Differential involvement of cAMP/PKA-, PLC/PKC- and Ca2+/calmodulin-dependent pathways in GnRH-induced prolactin secretion and gene expression in grass carp pituitary cells.

Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg6, Pro9, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca2+ by Ca2+ influx via L-type voltage-sensitive Ca2+ channel (VSCC) with subsequent CaM expression and NFAT2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca2+/CaM/CaMK-II pathways but not the signalling events via IP3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca2+/CaM/CaN/NFAT2 signalling but not PLC/IP3/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca2+/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca2+/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca2+/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT2 signalling.

The effect of miR-23b-3p on regulating GH by targeting POU1F1 in Yanbian yellow cattle.

This study aimed to evaluate the effect of miR-23b-3p on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. The mRNA and protein levels of GH and miR-23b-3p target genes were measured by real time fluorescence quantitative PCR (qPCR) and Western blot, respectively. The target relationship of miR-23b-3p was validated by double luciferase reporter gene system. The results showed that GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-23b-3p-mi group than in the NC group (P<0.01), while GH mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.05). The result of bioinformatics analysis and double luciferase reporter gene system validation proved that miR-23b-3p targeted 3'UTR of pituitary specific transcription factor 1 (POU1F1). POU1F1 mRNA and protein levels were lower miR-23b-3p-mi group than in the NC group (P<0.01), while POU1F1 mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.01). These results demonstrated that miR-23b-3p could regulate GH expression in pituitary cells by regulating POU1F1 gene.

PPP2R2A promotes Hu sheep pituitary cell proliferation and gonadotropin secretion associated with prolificacy.

The anterior pituitary plays a critical role in the endocrine system, contains gonadotrophs, which regulate reproductive efficiency by secreting follicle-stimulating hormone (FSH) and luteinizing hormone (LH). PPP2R2A is a serine-threonine phosphatase that regulates reproductive functions in both females and males, its function in pituitary cells remain unclear. Hu sheep is a highly prolific breed, which makes it suitable for studying reproductive mechanisms. In this study, the relative abundances of PPP2R2A mRNA expression were higher in the pituitary of high-prolificacy (HF) Hu sheep compared to those of low-prolificacy (LF) Hu sheep. Additionally, we demonstrated that PPP2R2A promotes pituitary cell proliferation and gonadotropin secretion using the EdU assay and ELISA, respectively. Moreover, it inhibits pituitary cell apoptosis using flow cytometry. Furthermore, PPP2R2A may affect pituitary cell function by regulating the AKT/mTOR signaling pathway. In summary, our findings suggest that PPP2R2A may play a role in regulating pituitary function and influencing the secretion of gonadotropins.

EZH2 Gene Knockdown Inhibits Sheep Pituitary Cell Proliferation via Downregulating the AKT/ERK Signaling Pathway.

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2's abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHß, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

Estradiol and progesterone regulate NUCB2/nesfatin-1 expression and function in GH3 pituitary cells and THESC endometrial cells.

Estradiol (E2) and progesterone (P4) are essential sex steroid hormones that play critical roles in the pituitary gland and uterus. Recently, nesfatin-1, a polypeptide hormone that regulates appetite and energy homeostasis in the hypothalamus, was found to be expressed in the pituitary gland and uterus. In this study, we aimed to investigate the relationship between these two steroid hormones and the expression and function of nesfatin-1 in the pituitary gland and uterus using GH3 cells, a lacto-somatotroph cell line, and THESC cells, an endometrial stromal cell line. First, we verified the presence of nesfatin-1 and nesfatin-1 binding sites in GH3 and THESC cells. E2 increased the mRNA expression of NUCB2, the gene encoding the nesfatin-1 protein, in GH3 cells, while P4 had no significant effect. In THESC cells, NUCB2 mRNA expression was decreased by E2 but increased by P4. In addition, nesfatin-1 significantly increased growth hormone (GH) and prolactin (PRL) mRNA expression in GH3 cells, and E2 enhanced this effect. In THESC cells, nesfatin-1 significantly increased the mRNA expression of insulin-like growth factor binding protein 1 (IGFBP1) and PRL, which are decidualization marker genes, and P4 further enhanced this effect. These results suggest that nesfatin-1 may act as a local regulator of GH and PRL production in the pituitary gland and decidualization in the uterus, modulating its effects in response to E2 and P4.

Small GTPase control of pituitary hormone secretion: Evidence from studies in the goldfish (Carassius auratus) neuroendocrine model.

The secretion of vertebrate pituitary hormones is regulated by multiple hypothalamic factors, which, while generally activating unique receptor systems, ultimately propagate signals through interacting intracellular regulatory elements to modulate hormone exocytosis. One important family of intracellular regulators is the monomeric small GTPases, a subset of which (Arf1/6, Rac, RhoA, and Ras) is highly conserved across vertebrates and regulates secretory vesicle exocytosis in many cell types. In this study, we investigated the roles of these small GTPases in basal and agonist-dependent hormone release from dispersed goldfish (Carassius auratus) pituitary cells in perifusion experiments. Inhibition of these small GTPases elevated basal LH and GH secretion, except for Ras inhibition which only increased basal LH release. However, variable responses were observed with regard to LH and GH responses to the two goldfish native gonadotropin-releasing hormones (GnRH2 and GnRH3). GnRH-dependent LH release, but not GH secretion, was mediated by Arf1/6 GTPases. In contrast, inhibition of Rac and RhoA GTPases selectively enhanced GnRH3- and GnRH2-dependent GH release, respectively, while Ras inhibition only enhanced GnRH3-evoked LH secretion. Together, our results reveal novel divergent cell-type- and ligand-specific roles for small GTPases in the control of goldfish pituitary hormone exocytosis in unstimulated and GnRH-evoked release.

Contributions of Andrée Tixier-Vidal (1923-2021) to modern cell biology.

This article illustrates the main stages of the scientific career of Dr Andrée Tixier-Vidal, a pioneer in cell biology research in France. She made important discoveries in the field of hormone secretion and neuronal morphogenesis. She played a key role in developing pituitary and neuronal cultures and using electron microscopy to study cellular structures. Her scientific influence continues to irradiate through her students and collaborators.

The Novel Competing Endogenous Long Noncoding RNA SM2 Regulates Gonadotropin Secretion in the Hu Sheep Anterior Pituitary by Targeting the Oar-miR-16b/TGF-ß/SMAD2 Signaling Pathway.

Pituitary gonadotropins play a pivotal role in reproduction. Long noncoding RNAs (lncRNAs) have been identified as important regulators in the hypothalamic−pituitary−ovarian (HPO) axis associated with reproduction. However, the contributions of lncRNAs to pituitary gonadotropin secretion remain largely unknown. Therefore, this work was performed to uncover the functional mechanisms of the novel lncRNA TCONS_00083279 (lncRNA SM2) and its potential targeting pathway oar-miR-16b/TGF-beta/SMAD2, which is associated with gonadotropin secretion in sheep pituitary cells. In the present study, the lncRNA SM2 showed high expression levels in the sheep pituitary gland, and it was located in both the nucleus and the cytoplasm of pituitary cells. lncRNA SM2 knockdown inhibited pituitary cell proliferation and FSH and LH secretion. The function of the lncRNA SM2 was sponged by oar-miR-16b, and this regulated the growth and gonadotropin secretion of pituitary cells by modulating SMAD2, as shown by the dual-luciferase reporter assay. FSH and LH levels were both upregulated by SMAD2 overexpression. Moreover, the levels of the lncRNA SM2, SMAD2 and TGFR1, as well as FSH and LH, in sheep pituitary cells increased significantly under gonadotropin-releasing hormone (GnRH) stimulation (p < 0.05). This work illustrates that the lncRNA SM2 regulates gonadotropin secretion in the Hu sheep anterior pituitary by targeting the oar-miR-16b/TGF-ß/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction.

The effect of miR-93 on GH secretion in pituitary cells of Yanbian yellow cattle.

Yanbian yellow cattle breeding is limited by slow growth. We previously found that the miRNA miR-93 was differentially expressed between the blood exosomes of Yanbian yellow cattle and Han Yan cattle, which differ in growth characteristics. In this experiment, we evaluated the effects of miR-93 on growth hormone (GH) secretion by pituitary cells of Yanbian yellow cattle using qPCR, Western blot, Targetscan and RNA hybrid analysis software and Dual-Luciferase reporter gene system. The results showed that miR-93 targeted 3' UTR of GHRHR(growth hormone releasing hormone receptor); GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-93-mi group than in the NC control group (p < 0.01), while GH mRNA and protein levels were higher in the miR-93-in group than in the iNC control group, but the difference was not significant (p > 0.05); GHRHR mRNA and protein levels were significantly lower in the miR-93-mi group than in the NC control group (p < 0.01), while GHRHR protein levels were significantly higher in the miR-93-in group than in the iNC control group (p < 0.05), but there was no significant difference about GHRHR mRNA level between two groups (p > 0.05). These results prove that miR-93 regulates GH secretion in pituitary cells via GHRHR.

Differential Regulation of the Expression of the Two Thyrotropin Beta Subunit Paralogs by Salmon Pituitary Cells In Vitro.

We recently characterized two paralogs of the thyrotropin (TSH) beta subunit in Atlantic salmon, tshßa and tshßb, issued from teleost-specific whole genome duplication. The transcript expression of tshßb, but not of tshßa, peaks at the time of smoltification, which revealed a specific involvement of tshßb paralog in this metamorphic event. Tshßa and tshßb are expressed by distinct pituitary cells in salmon, likely related to TSH cells from the pars distalis and pars tuberalis, respectively, in mammals and birds. The present study aimed at investigating the neuroendocrine and endocrine factors potentially involved in the differential regulation of tshßa and tshßb paralogs, using primary cultures of Atlantic salmon pituitary cells. The effects of various neurohormones and endocrine factors potentially involved in the control of development, growth, and metabolism were tested. Transcript levels of tshßa and tshßb were measured by qPCR, as well as those of growth hormone (gh), for comparison and validation. Corticotropin-releasing hormone (CRH) stimulated tshßa transcript levels in agreement with its potential role in the thyrotropic axis in teleosts, but had no effect on tshßb paralog, while it also stimulated gh transcript levels. Thyrotropin-releasing hormone (TRH) had no effect on neither tshß paralogs nor gh. Somatostatin (SRIH) had no effects on both tshß paralogs, while it exerted a canonical inhibitory effect on gh transcript levels. Thyroid hormones [triiodothyronine (T3) and thyroxine (T4)] inhibited transcript levels of both tshß paralogs, as well as gh, but with a much stronger effect on tshßa than on tshßb and gh. Conversely, cortisol had a stronger inhibitory effect on tshßb than tshßa, while no effect on gh. Remarkably, insulin-like growth factor 1 (IGF1) dose-dependently stimulated tshßb transcript levels, while it had no effect on tshßa, and a classical inhibitory effect on gh. This study provides the first data on the neuroendocrine factors involved in the differential regulation of the expression of the two tshß paralogs. It suggests that IGF1 may be involved in triggering the expression peak of the tshßb paralog at smoltification, thus representing a potential internal signal in the link between body growth and smoltification metamorphosis.

Production of two recombinant insulin-like growth factor binding protein-1 subtypes specific to salmonids.

Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.

The effect of miR-10b on growth hormone in pituitary cells of Yanbian yellow cattle by somatostatin receptor 2.

