| Amphibian metamorphosis is accurately regulated by thyroid hormones(THs).Several environmental stressors(i.e.temperature,perchlorate and exogenous T4)have been shown to disrupt the normal action of THs,and affect growth and development of amphibians.Temperature,a major abiotic factor,affects the growth and development of amphibians by altering THs levels.In generally,amphibians reared at relatively warmer temperatures display faster growth rates and shorter larval periods when compared with conspecifics reared at colder temperatures.Perchlorate,a chemical contaminant,effectively decreases or blocks THs synthesis through inhibition of the sodium-iodide symporter,leading to change in growth and development of amphibians.Additionally,adding exogenous T4 to the rearing water of the tadpoles can increase THs levels and induce metamorphic changes precociously.Studies have shown the impacts of environmental stressors(i.e.temperature,perchlorate and exogenous T4)on the growth and development of amphibians by changing THs levels.Skeletons play an important role in forming skeleton,organ protection and mineral storage.In the life history of amphibians,the development of limb bones can contribute amphibians to complete the transformation from aquatic to terrestrial environment.Most of the bones develop through a process known as endochondral ossification,which is tightly regulated by various specific molecules such as systemic factors,transcription factors,locally produced growth factors and extracellular matrix(ECM)components secreted by the chondrocytes.THs,systemic factors,regulate proliferation,hypertrophy and apoptosis of chondrocytes in growth cartilage.Runt-related transcription factors(Runx)regulate early and late differentiation of osteoblast and chondrocyte.Vascular endothelial growth factors(VEGF),a member of locally produced growth factors,could facilitate vascular invasion of growth cartilage by binding to VEGF receptors during the endochondral ossification.Additionally,matrix metalloproteases(MMPs)directly participate in the remodeling of the extracellular matrix(ECM).However,relatively little is known about the effects of environmental stressors(i.e.temperature,perchlorate and exogenous T4)on skeletal development via altering THs levels.In this study,B.gargarizans tadpoles have been used as an excellent test animal to examine whether environmental stressors(i.e.temperature,perchlorate and exogenous T4)may affect the endochondral ossification of B.gargarizans tadpoles by altering THs level.The present study consists of two parts.The first part investigated the effect of temperature on the growth and development as well as endochondral ossification in tadpoles:B.gargarizans tadpoles were reared at three different water temperatures(15?,22? and 29?)from Gosner stage 28 to 46.We examined the effects of temperature on survival,growth,development,metamorphic duration,size and skeletal ossification at Gosner stage 40,42,and 46,as well as thyroid tissue of tadpoles reached to metamorphic climax(Gs42)by using hematoxylin and eosin(HE)staining methods.Besides,we examined the transcription levels of endochondral ossification-related genes in the hind limb at metamorphic climax(Gs42)via RT-qPCR technology.The second part studied the effects of perchlorate and exogenous T4 on the growth and development as well as endochondral ossification in tadpoles:B.gargarizans larvae from Gosner stage 28-46 were exposed to 250 ?g/L NaClO4 and 10 ?g/L T4 treatments.We measured the survival,growth,development and metamorphic duration of tadpoles at Gosner stage 40,42 and 46,and evaluated histological characteristics of thyroid glands in tadpoles at metamorphic climax(Gs42)treated with 250 ?g/L NaClO4 and 10?g/L T4.Furthermore,we assessed that the effects of perchlorate and exogenous T4 on skeletal ossification of tadpoles at Gosner stage 40,42 and 46 with the alizarin redalcian blue double staining technique.Additionally,we investigated the transcription levels of endochondral ossification-related genes in the hind limb of tadpoles at metamorphic climax(Gs42)under 250 ?g/L NaClO4 and 10 ?g/L T4 treatments.The results of the first part are as follows:1.Morphological measurements showed that warm temperature-treated(29?)significantly decreased the total length,body length,weight and hind limb length of tadpoles,and shortened the metamorphic duration,while cold temperature(15?)led to an increase in body size and hind limb length,and prolonged metamorphic