Shigeharu Kudo

Sperm penetration and the formation of a fertilization cone in the common carp egg.

Sperm penetration and the formation of a fertilization cone in the micropylar canal of the egg of the common carp were examined by electron microscopy. The overwhelming majority of inseminated eggs fixed without immersion in fresh water showed that the first spermatozoon had penetrated into the ooplasm before the cortical reaction had occurred, and in many cases had formed a fertilization cone to plug the micropylar canal. At this stage the sperm head was usually located at the base of the cone, and the tail part did not participate in the formation of the cone. Inseminated eggs fixed soon after immersion in fresh water showed that the elevation of the fertilization membrane and the simultaneous recession of the fertilization cone often permitted the penetration of a few supernumerary spermatozoa into the perivitelline space near the micropylar canal, but polyspermic fertilization was never observed. The mechanism of the block to polyspermy in the egg of the common carp is discussed in connection with the fertilization cone.

Morphology, composition and fertilization of carp eggs: a review

Abstract This paper is a review of the literature on eggs of carp (Cyprinus carpio L.) and some related species and includes data taken from the former USSR and eastern European countries. Reported data relate to fecundity, egg morphology and composition, and fertilization. Information is also available on the ovarian fluid composition with characterization of proteins and lipids. The fecundity of common carp is very high, ranging from 100 000 to 300 000 eggs · kg−1 body weight per oogenetic cycle, with one cycle/year in the case of females reared in an outside natural pond environment. The diameter and weight of the eggs are in the range of 1.24–1.42 mm and 0.86–1.41 mg, respectively. The ooplasm includes large amounts of yolk and various organelles (endoplasmic reticulum, Golgi apparatus, mitochondria), cortical granules and alveolae. The main characteristics of cyprinid ovarian fluid are an osmotic pressure of about 300 mOsm, a Mg2+ concentration of 2.58 mM and a high ovarian fluid pH of 9. Energy in the form of ATP which is necessary for egg metabolism originates from glycolytic and oxidative reactions. The egg has a relatively thick vitelline envelope (VE) or zona radiata which is reorganized into the fertilization envelope (FE) after fertilization. The micropyle located at the animal pole is a funnel-shaped structure leading spermatozoa to the ooplasmic surface on which a fertilization cone develops after fertilization. A site of sperm attachment is identified on the plasma membrane at the level of the internal aperture of the micropylar canal. Major differences are observed between the two outermost VE and FE layers, as revealed by electron microscopy, enzyme or carbohydrate cytochemistry, and imunohistochemistry. FE extracts have strong bactericidal and fungicidal effects.

Binding of rainbow trout sperm to egg is mediated by strong carbohydrate-to-carbohydrate interaction between (KDN)GM3 (deaminated neuraminyl ganglioside) and Gg3-like epitope

KDNα2→3Galβ4Glcβ1Cer [(KDN)GM3] is a major (≈90%) component of total gangliosides found in sperm of rainbow trout (Oncorhynchus mykiss) and was shown to be present prominently at the sperm head by immunochemical staining with its specific mAb kdn3G. Liposomes containing (KDN)GM3 adhere specifically to GalNAcβ4Galβ4Glcβ1Cer (Gg3Cer)-coated plastic plates. Interaction between (KDN)GM3 and Gg3Cer was much stronger than that previously observed between Neu5Acα2→3Galβ4Glcβ1Cer and Gg3Cer. (KDN)GM3–Gg3Cer interaction did not require the presence of Ca2+ and Mg2+, but was enhanced in the presence of Mn2+. Fresh trout sperm adhered specifically to Gg3Cer-coated plates under physiological conditions, and the binding was inhibited by pretreatment of sperm with mAb kdn3G. The presence of Gg3 or Gg3-related epitope structure in the specific area surrounding the micropyle, through which sperm enter the egg, was confirmed by immunogold labeling under electron microscopy. These findings suggest that initial sperm-egg adhesion during the process of fertilization occurs when sperm adhere to the area surrounding the micropyle through specific interaction between (KDN)GM3 on the sperm head and Gg3 epitope (GalNAcβ4Galβ1→) expressed at a defined region of the egg surface membrane.

Localization of polysialoglycoprotein as a major glycoprotein component in cortical alveoli of the unfertilized eggs of Salmo gairdneri

Polysialoglycoprotein (PSGP, 200 kDa), first isolated by S. Inoue and M. Iwasaki in 1978 (Biochem. Biophys. Res. Commun. 83, 1018-1023) from unfertilized eggs of rainbow trout, has been shown to comprise a unique class of glycoproteins associated with the exocytosis of cortical alveoli. In 1986, 200-kDa PSGP was shown to undergo proteolytic depolymerization to 9-kDa PSGP on egg fertilization (activation) and there was an indication that 200-kDa PSGP may possibly be a component of cortical alveoli (J. Biol. Chem. 261, 5256-5261). In this paper we present evidence demonstrating that PSGP is actually a component of cortical alveolus. First, a cortical alveolus-rich fraction (CA fraction) was obtained by low-speed centrifugation of the homogenate of unfertilized eggs of rainbow trout. The 200-kDa PSGP was found to be a major component extractable with buffered saline from the CA fraction by chemical analysis of isolated materials. Treatment of the eggs to induce parthenogenetic activation resulted in all cases in the loss of both cortical alveoli and PSGP in the CA fraction. Second, perivitelline space fluid was isolated from the activated eggs of rainbow trout and analyzed, and 9-kDa PSGP was confirmed to be present as a major proteinaceous component. Third, following incubation of the eggs in water for activation, the time course of the appearance of 9-kDa PSGP and the breakdown of 200-kDa PSGP was observed. The formation of 9-kDa PSGP was detected in the eggs after 1 min of incubation and its level rose rapidly, attaining a maximum at 7 min after incubation. During this period, there was a concomitant fall in the level of 200-kDa PSGP. This formation and rapid increase in 9-kDa PSGP correspond directly to the time course of cortical alveolus exocytosis in activated chum salmon eggs recently studied by scanning electron microscopy.

