Full-term development of in vitro-matured, vitrified and fertilized bovine oocytes

In vitro-matured bovine oocytes were vitrified in a mixture of 2 M-dimethyl sulphoxide (DMSO), 1 M-acetamide and 3 M-propylene glycol dissolved in mTCM199. After vitrification and thawing, the oocytes were exposed to 2-0.1M-sucrose solution in 1 or 12 steps to remove the cryoprotectants. Then the oocytes were fertilized in vitro and co-cultured with a monolayer of cumulus cells for 7 days. Nine of 88 inseminated oocytes developed to the blastocyst stage. Three blastocysts were transferred to 3 recipients, resulting in 2 pregnancies.     

Enhancement by cytochalasin B of ouabain-stimulated catecholamine secretion from cultured bovine adrenal chromaffin cells: possible relation to alteration in Na+/K+-pump activity

1. Catecholamine secretion evoked by ouabain from cultured bovine adrenal chromaffin cells has previously been shown to be markedly enhanced by pretreatment of the cells with cytochalasin B (Morita et al., 1988). To elucidate a possible mechanism of this enhancement, the stimulatory action of ouabain on Ca2+ influx as well as catecholamine secretion was then examined in the cells pretreated with or without cytochalasin B. The effect of cytochalasin B pretreatment on the inhibitory action of ouabain on the Na+/K+ pump was also examined by measuring 86Rb+ uptake into the cells. 2. Pretreatment of the cells with cytochalasin B caused enhancement of ouabain-induced catecholamine secretion, and this enhancement was accompanied by the elevation of ouabain-stimulated 45Ca2+ uptake into the cells. The inhibitory action of ouabain on 86Rb+ uptake was significantly enhanced by pretreatment of the cells with cytochalasin B under the same conditions. 3. These results indicate that the enhancement of ouabain-induced catecholamine secretion caused by cytochalasin B pretreatment may be due to the increase in ouabain-stimulated Ca2+ influx into the cells and, furthermore, suggest the possibility that this increase in Ca2+ influx may be attributed to the potentiation of the inhibitory action of ouabain on the Na+/K+ pump in the adrenal chromaffin cell. Thus, the present study provides an evidence for a possible role of microfilaments as one of the intrinsic factors modulating the plasma membrane functions.     

Developmental changes in the incidence of chromosome anomalies of bovine embryos fertilized in vitro.

In total, 196 two- to 32-cell bovine embryo and 104 blastocysts were obtained by the in vitro fertilization of follicular oocytes matured in vitro, and 15 blastocysts fertilized in vivo were used. Chromosomal anomalies in these embryos and the inner cell mass (ICM) separated immunologically were investigated. Chromosomal anomalies were observed in 12.1% (5/41) of 2-cell embryos, 20.0-36.4% of 4- to 16-cell embryos, 7.1% (1/14) of 17- to 32-cell embryos, 44.2% (15/34) of blastocysts, and 18.6% (13/70) of ICM cells derived from in vitro fertilization. These anomalies were mainly 3N and 4N at 2-cell stage, 1N and 1N/2N at 4- to 32-cell stages, and 2N/4N in blastocysts and in their ICM cells. Chromosomal anomalies of blastocysts from in vivo fertilization and their ICM were observed in 20.0% (1/5) of blastocysts and 33.3% (3/9) of ICM cells and these compositions were mainly 2N/4N. These results indicate that the abnormalities at early and blastocyst stages of embryos derived from in vitro fertilization were caused by abnormal fertilization in vitro and abnormal cleavage, respectively. Furthermore, a definite location of the chromosomal anomalies was observed in the trophectoderm of blastocysts derived from in vitro fertilization.     

