inform breeding programs of potential genetic gains achievable for resistance to BoHV-1 infection in cattle.     

Inhibition of mastitic bacteria by bovine milk apo-lactoferrin evaluated by in vitro microassay of bacterial growth

An in vitro microassay was developed to evaluate antimicrobial properties of bovine apo-lactoferrin. The growth of coliform, staphylococcal, and streptococcal bacterial strains in a defined synthetic medium was inhibited by bovine apo-lactoferrin (.5 to 30.0 mg/ml). Addition of iron-saturated lactoferrin to the synthetic medium did not inhibit growth of test strains. Inhibition by apo-lactoferrin was greater for coliform than Gram-positive strains for all concentrations of apo-lactoferrin evaluated. No concentration of apo-lactoferrin proved bactericidal for either coliform or Gram-positive strains. Inhibition of two coliform strains by apo-lactoferrin (10 mg/ml) was abolished by addition of ferric iron to the assay system, indicating an iron-dependent nature of apo-lactoferrin induced inhibition of bacteria. Bicarbonate supplementation of the growth system containing apo-lactoferrin (1 mg/ml) increased inhibition of three coliform strains by apo-lactoferrin. Addition of increasing concentrations of citrate (2.0 mg/ml) to an assay system containing apo-lactoferrin (5 mg/ml) resulted in a concomitant reduction of growth inhibition of three coliform strains. These data indicate a potential relationship between the molar ratio of citrate to lactoferrin of the lacteal secretion and its capacity to inhibit coliform strains associated with mastitis.     

Inhibition of L-alanine uptake into bovine jejunal brush border membrane vesicles by L-amino acids

Jejunal epithelial cells from slaughtered Holstein cows were fractionated to obtain purified brush border membranes from which membrane vesicles were prepared for use in amino acid uptake studies. Uptake of alanine was determined by incubation of vesicles with a solution containing radiolabelled alanine, isolation of vesicles and accumulated alanine by filtration, and detection of accumulated alanine by liquid scintillation counting. Uptake studies were conducted under conditions shown to provide linear rates of accumulation. Sodium-dependent active transport was determined as the difference between uptake measured in the presence and absence of sodium in the extravesicular buffer. Inhibition of alanine uptake increased with increasing extra-vesicular inhibitor concentration until a plateau value was reached. Inhibition of sodium-dependent alanine uptake by 100 mM glycine was 72%; 25 mM isoleucine, valine, or methionine completely inhibited initial alanine uptake. These results indicate the existence of at least two sodium-dependent transport systems, one capable and one incapable of accepting glycine for transport. At concentrations designed to represent expected concentrations of free amino acids in intestinal digesta, several equimolar mixtures (.2 to 5 mM) of 20 amino acids inhibited alanine uptake, suggesting that significant interaction among amino acids for uptake may be occurring under in vivo conditions.     

Pre-weaning plane of nutrition and Mannheimia haemolytica dose influence inflammatory responses to a bovine herpesvirus-1 and Mannheimia haemolytica challenge in post-weaning Holstein calves

The objectives of this study were to determine whether plane of nutrition (PON) of milk replacer previously provided to calves, and dosage level of Mannheimia haemolytica (MH), influenced inflammatory responses to a combined viral-bacterial respiratory challenge. Holstein calves (1 d of age; n = 30) were assigned to treatments in a 2 × 3 factorial with pre-weaning PON and MH dose as main effects (n = 5 per treatment). Calves were fed either a low (LPN; n = 15) or a high PON (HPN; n = 15) from birth through weaning. Calves fed LPN were fed 436 g of dry matter (DM) per day of milk replacer until weaning, and HPN calves were fed 797 g of DM per day of milk replacer from d 1 to 10 and 1,080 g of DM per day from d 11 until weaning. Calf starter and water were offered ad libitum. Calves were step-down weaned beginning at d 54 and moved into an enclosed barn at d 70. Indwelling rectal temperature (RT) recording devices and jugular catheters were inserted at d 80. Calves were challenged with 1.5 × 10⁸ plaque-forming units (pfu) per mL of bovine herpesvirus-1 (BHV-1) in each nostril at d 81 and with either 10⁶, 10⁷, or 10⁸ cfu of MH at d 84. Blood samples were collected at varying intervals relative to BHV-1 and MH challenges. Four LPN calves either died or were euthanized soon after the 144-h observation period, whereas all HPN calves survived the entire observation period. As dosage of MH administered increased, acute and systemic inflammatory responses increased. Higher doses of MH resulted in increased leukocyte, neutrophil, and haptoglobin concentrations in infected calves. Data from the current study suggest that the highest dose, 10⁸ cfu, triggered weaned calves' acute disease response, whereas the lower doses, 10⁶ and 10⁷ cfu, caused more moderate inflammation and disease. The effects of PON on inflammation responses to the disease challenge indicated that calves previously fed the LPN diet had more severe pathophysiological responses. Calves fed LPN showed higher peripheral neutrophil and leukocyte counts and serum haptoglobin concentrations following the BHV-1 challenge. Additionally, following the MH challenge, LPN calves had higher peripheral neutrophil counts, neutrophil-to-lymphocyte ratios, and serum tumor necrosis factor-α concentrations. These data demonstrate that higher doses of MH increase the acute inflammatory response and prolong inflammation, and that calves previously fed LPN responded more severely to the combined viral-bacterial respiratory challenge.     

