The presence or absence of sperm-borne oocyte-activating factor (SOAF) in the Antarctic minke whale haploid spermatogenic cells was determined by assessing the meiosis resumption of microinseminated mouse oocytes. The relative capacity of mature spermatozoa from mouse, cattle and whale to resume the meiosis of BDF1 mouse oocytes was, respectively, 90.5, 84.6 and 76.5%, while nuclear changes in nontreated or buffer-injected oocytes did not occur after 90min culture. In the whales, the late-stage elongating spermatids as well as the testicular spermatozoa triggered the meiosis resumption of mouse oocytes at similar rates (oocyte activation rates; 68.0 and 62.5%, respectively). The oocyte activating capacity of the early-stage elongating spermatids was significantly lower (25.0%), and the round spermatids did not activate mouse oocytes at all. This result suggests that the SOAF activity in the Antarctic minke whales is acquired during the early phase of spermiogenesis.

Production efficiency of transgenic rats was compared directly between the routine pronuclear microinjection of exogenous DNA solution (PNMI-Tg method) and the ooplasmic injection of sperm cells exposed to exogenous DNA solution (ICSI- Sciences, Okazaki, Japan Pronuclear DNA microinjection is the most convenient approach to produce transgenic animals (PNMI-Tg method). In general, the proportion of microinjected zygotes developing into transgenic offspring is <5% in rodents [1,3] and <1% in large domestic species [13,16]. Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg method), in which <7% of inseminated oocytes develop into transgenic mice [12,14] and rats [5,8]. Various factors, including the characteristics of exogenous DNA (component, chain length, preparation batch, and concentration of DNA), operator (experience of technician) and strain (genetic background), are known to influence the production efficiency of transgenic rodents [2,3]. The present study was undertaken to compare the production efficiency of transgenic rats between the PNMI-Tg method and the ICSI-Tg method, using six DNA constructs and five rat strains under the equal contribution of three operators.Six different exogenous DNA (3.0-11.7 kb) were prepared as described previously [5,8] and coded as A to F according to their size order. Exogenous DNAs with this size range have no adverse effect on the developmental potential of injected oocytes/embryos [7]. In brief, each linearized plasmid DNA was separated by 1.2% agarose gel electrophoresis, extracted from the gel using the QIAEX II (Qiagen Inc., Valencia, CA, USA), dissolved in 10 mM Tris-HCl (pH 7.6) / 0.1 mM EDTA, and stored at 4°C until use. Two strains of donor rats (Crlj:WI and BN/Crlj) were purchased from Charles River Japan, Inc. (Kanagawa, Japan) and three strains (Slc:SD, Slc:DLF1 and SDR/Slc) were from Japan SLC, Inc. (Shizuoka, Japan).The PNMI-Tg method routine for production of trans--Note-

Interspecies microinsemination assay was applied to examine the ability of minke whale haploid spermatogenic cells to induce Ca2+ oscillations and oocyte activation. Populations of round spermatids (RS), early-stage elongating spermatids (e-ES), late-stage elongating spermatids (1-ES) and testicular spermatozoa (TS) were cryopreserved in the presence of 7.5% glycerol on board ship in the Antarctic Ocean. Repetitive increases of intracellular Ca2+ concentration occurred in 0, 65, 81 and 96% of BDF1 mouse oocytes injected with the postthaw RS, e-ES, 1-ES and TS, respectively. A normal pattern of the Ca2+ oscillations was observed in 26-47% of the responding oocytes. Most oocytes that exhibited Ca2+ oscillations, regardless of the oscillation pattern, resumed meiosis (83-94%). These results indicate that whale spermatogenic cells acquire SOAF activity, which is closely related to their Ca2+ oscillation-inducing ability at the relatively early stage of spermiogenesis.

SummaryUsing an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against α-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.

Polymer powders, poly(methyl methacrylate) (PMMA) and polymmide (nylon 6, PA), were treated by four kinds of non-polymer-forming plasmas, Ar, Oz, CF4, and CCl4. The greatest weight loss was observed in Oz-plasma treatment due to its effective etching. In CF4 plasma at the flow rate of 10 cm3(STP)/min, the molecular weights of PMMA and PA were reduced effectively. From the analysis by X-ray photoelectron spectroscopy, a new peak that may be assigned to acid fluoride was found, which was resulted from the decomposition of ester and amide groups in PMMA and PA, respectively. In CCl4 plasma, polymerization took place preferentially.