Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.     

THE RELATION BETWEEN THE INTRACELLULAR RIBONUCLEIC ACID DISTRIBUTION AND AMINO ACID INCORPORATION IN THE LIVER OF THE DEVELOPING CHICK EMBRYO

The RNA-P and DNA-P content of the nucleus and the RNA-P content of the whole cell of the livers of 8- to 20-day chick embryos and of adult fowls have been determined. The DNA-P content of the liver nuclei was slightly higher in the 8- and 10-day embryo than in all the other stages examined. A significant decrease in the RNA content of the cell occurred during embryonic development. The RNA content of the adult cell was the same as that of the 14- to 16-day embryo. The proportion of the cellular RNA contributed by the nucleus also decreased during development. In respect to both nuclear RNA content and distribution of RNA between nucleus and cytoplasm, the adult resembled the 8- to 12-day embryo. Examination of the fine structure of the cell showed that, as development progressed, free ribosomes decreased in number and the rough membranes increased. Slices of 8-, 14-, and 20-day embryonic livers and of adult livers were incubated with ¹⁴C-leucine, and the amount of labeled amino acid incorporated into whole tissue protein and into the proteins of the subcellular fractions was measured. Embryonic liver incorporated ¹⁴C-leucine 15 to 30 times more rapidly than adult liver. The microsomal protein was always more highly labelled than the protein in any other subcellular fraction; however, in the 8-day embryonic and the adult liver the proportion of total counts found in the nuclear fraction was considerably higher than in the 14- or 20-day embryonic liver. The significance of an apparent correlation between the proportion of the cell's RNA contributed by the nucleus and the proportion of total counts in the nuclear fraction is discussed.     

Stress and relapse of breast cancer.

To elucidate the association between stressful life events and the development of cancer the influence of life stress on relapse in operable breast cancer was examined in matched pairs of women in a case-control study. Adverse life events and difficulties occurring during the postoperative disease free interval were recorded in 50 women who had developed their first recurrence of operable breast cancer and during equivalent follow up times in 50 women with operable breast cancer in remission. The cases and controls were matched for the main physical and pathological factors known to be prognostic in breast cancer and sociodemographic variables that influence the frequency of life events and difficulties. Severely threatening life events and difficulties were significantly associated with the first recurrence of breast cancer. The relative risk of relapse associated with severe life events was 5.67 (95% confidence interval 1.57 to 37.20), and the relative risk associated with severe difficulties was 4.75 (1.58 to 19.20). Life events and difficulties not rated as severe were not related to relapse. Experiencing a non-severe life event was associated with a relative risk of 2.0 (0.62 to 7.47), and experiencing a non-severe difficulty was associated with a relative risk of 1.13 (0.38 to 3.35). These results suggest a prognostic association between severe life stressors and recurrence of breast cancer, but a larger prospective study is needed for confirmation.