Inhibition of cholesterol biosynthesis and acetyl-coenzyme A synthetase by bovine milk and orotic acid.

Pasteurized, homogenized bovine milk or orotic acid in solution at final concentrations varying from 3.3 μM to 322 μM in rat liver homogenates inhibited the incorporation of [1-Carbon 14] acetate but not [1-Carbon 14] acetyl-coenzyme A, 3-hydroxy-3-methyj-[Carbon 14] glutaryl-coenzyme A or [5-hydrogen 3] mevalonic acid into cholesterol. Milk inhibited cholesterol biosynthesis up to 72 ± 10% before acetyl-coenzyme A formation, thus indicating that acetyl-coenzyme A synthetase is the affected enzyme. Kinetics of the inhibition were studied with acetyl-coenzyme A synthetase purified from yeast. From a Line-weaver-Burk plot of the inhibition of yeast acetyl-coenzyme A synthetase, orotic acid is a noncompetitive inhibitor of acetyl-coenzyme A synthetase. A Michaelis constant was 6.0 × 10^?4 M for acetate with the yeast enzyme while a K_i was 6.6 × 10^?5 M for orotic acid. The approximate point of 50% inhibition with rat liver enzyme was 7 × 10^?6 M orotic acid indicating the mammalian enzyme may be more sensitive to the orotic acid. Nicotinic acid also inhibited the yeast enzyme. Fifty percent inhibition required a relatively high final concentration–about 12 mM. Neither raw milk nor pasteurized, homogenized milk inhibited 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase that had been partially purified from rat liver.     

Vitamin D binding factors in bovine blood.

Both 25 [25,26-hydrogen-3] hydroxycholecalciferol and [1alpha,2alpha-hydrogen-3] cholecalciferol were added to bovine plasma in vitro. Analysis by gel-filtration and ion exchange chromatography, electrophoresis, ultracentrifugation, competitive binding specificity studies, and plasma stripping showed that vitamin D circulated with a protein of alpha-globulin mobility. This globulin had a much higher affinity for 25-hydroxycholecalciferol while vitamin D3 appeared to be associated first with an alpha-lipoprotein and with time because associated with the alpha-globulin. This alpha-globulin had a molecular weight of approximately 70,000 as determined by gel-filtration. Cholecalciferol appeared to bind tightly to the alpha-lipoprotein and resisted being stripped from the plasma. Thus, alpha-globulin appears to be the major carrier of vitamin D in the blood while the alpha-lipoprotein may aid in the transfer of cholecalciferol from the gut to the liver via the lymph system.     

Inhibition of fatty acid synthesis in bovine mammary homogenate by palmitic acid is not a detergent effect

Supplemental fat fed to dairy cows affects the fat composition of milk by reducing the yield of mammary synthesized fatty acids. The effect has been attributed to a potential allosteric inhibition of acetyl coenzyme-A, a key enzyme in fatty acid synthesis. In vitro experiments have demonstrated an inhibition of fatty acid synthesis when long-chain fatty acids are added to incubations. However, in vitro inhibition can result from a nonspecific detergent effect arising from an inherent physical property of fatty acids. An allosteric role for palmitic acid has not been tested in bovine mammary tissue. The objective of this experiment was to test the hypothesis that palmitic acid is an allosteric inhibitor of fatty acid synthesis in mammary tissue. We tested for a detergent effect by including a synthetic detergent, sodium dodecyl sulfate, under identical incubation conditions. A subcellular supernatant fraction of mammary tissue was used for incubations in the present experiment. The incubation system produced free fatty acids in a linear fashion for time and protein content. Results indicated that fatty acid synthesis was affected by the addition of palmitic acid to the incubations but not by caprylic acid, a short-chain fatty acid. Sodium dodecyl sulfate did not affect fatty acid synthesis at the concentrations used. The results of the present experiment indicate that palmitic acid inhibited fatty acid synthesis, and the effect was not the result of a detergent effect.     

