Low-temperature-induced disruption of reproductive axis and sperm vitality via stress axis in Monopterus albus.

The ricefield eel (Monopterus albus) is inherently timid and highly sensitive to stress. Our previous studies have shown that low-temperature weather could significantly affect the sperm vitality of ricefield eels. This study aims to investigate the regulatory mechanism of low-temperature effects on testicular function and sperm vitality in ricefield eels. The ricefield eels were initially reared at low (10 °C) and normal (25 °C) temperatures for 24 h. Low temperatures were found to induce the expression of pituitary pro-opiomelanocortin (POMC) and testes insulin-like growth factor-binding protein 1 (IGFBP1) mRNA expression, suggesting that the reduction in sperm vitality could be attributed to the activation of the stress axis. Moreover, the results indicated a significant decrease in sperm occupancy and count in the testes, along with a reduced percentage of motile sperm. Subsequent transcriptome analysis showed substantial inhibition of reproductive hormone genes (gnrh1, lh, and fsh) in the brain and pituitary, and downregulation of meiosis-related genes (dmc1, rec8, and sycp3) in the testes. These findings suggest that low temperatures might disrupt testicular development and spermatogenesis by inhibiting the reproductive axis. Metabolomics analysis then demonstrated a significant reduction in the levels of metabolites related to glycolysis, fatty acid metabolism, and the tricarboxylic acid (TCA) cycle in the testes after low-temperature treatment. Interestingly, the expression of zona pellucida sperm-binding proteins 3 and 4 (ZP3 and ZP4), which may affect sperm vitality and spermatogenesis, was significantly induced by low temperatures in the testes. In conclusion, these findings suggested that low temperatures might affect testicular function and sperm vitality by simultaneously activating the stress axis and inhibiting the reproductive axis and energy metabolism in the testes.

Characterization and Functional Analysis of the 17-Beta Hydroxysteroid Dehydrogenase 2 (hsd17b2) Gene during Sex Reversal in the Ricefield Eel (Monopterus albus).

In mammals, 17-beta hydroxysteroid dehydrogenase 2 (Hsd17b2) enzyme specifically catalyzes the oxidation of the C17 hydroxyl group and efficiently regulates the activities of estrogens and androgens to prevent diseases induced by hormone disorders. However, the functions of the hsd17b2 gene involved in animal sex differentiation are still largely unclear. The ricefield eel (Monopterus albus), a protogynous hermaphroditic fish with a small genome size (2n = 24), is usually used as an ideal model to study the mechanism of sex differentiation in vertebrates. Therefore, in this study, hsd17b2 gene cDNA was cloned and its mRNA expression profiles were determined in the ricefield eel. The cloned cDNA fragment of hsd17b2 was 1230 bp, including an open reading frame of 1107 bp, encoding 368 amino acid residues with conserved catalytic subunits. Moreover, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis showed that hsd17b2 mRNA expressed strongly in the ovaries at early developmental stages, weakly in liver and intestine, and barely in testis and other tissues. In particular, hsd17b2 mRNA expression was found to peak in ovaries of young fish and ovotestis at the early stage, and eventually declined in gonads from the late ovotestis to testis. Likewise, chemical in situ hybridization results indicated that the hsd17b2 mRNA signals were primarily detected in the cytoplasm of oogonia and oocytes at stage I-II, subsequently concentrated in the granulosa cells around the oocytes at stage Ⅲ-Ⅳ, but undetectable in mature oocytes and male germ cells. Intriguingly, in ricefield eel ovaries, hsd17b2 mRNA expression could be significantly reduced by 17ß-estradiol (E2) or tamoxifen (17ß-estradiol inhibitor, E2I) induction at a low concentration (10 ng/mL) and increased by E2I induction at a high concentration (100 ng/mL). On the other hand, both the melatonin (MT) and flutamide (androgen inhibitor, AI) induction could significantly decrease hsd17b2 mRNA expression in the ovary of ricefield eel. This study provides a clue for demonstrating the mechanism of sexual differentiation in fish. The findings of our study imply that the hsd17b2 gene could be a key regulator in sexual differentiation and modulate sex reversal in the ricefield eel and other hermaphroditic fishes.

Expression of Tshb and Tshr in the ricefield eel Monopterus albus: Potential paracrine/autocrine roles in gonads.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.

The ontogenic gonadal transcriptomes provide insights into sex change in the ricefield eel Monopterus albus.

BACKGROUND: The ricefield eel is a freshwater protogynous hermaphrodite fish and has become an important aquaculture species in China. The sex change of ricefield eel is impeding its aquaculture practice, particularly the large-scale artificial breeding. Many studies including transcriptomes of mixed gonadal samples from different individuals have been aimed to elucidate mechanisms underlying the sex change. However, the key physiological factors involved in the initiation of sex change remain to be identified.  RESULTS: The present study performed transcriptomic analysis on gonadal samples of different sexual stages obtained through biopsy from the same fish undergoing sex change. A total of 539,764,816 high-quality reads were generated from twelve cDNA libraries of gonadal tissues at female (F), early intersexual (EI), mid-intersexual (MI), and late intersexual (LI) stages of three individual sex-changing fish. Pairwise comparisons between EI and F, MI and EI, and LI and MI identified 886, 319, and 10,767 differentially expressed genes (DEGs), respectively. Realtime quantitative PCR analysis of 12 representative DEGs showed similar expression profiles to those inferred from transcriptome data, suggesting the reliability of RNA-seq data for gene expression analysis. The expression of apoeb, csl2, and enpp2 was dramatically increased and peaked at EI while that of cyp19a1a, wnt4a, fgf16, and foxl2a significantly downregulated from F to EI and remained at very low levels during subsequent development until LI, which suggests that apoeb, csl2, enpp2, cyp19a1a, wnt4a, fgf16, and foxl2a may be closely associated with the initiation of sex change of ricefield eels. CONCLUSIONS: Collectively, results of the present study confirmed that the down-regulation of female-related genes, such as cyp19a1a, wnt4a, fgf16, and foxl2a, is important for the sex change of ricefield eels. More importantly, some novel genes, including apoeb, csl2, and enpp2, were shown to be expressed with peak values at EI, which are potentially involved in the initiation of sex change. The present transcriptomic data may provide an important research resource for further unraveling the mechanisms underlying the sex change and testicular development in ricefield eels as well as other teleosts.

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 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.

Oxytocin-like signal regulates Lh cells directly but not Fsh cells in the ricefield eel Monopterus albus†.

The synthesis and release of LH and FSH in the pituitary of vertebrates are differentially regulated during gonadal development and maturation. However, the underlying neuroendocrine mechanisms remain to be fully elucidated. The present study examined the possible involvement of isotocin (Ist), an oxytocin-like neuropeptide, in the regulation of Lh and Fsh in a teleost, the ricefield eel Monopterus albus. The immunoreactive isotocin receptor 2 (Istr2) was shown to be localized to Lh but not Fsh cells. In contrast, immunoreactive isotocin receptor 1 (Istr1) was not observed in either Lh or Fsh cells in the pituitary. Interestingly, Lh cells in female ricefield eels expressed Istr2 and secreted Lh in response to Ist challenge stage-dependently and in correlation with ovarian vitellogenesis. Moreover, Ist decreased Lh contents in the pituitary of female fish, indicating its stimulatory roles on Lh release in vivo. The induction of Lh release by Ist in dispersed pituitary cells was blocked by a PLC or IP3R inhibitor but not by a PKA or PKC inhibitor, indicating the involvement of the IP3/Ca2+ pathway. Collectively, the above results indicate that isotocin may bind to Istr2 to stimulate Lh release via the IP3/Ca2+ pathway, and play important roles in the ovarian maturation in ricefield eels. Furthermore, the present study suggests a novel neuroendocrine mechanism underlying the differential regulation of Lh and Fsh in vertebrates.

Metabonomic Insights into the Sperm Activation Mechanisms in Ricefield Eel (Monopterus albus).

In fish, sperm motility activation is one of the most essential procedures for fertilization. Previous studies have mainly focused on the external environmental effects and intracellular signals in sperm activation; however, little is known about the metabolic process of sperm motility activation in fish. In the present study, using ricefield eel (Monopterus albus) sperm as a model, metabonomics was used to analyze the metabolic mechanism of the sperm motility activation in fish. Firstly, 529 metabolites were identified in the sperm of ricefield eel, which were clustered into the organic acids, amino acids, nucleotides, benzene, and carbohydrates, respectively. Among them, the most abundant metabolites in sperm were L-phenylalanine, DL-leucine, L-leucine, lysolecithin choline 18:0, L-tryptophan, adenine, hypoxanthine, 7-Methylguanine, shikimic acid, and L-tyrosine. Secondly, compared to pre-activated sperm, the level of S-sulfo-L-cysteine and L-asparagine were both increased in the post-activated sperm. Ninety-two metabolites were decreased in the post-activated sperm, including quinic acid, acetylsalicylic acid, 7,8-dihydro L-biopterin, citric acid, glycylphenylalanine, and dihydrotachysterol (DHT). Finally, basing on the pathway analysis, we found that the changed metabolites in sperm motility activation were mainly clustered into energy metabolism and anti-oxidative stress. Fish sperm motility activation would be accompanied by the release of a large amount of energy, which might damage the genetic material of sperm. Thus, the anti-oxidative stress function is a critical process to maintain the normal physiological function of sperm.

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.

Transcriptome analysis of blood for the discovery of sex-related genes in ricefield eel Monopterus albus.

The blood acts as a transfer channel for a variety of factors in the whole body. The ricefield eel (Monopterus albus) is a protogynous hermaphrodite vertebrate. Until now, no research has reported an analysis of the blood transcriptome during the process of sexual development in the ricefield eel. In this study, the transcriptome sequencing of blood samples from male and female ricefield eels was completed with a total of 34.70 Gb clean data. The clean data of each sample all reached 5.23 GB, and the percent of the Q30 basic group was 88.62% and above. A total of 106,369 unigenes were obtained after assembly, including 13,296 unigenes with a length of more than 1 kb. Further functional annotation analysis showed that there are 28,522 unigenes that can be annotated. The annotations of genes with differential expression revealed that there were 563 genes with significant differential expression in the blood of male and female ricefield eels, including 91 upregulated genes and 472 downregulated genes. Among which, 14 genes may be closely related to sex differentiation, the qPCR was used to confirmed the expression pattern of those genes and result shown that 11 genes were downregulated and 3 genes were upregulated, consistent with the results of our RNA-Seq analysis. This blood transcript dataset will open future research avenues on ricefield eel sex development and differentiation.

Expression and regulation of Smad2 by gonadotropins in the protogynous hermaphroditic ricefield eel (Monopterus albus).

Smad2, a receptor-activated Smad, plays a critical role in regulating gametogenesis. In this study, a smad2 homologue was identified and sequenced from ricefield eel ovary cDNA, and its mRNA and protein expression levels were analysed during oocyte development. The cDNA sequence of ricefield eel smad2 consisted of 1863 bp encoding a 467-amino acid protein that had high sequence homology with Smad proteins in other teleosts, especially in Poeciliopsis prolifica. The results of real-time quantitative PCR (RT-qPCR) analysis revealed that smad2 is expressed in the ovary during gonad development, increased continuously until the early vitellogenic stage in the ovaries, and then decreased with ovary maturation. Smad2 protein immunoreactivity was localized in the cytoplasm of follicular cells, oogonia, and primary growth stage oocytes. In vitro experiments revealed that follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) promoted smad2 expression in ovary tissue in a time- and dose-dependent manner, respectively. In summary, Smad2 plays a potentially vital role in ricefield eel ovary development.

