ABSTRACT
Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.
Subject(s)
Arthropod Proteins/pharmacology , Carrier Proteins/pharmacology , Drug Design , Invertebrate Hormones/pharmacology , Models, Molecular , Penaeidae/drug effects , Reproductive Control Agents/pharmacology , Vitellogenesis/drug effects , Amino Acid Sequence , Animals , Antibodies, Neutralizing/pharmacology , Aquaculture , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/metabolism , Biological Assay , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Conserved Sequence , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/pharmacology , Eye , Female , Invertebrate Hormones/chemistry , Invertebrate Hormones/genetics , Invertebrate Hormones/metabolism , Neurosecretory Systems/cytology , Neurosecretory Systems/drug effects , Neurosecretory Systems/physiology , Penaeidae/cytology , Penaeidae/physiology , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Reproductive Control Agents/antagonists & inhibitors , Reproductive Control Agents/chemistry , Reproductive Control Agents/metabolism , Sequence Alignment , Structural Homology, Protein , Thioredoxins/chemistry , Thioredoxins/genetics , Thioredoxins/metabolism , Thioredoxins/pharmacology , Vitellins/antagonists & inhibitors , Vitellins/genetics , Vitellins/metabolism , Vitellogenins/antagonists & inhibitors , Vitellogenins/genetics , Vitellogenins/metabolismABSTRACT
BACKGROUND AND PURPOSE: Arsenic exposure frequently leads to reproductive failures by disrupting the rat uterine histology, hormonal integrity and estrogen signaling components of the rat uterus, possibly by generating reactive oxygen species. All-trans retinoic acid (ATRA) was assessed as a prospective therapeutic agent for reversing reproductive disorders. EXPERIMENTAL APPROACH: Rats exposed to arsenic for 28 days were allowed to either recover naturally or were treated simultaneously with ATRA for 28 days or treatment continued up to 56 days. Hematoxylin-eosin double staining was used to evaluate changes in the uterine histology. Serum gonadotropins and estradiol were assayed by ELISA. Expression of the estrogen receptor (ERα), an estrogen responsive gene vascular endothelial growth factor (VEGF), and cell cycle regulatory proteins, cyclin D1 and CDK4, was assessed by RT-PCR, immunohistochemistry and western blot analysis. KEY RESULTS: ATRA ameliorated sodium arsenite-induced decrease in circulating estradiol and gonadotropin levels in a dose- and time-dependent manner, along with recovery of luminal epithelial cells and endometrial glands. Concomitant up regulation of ERα, VEGF, cyclin D1, CDK4 and Ki-67 was also observed to be more prominent for ATRA-treated rats as compared to the rats that were allowed to recover naturally for 56 days. CONCLUSIONS AND IMPLICATIONS: Collectively, the results reveal that ATRA reverses arsenic-induced disruption of the circulating levels of gonadotropins and estradiol, and degeneration of luminal epithelial cells and endometrial glands of the rat uterus, indicating resumption of their functional status. Since structural and functional maintenance of the pubertal uterus is under the influence of estradiol, ATRA consequently up regulated the estrogen receptor and resumed cellular proliferation, possibly by an antioxidant therapeutic approach against arsenic toxicity.
