Expression Characteristics Of Inhibitors Of Apoptosis Proteins(IAP) In Gonads From Hyriopsis Schlegelii

Hyriopsis schlegelii originated in Lake Biwa of Japan;in 1997 it was introduced to China for production of pearls.Our previous research showed that during the process of gonad development in Hyriopsis schlegelii there was obvious gender differentiation after the age of 12 months,but hermaphroditism was observed at the age of 28 months.The individual,which of female gonad cells began to apoptosis and at last was degraded completely,developed into a male,vice versa.It’s demonstrated that there could be some mechanisms to regulate the apoptosis of germ cells,which ultimately determine sex differentiation of the Hyriopsis schlegelii.In order to reveal this scientific problem this thesis was based on transcriptome database of T.pandanus.Database was screened and the expression of two inhibitor apoptosis protein(IAP)was found to express higher m RNA on the database.It was noticed that the two IAPs may be involved in the apoptosis of gonad cells and gonads degeneration.Therefore,the present study was aimed and work has been carried out to understand the function of IAP.For morphological studies the mature gametes were collected from Hyriopsis schlegelii and its microstructure and ultrastructure was observed under light microscope and scanning electron microscopy.Results showed that the structure of eggs were round or oval with 245.6 ±26.8 μm length and 213.4±44.3μm width in shape.Under the observation of electron microscopy scanning several serosa fold and fertilization hole were seen on the surface of eggs.The flagellum type sperms structure with length of 42.47±3.57μm including a bullet shape head(2.99±0.22μm),the middle part containing five mitochondria with circular permutation and flagella structure tail with thickness of(38.73±3.55μm)were observed.Hermaphrodite phenomenon was observed in the tissue of spermary at age of 26 to 32 months.Follicular counted were more than 90% in male and small proportion in female gonads of the hermaphrodite individuals.Follicular of both individuals were separated by connective tissue in gonads and the two reproductive cell developed unsynchronously.Two IAPs(defined as Hs IAP-1 and Hs IAP-2)genes were cloned from Hyriopsis schlegelii and submitted to National center for Biotechnology Information(NCBI)with(accession No.KY123703 and KY123704).The full length of Hs IAP-1 was 2458 bp contained an open reading frame of 1722 bp encoding 573 amino acids.The structure of Hs IAP-1 protein contains two BIR domains with a RING.The full length of Hs IAP-2 was 3012 bp contained an open reading frame of 1209 bp encoding 402 amino acids.The structure of Hs IAP-2 protein contains one BIR domain without RING.The conserved sequence of CX2CX16HX6 C was shared by BIR domains of Hs IAP-1 and Hs IAP-2.The bioinformatics analyses were performed for Hs IAP-1 and Hs IAP-2 genes.Phylogenetic trees revealed that two Hs IAP proteins were closely related to lower invertebrates and the results were consistent with their taxonomic status.In addition,the first designated as(Hiap-1-1)and the second BIR structure(Hiap-1-2)of Hs IAP-1 domain gathered with vertebrate BIR1 domain and BIR3 domain,respectively.However,the BIR of Hs IAP-2(Hiap-2)was clustered with invertebrates BIR3 domain.All these indicated that three BIR domains might have different functions.Furthermore,the fluorescent quantitative PCR analysis and prokaryotic expression of IAP were performed.The results showed that Hs IAP-1 was expressed in all examined tissues.The highest expression was observed in hepatopancreas,the moderate expression in gills,and the lowest expression in gonad tissues of male and female.Quantitative results of light fixation showed that asiap-1 was ubiquitously expressed in different tissues of mussel,among which the highest expression was checked in hepatopancreas,the second highest expression was checked in gill and the glands both female and male,while in blood cells the lowest expression was checked.The difference was checked in expression of Hs IAP-1 in gonads of five different age wise groups.The results showed that the expression of Hs IAP-1 in the female and male gonads decreased at the 1st,2nd and 3rd age,and then increased significantly at the 4th and 5th age.This result may be due to the different stages of gonadal development,and the regulatory effect of Hs IAP-1 in female gonads is greater than that in male gonads.During the breading season the expressions of Hs IAP-1 in gonad were observed in different year age.The results showed that the expression of Hs IAP-1 in female and male gonads decreased at 1 to 3-year old,and then increased significantly at 4 and 5-year old.This result may be due to periodic differences in gonadal development,and stronger Hs IAP-1 regulation in female gonads than that in male gonads.Purified Hs IAP-1 recombinant protein was obtained using the prokaryotic expression affinity system.The protective effect of the protein on cells was analyzed in vitro.It was observed the effect of Hs IAP-1 on the apoptosis of Hela cells induced by H2O2 stimulation.Flow cytometry analysis showed that Hs IAP-1 can inhibit the occurrence of apoptosis.The degree of inhibition of apoptosis increased with the higher proteins concentration.At the same time the activity of caspase-3 protein under the similar experimental conditions was tested,and the results showed that the protein expression of caspase-3 gradually decreased with the increase of Hs IAP-1 protein concentration.Results indicated that Hs IAP-1 protein can attenuate the effect of H2O2 on Hela cells and slow the apoptotic rate,and the mechanism may be related to the mitochondrial apoptotic pathway associated with caspase-3 protein.