A Type IIb, but Not Type IIa, GnRH Receptor Mediates GnRH-Induced Release of Growth Hormone in the Ricefield Eel.

Multiple gonadotropin-releasing hormone receptors (GnRHRs) are present in vertebrates, but their differential physiological relevances remain to be clarified. In the present study, we identified three GnRH ligands GnRH1 (pjGnRH), GnRH2 (cGnRH-II), and GnRH3 (sGnRH) from the brain, and two GnRH receptors GnRHR1 (GnRHR IIa) and GnRHR2 (GnRHR IIb) from the pituitary of the ricefield eel Monopterus albus. GnRH1 and GnRH3 but not GnRH2 immunoreactive neurons were detected in the pre-optic area, hypothalamus, and pituitary, suggesting that GnRH1 and GnRH3 may exert hypophysiotropic roles in ricefield eels. gnrhr1 mRNA was mainly detected in the pituitary, whereas gnrhr2 mRNA broadly in tissues of both females and males. In the pituitary, GnRHR1 and GnRHR2 immunoreactive cells were differentially distributed, with GnRHR1 immunoreactive cells mainly in peripheral areas of the adenohypophysis whereas GnRHR2 immunoreactive cells in the multicellular layers of adenohypophysis adjacent to the neurohypophysis. Dual-label fluorescent immunostaining showed that GnRHR2 but not GnRHR1 was localized to somatotropes, and all somatotropes are GnRHR2-positive cells and vice versa at all stages examined. GnRH1 and GnRH3 were shown to stimulate growth hormone (Gh) release from primary culture of pituitary cells, and to decrease Gh contents in the pituitary of ricefield eels 12 h post injection. GnRH1 and GnRH3 stimulated Gh release probably via PLC/IP3/PKC and Ca2+ pathways. These results, as a whole, suggested that GnRHs may bind to GnRHR2 but not GnRHR1 to trigger Gh release in ricefield eels, and provided novel information on differential roles of multiple GnRH receptors in vertebrates.

Isotocin Regulates Growth Hormone but Not Prolactin Release From the Pituitary of Ricefield Eels.

The neurohypophyseal hormone oxytocin (Oxt) has been shown to stimulate prolactin (Prl) synthesis and release from the adenohypophysis in rats. However, little is known about the functional roles of Oxt-like neuropeptides in the adenohypophysis of non-mammalian vertebrates. In this study, cDNAs encoding ricefield eel oxytocin-like receptors (Oxtlr), namely isotocin (Ist) receptor 1 (Istr1) and 2 (Istr2), were isolated and specific antisera were generated, respectively. RT-PCR and Western blot analysis detected the presence of both Istr1 and Istr2 in the brain and pituitary, but differential expression in some peripheral tissues, including the liver and kidney, where only Istr1 was detected. In the pituitary, immunoreactive Istr1 and Istr2 were differentially distributed, with the former mainly in adenohypophyseal cell layers adjacent to the neurohypophysis, whereas the latter in peripheral areas of the adenohypophysis. Double immunofluorescent images showed that immunostaining of Istr1, but not Istr2 was localized to growth hormone (Gh) cells, but neither of them was expressed in Prl cells. Ist inhibited Gh release in primary pituitary cells of ricefield eels and increased Gh contents in the pituitary gland of ricefield eels at 6 h after in vivo administration. Ist inhibition of Gh release is probably mediated by cAMP, PKC/DAG, and IP3/Ca2+ pathways. In contrast, Ist did not affect either prl gene expression or Prl contents in primary pituitary cells. Results of this study demonstrated that Ist may not be involved in the regulation of Prl, but inhibit Gh release via Istr1 rather than Istr2 in ricefield eels, and provided evidence for the direct regulation of Gh cells by oxytocin-like neuropeptides in the pituitary of non-mammalian vertebrates.

