Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Karyotypic study of some species of family Mochokidae (Pisces, Siluriformes): evidence of female heterogamety

The relationship between the specific anatomy of tilapia gametes and their function was studied in the sequence of events which follow artifical fertilization. Scanning electron microscopic observations showed that some events of the fertilization process, involving spermatozoon migration through the micropylar canal until reaching the villous plasma membrane of the eggs and its penetration into the egg cytoplasm, occur very rapidly (fractions of a second). However, the spermatozoon tail remains outside for about 1–2 min. Then, following the zygote formation and the elevation of the chorion after its separation from the plasma membrane, numerous sperm cells could be found in the vicinity of the micropyle. This cell mass, which seemed to be trapped by a network of microfilaments, was suggested to be the result of the evacuation of excess sperm cells throughout the micropylar canal. The significance of these results for sperm and egg plasma membrane interaction and for prevention of polyspermy are discussed.     

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration. © 1996 Wiley-Liss, Inc.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Upon fertilization, ascidian eggs release a cell surface glycosidase used in the block to polyspermy and undergo cortical contractions resulting from increased intracellular calcium levels. The glycosidase is released by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Intact Ascidia ceratodes eggs cleave 4-methylumbelliferyl-phospho-choline when it is added to seawater. This yields highly fluorescent 4-methylumbelliferone. Authentic phospholipase C but not phospholipase D can cleave this substrate. Thus, the authors believe that cleavage of the substrate is specific for PLC activity. Eggs incubated in the fluorogenic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Glycosidase release and PLC activity were stimulated by ionomycin. Octylglucoside or Triton X-100 extracts of ascidian eggs had two forms of phospholipase activity as shown by ion affinity chromatography: PL1 eluting at 0.25 mol/L NaCl and PL2 eluting at 0.6mol/L NaCl. The PL1 appeared to be isolated as a single protein. When surface proteins were labeled with non-penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that half the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.