Kooichi Konishi

Cloning of cDNA encoding vitellogenin and its expression in red sea urchin, Pseudocentrotus depressus

Abstract Both male and female red sea urchins, Pseudocentrotus depressus, accumulate a large quantity of the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before the initiation of game-togenesis. To examine the accumulation mechanism of this protein in the gonad, we cloned full-length cDNA encoding vitellogenin (Vg; the MYP precursor in the coelomic fluid), and investigated its expression in various tissues of immature adults. The nucleotide sequence of Vg contains an open reading frame of 4050 bp encoding 1349 amino acids. The deduced amino acid sequence near the N-terminal showed 25% homology to the vertebrate transferrin family. Vitellogenin mRNA was detected in the ovary, testis, stomach, intestine and rectum by Northern blot analysis, with the highest level of mRNA expression in the gonad. Weak expression was also detected in the esophagus and coelomocytes by RT-PCR. In situ hybridization demonstrated that nutritive phagocytes, which exclusively fill the lumina of the immature gonad, contained Vg mRNA. These results suggested that the MYP stored in the immature gonads is synthesized and accumulated mainly within the nutritive phagocytes.

Non-reductional Spermatozoa in Three Shell Color Types of the Freshwater Clam Corbicula fluminea in Taiwan

Abstract We found three distinct shell color types in samples of the freshwater clam Corbicula fluminea Müller collected at Hou Don, Keelung, Taiwan. DNA microfluorometric analysis revealed that these three types consisted of both diploids and triploids. DNA microfluorometry on sperm and somatic cells showed that both diploid and triploid produced non-reductional spermatozoa. These characteristics are similar to triploid C. leana Prime sampled in Japan. These findings suggest that Corbicula fluminea at different ploidy levels may be reproducing by androgenesis as already shown in C. leana from Japan.

The Major Yolk Protein is Synthesized in the Digestive Tract and Secreted Into the Body Cavities in Sea Urchin Larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc‐binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT‐PCR in the early 8‐arm pluteus stage and its expression persisted until after metamorphosis. Real‐time RT‐PCR revealed that MYP mRNA increased exponentially from the early 8‐arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4‐arm stage and that newly synthesized CFMYP was present at and after the mid 8‐arm stage. In the late 8‐arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel‐derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8‐arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults. Mol. Reprod. Dev. 76: 142–150, 2009.

On the prezoeal stage: observations on three Pagurus species (Decapoda, Anomura)

Observation on prezoeas of three Pagurus species inhabiting the coasts of Hokkaido, P. lanuginosus de Haan, P. middendorffii Brandt, and P. brachiomastus (Thallwitz), were carried out using light and scanning electron microscopy. Hatching was produced at laboratory conditions of 17·5–18·2 °C and 32·5–33·2 ppt salinity, and after a short duration of 5–10 min, most of prezoeas moulted to the first zoeal stage. Prezoeal appendages are described and figured; previous observations on prezoeas in allied groups are summarized.

Ultrastructure of the prezoeal cuticle and integument of the first zoea of Pagurus brachiomastus (Thallwitz) (Anomura, Paguridae) just after hatching

Transverse sections of the prezoea stage of Pagurus brachiomastus (Thallwitz) were made to determine the fine structure of the prezoeal cuticle and the exoskeleton of the first zoea, by using transmission electron microscopy. The thin cuticle covering the newly hatched prezoea is 15–17 nm thick, which is approximately 24 times thinner than the zoeal cuticle. High resolution revealed a trilaminar structure of the prezoeal cuticle, which is composed of an outer layer, approximately 4·4 nm thick, a mid lighter layer, approximately 3·5 nm thick, and an inner layer, approximately 7·7 nm thick. During the prezoeal stage, the inner zoeal exoskeleton consists of an epicuticle and an exocuticle, measuring approximately 0·36 μm thick. At this stage, there were no indications of the formation of endocuticle.

Does Light-Induced Relief of Cytochrome c Oxidase from CO-Induced Inhibition Result in Photo-Reactivation of CO-Inhibited Respiration in Sperm of Sea Urchin, Oyster and Fish

Abstract Light irradiation, at a light fluence rate sufficient for the strong photo-reactivation of the CO-inhibited cytochrome c oxidase in mitochondria isolated from the sperm of fish, oyster and sea urchin, weakly activated the CO-inhibited respiration only in the sea urchin sperm, with peaks of photo-reactivation corresponding to those in the absorption spectrum of CO-bound cytochrome aa3. NADH cytochrome c reductase was inhibited by CO, weakly in mitochondria from sea urchin sperm and completely in those from fish and oyster sperm. The CO-induced complete inhibition of cytochrome c reduction in fish and oyster sperm probably does not allow the photo-reactivation of CO-inhibited cytochrome c oxidase to augment CO-blocked respiration. At a light fluence rate higher than that mentioned above, photo-activation of NADH cytochrome c reductase, found in the sperm of oyster and sea urchin, occurred even in the presence of CO in mitochondria isolated from sea urchin sperm and strongly activated CO-inhibited respiration in sea urchin sperm, with peaks corresponding to those in the absorption spectrum of reduced cytochrome b. The acceleration of cytochrome c reduction due to the photo-activation of this complex enzyme in sea urchin sperm probably induces another activation of CO-inhibited respiration at the high light fluence rate.