Characterization of the Mechanism of Inhibin α-Subunit Gene in Mouse Anterior Pituitary Cells by RNA Interference

Abstract

Inhibin, a member of the transforming growth factor-β [TGF-β] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary–gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of β-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-β concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-β in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinβ which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

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

“…Inhibin primarily provides negative feedback regulation of FSH production. Inha knock‐out rats exhibited reduced expression of TGFBR3 , which disrupted inhibin‐TGFBR3 signaling and therefore increases the secretion of FSH in the rat anterior pituitary cells (Han et al, ). Also, although Inhbb expression was up‐regulated after Inha silencing, the secretion of Inhibin β is still decreased (Han et al, ), indicating how important their co‐production is.…”

“…Inha knock‐out rats exhibited reduced expression of TGFBR3 , which disrupted inhibin‐TGFBR3 signaling and therefore increases the secretion of FSH in the rat anterior pituitary cells (Han et al, ). Also, although Inhbb expression was up‐regulated after Inha silencing, the secretion of Inhibin β is still decreased (Han et al, ), indicating how important their co‐production is. Interestingly, inhibin vaccines that attenuate its activity resulted in higher fertility and ovulation rates in sheep, cattle, and rats (Dan, Liu, Han, Liu, & Yang, ; Han et al, ; Takedomi et al, ).…”

“…Three siRNA target sites were selected from the siRNA online program (http://rnaidesigner.thermofisher.com) at positions 213, 477, and 909 in the coding region (Table ). The typical structure of the short‐hairpin RNA was described previously (Han et al, ), and contained a restriction site at its 5′ end, a 19‐base sense strand, 9 bases for the hairpin loop, a 19‐base antisense strand, 6 bases for the terminator, and 6 bases corresponding to a unique HindIII restriction site (allowing for identification of the recombinant plasmids by restriction enzyme digestion [Figure ]). Two complementary oligonucleotides were synthesized for hybridization and cloning.…”

GDF9 affects the development and tight junction functions of immature bovine Sertoli cells

“…Numerous studies have shown that Cyclin E is required for cells to transit from G1 to S phase (Liszewska, Rekawiecki, & Kotwica, 2005). Bcl-2 is an inhibitor of apoptosis, and its expression level determines the apoptotic fate of various cells (Han et al, 2013;Liszewska et al, 2005). Caspase-3 has been shown to promote cell apoptosis in most cells, and its activation often serves as an irreversible apoptotic signal (Mruk & Cheng, 2000).…”

Overexpression of C/EBPβ Affects The Cell Cycle Regulators and Spermatogenesis Related Genes Expression And Function of Bovine Sertoli Cells

“…Caspase‐3 has been shown to promote cell apoptosis in most cells, and its activation often serves as an irreversible apoptotic signal (Han et al. ). In this study, we found Caspase‐3 mRNA levels to be significantly upregulated upon overexpression of CEBP β .…”