Ultrastructure and Ultracytochemistry of Fertilization Envelope Formation in the Carp Egg

This paper describes ultrastructural and ultracytochemical events occurring during the process of transformation of the vitelline envelope (VE) into the fertilization envelope (FE) in the egg of Cyprinus carpio. The VE is composed of four layers, except the micropylar region. The outermost (first) layer can be subdivided into a double layer (upper and lower halves) by cytochemical differences. The upper half is more protein‐rich and positive for acid phosphatase (AcPase) activity, while the lower one is more carbohydrate‐rich and negative for AcPase.

Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilisation envelopes of rainbow trout eggs.

Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.

Response to Sperm Penetration of the Cortex of Eggs of the Fish, Plecoglossus altivelis

The response of the egg to sperm penetration was examined in eggs of the fish, Plecoglossus altivelis, by scanning electron microscopy. Eggs responded to sperm penetration by forming a fertilization cone at a “sperm entry site”, which is a specialized structure in the egg surface under the micropyle. Within one minute, the fertilization cone showed dramatic morphological changes from its earliest appearance, through full two‐storied growth to its marked recession. The sperm entry site in the egg surface is discussed as a morphologically specialized organ responsible for the entrance of a fertilizing spermatozoon. The morphological characteristics of the egg and sperm are also described.

A novel ex vivo method for assaying adhesion of cancer cells to the peritoneum

A novel ex vivo method to determine the cell adhesion of cancer cells to the peritoneum was described. The wells of a microtiter plate were filled with cell suspension and sealed using mouse peritoneum. The peritoneum was fixed using a plastic cover and the plate was turned upside down and incubated for cell adhesion. After incubation for 80 min, the plate was centrifuged and non-adherent cells were assayed by MTT assay. Human cancer cells (MKN28, MKN45, MKN74, KM12C and KM12SM) adhered to the mouse peritoneum as well as cells from mouse (Colon26) and the ratio of cells attached to the peritoneum was estimated to be between 12.8 and 66.4%. This simple method could be useful to investigate the adhesion molecule associated with peritoneal dissemination.

ENZYMO-CYTOCHEMICAL OBSERVATIONS ON THE CORTICAL CHANGE IN THE EGGS OF CYPRINUS CARPIO AND CARASSIUS AURATUS

Acid phosphatase (AcPase) and cholinesterase (ChE) activities were examined by ultracytochemical techniques in the mature unfertilized and the fertilized eggs of Cyprinus carpio and Carassitus auratus, to reveal the differences among three kinds of structures, cortical alveoli, CA‐ and CB‐granules, which discharge their contents on fertilization into the perivitelline space. Deposits of the reaction product for AcPase activity are localized on the plasmalemma of unfertilized eggs, in the cortical alveoli, cytoplasmic matrix, lamellae of the Golgi apparatus and sometimes in multivesicular bodies but not in CA‐ and CB‐granules, mitochondria, rough endoplasmic reticulum or on the plasmalemma of fertilized eggs. Deposits of the reaction product for ChE activity are localized on the inner surface of the plasmalemma, in the cytoplasmic matrix, in mitochondria and on a small number of tubular or cisternal membranes of the endoplasmic reticulum in mature unfertilized eggs, and on the outer surface of the limiting membrane of CB‐granules and on membranous structures (possibly Golgi lamellae) associated with their formation, in fertilized eggs. The deposits on the plasmalemma rapidly disappear almost completely, with discharge of the cortical alveoli soon after fertilization, but they are again seen on the inner surface of the plasmalemma when emiocytotic discharge of the CB‐granules begins about 10 min after fertilization.

ULTRASTRUCTURAL OBSERVATIONS ON THE DISCHARGE OF TWO KINDS OF GRANULES IN THE FERTILIZED EGGS OF CYPRINUS CARPIO AND CARASSIUS AURATUS

This paper describes mainly ultrastructural evidence on the discharge of two kinds of granules, CA and CB, as well as the cortical alveoli in an egg of the common carp and the goldfish. The cortical alveoli (about 2–28 μ in diameter) and CA‐granules (about 0.4–2 μ in diameter) are located in the cortical cytoplasm of the mature egg, and the latter is distinguishable in size and texture from the former which contains an eccentric core at the very least as a basal internal structure. The CB‐granules (about 100–950 mμ in diameter) appear in small or large clusters in the cortical cytoplasm after fertilization, being formed in connection with constriction or pinching‐off of dilated tubular elements. After fertilization the cortical alveoli, CA‐ and CB‐granules are discharged at different times. The new plasmalemma of a fertilized egg appears to be a joint production of the original plasmalemma and the limiting membranes of the cortical alveoli, CA‐ and CB‐granules.