| Historically, somatostatin and its receptors have been extensively manipulated in the pituitary. The logic behind this is the presence of somatotrophs in the anterior pituitary which releases growth hormones (GH). Advances in the manipulatory techniques revealed novel biological functions of these receptors particularly their constitutive actions in controlling physiological functions and cure of cancerous growth. Cell specific somatostatin receptor signaling enabled in understanding the pituitary hormone control and cell proliferation. However, current knowledge of these receptors in gonads is limited.1. Somatostatin and its receptors:Functional regulation in regulating apoptosis and cell growth of mouse Sertoli cellsRecently, Sertoli cells have been ascertained as the target for the regulatory peptide somatostatin (SST). Therefore, the present study investigated the expression of somatostatin receptors, their age-related alterations and homologous regulation by in vitro treatment with SRIF14on mice Sertoli cells; furthermore, it dealt with SRIF14action on growth progression, apoptotic activity and related gene expressions in these cells. We found that mice Sertoli cells expressed all SST1-5receptors with differential intensities. Age-related real-time PCR of all somatostatin receptors identified abundance of SSTR2and SSTR5mRNA level during Sertoli cell developmental period. Furthermore, higher level of these two receptors was independent of SRIF14, as treatment with SRIF14failed to induce both receptor expressions when compared with control. Somatostatin treatment elicited a dose-dependent decrease in forskolin stimulated cAMP production. Low (100pM and10nM) and high dosage (lμM) groups of SRIF14significantly promoted apoptosis, while all treatment groups led to dose dependent cessation (P<0.05) of G1phase of cell cycle as further validated by increase in casp3, decrease in bc12, elevation of P21(all by western blot) and decrease in Igfl expressions, similarly, SST treatment caused a dose dependent suppression in the mRNA level of kitl gene, which is important in the regulation of spermatogenesis. These findings suggest that somatostatin and its receptors (SSTR2and SSTR5) are important markers in the regulation and development of Sertoli cell; furthermore, it portrays physiological inhibitory role in Sertoli cell development by inducing apoptosis and cell cycle arrest. 2. Constitutive activation of Somatostatin receptor2and5and their interaction with follicle-stimulating hormone in regulating immature mouse Sertoli cells apoptosisSomatostatin receptor2(SSTR2) and somatostatin receptor5(SSTR5) have been reported to be the dominant mediators of Sertoli cells (SCs) apoptosis in immature mouse. However, their constitutive activation and interaction with follicle-stimulating hormone (FSH) remain unclear so far. In the present study, we differentially silenced SSTR2and SSTR5via shRNA and analyzed the transfection efficiency. Transient transfection via shRNA reduced the mRNA and protein expressions of SSTR2and SSTR5up to54%and62%respectively. Mouse SSTR2>SSTR5knockdown restrained cAMP accumulation (145%and89%for most efficient vectors, respectively) and ERK1/2phosphorylation. In addition, receptor knockdown-related cAMP production was partially blocked by purtussis toxin. Both of these receptors showed constitutive activity in mouse SCs, decreasing cell apoptosis (SSTR2=SSTR5, P<0.05) by down-regulating caspase-3and up-regulating Bcl-2protein level. Interestingly, we found that apoptosis in these transfected cells was more strongly decreased (averaging5.5%, P<0.05) on FSH treatment compared to control (9.6%) with relative decrease in apoptotic protein expressions. These results show that SSTR2and SSTR5appear to behave constitutively in regulating SCs apoptosis, independently to its ligand. Furthermore, these receptors may play a crucial role as local inhibitors of FSH on SCs apoptosis during the critical period of testicular development.3Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2on regulation of apoptosis and steroidogenesis in bovine granulosa cellsIn the present study, we employed primary bovine culture of granulosa cells (GCs) as a cellular model to study the potential involvement of somatostatin receptor2(SSTR2) in ovarian function. The results showed that bovine GCs expressed SST2receptor and further found that SSTR2was possibly regulated by follicle-stimulating hormone (FSH), as a significant increase in protein level of SSTR2was observed in FSH-treated GCs. For further analysis, endogenous SSTR2expression was disrupted using small inhibitory RNA (siRNA) and the efficacy of differential silencing of endogenous SSTR2expression was measured both at transcriptional and translational levels. Transient blockage of SSTR2evidenced its constitutive action on GCs, as it significantly increased level of cAMP (2.4-folds) and basal progesterone production (-2-fold,P<0.05) with significant increase (P<0.05) in mRNA levels of StAR and P450ssc without altering estradiol concentration and aromatase mRNA expression. Furthermore, silencing of SSTR2reduced GCs apoptosis (52.5%, P<0.05) and increased cell proliferation, which was further corroborated by up-regulation in protein expressions of B-cell leukemia/lymphoma2(Bcl-2), inhibition of caspase3and mRNA level ofbcl2-associated-X protein (Bax). These results provide evidence that SSTR2subtype controls GCs apoptosis, proliferation and hormonal secretions through selective constitutive action, independently of somatostatin (SST). Given the local inhibitory actions of SSTR2on the gonads, we further found that apoptosis in ssRNAi-2transfected cells decreased (6.8%vs1.9%, P<0.05) more strongly on FSH treatment. Apoptotic protein expressions and steroid hormone mRNA levels were correlated with a relative decrease in apoptosis and increase in progesterone production. Our results suggest that SSTR2may play a crucial role as a local inhibitor of FSH action on GCs apoptosis and steroidogenesis. |
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