Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Effect of membrane additives on vesicle fusion

A large variety of alkyl derivatives were found to either slow or block the low-temperature induced fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles (DPPC SUV) when incorporated into the SUV bilayer at five mol%. Only corn oil was fusogenic.     

Effects of bilayer thickness on the activity of diacylglycerol kinase of Escherichia coli.

We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max). In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v(max) with no effect on K(m). In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.     

Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages

Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.