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.
Highlights
Inhibin, a disulfide-linked dimer, plays an important role in the modulation of whole reproductive axis and encompasses all reproductive events [1]
The results showed that all three plasmids were able to down-regulate inhibin a-subunit expression, while all three parameters in agreement depicted the higher down regulation of mRNA and protein expression by pshRNA-2 (60% down regulation) as compared to other plasmids ascertained the inhibition of expression at protein level
The activin B (ACVB) concentration was increased significantly (P,0.05) in APCs at 24 and 48 h post transfection. These results showed that decrease in INHB and and increase in ACVB concentrations can be associated with increase in FSH concentration in APCs transfected with pshRNAi-2 vector
Summary
A disulfide-linked dimer, plays an important role in the modulation of whole reproductive axis and encompasses all reproductive events [1] In females, it is produced by ovarian follicles, and serves as a major antagonist in the control of FSH release. Presence of beta glycan implicates its autocrine exertion in pituitary cells, thereby, turns out to be a regulator of reproductive cycles [1]. This raises the question of whether production of inhibin in pituitary cells has functional association with local different steroids and their receptors [11,12]
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