Mechanism of oocyte maturation and ovulation and its application to seed production in the Japanese eel.

Reduction in eel resources and catches of glass eels as seedlings for aquaculture have been a serious concern in recent years in both Europe and East Asia. Thus, technical advancement to produce eel seeds for artificial cultivation is most desired. Fundamental information on oocyte maturation and ovulation and its application to artificial induction of sexual maturation are needed to produce good quality seeds of the Japanese eel. This review introduces hormonal mechanisms of cytoplasmic maturation (such as hydration, lipid coalescence, and clearing of the ooplasm) and the maturational competence (the ability to respond to maturation-inducing steroid) and nuclear maturation (germinal vesicle breakdown). In addition, previous and newly developed methods for induction of spawning have been described.

Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis.

In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.

Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region.

Cytochrome P450 aromatase (CYP19) converts androgens to estrogens. Unlike mammals, teleosts have two CYP19 genes, expressed differentially in ovary (CYP19A1) and neuronal tissues (CYP19A2). The primary purpose of this study was to demonstrate the potential involvement of CYP19A2 in the reproductive endocrinology of teleosts. Channel catfish CYP19A2 (ccCYP19A2) cDNAs were isolated from the brain using a PCR-based strategy. The ccCYP19A2 cDNA putatively encodes 500 amino acids which conferred aromatase activity in transfected COS-7 cells. Additionally, an alternatively spliced transcript was isolated which lacks the first 122 amino acids and is catalytically inactive. The brain and the pituitary were predominant sources of ccCYP19A2 transcript and the abundance in both tissues acutely increased prior to spawning. This preovulatory induction of ccCYP19A2 gene in the pituitary is remarkably similar to the pattern of gene expression for luteinizing hormone-beta (LHbeta). Estradiol-17beta (E(2)) and testosterone enhanced the transcript abundance of ccCYP19A2 and LHbeta in catfish pituitary cells cultured in vitro but the stimulatory effects of testosterone were abolished by an aromatase inhibitor, indicating an important role of E(2), the product of CYP19A2 activity, in the regulation of CYP19A2 and LHbeta. Structural and functional analysis of the 5'-flanking region of the gene suggested that the sequence from -1076 to - 435 bp is critical for the basal promoter activity in the pituitary. This report demonstrates that CYP19A2 functions as an important factor in the reproductive endocrinology of teleosts through the brain-pituitary-gonadal axis.

Ovarian mitochondrial cytochrome b mRNA levels increase with sexual maturity in freshwater eels (Anguilla spp.).

Differential display of mRNA was used to identify an upregulated gene in ovaries of artificially maturing Japanese eels (Anguilla japonica). Accordingly, mitochondrial (mt) cytochrome b, whose transcript levels increased from early to late vitellogenesis, was isolated, cloned and sequenced. Temporal trends in artificially maturing eels were compared with those in naturally and artificially maturing New Zealand eels (longfinned eel, Anguilla dieffenbachii; shortfinned eel, Anguilla australis) by Northern blot and in situ hybridization analysis to rule out any experimental artifacts. An increase in ovarian mt cytochrome b signals was seen when comparing immature and midvitellogenic longfinned eels, but not immature and early vitellogenic shortfinned eels, from the wild. Long-term captivity yielded reduced target mRNA levels, but abundance increased after hormonal induction of vitellogenesis. These results imply that the increase in mt cytochrome b mRNA levels during artificial maturation reflects natural development, although its onset appears to be brought forward during artificial maturation in the Japanese eel. It is suggested that increased mt cytochrome b mRNA levels result from both mitochondrial replication and increased transcription, and that they reflect the build-up of machinery for enhanced ATP-synthesis at some stage of oogenesis and/or early zygote development.

The 5'-flanking regions of CYP19A1 and CYP19A2 in zebrafish.

This report describes the structure of the 5'-flanking regions of both the CYP19A1 and A2 genes that were isolated from the genome of the zebrafish (Danio rerio). Consensus sequences of three cAMP-responsive elements (CRE), an aryl hydrocarbon-responsive element (AhR/Arnt), a steroidogenic factor 1 (SF-1) site, and a TATA box were observed in the 5'-flanking region of CYP19A1. In contrast, the 5'-flanking region of CYP19A2 was located upstream of an untranslated exon and possessed consensus sequences of a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha heterodimer-responsive element (PPARalpha/RXRalpha), and a TATA box. Primer extension analysis revealed that the predominant transcription initiation sites for CYP19A1 and A2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively. These analyses indicate that substantially different regulators, including a variety of environmental xenobiotics, control the expression the two CYP19 genes.

20beta-Hydroxysteroid dehydrogenase of the Japanese eel ovary: its cellular localization and changes in the enzymatic activity during sexual maturation.

In many species of teleost, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) is a key steroidogenic enzyme in the production of the oocyte maturation-inducing steroid (MIS), 17alpha, 20beta-dihydroxy-4-pregnen-3-one. In this study, 20beta-HSD in the ovary of the Japanese eel was biochemically characterized using a cell-free system. 20beta-HSD activity was located mainly in the membrane-bound fractions of the mitochondria and microsome, with lower levels detected in the cytosolic fraction. The enzymatic activity of membrane-bound 20beta-HSD was strikingly enhanced by treatment of eels with gonadotropin-rich salmon pituitary homogenate. The activity of eel ovarian mitochondrial 20beta-HSD in the presence of different solubilizing detergents was then assessed. Mitochondrial fractions solubilized by sodium deoxycholate and octhylthioglucoside retained approximately 30% of 20beta-HSD activity when compared to those of nontreated mitochondria. These results suggest that Japanese eel ovarian 20beta-HSD is composed of membrane-bound and soluble activities, and that the membrane-bound component is stimulated by gonadotropin.

Cloning of 17beta-hydroxysteroid dehydrogenase-I cDNAs from Japanese eel ovary.

17beta-hydroxysteroid dehydrogenase-I (17beta-HSD-I) is a key steroidogenic enzyme for estradiol-17beta (E(2)) production. cDNAs encoding 17beta-HSD-I were cloned for the first time in lower vertebrates from the ovary of a teleost, the Japanese eel. The deduced amino acid sequence from these cDNAs was approximately 50% identical to mammalian 17beta-HSD-Is. 17beta-HSD-I mRNA was not detected in previtellogenic ovaries by Northern blotting. However, transcript abundance increased in early vitellogenic ovaries obtained from fish artificially matured by gonadotropic treatment, but thereafter did not appear to change further. Recombinant 17beta-HSD-I expressed in human kidney 293 cells selectively converted estrone to E(2), but androstenedione, testosterone, or E(2) were not converted to any other steroids. Although it is widely accepted that E(2) is produced from testosterone in other species of teleosts, the substrate specificity of eel 17beta-HSD-I suggests that a steroidogenic pathway for production of E(2) from androstenedione via estrone exists in the Japanese eel ovary.

Molecular cloning and characterization of Japanese eel ovarian P450c17 (CYP17) cDNA.

As a first step in investigating the mechanism underlying the steroidogenic shift from the production of ovarian androgens (vitellogenic stage) to that of 17alpha-hydroxylated progestins (maturational stage) in Japanese eel during induced oogenesis, a cDNA encoding Japanese eel (Anguilla japonica) ovarian P450c17 (CYP17: steroid 17alpha-hydroxylase/C(17-20) lyase) was cloned and sequenced. This cDNA contained the complete coding region representing 510 amino acid residues, which showed high sequence homology to those of rainbow trout (74%) and mammals (45-55%). The protein encoded by this cDNA possessed high enzymatic activities of 17alpha-hydroxylase and C(17-20) lyase, thus quickly converting pregnenolone and progesterone to their respective delta(4) and delta(5) C19 products. P450c17 produced a single transcript of 2.4 kb in length, as assessed by Northern blot. Transcript levels of this enzyme significantly increased throughout artificially induced ovarian development. Considering this together with the previous data showing that C(17-20) lyase activity decreased from the vitellogenic to the maturational stage, whereas 17alpha-hydroxylase activity increased, the present data suggest that changes in C(17-20) lyase activity (the production of androgens) do not depend on transcriptional changes of the P450c17 gene.