DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 ± 0.16) and testicular spermatozoa (4.03 ± 0.34), and then decreased sharply (p &#60; 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 ± 0.03). However, the ratio was not different among corpus (0.69 ± 0.01), cauda epididymis (0.68 ± 0.11) and ejaculated spermatozoa (0.63 ± 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p &#60; 0.01) during passage of spermatozoa from testis (4.74 ± 0.16) through epididymis (caput, 2.72 ± 0.08; corpus, 1.07 ± 0.03; cauda, –0.05 ± 0.05; ejaculated, 0.08 ± 0.03). The acridine orange red/green fluorescence ratio increased (p &#60; 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.</p

The Influence of End Frictions on Stresses in Compressed Specimens

The present paper concerns the influences of end frictions on stresses in compressed rectangular and cylindrical specimens. In numerical calculations, the finite element method was employed. The following conclusions are made on the influences of the end frictions on stresses in the compressed specimens. (i) The more the end friction between the end of the specimen and the platten is reduced the more uniform stresses are developed in the specimen. (ii) When Poissoin's ratio is 1/6 and the coefficients of the end friction are approximately larger than 0.25, no lubrication can be practically expected, in other terms stresses in specimens with such coefficients of end friction are practically the same as stresses in specimens completely restrained at the ends. (iii) Stresses in both rectangular and cylindrical specimens are similar. The only difference is that the deviations of the axial stresses for various coefficients of frictions from the average are larger in cylindrical specimens than those in rectangular ones. (iv) Stresses in the mid-height region of the specimens are not so sensitive to the end friction as the compressed ends. Uniformity of stresses depends on the width-height or radius-height ratios as well as the end frictions. (v) For sufficiently small coefficients of the end friction, some portions of the end of the specimen slide and the shear stresses on the end become terrace-like and axial stressses become more or less uniform

Clustering of Emission-line Stars in the W5E HII region

We have made a new survey of emission-line stars in the W5E HII region to investigate the population of PMS stars near the OB stars by using the Wide Field Grism Spectrograph 2 (WFGS2). A total of 139 H-alpha emission stars were detected and their g'i'-photometry was performed. The spatial distribution of them shows three aggregates, i.e., two aggregates near the bright-rimmed clouds at the edge of W5E HII region (BRC 13 and BRC 14) and one near the exciting O7V star. The age and mass of each H-alpha star were estimated from the extinction corrected color-magnitude diagram and theoretical evolutionary tracks. We found, for the first time in this region, that the young stars near the exciting star are systematically older (4 Myr) than those near the edge of the HII region (1 Myr). This result supports that the formation of stars proceed sequentially from the center of HII region to the eastern bright rim. We further suggest a possibility that the birth of low mass stars near the exciting star of HII region precede the production of massive OB stars in the pre-existing molecular cloud.Comment: 16 pages, 7 figures, 3 tables, accepted for publication in PAS

Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus–oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitromatured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozenthawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.</p

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.</p

Develop to Term Rat Oocytes Injected with Heat-Dried Sperm Heads

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term