Effects Of Octylphenol On Expression Of PRL And PRLR Of Rana Chensinensis Tadpoles

Endocrine disrupting compounds (EDCs) was a kind of exogenous chemicals that could influence animal endocrine system by interacting with endogenous hormone synthesis, release, transport and metabolism. The development and reproduction of normal function of aquatic animals such as fish and amphibians could affect by EDCs, thus further affecting survival in the wild. Alkylphenol polyethoxylates (APEs) was a kind of EDCs and one of the classes of nonionic surfactants, most widely used in the manufacturing of plastics, cleaning agents, agrochemicals, textiles, pulp and paper, cosmetics and food products. Octylphenol (OP), with the characteristics of easy to bioaccumulation and hard-degradation and widespread in water, soil, sludge, and sediment, belonging to a kind of endocrine disrupting chemicals (EDCs). OP is one of the degradation products of alkylphenol polyethoxylates (APEs), regulating endocrine hormone levels by interfering endocrine activities. Prolactin (PRL), a protein hormone synthesized and secreted by the anterior pituitary in vertebrates, has a wide range of physiological function through binding prolactin receptor (PRLR). In amphibian, PRL involved in the process of growth and development, osmoregulation and reproduction, and involved in remodeling of several tissues and organs in tadpoles metamorphosis.Due to the investigation of amphibians PRL and PRLR reported mostly in molecular cloning, signaling pathways, etc, the information on the effects of EDCs on PRL and PRLR expression are inadequately. In order to investigate the mechanism of OP disturbed PRL in amphibian metamorphosis, the microstructure of the liver, kidney, skin and tail tissues of Chinese brown frogs R. chensinensis tadpoles at Gosner stage 43 were observed by paraffin section through HE staining, and then the expressions of PRLR were detected in liver, kidney, skin and tail tissues respectively by in situ hybridization. In addition, the tadpoles at Gosner stage 29 were exposed to 10-8,10-7, 10-6 mol/L OP for 20,30,40 and 50 d respectively, the levels of PRL and PRLR mRNA in brain, and the levels of PRLR mRNA in liver, kidney, skin and tail were detected by quantitative RT-PCR under different OP concentrations and different exposure time, then analyzed the possible influencing mechanism. The main experimental results and analysis are as follows:1. Tissue section observation results shown that in the liver tissue of tadpoles at Gosner stage 43 under the normal development condition, hepatocytes were lined funicular in the hepatic tissue, and the hepatic sinusoidal were between the hepatic cords. In kidney, the renal glomerular and renal tubules have formed and the structures of them were clearly observed. The skin tissue was containing one or two layers of epidermis and a handful of loose connective tissue. In tail tissue, there were vertical, horizontal and diagonal line arrangement of skeletal muscle fibers and piles of adipocyte. These indicated that PRL combined with PRLR may involve in development of hepatic tissue, osmoregulation in kidney and skin, degradation process of the tail and inhibit tail resorption during R. chensinensis larva stage.2. PRLR positive reaction products were localized in hepatic sinusoidal by in situ hybridization. In addition, abundant PRLR mRNA signals were localized in the kidney tubules epithelial cells and skin epidermal cells. In tail, the signals were weakly expressed in adipocyte, muscle extracellular connective tissues and blood vessels, but there were no PRLR mRNA signals expressed in the skeletal muscle cells.3. The detection results of qRT-PCR shown that there were no significant increase of PRL and PRLR mRNA in brain, and PRLR mRNA in liver, kidney, skin and tail tissues of the tadpoles with different OP treatment concentrations for 20 d when compared to the control. However, exposed 30,40 and 50 d, compared with the control, the levels of PRL and PRLR mRNA were significantly increased with the similar trends in brain. Exposure OP for 40 and 50 d, the levels of PRLR mRNA were significantly increased in liver, kidney, skin. These indicated that the transcription of PRL and PRLR can be up-regulated after chronically exposure to lower OP concentration. Expoeure for 40 d, OP also up-regulated the levels of PRLR mRNA compared with the control in tail, and either in control or exposure OP for 40 d, the levels of PRLR mRNA were increased climax which was associated with the tadpoles reached metamorphic climax. These indicated that OP affected the functions of PRL via disturbing the expression of PRLR, leading to the degradation process of the tail delay.4. Compared OP exposure for 50 d with those for 40 d, the up-regulated of PRLR mRNA was decreased in brain, liver and skin. However, the changes were not obviously in kidney. These implied that PRLR expression were different in various organs, which was associated with the ability to adapt to the OP interference.5. Compared the relative expression values of PRLR mRNA in brain, liver, skin and tail tissues under different OP treatment concentrations, the expressions of PRLR were strongest in 10-6 mol/L group and the dose dependent effect was displayed.6. In kidney, the relative values of PRLR mRNA in 10-7 mol/L were similar with those of in 10-6 mol/L. It indicated that kidney was more sensitive to OP than other tissues.The results shown that the distribution of PRLR mRNA in liver, kidney, skin and tail of tadpoles suggested that PRL and PRLR were related to remodeling of tissues and organs during metamorphosis. OP was considered as a common contaminant in aquatic environment and could disturb the expression of PRL and PRLR, further influencing development and delay metamorphosis of tadpoles. The expressional change of PRL and PRLR after exposure to OP were similar with the expressional patterns of PRL and PRLR of development process, and the sub lethal concentration of OP in our lab up-regulated genes transcription was only changed the PRL and PRLR mRNA levels, while not change its original trends, thus further affecting the growth and development, rather than stressed the survival of tadpoles in the wild aquatic environment.