Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

Participation of Opioid Peptides in Sucking‐induced Oxytocin and Prolactin Secretions in Lactating Goats

The role of opioid peptides in the secretion of oxytocin (OT) and prolactin (PRL) induced by sucking was studied in goats. Seven goats were isolated with their kids (four singletons and three twins) in individual corrals 3–4 weeks after parturition. On day 1 of the experiment, the kids were separated from the does for 7 h and were weighed before and 15 min after being reunited with their mothers to assess the amount of milk obtained by sucking. The does were blood‐sampled 10 min before and at the end of the sucking period. On day 2, a similar protocol was followed, but naloxone was given immediately after the first blood sample. On day 3, the protocol was repeated but saline vehicle was injected instead of naloxone. On day 5, the naloxone experiment was repeated as on day 2. Milk ejection was evaluated as the difference in the weight of the kids before and after sucking for 15 min, and the maternal serum levels of OT and PRL were measured by radioimmunoassay. A significant decrease in the weight gain of the kids was obtained when the mothers were treated with naloxone on day 2. Consistently, serum levels of OT and PRL induced by sucking were significantly reduced; indicating that sucking‐induced OT secretion for milk ejection in lactating goats is facilitated by opioid peptides. In a second experiment performed in the same animals 10 days later, the administration of OT, immediately after naloxone administration, prevented the decrease in the weight gain induced by naloxone, suggesting that the effect of the opioid antagonist on milk ejection in goats is a result of a reduced OT secretion. The results of this study confirm the importance of sucking‐induced OT secretion for milk ejection in lactating goats, and indicate that OT and PRL secretion are regulated by opioid peptides in this species.     

Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献   

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and rhodamine 123; protocol 2: PI, FITC‐PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC‐PSA and CMXRos; and protocol 4: PI, H342, FITC‐PSA and JC‐1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility ≥80% and abnormal morphology ≤10%. The semen was diluted in Modified Tyrode’s medium (TALP) (25 × 10⁶ spermatozoa/ml) and split into two aliquots, one sample was flash‐frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash‐frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R² = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R² = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R² = 0.84, 0.93 and R² = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC‐1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.     

A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein

The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of bicoid mRNA and the posterior localization of oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive bicoid and oskar localization have been elusive, oocyte microtubules are known to be essential. Here we report that the plus end-directed microtubule motor kinesin I is required for the posterior localization of oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I.     

Lunar surface magnetic fields and their interaction with the solar wind: results from lunar prospector

The magnetometer and electron reflectometer experiment on the Lunar Prospector spacecraft has obtained maps of lunar crustal magnetic fields and observed the interaction between the solar wind and regions of strong crustal magnetic fields at high selenographic latitude (30 degreesS to 80 degreesS) and low ( approximately 100 kilometers) altitude. Electron reflection maps of the regions antipodal to the Imbrium and Serenitatis impact basins, extending to 80 degreesS latitude, show that crustal magnetic fields fill most of the antipodal zones of those basins. This finding provides further evidence for the hypothesis that basin-forming impacts result in magnetization of the lunar crust at their antipodes. The crustal magnetic fields of the Imbrium antipode region are strong enough to deflect the solar wind and form a miniature (100 to several hundred kilometers across) magnetosphere, magnetosheath, and bow shock system.     

The Solar Acoustic Spectrum and Eigenmode Parameters

The Global Oscillation Network Group (GONG) project estimates the frequencies, amplitudes, and linewidths of more than 250,000 acoustic resonances of the sun from data sets lasting 36 days. The frequency resolution of a single data set is 0.321 microhertz. For frequencies averaged over the azimuthal order m, the median formal error is 0.044 microhertz, and the associated median fractional error is 1.6 x 10(-5). For a 3-year data set, the fractional error is expected to be 3 x 10(-6). The GONG m-averaged frequency measurements differ from other helioseismic data sets by 0.03 to 0.08 microhertz. The differences arise from a combination of systematic errors, random errors, and possible changes in solar structure.     

Direct demonstration of milk as an element of archaeological economies

The stable carbon isotope (delta13C) compositions of individual fatty acid components of remnant fats preserved in archaeological pottery vessels show that dairying was a component of archaeological economies. Characteristic delta13C values arise from biases in the biosynthetic origins of the C18:0 fatty acids in milk and adipose fat. Milk and adipose fat from animals raised on similar pastures and fodders have distinct isotopic signatures.