Biochemical and Immunochemical Characterization of Two Discrete Vitellogenin Proteins and Their Derived Lipovitellins in the Inshore Hagfish (Eptatretus burgeri)

Vitellogenesis has been extensively studied in oviparous vertebrates, including teleost fishes, while not much is known with regard to jawless hagfishes, modern representatives of the most primitive vertebrate class. This study aimed to characterize vitellogenin (Vtg) and yolk protein (YP) in the inshore hagfish (Eptatretus burgeri) as an initial step to understand vitellogenesis in this species. A putative Vtg fraction was purified from the serum of female hagfish by combinations of hydroxylapatite and ion-exchange chromatography, followed by gel filtration. The purified fraction appeared to contain two distinct Vtgs (Vtg1 and Vtg2) and exhibited biochemical properties resembling those previously reported for teleost Vtgs; these appeared to be female-specific serum proteins and high-molecular-weight proteins in gel filtration (similar to 505 kDa as the mixture fraction of both Vtgs) and in SDS-PAGE (Vtg1 and Vtg2; similar to 210 kDa and similar to 195 kDa, respectively). A major YP was also purified from hagfish eggs by combinations of hydroxylapatite chromatography and gel filtration; the apparent native mass of the purified YP was unusually large (>669 kDa). The purified YP consisted of four polypeptides in SDS-PAGE; the peptide pattern indicated that it consisted of two lipovitellins (Lv1 and Lv2) giving rise to two sets of heavy chains (similar to 116 kDa and similar to 106 kDa, respectively) and two light chains (similar to 32 kDa and similar to 28 kDa, respectively). Additional immunological analysis, N-terminal amino acid sequencing and cDNA cloning firmly confirmed the precursor-product relationship between hagfish Vtgs and Lvs

Localization of major yolk protein in the digestive tract of the sea urchin Strongylocentrotus intermedius

In the present study, we examined the localization of the major yolk protein (MYP) in the intestine of the sea urchin Strongylocentrotus intermedius. First, partial MYP complementary DNA was isolated from the sea urchin intestine. The expression level of MYP messenger RNA (mRNA) along the sea urchin digestive tract is highest in the intestine, so we performed in situ hybridization and immunohistochemical analysis using this tissue. No MYP mRNA was detected in the luminal epithelium, connective tissue, muscle tissue, or coelomic epithelium by in situ hybridization analysis. Positive immunohistochemical staining was observed in the luminal epithelium, inner epithelium and connective tissue, the signal being strongest in the latter. We conclude that MYP synthesized in the inner epithelial cells is moved to and stored in connective tissue and the luminal epithelium, before being secreted into the body cavity and the inner digestive cavity of the sea urchin

Quantitative Changes of Major Yolk Protein in the Coelomic Fluid and Gonads of the Sea Urchin, Mesocentrotus nudus, during the Reproductive Cycle

Both female and male sea urchins accumulate the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before gametogenesis. Additionally, the most abundant protein in the coelomic fluids of both sexes is MYP. In females, MYP in the coelomic fluid is taken up by the nutritive phagocytes and then transported into growing oocytes. In this study, quantitative changes of MYP in the coelomic fluid of both sexes were examined during the reproductive cycle of the wild sea urchin, Mesocentrotus nudus. In females but not males, positive correlation between MYP level and the gonadosomatic index was observed. These results indicate that MYP in the coelomic fluid is suitable as a biomarker of the onset of puberty and progression of maturity in female sea urchins

Molecular Cloning and Characterization of the Expression Profiles of Vitellogenin Transcripts in the Dojo Loach (Misgurnus anguillicaudatus) in Response to 17 alpha-ethinylestradioland 17 beta-estradiol Administration

The gene, vitellogenin (vtg) was cloned and characterized in the dojo loach (Misgurnus anguillicaudatus), an indigenous freshwater species in East Asia, in order to develop tools for detecting the effects of estrogenic endocrine-disrupting chemicals (EEDCs). Full-length cDNAs encoding seven distinct vtg transcripts (vtg1-7) were obtained. The corresponding deduced amino acid sequences (Vtg1-7) were divided into two types; type I (Vtg1-6; 89-99% identical), which contained both lipovitellin (Lv) and phosvitin (Pv), and type II (Vtg7), which contained Lv alone. Phylogenetic analysis revealed that the type I and type II Vtgs in the loach could be classified as VtgAo1 and VtgC types, respectively. Immuno-biochemical analyses using type-specific Vtg antisera revealed that VtgAo1 proteins appeared to be the major Vtg type in this species. Males were administered (aqueous exposure) either 17 beta-estradiol (E2) or 17 alpha-ethinylestradiol (EE2), the results from which were used to determine that hepatic vtgAo1 expression was estrogen-sensitive. The precise classification of the loach vtg/Vtg products, as well as their induction profiles following the estrogenic stimulation, provide a basis for their use as sensitive biomarkers when EEDC activities are evaluated in the freshwater environments in East Asia

Hepatic estrogen-responsive genes relating to oogenesis in cutthroat trout (Oncorhynchus clarki) : The transcriptional induction in primary cultured hepatocytes and the in vitro promoter transactivation in responses to estradiol-17 beta

Estradiol-17 beta (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg: chgH alpha, chgH beta, chgL; vtg: vtgAs, vtgC; esr: esr1a, esr1b, esr2a, esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a, were increased by E2 treatment (10(-6) M) at 24 and 72 h post initiation (hpi), but esr1b, esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10(-11)-10(-6) M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10(-11) M for chgHa at 24 hpi, as 10 -9 M for vtgC at 72 hpi, and as 10(-19) M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esrla mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10(-9 )M at 24 hpi to 10(-7) M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg, vtg and esrl a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHa > vtgAs > chgH5 > chgL > vtgC > esr1a when mediated by Esr1a, chgHO > chgHa > chgHL > vtgAs > vtgC > esr1a by Esr1b, chgHO > chgL > chgHa > vtgAs > vtgC > esr1a by Esr2a, and chgHO > chgHa > vtgAs > chgL > vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg, vtg and esr1a genes in the estrogen-exposed hepatocytes

Efficient Artificial Fertilization and Ovulated Egg Preservation in Kawakawa <i>Euthynnus affinis</i>

Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks’ solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks’ solution at 20 °C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding