Molecular cloning and characterization of Sirt1 and its role in the follicle of juvenile Chinese soft‐shelled turtle (Pelodiscus sinensis)

Abstract

Sirt1 is a member of the Sirtuins family that regulates ovarian senescence, follicular development, and oocyte maturation in vertebrates. To understand its role in the ovary of Pelodiscus sinensis, we cloned the full-length cDNA of Ps-Sirt1 and characterized its potential function by intraperitoneally injecting agonist (resveratrol) and antagonist (EX527) in the female juvenile turtle. The full-length cDNA of Ps-Sirt1 was 2106 bp, comprising 203 bp 5′UTR, a 226 bp 3′UTR, and a 1677 bp ORF encoding 558 amino acids. The calculated molecular weight of predicted protein was 63 kDa, and the isoelectric point was 4.65. The predicted protein comprised a conserved Sir2 domain. Amino acid sequence alignment and phylogenetic analyses showed that Ps-Sirt1 was most closely related to turtles, and distantly related to fish. Expression pattern analysis showed Ps-Sirt1 was highest expressed in ovary, followed by testis, liver, heart, and brain. In the ovarian differentiation processes, Sirt1 showed significantly higher expression at embryonic stage 15 and 21. In the testis differentiation process, Sirt1 expression was downregulated at embryonic stages 15–19. Activated and inactivated Sirt1 decreased the number of primordial follicles in juvenile turtles. Bcl2, Bax, mTOR, and rpS6 expressions were up-regulated, whereas GnRH, Fshb, p50, and p65 were down-regulated after agonist treatment. The inaction of Sirt1 with antagonist up-regulated GnRH, Fshb, p65, p53, Foxo3a, Bcl2, Bax, mTOR, and rpS6, but down-regulated p50. In summary, Sirt1 might be involved in the ovarian follicle development of P. sinensis.