Tatsuya Unuma

A Protein Identical to the Yolk Protein Is Stored in the Testis in Male Red Sea Urchin, Pseudocentrotus depressus

Female sea urchins store the major yolk protein (MYP) in ovarian nutritive phagocytes before vitellogenesis. Using immunological procedures, we detected MYP in the testicular nutritive phagocytes of Pseudocentrotus depressus, the red sea urchin, and then compared the distribution of MYP between sexes during gametogenesis. MYP was purified from unfertilized eggs by ion exchange chromatography (Q Sepharose) and gel filtration (Superdex 200), and an antiserum (anti-MYP) was raised against MYP. Immunoblot analysis demonstrated that immature testes, as well as ovaries, contained a large quantity of MYP. Immunohistochemistry showed that MYP was distributed in the nutritive phagocytes occupying the follicular lumen in both males and females. In both sexes, as gametogenesis proceeded, the nutritive phagocytes degenerated and the gonadal lumen filled with gametes. MYP accumulated in ripe ova as a yolk protein in the mature ovary. In contrast, MYP was not detected in mature testes, because stored spermatozoa did not react with anti-MYP. We conclude that in male P. depressus, MYP is stored in the testicular nutritive phagocytes and utilized as the nutrient source for spermatogenesis.

The influence of dietary protein and fat levels on tissue free amino acid levels of fingerling rainbow trout (Oncorhynchus mykiss).

The influence of six semi-purified diets having different nutrient composition on the tissue free amino acid (FAA) levels of fingerling rainbow trout was examined. Four balanced amino acid diets (casein:gelatin=6:1) with combinations of two protein levels (35% (LP) and 50% (HP)) and two fat levels (10% (LF) and 20% (HF)), and two imbalanced high-protein (50%) diets (IMB-HP, casein:gelatin=1:1) with two fat levels (LF and HF), were each fed to duplicate groups (30 fish/group, 10 g/fish) for 6 weeks at 15°C. FAA in plasma, liver, dorsal white muscle and brain at 12 h after the last feeding were compared. Percentage weight gain of the HPHF diet group was the highest, but was not different (P>0.05) from the others except for the IMB-HPLF diet group (P<0.05). Levels of individual amino acids in the whole body protein were not markedly different among the treatments. Most of the free essential amino acid (EAA) levels in the tissues of the HP diet fed fish were higher than in those of the LP diet group. The levels of free phenylalanine and tyrosine of the HF group tended to be higher, and those of taurine lower, than the LF group. Most of the free EAA levels of the IMB diet group were lower even than the LP group. Correlations in EAA patterns between a given diet and tissue were high in plasma, but very low in muscle and brain. These findings suggest that not only dietary protein level and amino acid profile, but also dietary fat level affects the levels of certain FAA of rainbow trout tissues.

Molecular Cloning and Ovarian Expression Profiles of Thrombospondin,a Major Component of Cortical Rods in Mature Oocytes of Penaeid Shrimp,Marsupenaeus japonicus

Abstract In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.

Biochemical composition of eggs in relation to egg quality in the Japanese eel, Anguilla japonica

AbstractThis paper presents the relationship between egg quality and egg biochemical composition of cultured and wild Japanese eel, Anguilla japonica. Eggs were obtained by artificialinduction of maturation. Fertilization and hatching rates were used as characteristics of egg quality. Egg quality characteristics showed large variation; fertilization rate, 0–96; hatching rate, 0–84%. The biochemical composition also showed a large variation. There was no marked relationship between egg quality and fatty acid contents of eggs, except for n-6 highly unsaturated fatty acids (HUFA). Both the fertilization and hatching ratesincreased proportionally withincreases of the α-tocopherol g(α-Toc) contentin eggs. A more significant correlation was found between the amount of α-Toc relative to the amount of HUFA and egg quality. The results of this study show that the egg quality of Japanese eel is affected by the α–Toc level, andin particular, the ratio of α-Toc to HUFAin the eggs. Abbreviations: BHT – butylhydroxytoluene; EFA – essential fatty acids; FAME – fatty acid methyl esters; HPLC – high performance liquid chromatography; HUFA – highly unsaturated fatty acids; NADH – nicotinamide adenine dinucleotide; NADPH - nicotinamide adenine dinucleotide phosphate; ROS – reactive oxygen species; α-Toc –α-tocopherol.