time compared to ambient temperature(22?).This result indicates that temperature may affect the growth and development of B.gargarizans tadpoles.2.Histopathological studies for thyroid tissue showed that warm temperature significantly reduced the colloid area in thyroid gland,while cold temperature resulted in a significant increase in the colloid area.The colloid content in thyroid follicles determines the secretory activity of THs.It thus suggests that warm temperature may induce an increase in the ability of THs secretion as reflected by decreased in colloid area,while cold temperature may significantly increase the colloid area,resulting in the reduction of THs secretion.3.The results of alizarin red-alcian blue double staining indicated that warm and cold temperatures inhibited the degree of ossification in B.gargarizans tadpoles.The hind limb bones of tadpoles exhibited the highest degree of ossification at ambient temperature,followed by cold temperature and finally at warm temperature.It has been verified that THs affected the skeletal development in tadpoles.This suggests that altering THs levels(increase or decrease)may inhibit the ossification degree of tadpoles.4.According to RT-qPCR technology,warm and cold temperatures significantly downregulated the transcript levels of endochondral ossification-related genes TR?,MMP9,MMP13 and Runx3,while significantly upregulated Runx2,VEGFA,VEGFB,VEGFC,VEGFR-1,VEGFR-2 and VEGFR-3 levels in the hind limb of B.gargarizans tadpoles.This indicates that temperature restrains the endochondral ossification in B.gargarizans tadpoles.The results of the second part are as follows:5.Morphological results illustrated that 250 ?g/L NaClO4 treatments resulted in increased body size and delayed the developmental stage in B.gargarizans tadpoles.Whereas 10 ?g/L T4 treatments led to decreased body size and accelerated metamorphic duration,and reduced the survival rate of tadpoles.In addition,250 ?g/L NaClO4 and 10 ?g/L T4 treatments inhibited hind limb growth in B.gargarizans tadpoles.Thus,perchlorate and exogenous T4 may affect the growth and development of B.gargarizans larvae.6.Histological studies of thyroid tissue suggested that exposure to 250 ?g/L NaClO4 significantly reduced the follicle size and follicular epithelial cells height,and led to the follicular epithelial cell hyperplasia.10 ?g/L T4 exposure significantly decreased the thyroid gland size,follicle size,colloid area,follicular epithelial cells height and the number of follicles.This indicates that perchlorate and exogenous T4 may damage thyroid tissue in B.gargarizans tadpole.7.The double staining results indicated that 250 ?g/L NaClO4 and 10 ?g/L T4 treatments inhibited the degree of ossification in B.gargarizans larvae.At Gs46,whole the hind limb bones of tadpoles were fully ossified in the control groups,while hind limb bones at 250 ?g/L NaClO4 and 10 ?g/L T4 treatments were still partially cartilage.Studies suggested that perchlorate effectively deceases THs synthesis by inhibition of the sodium-iodide symporter,while adding exogenous T4 can increase THs levels in tadpoles.It thus suggests that increased or decreased THs levels may inhibit tadpole ossification after perchlorate and exogenous T4 treatments.8.RT-qPCR results showed that 250 ?g/L NaClO4 and 10 ?g/L T4 treatments significantly changed the expression levels of endochondral ossification-related genes in the hind limb of B.gargarizans tadpoles.Exposure to 250 ?g/L NaClO4 and 10 ?g/L T4 significantly decreased MMP9,MMP13.and Runx3 expression levels.250?g/L NaClO4 significantly decreased the expression levels of TR?,Dio2 and Dio3 genes,while 10?g/L T4 treatments significantly increased Runx1,Runx2,VEGFA,VEGFB,VEGFC,VEGFR-1,VEGFR-2,VEGFR-3,TR?,TR?,Dio2 and Dio3 levels.This indicates that perchlorate and exogenous T4 inhibit the endochondral ossification in B.gargarizans tadpoles.In conclusion,environmental stressors(i.e.temperature,perchlorate and exogenous T4)affect the growth and development of B.gargarizans tadpoles,cause damage to thyroid tissue,and inhibit the ossification of tadpoles.Also,the present study suggests that temperature,perchlorate and exogenous T4 exert their effect on endochondral ossification of B.gargarizans larvae by altering THs levels.These results will provide experimental references for further studies on the effects of environmental stressors on endochondral ossification in amphibians. |
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