Induction of the Acrosome Reaction of Sea Urchin Spermatozoa by Means of Urea

Urea is an effective reagent for inducing the acrosome reaction of spermatozoa in sea urchins. Urea-treated spermatozoa are capable of fertilizing eggs in Ca-deficient sea water. The pH of the urea solution is an important factor affecting the induction of the acrosome reaction. The reaction occurs at a high percentage in urea Solution at pH's higher than 7.8, while the reaction is almost completely suppressed at pH 7.2. Ca⁺⁺ is also an important factor for the induction of the reaction, although the minimum concentration required is very low.
The acrosomal filament formed in urea solution is similar in shape to that formed in egg-water, when fixed after 10 seconds' urea-treatment. The acrosome granule material is found around the basal portion of the acrosomal filament.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. I. FERTILIZATION IN Ca-FREE SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA. *

When spermatozoa are treated with egg-water and undergo the acrosome reaction, their fertilizing capacity is lost within 5 min. However, if insemination is carried out within 4 min after the egg-water treatment, there is no difference in fertilizing capacity between spermatozoa treated with egg-water and non-treated ones. With such spermatozoa, eggs can be fertilized even in the virtual absence of calcium, whereas with spermatozoa treated with Ca-free egg-water, no fertilization occurs under the same conditions. It is postulated that in normal fertilization the acrosome reaction has occurred before the attachment of the gametes. The failure of fertilization with normal spermatozoa in Ca-free sea water may be due to the failure of occurrence of the acrosome reaction.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA*

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5–6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.     

EFFECT OF TRYPSIN ON THE FERTILIZING CAPACITY OF SEA URCHIN SPERMATOZOA TREATED WITH EGG-WATER*

The effect of trypsin on the fertilizing capacity of spermatozoa was studied with 6 species of sea urchins. Trypsin has no harmful effect on intact spermatozoa. However, spermatozoa which have undergone the acrosome reaction in egg-water lose the fertilizing capacity when treated with trypsin-sea water. Electron- microscopical examination revealed that trypsin does not produce any morphologically noticeable effect on intact spermatozoa, but does dissolve the material covering the acrosomal tubule of the spermatozoa which have undergone the acrosome reaction. It is likely that the loss of this material is closely correlated with the loss of fertilizing capacity of spermatozoa by the trypsin treatment.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

INDUCTION OF THE ACROSOME REACTION ON THE EGG SURFACE OF DE-JELLIED SEA URCHIN EGGS BEFORE AND AFTER FERTILIZATION*

The capacity of the surface of sea urchin eggs to induce the acrosome reaction was assayed by estimating the rate of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs before and after fertilization. DTT-disruption of the vitelline coat did not eliminate the acrosome reaction-inducing capacity. This capacity was retained after fertilization in eggs of both H. pulcherrimus and A. crassispina. The acrosome reaction-inducing capacity of the eggs was markedly decreased by treatment with trypsin. The low capacity of the trypsin-treated eggs was maintained after fertilization in H. pulcherrimus, but in A. crassispina the capacity returned to the pre-trypsin treatment level after fertilization. Fertilized eggs from which the fertilization membrane was mechanically removed retained the inducing capacity to a considerable extent, independent of the presence or absence of the hyaline layer, but the capacity diminished rapidly as cleavage proceeded. It was concluded from these data that the acrosome reaction of spermatozoa actually occurred at the surface of de-jellied eggs and that the inducing substance resides in the plasma membrane in addition to the fertilization membrane. A chemical difference between the inducing substance of egg surface and jelly substance is discussed.     

SPERMIOGENESIS IN BARNACLES WITH SPECIAL REFERENCE TO ORGANIZATION OF THE ACCESSORY BODY*

Ultrastructural changes during spermiogenesis in the barnacles, Balanus amphitrite albicostatus, Balanus tintinnabulum rosa, Balanus trigonus and Tetraclita squamosa japonica, and organization of the sperm with special reference to the accessory body were studied. The Golgi complex organizes both the acrosome and the accessory body at different stages during spermiogenesis; the former is formed at the mid-spermatid stage and the latter is formed at the late spermatid stage. The arrangement of the components in the mature filiform sperm is quite unique, with the acrosome, the basal body just behind the acrosome, the axial filament parallel to a long nucleus, and a slender long mitochondrion behind the nucleus. The sperm in the anterior and posterior half of the ejaculatory duct differ from each other in form in that the sperm in the anterior duct are not equipped with the accessory body and the sperm in the posterior duct are. The accessory body can be artificially broken down by some treatments (1 M urea, alkaline sea water: pH 9.0-9.7, low ionic concentration of sea water). The loss of the accessory body from the sperm is assumed to be related to the ferti-lizability of the sperm.     

Regulation of Sperm Motility in Starfish. I. Initiation of Movement

Starfish seminal plasma has such characteristics as higher concentration of potassium ions, higher osmolality, and lower pH compared with those of sea water. These factors independently inhibited the movement of spermatozoa. Sperm movement was recorded by taking photographs of the swimming paths under a dark field microscope. Spermatozoa either did not move or swam with decreased beat frequency in isolated seminal plasma or in the presence of certain fractions of seminal plasma obtained by gel-filtration or by partitions. In normal sea water (containing calcium ions), a low pH of less than 6 resulted in a decrease in the number (%) of swimming spermatozoa, though the speed of propulsion and the beat frequency was not affected. On the other hand, in a calcium-free sea water, a large proportion of spermatozoa moved in both low and high pH conditions, but in low pH the speed of propulsion was reduced and many spermatozoa (31 %) swam in smaller circles.     

