Inhibition of acrosin by oviductal secretory immunoglobulin A

A major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α-chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.     

The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster

Taurine and hypotaurine were examined for their efficacy in replacing sperm motility factor (SMF), prepared from bovine adrenal cortex, for in vitro fertilization in the golden hamster. Combinations of these amino acids at concentrations of 0.001, 0.01, 0.1, and 1 mM together with 16 μM isoproterenol (a catecholamine β-agonist) were added to the sperm incubations. After three hours of sperm preincubation, oviductal eggs were added to the sperm suspensions and examined for penetration and stage of fertilization after three or five hours of culture. At 0.001 mM, neither taurine or hypotaurine was capable of maintaining motility of hamster sperm for four to 4½ hours or of inducing fertilization. With all other concentrations, both amino acids were found to maintain motility of sperm as well as SMF. Hypotaurine stimulated motility to a greater extent than taurine and both required isoproterenol for the greatest motility. A low proportion of cumulus-free ova were fertilized when sperm were preincubated with either amino acid alone over the range of 0.01 to 1 mM; however, over 80% fertilization was consistently obtained when isoproterenol was also present during sperm incubation. Proportions of ova fertilized with taurine or hypotaurine present during sperm preincubation were comparable to those achieved with SMF. The possibility that taurine or hypotaurine is the sperm motility factor is discussed. After three hours of sperm/egg incubation, a lag in the early events of fertilization was observed in experimental groups treated with one of the amino acids (0.01 mM) alone compared with groups treated with isoproterenol present. However, if sperm/egg incubation was extended from three to five hours, no increase in number of eggs penetrated was found. Therefore, the delay observed at three hours was considered a function of fewer numbers of capacitated sperm present in the absence of isoproterenol rather than of the need for an extended capacitation time.     

Evidence for conformeric states of Rhodopsin

Spectrophotometric measurements of metarhodopsin II appearance are made on five different kinds of rhodopsin preparations. Although the preparations differ greatly in their rhodopsin: phospholipid ratio, the meta II kinetics in all of them are strikingly similar in certain respects. Meta II appearance kinetics in all of the preparations are best described by two and only two exponentials. The ratio of these two rates is always about 5. The fast fraction: slow fraction ratio depends upon temperature. These fractions are reversibly convertible in the dark, and are interconverted on a time-scale which is long compared to the meta II appearance rate. It is shown that the kinetics of the earlier step in the bleaching sequence, viz., lumi- meta I, is also described by double exponentials. Again the ratio of rates is ca. 5 and the fast-slow fractions correspond to those found in the meta I meta II step. It is proposed that these facts support an hypothesis for the existence of two conformeric states of rhodopsin which are in thermal equilibrium. Thermodynamic parameters associated with this proposed equilibrium are presented.     

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycorrhizal status of herb-layer plants in a fertilized oak-pine forest

In the years 1986–1988 investigations were carried out on the influence of fertilizers on the mycorrhizal status of herb-layer plants of a mixed oak-pine forest in the Niepolomice Forest (Southern Poland). The site enrichment with N, P, K altered the frequency of mycorrhizal infection in most species investigated and decreased the percentage of colonized root cells. Some species disappear after fertilization (e.g. Vaccinium myrtillus L.). Among the species vigorously expanding after the treatment, mainly nonmycorrhizal plants (e.g. Festuca gigantea (L.) Vill.) were found. Some species (e.g. Milium effusum L.) favourably reacting to mineral fertilization, lost their mycorrhizae.     

Effects of legumes on soil physical quality in a maize crop

The effect of intercropped legumes and three N fertilizer rates in a continuous maize (Zea mays L.) cropping system on the physical properties of two soils were investigated for three years. The legumes, being a mixture of alfalfa, clover and hairy vetch, had a significant cumulative effect on some physical properties of both soil. The lowest stability and smallest mean weight diameter of soil aggregates were associated with monoculture maize plots. Aggregate size and stability were not affected by N fertilization at any of the rates of 0, 70, and 140 kg ha^-1 in intercropped plots, except that aggregate stability was actually reduced by N fertilization in one soil, the Ste. Rosalie clay. In maize plots in both soils, stability and size of soil aggregates were significantly increased with increased added N. Intercropped legumes significantly decreased dry bulk density and soil penetration resistance. Added N had no measurable influence on these compaction factors. Soil water properties were not significantly affected by either intercropping or N fertilization. Positive effects noted on soil aggregation and other physical properties in intercropped plots are the result of enhanced root activity, or incorporation of legumes as green manure, or both. Improvement of soil structure in maize plots associated with increasing N application was the result of increased maize-root residues.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Temperature,hydrochemistry and plankton in Wicken Brickpits, 1930–1931

Temperature, selected chemical constituents and plankton were analysed from three depths, fortnightly, by day and occasionally by night, in two flooded brickpits, between May 1930 and August 1931.Br was 3.3 m deep, with clear water and little weed; III was only 1.8 m deep, with thick submerged weed, more sheltered than Br and becoming eutrophic. Neither had direct inflow nor outlet. Both conformed to the second order for dimictic lakes, with summer and winter stratification, leading, for varying periods, to stagnation marked by pH between 6.6 and 7.2 and O₂ much depleted or absent at the bottom.During stagnation, release of SiO₂, soluble inorganic phosphate-P, ammonium ions and bases from the bottom was shown by sampling water close to the mud. This finding preceded the definition of redox potential by others.Thermistor temperature measurements, used for the first time, showed comparable summer gradients in both ponds: on windy days these were usually sigmoid, with discontinuity at various depths, or else nearly isothermal; on calm days, they were exponential, when transmission of heat, from surface to bottom, was apparently due to radiation alone, without water circulation. Stability was then high.Diatoms were abundant in both ponds; but planktonic Rotifera and Crustacea differed in the two, apart fromDiaptomus gracilis, which was dominant in both.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.