Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs

In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD-1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Knowledge based diagnosis of inoculum properties and sterilization time in lactic acid fermentation

Summary A knowledge based system has been shown to be a powerful tool for diagnosing microbial activities during a fermentation process. Knowledge about lactic acid fermentation was collected by an experimental study ofLactobacillus casei. The effects of the inoculum properties and sterilization time on the cultivation were expressed in a form of a fuzzy rule-based knowledge network. The system was able to detect abnormal inoculum or sterilization conditions which caused malfunctions in the cultivations.     

Utility of the hot-wire spirometer

A comparative study has been carried out to investigate the performance of the hot-wire spirometer and the Rollingseal spirometer; the reproducibility of the data generated by an individual examiner; and the validity of data obtained by an "inexperienced examiner" using the hot-wire spirometer. The following results were obtained: 1. The experimental data given by the hot-wire spirometer were about 5-6% higher for FVC and FEV1, and about 10-12% higher for PFR, V50, and V25, respectively, when compared to the data generated by the Rollingseal spirometer. 2. The reproducibility of the data produced by the hot-wire spirometer operated by an "experienced examiner" was good, as the percent difference was about +/- 3%. 3. Reliable data were obtained with the spirometer even by an "inexperienced examiner" if he/she has gone through an on-site training concerning instrumentation and measurement methods for spirography.     

Glucosylation of Salicyl Alcohol by Gardenia jasminoides Cell Cultures

Cultured cells of Gardenia jasminoides produced both salicinand isosalicin from exogenously supplied salicyl alcohol. Theglucosylation activity of the cells was highest in the exponentialphase of growth and ca. 70% of the added substrate was convertedto the glucosides within 4 days. The rate of glucosylation wasalso dependent on the medium composition such as auxin and sucroseconcentrations. The ratio of salicin to isosalicin formed fromsalicyl alcohol was influenced by the growth stage of the culturedcells. Salicin was converted to isosalicin when exogenouslyadded to the culture. (Received October 11, 1985; Accepted March 10, 1986)     

Production of monozygotic twins following the transfer of bisected embryos in the goats

Embryos at the morula, blastocyst and hatched blastocyst stage were obtained from superovulated and naturally ovulated Japanese native goats. They were bisected into halves with a glass needle, and transferred immediately or after culture (for morula) to recipients. None of five does which received bisected morula became pregnant. Three of nine goats became pregnant after transfer of bisected hatched blastocysts, six of eleven recipients became pregnant. Four of them produced monozygotic twins and the remaining two produced singles. The present study demonstrated that the hatched blastocyst is suitable for bisection in the goat.     

Improvement of first-service pregnancy rate in cows with gonadotropin-releasing hormone analog

The effect of an intramuscular injection of a new analog of gonadotropin-releasing hormone (GnRH), fertirelin, on the first-service pregnancy rate in cows was investigated by a double blind experiment. A total of 1,194 cows was injected intramuscularly either with 100 mug of GnRH or placebo (physiological saline solution) at the time of first insemination postpartum. Pregnancy rate (number of cows calved/ number of cows serviced) was 57.2 % in 605 cows treated with GnRH, while the performance was 49.7 % in 589 cows of the placebo group. The difference of pregnancy rates in both groups was significant (P<0.05). GnRH injected at insemination was effective, especially in cows at the first and third lactations, cows at 101 days postpartum or later, cows with daily milk yield of 26-30 kg, and also in cows from the area where a regional average fertility was relatively low.     

Cortical vesicle exocytosis in isolated cortices of sea urchin eggs: Description of a turbidometric assay and its utilization in studying effects of different media on discharge

Cortices of unfertilized sea urchin eggs can be isolated in suspension and will discharge the attached cortical vesicles (CVs) in response to calcium. We describe a simple turbidometric assay for monitoring the Ca²⁺-induced discharge of these vesicles and also compare the discharge of vesicles isolated in a high salt medium (primarily KCl) with a medium more closely simulating the internal milieu of the cell (primarily potassium gluconate and glycine). Discharge in response to calcium is similar in both media, requiring approximately 6 μM calcium for one-half maximal discharge. There are, however, significant differences in morphology and protein composition of the two types of preparations (more proteins present in the glycine cortices) and also in the rate of discharge of the vesicles in response to calcium (KCl cortices with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 6 sec as opposed to 30 sec in the glycine cortices). The glycine cortices gradually lose their ability to respond to calcium but retention of calcium sensitivity is considerably aided by inclusion of ATP in the media; ATP has no apparent effect on discharge of the KCl cortices. The glycine cortices, as opposed to the KCl cortices, exhibited variation in calcium sensitivity during the breeding season and in the number of vesicles which would not break down in response to added calcium (referred to as refractory vesicles). The question of which type of cortex preparation most closely simulates the in vivo situation is discussed, and the view is presented that the glycine cortices most closely resemble the in vivo situation.