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca²⁺-free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca²⁺at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca²⁺levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca²⁺is somewhat lower than that of the reaction by jelly water. In Ca²⁺-free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca²⁺free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca²⁺-free artificial sea water. Even in the absence of extermal Ca²⁺, preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca²⁺-mobilization in spermatozoa.     

豚鼠精子在发生及顶体反应过程中细胞内Ca~(2+)的定位研究

实验利用焦锑酸钾法对豚鼠精子在发生及顶体反应过程中的Ca~(2+)定位作了较详细的研究。在精母细胞及精子细胞上都有Ca~(2+)分布,但睾丸中的精子上则无Ca~(2+)。成熟精子中Ca~(2+)主要定位于顶体帽的整个腹面及背面的两个特定区域。发生顶体反应的精子上Ca~(2+)则位于顶体外膜上或囊泡内,已发生顶体反应的精子中Ca~(2+)则位于顶体内膜上。     

Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization

Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0–96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process. Mol. Reprod. Dev. 79: 861–869, 2012. © 2012 Wiley Periodicals, Inc.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Progesterone induces activation in Octopus vulgaris spermatozoa

The purpose of the present study was to determine whether Octopus vulgaris spermatozoa are activated by progesterone stimulation. Spermatozoa were collected from the spermatophores in the Needham's sac of the male (MS) and from the spermathecae of oviducal glands of the female (FS). We used transmission (TEM) and scanning (SEM) electron microscopy to study the morphology of untreated, Ca2+ ionophore A23187 and progesterone-treated MS spermatozoa, and untreated FS spermatozoa. We showed that ionophore and progesterone stimulation of MS spermatozoa induce breakdown of the membranes overlapping the acrosomal region, exposing the spiralized acrosome. These modifications resemble the acrosome reaction observed in other species. FS stored in the spermathecae did not show the membranes covering the acrosomal region present in the MS spermatozoa. When ionophore and progesterone treatments were performed in Ca2+-free artificial sea water, no changes were observed, suggesting the role of external calcium in modifying membrane morphology. Lectin studies showed a different fluorescence distribution and membrane arrangement of FS-untreated spermatozoa with respect to the MS, suggesting that spermatozoa transferred in the female genital tract after mating, are stored in a pre-activated state. The plasma membrane of the untreated MS and FS spermatozoa was labelled with Progesterone-BSA-FITC, indicating the presence of plasma membrane progesterone receptor. Taken together these data suggest that progesterone induces an acrosome- like reaction in MS spermatozoa similar to that induced by calcium elevation. In addition progesterone may play a role in the pre-activation of spermatozoa stored in the female tract, further supporting the hypothesized parallelism between cephalopods and vertebrates.     

Chromosome changes during the in vitro ageing of MRC-5 human fibroblasts.

We have measured the release of fertilization acid from sea urchin eggs in specific cation-free media by activating the eggs with the calcium ionophore A23187. The fertilization acid is normal in calcium-free sea water and several other substituted media, indicating that specific cations present in sea water are not required for release of the acid. The fertilization acid may result from release of cortical granule contents, but we suggest the possibility that other cellular processes are involved.     

Influences of Ca and Mg ions on the egg survival of grey mullet, Mugil cephalus L.

Five kinds of artificial sea water with similar salinity, pH and osmolarity were prepared with different concentrations of calcium and magnesium ions. They were complete sea water, calcium-free sea water, half calcium-free sea water, magnesium-free sea water and half magnesium-free sea water. The fertilized eggs from induced spawnings of grey mullet Mugil cephalus , were transferred to these sea waters at two development stages, i.e. before the formation of 2-blastomere stage and at the gastrula stage. Survivals of fertilized egg in different sea waters were checked after several hours incubation, the percentage survival was used as an index of the importance of calcium and magnesium to the embryonic development. The results showed that calcium is indispensable to the embryonic development of mullet egg. Magnesium, however, is not the necessary element in the incubation medium.     

Sperm binding to an egg model composed of agarose beads

A sperm-binding factor of the eggs of the sea urchin Hemicentrotus pulcherrimus or Anthocidaris crassispina, was coupled to agarose beads. Upon contact with these beads, the spermatozoa exhibited an acrosome reaction and bound to them. No binding occurred to BSA-coupled beads or to beads missing active groups. When coupled beads were pretreated with a sperm factor, which is the probable counterpart to the sperm-binding factor, they could no longer bind sperm. Reciprocal cross-binding between the above two species was not successful, but H. pulcherrimus sperm became capable of binding to A. crassispina beads if they first underwent acrosome reaction in egg water. Sperm of two of seven other echinoderms examined was able to bind to the coupled beads.     

Initiation of cleavage in starfish eggs by the injection of triton-treated spermatozoa

Triton X-100-treated spermatozoa were injected into immature (fully grown, germinal vesicle stage) or mature (pronuclear stage) oocytes of the starfish, Asterina pectinifera, to study relation between initiation of cleavage and cortical reaction. Immature oocytes into which Triton X-100-treated spermatozoa were injected were treated with 1-methyladenine. Such immature oocytes initiated cleavage after completion of meiosis without formation of the fertilization membrane. The same results were obtained when Triton X-100-treated spermatozoa were injected into mature oocytes. Control oocytes into which only calcium-free sea water was injected did not cleave. These results indicate that the initiation of cleavage is independent of the cortical reaction but dependent on the existence of spermatozoa (spermatozoon) in the egg cytoplasm.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus , became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).