环境温度对人工饲料育家蚕体温和呼吸量的影响

本文以全龄人工饲料饲育的5龄幼虫为材料,调查了二化性和多化性家蚕品种在不同气温条件下的体温及呼吸量的差异.蚕是变温动物,体表温度及体内温度和环境气温虽大致相同,但也有一些微小的差异,体内温度>体表温度>气温.在0-30℃范围内,体内温度比气温高不到1℃.0℃时,5龄蚕仍有微弱的呼吸作用,呼吸强度随体温上升而缓慢上升,超过10℃后,上升速度加快.多化性品种在25—36℃范围内,随温度上升呼吸量增加速度有比二化性品种慢的倾向,这可能与多化性品种抗高温性有关.另外,饷食前后的呼吸量变化相当大,饷食后呼吸量急速上升,约1小时后达起蚕的2.4倍左右,以后相对稳定.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, -actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic -actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads

We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7-8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate.     

Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida

The time for solubilization of the bovine zona pellucida in a hypotonic buffer containing 5% (v/v) beta-mercaptoethanol and 7 mol urea l-1 increased by 10% after fertilization. Coupling with a specific fluorescent thiol probe, monobromobimane (mBBr), was markedly greater in the zona pellucida of ovarian eggs compared with fertilized eggs, indicating that the cysteine residues in the zona pellucida of unfertilized eggs are oxidized to cystines during fertilization. After endo-beta-galactosidase digestion to remove N-acetyllactosamine repeats of the carbohydrate chains, three zona pellucida glycoproteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane groups in the three components and SDS-PAGE revealed that intramolecular disulfide bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were formed during fertilization, but oxidation of cysteine residues in ZPC was low. Specific proteolysis of ZPA during fertilization was also observed. These results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida structure, which is responsible for hardening of the zona pellucida.     

Involvement of abscisic acid in bulb dormancy of Allium wakegi Araki. II. A comparison between dormant and nondormant cultivars

The content of abscisic acid (ABA) in bulbs of two Allium wakegi Araki cultivars, Kiharabansei No. 1 (dormant type) and Ginoza (nondormant type), was similar and changed similarly during the development and storage of the bulbs. It increased during bulb development, reached a maximum shortly after bulb harvesting, and gradually decreased during bulb storage. The bulbs of Kiharabansei No. 1 showed dormancy correlated with the change in ABA content, but those of Ginoza did not show significant dormancy throughout the experimental period. The ABA content in the buds of dormant bulbs of Kiharabansei No. 1 did not change after planting of bulbs, but that of nondormant bulbs of Ginoza planted on the same day rapidly decreased after planting. Application of ABA to bulbs delayed sprouting of both cultivars, but dormant bulbs of Kiharabansei No. 1 had higher sensitivity to ABA than the bulbs of Ginoza or the bulbs of Kiharabansei No. 1 partly released from dormancy. These results suggest that the decrease in the ABA content after planting (watering) and low sensitivity to ABA are correlated with the nondormancy of Ginoza.     

Correction to: Rhamphocottus nagaakii (Cottoidea: Rhamphocottidae), a new species of grunt sculpin from the northwestern Pacific,with notes on the phylogeography of the genus Rhamphocottus

A new species of grunt sculpin, Rhamphocottus nagaakii inhabiting the northwestern Pacific, previously identified as Rhamphocottus richardsonii Günther 1874, is described based on genetic evidence and morphological differences. The new species can be distinguished based on morphometric characters related to the head, including head length [45.3–54.6% of standard length (SL)], postorbital head length (18.8–25.5% SL) and the length of pectoral-fin base (15.8–20.7% SL), which are smaller than in R. richardsonii (53.6–60.5% SL, 26.2–31.7% SL, and 19.5–25.2% SL, respectively). Genetic differences between two species markedly exceed levels for intra-specific differences. Rhamphocottus nagaakii is considered to have arisen from a common ancestor of the two species, which probably inhabited somewhere the North Pacific Rim around the Aleutian Archipelago. During a period of cooling in the Pliocene or the Miocene, R. nagaakii and R. richardsonii became separated to the southern regions of the northwestern and northeastern Pacific, and subsequently underwent speciation.

     

The autoregulation of nodulation mechanism is related to leaf development

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.