The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

植物细胞的遗传全能性与组织培养形态发生控制

引言自1902年德国植物学家Haberlandt提出植物的单个细胞可能具有分化的全能性的理论以来,人们才开始从事植物组织培养,以致今天广泛用来有意识地定向控制遗传变异和人工创造植物新类型的研究。到今据不完全统计,全世界约有1000种高等植物作过离体培养尝试。根据大量实验结果证明,植物单个细胞具有遗传的全能性,     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum

Production of bulblets of Muscari armeniacum through tissue culture is enhanced when 1 g/l activated charcoal is added to a modified Murashige and Skoog (MS) medium. Bulblet regeneration is direct from bulb scale explants with no intermediate callus growth. Bulblets can be transferred successfully to a greenhouse environment directly from aseptic culture.     

Anther and tissue culture-induced grain chalkiness and associated variants in rice

Rice, Oryza sativa, plants regenerated from anther culture with and without in vitro selection pressure were evaluated for chalky seed. Progeny evaluated included 21 spontaneously doubled haploids selfed 4 times, progeny from plants regenerated from S-aminoethylcysteine resistant callus selfed 4 times and backcrosses of both types to the parental type. All lines with in vitro histories had higher seed chalkiness than the controls both in the intensity of chalkiness and in the number of seeds expressing the character. The full range of intensity and amount of chalkiness was expressed in the progeny. The average intensity of anther/tissue culture-derived progeny was 4–5, based on a scale of 1 (translucent) to 10 (fully opaque), and the average amount of chalkiness within plants sampled was 50–75 percent. The chalky characteristic is transmitted from parent to offspring into a range of identifiable F₂ segregants. Disclaimer statement Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Cross reactivity and enzyme sensitivity of immunoaffinity purified Sm/RNP antigens

Immunoaffinity purified Sm/RNP antigens from buffalo and goat liver were studied to determine the role of RNA and proteins towards the antigenicity of Sm and RNP antigens. A more direct approach using enzyme-linked immunosorbent assay on nylon beads has been utilized to look into the problem. The effect of enzyme treatment and the role of RNA and protein fractions in influencing antigenicity have been described. RNA seems to be involved in the maintenance of RNP specific polypeptides in suitable conformation so as to keep them in solution. Removal of RNA leads to insolubilization of RNP specific polypeptides. Antibodies to Sm and RNP antigens have been shown to cross react with poly A containing heterogeneous nuclear ribonucleoprotein with no cross reactivity with thymus RNA or DNA.     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis