Two forms of G protein-coupled estrogen receptor 1 in the ricefield eel: Expression and functional characterization in relation to ovarian follicle development.

G protein-coupled estrogen receptor 1 (Gper1) mediates many rapid, non-genomic estrogenic effects in vertebrates, and plays an important reproductive role in the maintenance of oocyte meiotic arrest in teleost fishes. In the present study, two genes for Gper1, namely gper1a and gper1b, were identified in the genome of a teleost fish, the ricefield eel (Monopterus albus) through Blast and syntenic analysis. Although genes neighboring gper1b are of high synteny, ricefield eel Gper1b shares very low (around 15) percent identities with Gper1 homologues of other vertebrates. In transiently transfected HEK293T cells, both ricefield eel Gper1a and Gper1b responded to estradiol (E2) and estradiol-BSA (E2-BSA) challenges by activating pCRE but not pSRE luciferase reporters, which were abolished by G-15 and NF-449. The production of cAMP was also increased in HEK293T cells transfected with Gper1a or Gper1b expression construct after E2-BSA challenge, which was also abolished by G-15. Surprisingly, both Gper1a and Gper1b showed ligand-independent effects on pCRE luciferase reporters at higher transfected doses (10 ng). RT-PCR analysis showed that the transcript of gper1a is broadly expressed in tissues of both female and male fish while the expression of gper1b in tissues demonstrates obvious sexual dimorphism, with transcripts detected in all tissues examined in female whereas they were barely detectable in some peripheral tissues of male including the testis. In the ovary, the expression of both gper1a and gper1b was detected in the oocyte but not the follicular layer, with the mRNA levels increased during vitellogenesis, peaked at the late vitellogenic stage, and decreased precipitously at the full-grown and germinal vesicle breakdown (GVBD) stages. Moreover, E2 and E2-BSA induced cAMP production in the in vitro incubated follicles at mid-vitellogenic stage but not the GVBD stage, and the induction could be completely abolished by G-15, a Gper1 inhibitor. Taken together, these results suggest that both Gper1a and Gper1b may play important roles in the development and maturation of ovarian follicles in ricefield eels, possibly through inhibition of oocyte meiotic resumption.

Nr5a homologues in the ricefield eel Monopterus albus: Alternative splicing, tissue-specific expression, and differential roles on the activation of cyp19a1a promoter in vitro.

Nr5a (Fushi tarazu factor 1, Ftz-F1) homologues belong to the nuclear receptor superfamily, and are involved in the regulation of reproduction in vertebrates. Four genes encoding Nr5a homologues were present in the genome of ricefield eel, which are designated as nr5a1a, nr5a1b, nr5a2, and nr5a5 in the present study. Alternatively spliced transcripts were identified for nr5a1a and nr5a1b genes. Sequence analysis indicated that nr5a5 is possibly a paralog of nr5a2, and nr5a1b is lost during evolution in some teleosts including tilapia and medaka. Ricefield eel nr5a genes exhibit tissue-specific expression patterns, with nr5a1a and nr5a1b resembling that of the SF-1/Ad4BP (NR5A1) subfamily, and nr5a2 and nr5a5 resembling that of the NR5A2/LRH/FTF subfamily. Transcriptomic analysis revealed parallel expression profiles of nr5a1a, foxl2, and cyp19a1a in ovarian follicles during vitellogenesis, with peak values at the late vitellogenic stage. Real-time PCR indicated that the expression levels of nr5a1a and foxl2 in gonads were decreased significantly during the sexual transition from female to the late intersexual stage. In vitro transient transfection assay showed that Nr5a1a up-regulated ricefield eel cyp19a1a promoter activities synergistically with Foxl2. However, Nr5a1b, Nr5a2, and Nr5a5 could neither activate ricefield eel cyp19a1a promoter alone nor enhance the stimulatory effects of Foxl2 on cyp19a1a promoter activities. Collectively, the above data suggest that Nr5a homologues may have diverse and differential roles in the tissues of ricefield eels. The up-regulation of gonadal nr5a1a and foxl2 during vitellogenesis may be important for the ovarian development whereas their down-regulation during the sexual transition period may be important for the sex change process of ricefield eels, possibly through the regulation of cyp19a1a gene expression.

Two Foxo1 homologues in the orange-spotted grouper Epinephelus coioides: sequences, expression, and possible involvement in the activation of cyp19a1a expression in the ovary.

Foxo1, a member of Foxo transcription factor family, is involved in a number of physiological processes including metabolism, cell cycle progression, aging, and apoptosis. In the ovarian granulosa cell of mouse, Foxo1 is implicated to inhibit the expression of Cyp19a1, a gene encoding the aromatase that converts androgens into estrogens. Currently, the information about the expression and physiological relevance of Foxo1 homologues in the ovary of teleosts is scarce. In the present study, cDNAs encoding two forms of Foxo1, Foxo1a and Foxo1b, were isolated from the orange-spotted grouper. Phylogenetic analysis indicated that the orange-spotted groupers Foxo1a and Foxo1b were closely related to the counterparts of the ricefield eel. RT-PCR analysis showed that the orange-spotted groupers foxo1a and foxo1b were expressed in a wide range of tissues, with high levels detected in the brain regions, liver, and intestine. Quantitative real-time PCR analysis showed similar expression profiles for cyp19a1a, foxo1a, and foxo1b in the ovary during development from the primary growth to mature stages, with peak values detected at the vitellogenic stage. In situ hybridization detected mRNA of foxo1a, foxo1b, and cyp19a1a in granulosa cells surrounding vitellogenic oocytes. In vitro transfection showed that both Foxo1a and Foxo1b upregulated the orange-spotted grouper cyp19a1a promoter activities, possibly through the conserved Foxo binding site. Collectively, these results suggest that both Foxo1a and Foxo1b may be involved in the regulation of the ovarian functions in the orange-spotted grouper and the physiological roles of Foxo1 homologues in the ovary may be diversified in vertebrates.

Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus.

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.

Transcription factors Dmrt1a, Foxl2, and Nr5a1a potentially interact to regulate cyp19a1a transcription in ovarian follicles of ricefield eel (Monopterus albus).

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a, foxl2, and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10-50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100-200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well.

Paradiplozoon yunnanensis n. sp. (Monogenea, Diplozoidae) from Sikukia gudgeri (Cyprinidae, Barbinae) in southwest China.

Paradiplozoon yunnanensis n. sp. (Monogenea, Diplozoidae) is described from the gills of Sikukia gudgeri Smith, 1931 (Cyprinidae) collected from Jinghong Basin, a tributary of the international Lancang-Mekong River. This is the first diplozoid species from S. gudgeri and its description increases the number of Paradiplozoon species recorded in China to 25. The new species is distinguished from congeners by a combination of morphological and molecular features. The anterior end of the median plate is thickened in the marginal area and a narrow rectangular trapeze spur connects to the anterior jaw through two separate anterior joining sclerites. The posterior end of the median plate sclerite is invaginated with a smooth strip-shaped posterior joining sclerite. Comparison of a newly obtained sequence of rRNA ITS2 with 18 other congeneric sequences from GenBank provides support for separation of the new species.