Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2 on regulation of apoptosis and steroidogenesis in bovine granulosa cells
Introduction
Somatostatin (SST), a secretion from hypothalamus is the main inhibitor of pituitary hormones and their functions [1]. Dominantly, at pituitary level via portal venous system, it suppresses the secretions of growth hormone (GH) and luteinizing hormone (LH) [2]. Somatostatin has higher affinity with its five subtype receptors (SSTR1-5) encoded by separate independent genes and each receptor shares differential binding abilities with respective ligands [3]. The activation of these receptors is dependent on multiple cellular signaling pathways including inhibition of adenyl cyclase activity (i.e. cAMP), which preferably associated with SSTR2 and SSTR5 signaling pathways [4].
Earlier studies have confirmed the presence of SSTR2 and SSTR5 on GCs of mice, rats and human [5], [6], [7]. Particularly, SSTR2 protein was found to be localized both on GCs and oocyte. Previous studies have suggested the participation of SSTR2 in the regulation of ovarian function by using SST-specific ligands. Somatostatin (SST) inhibited not only FSH-induced progesterone production in rats but also suppressed the basal and LH-induced cAMP production in porcine GCs [8], [9]. Although readily evident in follicular fluid, the unavailability of SST mRNA is presumable that its ovarian expressions are short and thus, might be supplied by outer portion of gonads [2], [6].
Follicle-stimulating hormone (FSH) is believed to be the key regulator of programmed cell death and steroidogenesis in GCs. Studies on experimental FSHR knockout mice have proved the importance of FSH in the antrum formation and tertiary follicular development [10]. The direct FSH actions on GCs can either be potentiated or inhibited by certain local factors produced under the influence of this hormone. Ovarian follicular growth is governed by hypothalamic–pituitary axis and various local ovarian factors. Recently, administration of SST to mice resulted in the suppression of in vivo gonadotrophin-dependent follicular growth without affecting the atretic processes suggesting that SST has direct role in modulating follicular development [11]. In fact, BIM-23627, a SSTR2 antagonist, predicted that SST was involved in the maintenance of follicular population by reducing the recruitment of resting follicles [5]. Thus, it is likely, that SST receptors might uphold the fate of GCs and follicular maturation by revising local apoptotic factors. Furthermore, because FSH and SST impinge the same transduction (cAMP) pathway; we assume that SSTR2 plays a key role in control of FSH-induced GCs functions and steroidogenesis.
Many G protein coupled receptors (GPCRs) display constitutive activity independent of specific ligands. These receptors include histamine, dopamine, oxytocin and adrenoreceptors [12], [13]. In pituitary cells, SSTR2, SSTR3 and SSTR5 showed strong constitutive activity in controlling local signaling pathways and adrenocorticotropic (ACTH) secretions [3]. The mutation in constitutive active receptors resulted in loss of secretory disorders [13]. One possibility is to use the reverse antagonist against specific receptors to insight the possible functional disorder; however, reverse SST antagonist is not available yet [1].
In the present study, we searched for the presence of SSTR2 in bovine GCs by RT-PCR and western blotting and asked whether this receptor might be regulated by FSH secretion. Furthermore, on the basis of its inhibitory action against FSH in testicular development, we transiently blocked SSTR2 expression by RNAi to determine direct FSH action on GCs apoptosis, FSH-induced estradiol and progesterone production. In addition, by blocking SSTR2 expression, GCs apoptosis and basal hormonal levels were also tested in capacity with its constitutive action which has never been shown in an ovarian model.
Section snippets
Isolation and culture of bovine GCs
All procedures involving animals were approved by the Animal Care and Use Committee of Huazhong Agricultural University. Bovine ovaries were excised from local slaughter house (Wuhan abattoir, Hubei, China) in pre warmed (37 °C) phosphate buffer saline (PBS, Hyclone, UT, USA) containing penicillin (100 IU/mL) and streptomycin (100 μg/mL) (Pen-Strep, Invitrogen, Carlsbad, CA, USA) and transported and processed immediately (∼1 h). Granulosa cells were cultured from bovine ovaries as described
Expression of SSTR2 in bovine GCs
Before functional assessment of SSTR2, the receptor expression was validated in different direct and indirect approaches. At first, we used the primer pair specific to SSTR2 corresponding to cDNA of bovine GCs and detected the respective expected size of SSTR2 (lane 1, Fig. 1A) on agarose gel, which was further confirmed by sequencing and later matched with previously recorded mRNA sequence of bovine SSTR2 on NCBI. As a house keeping gene, GAPDH amplified in bovine GCs (lane 2). The chances of
Discussion
In the present study, we showed the constitutive activity of SSTR2 in regulating apoptosis and steroidogenesis of bovine GCs in the absence of exogenous SST ligand. Furthermore, we reported on SSTR2-mediated negative regulation of FSH action in bovine GCs. These novel biological roles for this regulatory peptide were revealed by its capacity to counteract FSH-induced cAMP production and progesterone release. Data reported here further suggested that SSTR2 was a putative candidate to function as
Funding
Supported by National Natural Science Foundation of China (No. 31001007) the Fundamental Research Funds for the Central Universities (No. 2011PY020 and 2012ZYTS049) and the earmarked fund for Modern Agro-industry Technology Research System (CARS-37-04B).
Conflict of interest
All authors have no conflict of interest.
Acknowledgments
Designed by: HR Conducted by: HR, DP Analyzed by: HR, Wrote the paper: HR. Liguo YANG was the major supervisor in this whole experiment. The authors are thankful to Shi Lei, Jiang Xu, Chong Zhenlu for ovarian collection from slaughter house and Xiao hui for gifting FSH during this experiment. The first author further gratitude the technical guidance of Dr. Muhammad Imran Hamid during the revision of this manuscript.
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