Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.     

Cytochemical behavior of rabbit spermatozoa chromatin in male and female genital tracts

Cytochemical parameters have been used to investigate the physicochemical modifications of rabbit spermatozoa chromatin in the male and female genital tracts. Analysis of these parameters revealed the progressive condensation of the DNA-protein complex along the male genital tract and its progressive decondensation along the female genital tract.     

Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa

Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.     

Nuclear and chromatin structure in rat spermatozoa

The nuclei of mature mammalian spermatozoa contain a highly ordered, lamellar substructure, presumably constituting the nucleoprotein of the haploid chromosomal complement. With a view toward constructing a plausible model of chromatin packing in sperm, we have determined some of the quantitative parameters associated with these “nuclear lamellae” in rat spermatozoa. Epididymal sperm from white, Sprague-Dawley rats were examined by conventional sectioning methods, freeze fracture of fixed and unfixed specimens, and by whole mount replica techniques. Fixation and glycerolation did not significantly alter nuclear structure as seen by freeze fracture. Numerical data obtained from cross fractures of sperm heads indicate that the number of lamellae are quite constant at 10.4 ± 1.8 and that the linear measure of the lamellae is 7.2 ± 2.3 μm per cross fracture. The total area of cross fracture, assuming an elliptical profile is 2.3 k 0.7 μm² and the thickness of the lamellae is 18.2 ± 3.5 nm with a range of 13.5 to 25.5 nm. An estimate of the total surface area of the nuclear lamellae could be made from measurements of projected nuclear area (from replicas and sections) as 173 ± 15 μm². From these data and the known amount of DNA in the rat sperm nucleus, a model can be proposed for the organization of the nucleoprotein in these lamellar sheets. It is suggested that the chromatin is arranged in a coiled-coil configuration closely associated together in a side-by-side fashion and continuous in extent. Approximate calculations based on this simple model are within a factor of 2 or 3 of predicting the correct amount of DNA in the sperm nucleus.     

Phenobarbital stimulation of housefly chromatin template activity

The effect of phenobarbital on the chemical composition and template activity for Escherichia coli RNA polymerase of housefly chromatin is examined. Chromatin isolated from the resistant Fc housefly strain after administration of phenobarbital has a much higher template activity as compared to control chromatin. The effect is minimal in a susceptible housefly strain. Phenobarbital treatment alters the composition of the RNA polymerase product which results in an increase of the $\text{(A + U)}\text{(C + G)}$ ratio with chromatin from the Fc strain as template, while the $\text{(A + G}\text{)}\text{(}\text{C + U)}$ and $\text{(A + U}\text{)}\text{(}\text{C + G)}$ ratios are decreased when chromatin of the susceptible strain is used.A substantial portion of the intracellular phenobarbital is physically bound to nuclei and microsomes, but most of it is associated with cytosolic macromolecules. Less than 0.2% is covalently bound to cellular macromolecules. Phenobarbital apparently binds to a small mol. wt cytosolic macromolecule(s) as shown by sucrose density gradient centrifugation and Sephadex G-25 column chromatography.     

Immunoferritin labeling of rabbit spermatozoa

Ferritin-conjugated goat IgG binds nonspecifically to rabbit sperm. This restricts use of ferritin-labeled goat antiglobulins as indirect labels in rabbit sperm antigen localization. Ferritin-conjugated Protein A does not bind nonspecifically to rabbit sperm and is a satisfactory substitute for indirect (“secondary”) labeling of sperm antigens.     

Occurrence of platelet-activating factor in rabbit spermatozoa

Spermatozoa obtained from rabbit ejaculate were analyzed for the presence of platelet-activating factor [PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)] by using standard HPLC and TLC procedures. Fractions corresponding to synthetic PAF (AGEPC) revealed PAF-like activity amounting to 0.35 +/- 0.06 pmol/10(8) cells (mean +/- SE) as determined by bioassays based on the release of [3H]serotonin from washed rabbit platelets. This activity was lost upon base-catalyzed methanolysis, but was restored to the original level after reacetylation. Analysis of the phosphatidylcholine (PC) fraction by GC-MS subsequent to base-catalyzed methanolysis showed that 1-O-alkyl-2-acylphosphocholine comprises about 12% of the PC fraction with alkyl chain lengths of 16:0 (88%) and 18:0 (12%).     

Arginine uptake by rabbit spermatozoa

The transport of l-arginine by rabbit spermatozoa was found to proceed by saturable, chemically specific mechanisms. Kinetic analysis of initial rates of transport at substrate concentrations from 1.0 μm to 1.0 μm indicate the presence of two saturable transport components. A low-affinity component has an apparent K_m of 0.64 μm and an apparent V of 43.4 nmol/10⁸ cells/30 s. A second, high-affinity component has an apparent K_m of 4.0 μm and an apparent V of 425 pmol/10⁸ cells/30 s. Rabbit spermatozoa actively transported l-arginine in a range of pH values from 6.5 to 10.5 with a pH optimum for the low-affinity component of 7.2–7.6 and a pH optimum for the high-affinity component of 7.8–8.0. Inhibitor studies indicate that the energization for transport may be dependent on ATP rather than on a pH gradient or transmembrane potential. Competition experiments with arginine analogs and amino acids suggest that the high- and low-affinity components may recognize the terminal guanidino group.