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Effects of macromolecules recovered from uterine luminal fluid on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

Day-4 mouse embryos grew well in culture media supplemented with macromolecular components of uterine fluids recovered on day 3, 4 or 5 of pregnancy and pseudopregnancy. Addition of these components to media during a 2-h pulse culture had no significant effect on the incorporation of glucose carbon by morulae/early blastocysts. However, various fractions of uterine luminal macro-molecules significantly increased the turnover of glucose carbon incorporated into acid-soluble and acid-insoluble glycogen, into nucleic acids and into proteins during a 24-h chase culture. These effects were due mainly to components with a molecular weight between 1000 and 10,000 Da and the activity was most marked in fluids collected on day 5 of pregnancy or pseudopregnancy. Oxidation of glucose during a 4-h incubation was inhibited in the presence of certain uterine macromolecules but most consistently by the large molecular weight component (greater than 300,000 Da). Some differences were noted in the inhibitory activity of macromolecules obtained from pregnant and pseudopregnant sources. There was little evidence of an effect of uterine-fluid components on lactate production from glucose.

Animals↗

Effects of oxygen concentration on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The utilization of the acid-soluble glycogen pool in pulse-labelled embryos was significantly enhanced during 24- and 48-h chase culture under low oxygen concentrations of 5, 2.5 and 1%. The lower the oxygen tension the greater was the turnover in the pool. The morphological development of embryos was equally as good at very low oxygen concentrations as when embryos were cultured in 5-20% oxygen. Reduction in oxygen concentration enhanced the oxidative utilization of substrate, as measured by rate of carbon dioxide production. The present study could provide an explanation for the discrepancy in glycogen content between mouse blastocysts developing in utero and in vitro and for the reported beneficial effects of low oxygen concentration during development of embryos in culture.

Animals↗

Precedence of cell-zona adhesion over cell-cell adhesion during marsupial blastocyst formation prohibits morula formation and ensures that both the pluriblast and trophoblast are superficial.

This study, based on 38 samples taken between the 16-cell stage on day 2.5 of gestation and the expanded 1.0-mm-diameter unilaminar blastocyst on day 6, describes the ultrastructural changes that occur in the conceptus of the marsupial Sminthopsis macroura in relation to cell-zonal adhesion initiated at the zygote stage and cell-cell adhesion initiated at the 16-cell stage, lineage allocation, extracellular matrix (ECM) secretion and embryo coat changes. In S. macroura, rather flattened pluriblast and rounded trophoblast cells appear as different cell types during the fourth division when nucleolar reticulation suggests activation of the zygotic genome in both cell types. The differences disappear nearly completely in blastocysts of 0.6-0.8 mm in diameter, but the two cell types then reappear as two distinct populations. The ECM varies depending on its location within the conceptus up to the stage of the expanding blastocyst. It is of rather granular appearance between the cell lining and zona pellucida and consists of patches of homogeneous material embedded in an electron-lucent substance in the cleavage cavity. Homogeneous ECM coats trophoblast but not pluriblast cells on blastocoelic surfaces. Transient structures such as 'myosin-like' fibrillar arrays, probably associated with exocytosis of ECM, and pearl string-like whorls are still present, but both disappear during further expansion of the 0.6- to 0.8-mm blastocyst. During blastocyst expansion, the patchy homogeneous ECM in the blastocoel changes structure and appears flocculent, while the continuous ECM coating trophoblast cells disappears. Pluriblast cells and yolk mass identify the embryonic pole and hemisphere, and the opposite hemisphere becomes abembryonic and is eventually fully lined by trophoblast cells. An increase in endocytotic, mainly coated vesicles at the apical, zona-orientated surface of both cell types is noticed and is probably responsible for uptake of the mucoid coat. In 1-mm blastocysts, numerous vesicles contain rod-shaped crystalline inclusions.

Animals↗

Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro.

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.

Animals↗

Fibronectin localization in the mouse embryo from the two cell stage to the morula stage.

Among the characters of fibronectin, the adhesiveness may play an important role in embryo implantation. It is concluded that, using the direct and indirect immunofluorescence techniques, fibronectin was not found on the zona pellucida, but on the embryo surface even from the two cell stage. The results imply that (i) fibronectin is not an initial differentiation marker for the stage of endoderm, which used to be postulated, (ii) the surface fibronectin which has been produced at least from the two cell stage is covered by the zona pellucida to mask its adhesiveness during the oviduct transport, and (iii) after hatching in the uterus cavity the surface fibronectin facilitates the embryo implantation.

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Micromanipulation of sheep morulae to produce monozygotic twins.

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.

Journal Article↗