SIRT3 Positively Regulates the Expression of Folliculogenesis- and Luteinization-Related Genes and Progesterone Secretion by Manipulating Oxidative Stress in Human Luteinized Granulosa Cells

Abstract

SIRT3 is a member of the sirtuin family and has recently emerged as a vital molecule in controlling the generation of reactive oxygen species (ROS) in oocytes. Appropriate levels of ROS play pivotal roles in human reproductive medicine. The aim of the present study was to investigate SIRT3 expression and analyze the SIRT3-mediated oxidative response in human luteinized granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemical analysis to localize SIRT3 expression. Hydrogen peroxide and human chorionic gonadotropin were used to analyze the relationship between ROS and SIRT3 by quantitative RT-PCR and Western blotting. Intracellular levels of ROS were investigated by fluorescence after small interfering RNA-mediated knockdown of SIRT3 in human GCs. To uncover the role of SIRT3 in folliculogenesis and luteinization, mRNA levels of related genes and the progesterone concentration were analyzed by quantitative RT-PCR and immunoassays, respectively. We detected the expression of SIRT3 in the GCs of the human ovary. The mRNA levels of SIRT3, catalase, and superoxide dismutase 1 were up-regulated by hydrogen peroxide in both COV434 cells and human GCs and down-regulated by human chorionic gonadotropin. Knockdown of SIRT3 markedly elevated ROS generation in human GCs. In addition, SIRT3 depletion resulted in decreased mRNA expression of aromatase, 17β-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase in GCs and thus resulted in decreased progesterone secretion. These results have the important clinical implication that SIRT3 might play a positive role in the folliculogenesis and luteinization processes in GCs, possibly by sensing and regulating the generation of ROS. Activation of SIRT3 function might help to sustain human reproduction by maintaining GCs as well as oocytes.

HAND2-mediated proteolysis negatively regulates the function of estrogen receptor α

“…The expression of each mRNA was normalised for RNA loading in each sample using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers and conditions for the amplification of GAPDH were as described previously (19). The PCR primers for GAPDH VEGF and ERα were as follows: GAPDH, forward 5'-TGC ACC ACC AAC TGC TTA GC-3' and reverse 5'-GGC ATG GAC TGT GGT CAT GAG-3; VEGF, forward 5'-CCA GCA GAA AGA GGA AAG AGG TAG-3' and reverse 5'-CCC CAA AAG CAG GTC ACT CA C-3; and ERα, forward 5'-TGT GCA ATG ACT ATG CTT CA-3' and reverse 5'-GCT CTT CCT CCT GTT TTT A-3'.…”

The Role and Application of Sirtuins and mTOR Signaling in the Control of Ovarian Functions

“…The ability of SIRT1 to promote ovarian cell proliferation and IGF-I release, as well as to promote FSH action on these processes (from [23]) is illustrated by Figure 1. Fu et al [27] reported the involvement of another SIRT—SIRT3—in the up-regulation of genes involved in human ovarian folliculo-, luteo-, and steroidogenesis: knockdown of SIRT3 resulted in decreased expression of aromatase, 17β-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase in granulosa cells, and decreased their progesterone release. Aside from mTOR, SIRTs can affect ovarian cell functions via changes in the production of transcription factors p53, NFkB [18,23,25], FOXL2 [28], Noch 3 [29], STAT3, steroid hormone receptors, and other intracellular signaling molecules [5], which in turn affect ovarian proliferation, apoptosis, and steroidogenesis.…”

“…At least the amount of SIRT3 mRNA in both mice and human oocytes was positively correlated with mitochondrial biogenesis [25], and SIRT3 knockdown markedly elevated reactive reactive oxygen species in human ovarian granulosa cells [27]. …”

Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women

“…Yeast Sir2 gene is a SIRT1 homolog that is involved in yeast life span extension, nonetheless, but a similar role for SIRT1 has been refuted [16]. In the reproductive system, SIRT1 plays a role in apoptosis of granulosa cells during follicular atresia [17,18] and has been related to preservation of follicular reserve and extension of ovarian lifespan [19].SIRT2 is located in the cytoplasm [9]. It transiently migrates into the nucleus to deacetylate α-tubulin and to modulate chromatin condensation and cell cycle regulation by deacetylating H3 and H4 histones [20,21].…”

“…Yeast Sir2 gene is a SIRT1 homolog that is involved in yeast life span extension, nonetheless, but a similar role for SIRT1 has been refuted [16]. In the reproductive system, SIRT1 plays a role in apoptosis of granulosa cells during follicular atresia [17,18] and has been related to preservation of follicular reserve and extension of ovarian lifespan [19].…”