During spermatogenesis, DNA in the sperm head becomes more tightly condensed as histones are replaced by protamine-like molecules. In this article, the question is asked whether, during the production of this highly differentiated cell, controls are imposed on the spatial organization of DNA within the nucleus. Heads from bull spermatozoa were isolated by a technique that removed the plasma membrane and acrosomal contents, and the DNA was induced to decondense by addition of 2-mercaptoethanol and trypsin. Under these conditions, decondensation was induced in all regions of the head. To determine whether there was any spatial restraint on packaging of the genome, three DNA probes were used (pl.709-512, containing an interspersed repetitive sequence; pCSIH, containing a copy of the major bovine centromeric statellite sequence; p18 s and p28 s, containing the 18 S and 28 S ribosomal genes) that might be expected to hybridize to different regions. Results showed that the interspersed repetitive probe hybridized to all regions of the head, whereas the ribosomal and centromeric probes hybridized to sequences that were largely confined to the equatorial region of the sperm. We conclude that organization of the genome in the bovine sperm nucleus is not random.
Microinjection of inositol 1,4,5-triphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6.8 but a full cortical reaction occurring at pH8.0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTP gamma S also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.
When sheep ovarian follicle cells are maintained in an O2-rich environment their cells are metabolically coupled, as monitored by observing the exchange of [3H]choline; choline metabolites were detected up to 4 mm from the explant under these control conditions. When the tissues were placed in a CO2-rich environment the cells became uncoupled physiologically and choline metabolites were no longer exchanged. The cells in these two states, coupled and uncoupled, were examined by freeze-fracture. The initial controls were characteristic of ovarian follicular tissue exhibiting large macular plaques with regular outlines composed of PF intra-membranous particles (IMPs), which were arrayed in rows with IMP-free aisles. With uncoupling, the junctional plaques became irregular at the periphery, they became loosely packed and IMPs began to ‘stream’ out laterally across the membrane. Ultimately they were reduced to negligible IMP clusters or free IMPs. Analyses of the IMPs with an image analyser confirmed that in the uncoupled state the gap-junctional IMPs were dispersed over the membranes. On return to an O2-rich environment, the cells became recoupled as monitored by physiological criteria and in freeze-fracture replicas IMPs reclustered into macular, albeit smaller, plaques. These results support the contention that with uncoupling, gap-junctional particles are free to move and hence may become dispersed over the membrane face, with the possibility of being re-utilized to form junctions anew when conditions for coupling are re-established.
Spinach leaf disks grown initially in the dark, show increased cell expansion and chloroplast replication when transferred to the light. These changes are accompanied by increases in the total amount of DNA and the incorporation of [3H]thymidine (3H-TdR). Autoradiography of EDTA-separated cells dried on to glass slides was used to follow changes in 3H-TdR incorporation in both chloroplasts and nuclei. Specificity of incorporation was confirmed by nuclease studies. DNA synthesis occurs in both the chloroplasts and nuclei, and is highest just prior to, and during the period of most rapid cell growth and chloroplast replication which occurs shortly after the transfer to the light. Light, however, appears to have a greater and more immediate effect on nuclear DNA synthesis. Though nuclear and chloroplast DNA syntheses follow similar patterns during disk growth, in a given cell, chloroplast DNA synthesis can be separate in time from nuclear DNA synthesis. The increased nuclear DNA synthesis is possibly required to support the increased population of chloroplasts, while chloroplast DNA synthesis is associated with chloroplast division. If the disks are not transferred to the light but kept in darkness, chloroplast 3H-TdR incorporation remains high, though chloroplast division is reduced. Epidermal cells in light-grown tissue also show 3H-TdR incorporation but low rates of chloroplast division. It would appear that chloroplast DNA synthesis in mesophyll cells from light-grown tissue shows a general relation to chloroplast division, but there does not appear to be an obligatory close coupling between the 2 processes.