Vitamin B12 binding proteins in bovine serum.

Examination of bovine serum by the diethylaminoethyl cellulose small column method revealed three proteins binding vitamin B12. The elution pattern suggested that they are similar to the three transcobalamins recognized in human serum. Distribution of unbound binding capacity among serum binders was assessed in serum from normal, ketotic, and B12- and Factor B-supplemented cows in early lactation. No major differences were observed among groups; however, cow serum displayed a pattern different from human serum. Mean total binding capacity of bovine serum for B12 as well as mean unbound binding capacity were lower than the corresponding means for human serum.     

Serine protease activity, bovine sperm protease, 66 kDa (BSp66), is present in hamster sperm and is involved in sperm-zona interaction

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head-head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.     

青霉素与牛血清白蛋白结合作用机理的光谱法研究

模拟牛乳的生理环境pH6.6、37℃水浴的条件下,运用荧光光谱、同步荧光光谱和圆二色光谱法研究了青霉素与牛血清白蛋白(Bovine Serum Albumin,BSA)的相互作用。青霉素对BSA的猝灭机制属于静态猝灭,并发生分子间非辐射能量转移。热力学数据显示,二者之间的作用力主要为氢键作用;同步荧光光谱表明,青霉素与蛋白质中接近色氨酸残基的区域发生了相互作用;圆二色光谱法研究蛋白二级结构结果显示,青霉素与蛋白质结合会改变蛋白质的构象,进一步说明了青霉素在牛乳会与蛋白质作用形成稳定复合物。     

Tripolyphosphate hydrolysis by bovine fast and slow myosin subfragment 1 isoforms

Polyphosphates are used in the meat industry to increase the water holding capacity of meat products. Tripolyphosphate (TPP) is a commonly used polyphosphate and it is metabolized into pyrophosphate and monophosphate in meat. The enzymes responsible for its metabolism have not been fully characterized. The motor domain of myosin (subfragment 1 or S1) is a likely candidate. The objectives of this study were to determine if bovine S1 hydrolyzes TPP, to characterize the TPPase activity of the fast (cutaneous trunci) and slow (masseter) isoforms, and to determine the influence of pH on S1 TPPase activity. S1 hydrolyzed TPP and in comparison with ATP as substrate, it hydrolyzed TPP 16–32% more slowly. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The V_max was 0.94 and 5.0 nmol Pi/mg S1 protein/min while the K_m was 0.38 and 0.90 mM TPP for slow and fast S1, respectively. Pyrophosphate was a strong inhibitor of TPPase activity with a K_i of 88 and 8.3 μM PPi for fast and slow S1 isoforms, respectively. Both ATPase and TPPase activities were influenced by pH with the activity being higher at low pH for both fast and slow S1 isoforms. The activity at pH 5.4 was 1.5 to 4-fold higher than that at pH 7.6 for the different isoforms and substrates. These data show that myosin S1 readily hydrolyzes TPP and suggest that it is a major TPPase in meat.     

Inhibition of human recombinant cytochromes P450 CYP1A1 and CYP1B1 by trans-resveratrol methyl ethers

CYP1A1 and CYP1B1 are the inducible forms of cytochrome P450 expressed in extrahepatic tissues, which are responsible for the biotransformation of polycyclic aromatic hydrocarbons, heterocyclic amines and estradiol to the carcinogenic intermediates. The aim of our research was to determine and compare the inhibitory effect of naturally occurring analogues of trans-resveratrol on the catalytic activities of human recombinant CYP1A1 and CYP1B1. Pinostilbene (3,4'-dihydroxy-5-methoxystilbene), desoxyrhapontigenin (3,5-dihydroxy-4'-methoxystilbene), and pterostilbene (3,5-dimethoxy-4'-hydroxystilbene) appeared to be very potent inhibitors of CYP1A1 catalytic activity with Ki values of 0.13, 0.16 and 0.57 microM, respectively. Results from this study indicate that trans-resveratrol analogues in which the hydroxy groups are substituted by methoxy groups exhibit a remarkably stronger inhibitory effect towards CYP1A1 in comparison to the parent compound. On the contrary, the potency of pinostilbene, desoxyrhapontigenin and pterostilbene towards CYP1B1 with Ki values of 0.90, 2.06 and 0.91 microM, respectively, was comparable to that of resveratrol. It appears that between these analogues, inhibition of CYP1A1 and CYP1B1 catalytic activities does not vary much regardless of the number and position of methylether substitution. The results suggest that the trans-resveratrol analogues: pinostilbene, desoxyrhapontigenin and pterostilbene, which occur in some food plants, might be considered as promising chemopreventive agents.