Magnesium Ion-Requiring Step in Fertilization of Sea Urchins*

Abstract

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jellytreated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT-or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.Divalent cations in sea water, i.e., calcium and magnesium, have been shown to be requisite for fertilization of sea urchins. With respect to external calcium ions, DAN (2) showed that they are required to induce the acrosome reaction of sea urchin spermatozoa. Furthermore, recently a small amount of external calcium ions has been shown to be required in the fertilization process following the binding of the spermatozoa with reacted acrosomes to eggs in the sea urchin, Hemicentrotus pulcherrimus (9).With respect to magnesium ions in sea water, CLARK (1) reported that polyspermy could be induced at high percentage rate in sea urchin eggs when high concentration of magnesium was added to sea water. Recently, it was found that fertilization in sea urchins does not take place in the virtual absence of external magnesium ions, even when spermatozoa pre-treated with dissolved egg-jelly are employed, showing that external magnesium ions are requisite at least for the fertilization process following the induction of the acrosome reaction in spermatozoa (7, 8). When spermatozoa were treated with egg-water containing dissolved jelly to undergo the acrosome reaction, their fertilizing capacity was lost within 5 min in natural sea water (1 1).

Sperm–oocyte interaction

“…A similar effect was observed by inseminating eggs in Mg z+ -free sea water (Sano et al 1980). Quercetin at 100^M blocks sperm incorporation in oocytes; furthermore, despite apparently normal attachments, the spermatozoa do not induce any electrical changes across the oocyte plasma membrane.…”

Sperm-egg fusion in the sea urchin is blocked in Mg2+-free seawater

“…In previous papers we have reported that magnesium ions in seawater are required for fertilisation of sea urchins (Sano & Mohri, 1976Sano et al, 1980;Mohri et al, 1982). In the absence of Mg 2 * the fertilisation rate was reduced to almost nil.…”

“…The sperm head was moving in 3CMFSW, but abruptly came to a standstill, perpendicular to the egg surface, immediately after addition of Mg 2+ . Even though a spermatozoon other than that being observed in focus caused the elevation of the fertilisation membrane in the other six cases, it is quite likely that they also had already attached to the egg surface in the absence of Mg 2+ (see also Sano et al, 1980). Could the sperm attached to the egg in MFSW resume entry into the egg when the egg was activated by some other means?…”

“…In a previous experiment (Sano et al, 1980) we obtained indirect evidence to indicate that the entry into the egg of a sperm which had attached to the egg surface but did not proceed further with fertilisation in the absence of Mg 2 " 1 " in seawater, would be resumed upon the addition of a sufficient amount of Mg 2 " 1 ". In the present experiment we succeeded in observing such a resumption of sperm entry by following microscopically the behaviour of a single fertilising spermatozoon.…”

Sperm–Oocyte Interaction

“…It has been shown that in the presence of Quercetin, an ATPase inhibitor, spermatozoa attach to sea-urchin eggs and undergo the acrosome reaction; however, sperm incorporation is inhibited (Eckberg & Perotti, 1983). A similar effect was observed by inseminating eggs in Mg z+ -free sea water (Sano et al 1980). Quercetin at 100^M blocks sperm incorporation in oocytes; furthermore, despite apparently normal attachments, the spermatozoa do not induce any electrical changes across the oocyte plasma membrane.…”