The ultrastructure of human spermatozoa located in the cumulus cells and the zona pellucida of a pro-nuclear egg, and in the zona pellucida of a two-cell egg, both fertilized in-vivo, has been analysed in order to understand how the human spermatozoon penetrates the investing coats of the oocyte. Among the 36 spermatozoa found in the cumulus cells, 31 were phagocytosed by cumulus cells and 5 were wedged in the matrix between the cells. These spermatozoa were acrosome-reacted and their equatorial segment was intact. Six of the seven spermatozoa found in the zona pellucida (four spermatozoa in the pronuclear egg and three in the two-cell egg) had lost the equatorial segment, while the other one was partially reacted. The sperm heads were located in slits with sharp edges. From these findings it was concluded that in the human (1) only few and normal spermatozoa seem to reach the cumulus cells after natural insemination, (2) the acrosome reaction probably occurs sometime before the spermatozoa reach the vicinity of the corona cells, (3) the reaction of the equatorial segment seems to occur during or before the initial phase of zona penetration, since the spermatozoa located in the matrix of the zona pellucida had no equatorial segment. No evidence of the presence of spermatozoa with an intact acrosome in the matrix of cumulus cells or with an intact equatorial segment in the zona pellucida were found.

A two-cell human embryo recovered from the Fallopian tube 82 h following the LH peak in plasma and 37 h after a single episode of intercourse was examined by transmission electron microscopy. At the time of recovery the embryo was denuded of cumulus cells, and both the zona pellucida and the two adjoining blastomeres were intact. The finding of two polar bodies in the perivitelline space, two nucleated blastomeres and remnants of the fertilizing sperm tail within the cytoplasm of one of them, were considered as evidences that the embryo was normally fertilized. Among the most conspicuous features found were the presence of very distinct desmosome-like structures between blastomeres, and the cytoplasmic cell organelles distribution in three areas referred as: a sub-cortical, a middle and a perinuclear bands. An outstanding feature was the extensive blebbing of the nuclear envelope. In general, the features seem to correspond to a normally developing two-cell embryo undergoing cleavage at a normal rate.

SUMMARYThe synthesis of the radiolabelled heterobifunctional reagent N-methyl-Nchloroformyl-N'-terbutoxycarbonyl-para-phenylenediamine A ( fig. 1) is presented.In particular, the use of tri-tritiated methyl iodide in defined reaction conditions allowed the synthesis of this probe A (fig.