Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos.

Certain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.

Properties of two multisubstate Cl- channels from human syncytiotrophoblast reconstituted on planar lipid bilayers.

We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation, and Mg2+-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated in most cases, a result consistent with the higher permeability for cations as compared with Cl- and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode techniques. In this paper, we report that Cl--selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role of the reported Cl- channels in transplacental ion transport and/or syncytium homeostasis remains to be determined.

Effect of trypsin inhibitors and concanavalin A on the fertilization of Bufo arenarum coelomic oocytes.

The effect of trypsin inhibitors (obtained from soybean, lima bean and ovomucoid) and Concanavalin A on fertilization in Bufo arenarum was tested. In order to study the effect of these substances at the level of the vitelline envelope of the oocytes, a new bioassay was designed. This bioassay employs coelomic oocytes to which some oviducal factors necessary for their fertility was added. Trypsin inhibitors block both the lytic effect of the acrosomal proteases on the vitelline envelope and fertilization. This indicates that the blockade of fertilization is a consequence of the inhibition of the lytic effect of the acrosomal proteases. Concanavalin A is effective as well in blocking the lytic effect of acrosomal proteases and fertilization. These effects are reversed by some sugar antagonists of the lectin, thus indicating that the effect of Concanavalin A is through its saccharide-binding capacity. These results suggested the involvement of glucosidic residues of the vitelline envelope in amphibian fertilization (the saccharide residues might be involved in the attack of the vitelline envelope by the acrosomal proteases). The possible mechanism of action of these substances is discussed.

EFFECT OF TRYPSIN INHIBITORS AND CONCANAVALIN A ON BUFO ARENARUM FERTILIZATION.

The presence of some trypsin inhibitors (soybean trypsin inhibitor and lima bean trypsin inhibitor) in the inseminating media, is effective in blocking Bufo arenarum fertilization. Ovomucoid does not significantly affect fertilization, even at concentrations as high as 10 mg/ml. Similarly the presence of Concanavalin A in the inseminating media results in fertility impairment. The possible mechanisms of action of these substances are discussed.