Cleavage site of a major yolk protein (MYP) determined by cDNA isolation and amino acid sequencing in sea urchin, Hemicentrotus pulcherrimus

The overall sequence of cDNA encoding vitellogenin (Vg), a precursor to major yolk protein (MYP), of Hemicentrotus pulcherrimus was determined. Its nucleotide sequence has an open reading frame of 4041 bp encoding 1346 amino acids. The amino acid sequence showed little similarity to other Vgs in vertebrates, insects or nematodes, but resembled members of the vertebrate and invertebrate transferrin family. The N-terminal amino acid sequence of the protein fragments dominant in the later embryonic stage was analyzed in order to determine the cleavage site of MYP. Determination of the cleavage site in MYP and analysis of MYP proteolysis in vitro suggested that MYP has a specific molecular shape to permit its proteolytic fragmentation at a definite site. The functional region of transferrin in MYP is conserved after proteolytic processing. Considering these results and those from other work, the protein called sea urchin Vg is not a true Vg. Therefore, a new name, echinoferrin, is proposed for this protein.

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.

Zinc‐binding property of the major yolk protein in the sea urchin − implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc‐binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc‐binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc‐binding site(s).

Control by the environmental concentration of ions of the potential for motility in Japanese eel spermatozoa

Abstract We examined the effects in vitro of the ionic composition of the incubation medium on the acquisition and loss of the potential for motility by the spermatozoa of the Japanese eel ( Anguilla japonica ). Milt were obtained from 10 males that had been artificially matured by repeated injections of hCG. The percentage of motile spermatozoa in the milt samples varied widely (mean, 53.4±11.8%; range, 7.4–91.5%) when samples were diluted with a hyperosmotic solution (450 mM NaCl buffered at pH 7.5). The percent motility increased significantly after a 60-min incubation in artificial seminal plasma (ASP), consisted of 149.3 mM NaCl, 15.2 mM KCl, 1.3 mM CaCl 2 , 1.6 mM MgCl 2 , and 20 mM NaHCO 3 , buffered with 20 mM TAPS–NaOH at pH 8.1, or in Ca 2+ - and Mg 2+ -free ASP (83.4±2.5% and 86.1±2.1%, respectively). A rapid decrease in motility was observed in K + -free ASP (1.8±0.7%) and in HCO 3 − free ASP (5.7±2.2%). The percent motility increased with increasing concentrations and decreased with decreasing concentrations of K + ions (0–30 mM) and HCO 3 − ions (0–20 mM) in the ASP. The cycle of acquisition and loss of the potential for motility could be repeated several times by changing the concentrations of K + or HCO 3 − ions in the ASP. These results strongly suggest that variations in the quality of milt obtained from artificially matured males might be related to the ionic constituents of the seminal plasma. The percent motility of eel spermatozoa could be controlled by adjusting the concentrations of K + and HCO 3 − ions in the isotonic incubation medium, irrespective of the initial potential for motility.

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis.

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real‐time reverse‐transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad. Mol. Reprod. Dev. 77: 59–68, 2010.

Induction of Sperm Maturation

Cultivated male Japanese eels [200-300 g body weight (bw)] are sexually immature and do not mature under normal culture conditions. Injecting them with gonadotropins, however, can easily induce maturation (Yamamoto et al. 1972; Miura et al., 1991) and spermiation (Ohta et al. 1996). Administration of androgens can also induce maturation even at their juvenile stage (Ohta and Takano 1996). Sperm cells that have completed their spermatogenesis in the testis are immotile when diluted with seawater, but they acquire the potential for motility upon transfer from the testicular lumen to the sperm duct (Ohta et al. 1997a). Because a relationship between motility and the capacity for the fertilization of teleost spermatozoa has been confirmed by many authors (Billard and Cosson 1992; Lahnsteiner et al. 1996), the acquisition of motility by spermatozoa can be defined as the functional maturation of the spermatozoa. We have developed techniques for acquiring adequate quantities of high-quality spermatozoa for successful artificial fertilization.