Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. β-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

The ability of Trypanosoma cruzi to induce erythrocyte membrane destabilization in vitro was studied. Epimastigote forms adhered to human erythrocytes and caused fusion or lysis of the red cells, depending on the conditions of the interaction. Red cells were fused in the presence of calcium, while haemolysis was induced in the absence of the cation. Dextran 60 C facilitated fusion but delayed lysis. Optimum pH and temperature for fusion were 7·4 and 37 °C, respectively. Lipid alterations were produced in the plasma membrane of the red cell during the interaction with the parasite. A Ca2+-independent increase of lysophospholipids and free fatty acids was common to both the lysis and fusion processes. A relative increase of 1, 2-diacylglycerides was unique to the fusion process and these changes were dependent on Ca2+. The transfer of free fatty acids and lysophospholipids from T. cruzi to erythrocyte membranes was demonstrated using parasites pre-labelled with radioactive phospholipids. Pre-treatment of parasites with exogenous phospholipase A2 abolished the fusogenicity, while lysis was increased. Neither fusion nor haemolysis occurred when the parasites were pre-treated with fatty acid free albumin, phospholipase A2 inhibitors or when these compounds were present in the medium during the parasite-erythrocyte interaction. Our results suggest that T. cruzi induces erythrocyte membrane destabilization in vitro by transfer of lipid material in a calcium independent manner and that this ion is necessary for other membrane alterations that lead to erythrocyte fusion.

The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1°C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread – as a result of cell fusion – to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2–3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte–zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the ‘virgin’ part of oolemma resistant to sperm penetration remains unknown.