Knockdown of CEBPβ by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis

et al. 2016

PLoS ONE

Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.

Section: Discussionsupporting

confidence: 88%

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation

Zhao

et al. 2016

PLoS ONE

“…To confirm these results, we checked the effect of resistin on the protein expression of cyclins A and B. Cyclins A and B are well established markers of porcine ovarian cell proliferation, growth and development (Kolesarova et al 2011, Zhen et al 2014. We observed that resistin has no effect on the protein expression of cell proliferation markers, cyclins A and B.…”

Section: Effect Of Resistin On Cell Proliferationmentioning

confidence: 70%

Resistin is a survival factor for porcine ovarian follicular cells

Rak¹,

Drwal²,

Wróbel³

et al. 2015

Reproduction

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10 ng/ml alone or in combination with FSHand IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways. Reproduction (2015) 150 343-355

“…TP53 expression in sheep GCs is suppressed by Melatonin, which reduces the negative effects on these cells caused by thermal stress (Fu et al, ). Up‐regulating CASP3 and BAX expression and down‐regulating BCL2 expression are consistently linked during the early phases of apoptotic induction in GCs from mice (Kadariya et al, ), bovine (Chong et al, ), and pigs (Zhen et al, ). As with CASP3 , Melatonin inhibits the activation of CASP3 , and modulates CASP3 protein activity (Baydas, Reiter, Akbulut, Tuzcu, & Tamer, ), possibly by controlling the upstream step of mitochondrial cytochrome c release that otherwise promotes its activation (Zhang et al, ).…”

Section: Discussionmentioning

confidence: 99%

The role of Melatonin receptor MTNR1A in the action of Melatonin on bovine granulosa cells

Liu

Molecular Reproduction Devel

et al. 2017

Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin β and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin β abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.