Bertrand Picheral

Fertilization of amphibian eggs: a comparison of electrical responses between anurans and urodeles.

In Pleurodeles waltl and Ambystoma mexicanum, which exhibit physiological polyspermy, the membrane potential in most eggs did not change in any consistent pattern during 45 min after fertilization; in some cases, a slow hyperpolarization began 5 to 15 min after insemination and continued for 10-15 min. These eggs then slowly depolarized, reaching a stable value of -10 to +10 mV, about 45 min after fertilization. Membranes of eggs activated by A23187 or by electrical stimulus showed a similar behavior. The diversity of responses does not correlate with the number of sperm fusing with the egg. Holding the membrane potential at a constant value between -40 and +40 mV during insemination did not prevent fertilization nor delay sperm-egg interactions. The fertilization or activation potential of Rana temporaria eggs consists of a rapid (1 sec) depolarization accompanied by a sudden decrease in membrane resistance. The activation potential can be triggered by A23187 and by calcium iontophoresis; its amplitude depends on the (Cl-)0 and to a lesser extent on the (Na+)0. Fertilization was prevented when the membrane potential was clamped above +15 mV. However, slowing the rise time (5 to 8 sec instead of 1 sec) and reducing the amplitude (10-20 mV instead of 40-60 mV) of the fertilization potential, both by injecting negative current, never induced polyspermy.

Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians☆

Fertilization of frog eggs by frog sperm is inhibited if the eggs membrane potential is positive (N.L. Cross and R.P. Elinson, 1980, Dev. Biol. 75, 187-198); however, fertilization of salamander eggs by salamander sperm does not depend on membrane potential (M. Charbonneau, M. Moreau, B. Picheral, J.P. Vilain, and P. Guerrier, 1983, Dev. Biol. 98, 304-318). Since salamander sperm can fertilize frog eggs, we have investigated whether this cross-fertilization is voltage dependent. If, during insemination with Notophthalmus sperm, Xenopus eggs were voltage clamped between +7 and +20 mV, fertilization proceeded in 7/10 (70%) of the clamped eggs, compared to 38/48 (79%) of the neighboring eggs. In control experiments in which voltage-clamped Xenopus eggs were inseminated with Xenopus sperm, fertilization proceeded in only 1/10 (10%) of the clamped eggs, compared to 59/60 (98%) of the neighbors. Similar results were obtained with cross-fertilization experiments between Notophthalmus sperm and Rana eggs. These experiments indicate that the voltage dependence of fertilization depends on the species of sperm.

Early Events in Anuran Amphibian Fertilization: An Ultrastructural Study of Changes Occurring in the Course of Monospermic Fertilization and Artificial Activation

In unfertilized frog eggs, the plasma membrane displays an animal vegetal polarity characterized by the presence of short microvilli in the vegetal hemisphere and long microvilli or ridge‐like protrusions in the animal hemisphere. The densities of microvilli are similar in the two hemispheres.

Anuran fertilization: a morphological reinvestigation of some early events

The morphological events dealing with the fertilization or activation process of the eggs of different Rana species are reported. Steps from sperm penetration to male pronucleus formation are described as well as the egg surface evolution until the second polar body formation. Depending on the species, a “fertilization body” (FB) rapidly differentiates at the site of sperm entry. This FB is either extruded or is reincorporated into the egg. In both cases a landmark persists. For the first time, it is clearly demonstrated with SEM that the cortical-granule exocytosis wave starts from the site of sperm entry. Just after the exocytosis wave occurs, the villi elongate and the movements of the pigment granules increase, reflecting a change in the egg cortex fluidity. This last phenomenon should be considered as new criteria of activation. The cell coat appears just as the cortical granule material is extruded; it is no longer possible to recognize the new membranes.

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI‐induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI‐induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1–2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50–105 sec) than in monospermic eggs (10–40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.

Polysaccharide Complexes in Full-Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone-Induced Maturation

Immature full‐grown oocytes of Pleurodeles waltlii contain large amounts of small electron‐dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24–36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22–24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9–10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.