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus.

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

Characteristics and phylogenetic studies of complete mitochondrial DNA based on the ricefield eel (Monopterus albus) from four different areas.

In this study, we cloned and sequenced the complete mitochondrial DNA of ricefield eel populations from four different areas (Guizhou province, Guangxi province, Hunan province, and Guangdong province) in China. The mitochondrial genome was 16,622 bp in length and deposited in the GenBank with accession numbers KP779622-KP779625. The organizations of the complete mitogenomes of the four ricefield eel were similar to those of other teleost species which contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a putative control region (CR). Most of these genes were encoded on the H-strand, except for the ND6 and eight tRNA genes, all other genes were encoded on the H-strand. Phylogenetic analyses using N-J computational algorithms showed that the ricefield eel was clustered with Mastacembelus armatus (KJ184553) and they belong to Synbranchiformes. The four ricefield eel populations could be divided into two groups: Guangxi province and the other population cluster together.

Expression and ontogeny of growth hormone (Gh) in the protogynous hermaphroditic ricefield eel (Monopterus albus).

Growth hormone (GH) is a single-chain polypeptide hormone mainly secreted by somatotropes of the anterior pituitary gland and is an important regulator of somatic growth in vertebrates including teleosts. In this study, a polyclonal antiserum against ricefield eel Gh was generated and the expression of Gh at the mRNA and protein levels was analyzed. Both RT-PCR and western blot analysis showed that Gh was predominantly expressed in the pituitary glands of ricefield eels. The immunoreactive Gh signals were localized to the multicellular layers of the adenohypophysis adjacent to the neurohypophysis in ricefield eels. Ontogenetic analysis showed that immunoreactive Gh signals could be detected in the pituitary glands of ricefield eel embryos as early as 3 days post-fertilization. During the sex change from female to male, the levels of the immunoreactive Gh signals in the pituitary glands of the ricefield eels peaked at the intersexual stage. These results suggest that Gh in the pituitary glands may be associated with embryonic development before hatching, as well as with the sex change in the adult ricefield eels, possibly via the classical endocrine manner.

THE DISTRIBUTON CHARACTERISTIC OF FIVE PEPTIDE HORMONES IN THE ENDOCRINE CELLS IN DIGESTIVE TRACT OF MONOPTERUS ALBUS / 解剖学报

Objective To investigate the localization and distribution of the five endocrine cells in the digestive tract mucosa of ricefield eel(Monopterus albus). Methods Using immunocytochemical technique of strept avidin-biotin-peroxidase complex(SABC) were used. Results At least 5 kinds of immunoreactive endocrine cells distributed in the digestive tract mucosa of M.albus. They were gastrin(Gas),somatostatin(Som),5-hydroxytryptamine(5-HT),insulin(Ins),and neurofilament (NF) immunoreactive endocrine(IRE) cells.Gas and Som-IRE cells distributed between stratified squamous epithelium and goblet cell in esophagus. A large number of Gas-IRE cells were found between gastric fundus epithelium and gastric glands, and only a few in the carcia. Ins, 5-HT and NF-IRE cells distributed in the epithelium pylorus and pyloric glandular tube respectively. No any immunoreactive positive reaction was found in the gut of M.albus.In addition, immunoreactive positive reaction of glucagons was not found in whole digestive tract.All immunoreactive endocrine cells were dark brown in color.Their morphology was irregular, cytoplasmic process was shorter and thicker, their nucleus showed an empty bubble.They distributed between esophageal epithelium and gastric epithelium or glandular epithelium, and cytoplasmic process extended to the gastric lumen and glandular cavity.Conclusion There is a complex endocrine function of the digestive system in ricefield eel (M.albus) at the lowest vertebrate.