甲壳动物高血糖激素家族生理功能研究进展

甲壳动物高血糖激素家族是甲壳动物特有的神经多肽激素家族,主要由眼柄的X-器窦腺复合体(XO-SG)合成与分泌,包括高血糖激素(CHH)、蜕皮抑制激素(MIH)、性腺抑制激素(GIH)和大颚器抑制激素(MOIH),协同调控着甲壳动物的生长、繁殖与蜕皮等生理生化过程.本文就目前CHH家族神经肽的功能研究,包括功能研究的方法、各个激素的功能以及分泌调控等研究进展作一综述.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

Secretion of Pem-CMG,a peptide in the CHH/MIH/GIH family of Penaeus monodon,in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.     

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.     

Induction of Ovarian Maturation and Spawning in <Emphasis Type="Italic">Penaeus monodon</Emphasis> Broodstock by Double-Stranded RNA

Ovarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3 μg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30 days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice.     

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the -factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.     

Effect of x-organ sinus gland extract on [(35)S] methionine incorporation to the ovary of the red swamp crawfish Procambarus clarkii

The presence of gonad-inhibiting hormone in the x-organ sinus gland complex was evaluated in female Procambarus clarkii. Elimination of gonad-inhibiting hormone by way of eyestalk removal resulted in a large acceleration of ovarian development. Daily injection of four sinus gland equivalents reduced ovarian growth of eyestalk-ablated females by about 50% on day 6. Use of the radiotracer [(35)S] methionine showed that gonad-inhibiting activity reached its peak effect between 12 and 24 h following sinus gland injection. Dose-response showed that at least two sinus gland equivalents were needed to significantly counter the accelerated growth induced by eyestalk ablation. The high dose of extract needed to cause significant inhibition was attributed to this delayed response, which subsequently may have required a relatively prolonged exposure to the hormone.     

Endocrine control of reproduction in two rhynchocoelan worms

This brief review of the reproductive endocrinology in two distant species of Rhynchocoela examines selected results of earlier studies on the heteronemertean, Lineus ruber, and reports current and unpublished research on the hoplonemertean, Amphiporus lactifloreus.

Experimental evidence is presented for existence in the brain of a gonad-inhibiting hormone (GIH) that controls reproduction in male and female worms of both species. In adults deprived of GIH during the quiescent phase of the reproductive cycle, sexual characteristics grow rapidly and precociously; GIH replacement therapy prevents the anticipated sexual development. In L. ruber, the removal of GIH stimulates macromolecular syntheses in sexual target cells of both sexes: RNA synthesis is promoted in young oocytes; DNA synthesis is increased in spermatogonia, and induced in primary spermatocytes; and protein synthesis is significantly increased in gametocytes and sex-specific skin glands. GIH might be a regulating hormone which would inhibit the secretion of sex-specific stimulating substances produced by cells in proximity to the gonads.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.