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H. SPIELMANN

Summary.

The development of rat zygotes in vitro to the two-cell stage occurred if lactate, phosphoenolpyruvate (PEP), pyruvate or oxaloacetate were present in the media. When rat and mouse zygotes were cultured in the same droplet of medium containing lactate or PEP, mouse zygotes did not develop to the two-cell stage but the rat zygotes cleaved.

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J. Schiffner and H. Spielmann

Abteilung Embryonal-Pharmakologie, Department of Pharmacology, Freie Universität Berlin, 1 Berlin 33, Thielallee 69/73, West Germany

Quantitative and qualitative aspects of protein synthesis during the preimplantation development of the mouse have been studied by several groups (Weitlauf & Greenwald, 1967; Brinster, 1971 ; Epstein & Smith, 1973, 1974). Because of methodological difficulties, however, little attention has been given to the problem of estimating the absolute amounts of protein found in mouse embryos at successive stages of development. The protein content of embryos from mice induced to superovulate has been determined for the first 5 days of development by a micro-Lowry technique (Brinster, 1967). The protein content decreased from 28 ng at the 1-celled stage to 20·5 ng at the morula stage, and there appeared to be a 15 % increase in total protein when the blastocyst was formed (23 ng). Using the same technique, Weitlauf (1973) reported a net increase of 52 % (23

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H. Spielmann, H.-G. Eibs, C. Mentzel and D. Nagel

Summary. The binding of antibodies against LDH-X by preimplantation mouse embryos was studied to detect LDH-X from spermatozoa in embryos after fertilization. Incubation of preimplantation mouse embryos with rabbit anti-mouse-LDH-X–IgG and then with peroxidase-labelled goat anti-rabbit IgG revealed a strong peroxidase staining of the zona pellucida of normal fertilized and unfertilized 1-cell ova. However, the reaction was significantly weaker with both fertilized and unfertilized 1-cell ova from females induced to superovulate and normal and superovulated blastocysts. Pure antibody against mouse LDH-X was obtained by affinity chromatography of the rabbit anti-mouse LDH-X–IgG on pure mouse LDH-X covalently bound to sepharose. The pure antibody against mouse LDH-X reacted immunochemically identically to anti-mouse LDH-X–IgG, but it was not bound by any stage of preimplantation mouse embryos. The IgG fractions which had passed through the affinity column during the purification procedure and which did not contain any anti-LDH-X activity were bound by the zonae of preimplantation mouse embryos in the same manner as was unpurified anti-mouse LDH-X–IgG. Histochemical studies indicated LDH activity only in the embryo proper, but not on the zona pellucida. It is concluded that LDH-X is not present in preimplantation mouse embryos.

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H. SPIELMANN, R. P. ERICKSON and C. J. EPSTEIN

Summary.

Lactate dehydrogenase and glucose-6-phosphate dehydrogenase in Day-1 and Day-4 preimplantation mouse embryos was assayed by immunotitration. For both enzymes, immunoreactive protein was found to decrease in parallel with enzyme activity, indicating that loss of activity is probably the result of enzyme degradation. The identification of embryonic lactate dehydrogenase as isoenzyme LDH-1 and of glucose-6-phosphate dehydrogenase as the form found in red cells is corroborated by the immunological data.

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H. Spielmann, Ursula Jacob-Mueller, Petra Schulz and Angelika Schimmel

Summary. The amount of ATP, ADP and AMP and also the adenylate energy charge and the ATP/ADP ratio were determined in preimplantation mouse embryos (strain NMRI) in vivo. The ATP content decreased from 0·64 pmol at fertilization to 0·21 pmol in late blastocysts. ADP decreased from 0·1 pmol in the zygote to 0·06 pmol in 4–8-cell embryos and increased again to 0·15 pmol in late blastocysts. AMP changed considerably at the 1-cell and the 2-cell stage and increased from 0·04 to 0·2 pmol between the 4-cell and the late blastocyst stage. These developmental changes between fertilization and implantation result in a continuous decline of the total amount of adenine ribonucleotides (from 0·79 to 0·64 pmol), of the adenylate energy charge (from 0·87 to 0·45) and of the ATP/ADP ratio (from 6·4 to 1·4). In C57BL embryos developing to the 2-cell stage in vivo or in vitro there was a decrease in ATP content, as in NMRI embryos in vivo whereas the ATP content remained unchanged in NMRI embryos during culture to the 2-cell stage (no further development in vitro). In blastocysts cultured for 24 h in media supporting differentiation during implantation (MEM and NCTC-109) the content of ATP and ADP increased but AMP remained constant. The total adenine ribonucleotide content rose to 1 pmol and the ATP/ADP ratio and adenylate energy charge remained unchanged. After 48 h of culture in the two media to late blastocysts there was a decrease in ATP, an increase in AMP and a decline in adenylate energy charge and ATP/ADP ratio, as occurs in vivo.