This study aimed to evaluate the effect of miR-10b on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. According to analysis of GH and somatostatin receptor 2 (SSTR2) mRNA and protein expression levels, we found that miR-10b targeted 3'UTR of SSTR2. Compared with the negative control (NC) group, GH mRNA transcription and protein expression in pituitary cells of Yanbian yellow cattle were significantly increased by adding miR-10b mimics (p < .01), while these were significantly decreased by adding miR-10b inhibitor (p < .05); compared with the NC group, SSTR2 mRNA transcription and protein expression were significantly inhibited by the addition of miR-10b mimics (p < .01), while these were significantly increased by the addition of miR-10b inhibitor compared with the iNC group (p < .05). This study suggested that miR-10b could regulate GH level by regulating SSTR2 gene expression in pituitary cells of Yanbian yellow cattle.

Novel Pituitary Actions of Epidermal Growth Factor: Receptor Specificity and Signal Transduction for UTS1, EGR1, and MMP13 Regulation by EGF.

Epidermal growth factor (EGF) is a member of the EGF-like ligands family, which plays a vital role in cell proliferation, differentiation, and folliculogenesis through binding with EGF receptors, including ErbB1 (EGFR/HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). In mammals, many functional roles of EGF have been reported in the ovaries and breasts. However, little is known about the functions of EGF in the pituitary, especially in teleost. In this study, using grass carp pituitary cells as the model, we try to examine the direct pituitary actions of EGF in teleost. Firstly, transcriptomic analysis showed that 599 different expressed genes (DEGs) between the control and EGF-treatment group were mainly involved in cell proliferation, cell migration, signal transduction, and transcriptional regulation. Then, we further confirmed that EGF could significantly induce UTS1, EGR1, and MMP13 mRNA expression in a time-and dose-dependent manner. The stimulatory actions of EGF on UTS1 and EGR1 mRNA expression were mediated by the MEK1/2/ERK1/2 and PI3K/AKT/mTOR pathways coupled with both ErbB1 and ErbB2 in grass carp pituitary cells. The receptor specificity and signal transductions for the corresponding responses on MMP13 mRNA expression were also similar, except that the ErbB2 and PI3K/AKT/mTOR pathway were not involved. As we know, MMP13 could release EGF from HB-EGF. Interestingly, our data also showed that the MMPs inhibitor BB94 could suppress EGF-induced UTS1 and EGR1 mRNA expression. These results, taken together, suggest that the stimulatory actions of EGF on UTS1 and EGR1 mRNA expression could be enhanced by EGF-induced MMP13 expression in the pituitary.

Concerted suppression of Ih and activation of IK(M) by ivabradine, an HCN-channel inhibitor, in pituitary cells and hippocampal neurons.

Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (Ih) and also reported to ameliorate inflammatory or neuropathic pain. However, to what extent this agent can perturb another types of membrane ion currents in neurons or endocrine cells remains to be largely unknown. Therefore, the Ih or other types of ionic currents in pituitary tumor (GH3) cells and in hippocampal mHippoE-14 neurons was studied with or without the presence of IVA or other related compounds. The IVA addition caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 0.64 µM and a KD value of 0.68 µM. IVA (0.3 µM) shifted the Ih activation curve to a more negative potential by approximately 8 mV, despite no concomitant change in the gating charge. Additionally, IVA was found to increase M-type K+ current (IK(M)) together with a rightward shift in the activation curve. In cell-attached current recordings, IVA (3 µM) applied to the bath increased the open probability of M-type K+ channels; however, it did not modify single-channel conductance of the channel. In current-clamp voltage recordings, IVA suppressed the firing of spontaneous action potentials in GH3 cells; and, further addition of linopirdine attenuated its suppression of firing. In hippocampal mHippoE-14 neurons, IVA also effectively increased IK(M) amplitude. In summary, both inhibition of Ih and activation of IK(M) caused by IVA can synergistically combine to influence electrical behaviors in different types of electrically excitable cells occurring in vivo.

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1-/- and heptaTRPC knockout mice.

Store operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca2+ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca2+ release and Ca2+ entry in responsive cells while GHRH and CRH only induce Ca2+ entry. SOCE has been shown to contribute to Ca2+ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1-/- or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca2+ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1-/- mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca2+-oscillations related to electrical activity were not affected in the Orai1-/- mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca2+ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1-/-. In addition, Ca2+ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca2+ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca2+-oscillations and Ca2+ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.

Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHß and LHß in anterior pituitary cells in vitro.

Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.

Regulation of BuShen ZhuYun Decotion on gonadotropin in rat pituitary cells / 医学研究生学报

Objective The mechanism of luteal phase defect remains unclear. To investigate the mechanism of BuShen ZhuYun Decoction on the gonadotropin secretion in the pituitary gland, we observed the effects of medicated serum of BuShen ZhuYun Decotion on the secretion of gonadotropin-follicle-stimulating hormone (FSH) and luteotropic hormone (LH) in rat pituitary cells.Methods The BuShen ZhuYun Decotion was administered to the female SD rats by gavage to prepare the serum containing BuShen ZhuYun Decoction. The CCK-8 method was used to detect the effect of cetrorelix acetate powder for injection, medicated serum and gonadotropin releasing hormone (GnRH) on cell activity. In the maximum non-toxic concentration, we used cetrorelix acetate powder for injection to block the GnRH receptor (GnRHR) in pituitary cells and established the GnRHR antagonistic model. Then we treat the model group with medicated serum (model group). Moreover, we established the blank group (normal pituitary cells), the cetrorelix group (intervented with cetrorelix for 6 hours), and medicated serum group (intervented with medicated serum for 24 hours). 20nmol/L GnRH was used to stimulate cells for 6h. The contents of FSH and LH in the supernatant of each group and the mRNA expression of FSHβ, LHβ and GnRHR were detected.Results Compared with that of the blank group, the supernatant levels of FSH and LH in the Cetrorelix group decreased significantly \[(3.91±0.36) mIU/mL vs (2.26±0.22) mIU/mL, (8.94±0.57) mIU/mL vs (3.35±0.59) mIU/mL, P<0.05)\]. In contrast, the levels of LH significantly increased \[(8.94±0.57) mIU/mL vs (10.79±0.60) mIU/mL, P<0.05)\]; Compared with the cetrorelix group, the levels of FSH and LH in both medicated serum group and model group increased significantly (P<0.05). Compared with the blank group, the mRNA level of FSH and LH in the cetrorelix group decreased significantly \[(0.95±0.23) mIU/mL vs (0.58±0.12) mIU/mL, (0.98±0.14) mIU/mL vs (0.27±0.21) mIU/mL, P<0.01) \], and the mRNA expression of GnRHR increased in the cetrorelix group \[(0.97±0.13) mIU/mL vs (1.77±0.26) mIU/mL, P<0.01) \]; The mRNA levels of FSH and LH in the medicated serum group were increased (P<0.05). Compared with the cetrorelix group, the mRNA expression of FSHβ mRNA and LHβ were both increased in the medicated serum group and model group (P<0.05), the mRNA expression of GnRHR decreased (P<0.01).Conclusion It is suggested that the therapeutic mechanism of BuShen ZhuYun Decotion may be related to the improvement of GnRH receptor expression.

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells.

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.

N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K+ channels.

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC50 = 3.5nM [control] vs.1.0nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.

Irisin stimulates gonadotropins gene expression in tilapia (Oreochromis niloticus) pituitary cells.

The link between energy metabolism and reproduction is well known in vertebrates. Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, plays an important role in energy homeostasis. However, biological actions of irisin on reproduction remain elusive. To address this gap, we examined the direct effects of irisin on luteinizing hormone ß (LHß) and follicle-stimulating hormone ß (FSHß) gene expression in tilapia pituitary cells. As a first step, the transcripts of FNDC5 were detected in the proximal pars distalis (PPD), but not in the rostral pars distalis (RPD) and neurointermediate lobe (NIL) of the tilapia pituitary by RT-PCR. In the tilapia pituitary, irisin immunoreactive signals were also detected in PPD region. In primary cultures of tilapia pituitary cells, irisin was effective in stimulating both LHß and FSHß mRNA levels in vivo and in vitro. In cultured pituitary cells of tilapia, removal of endogenous irisin by immunoneutralization using irisin antiserum inhibited LHß and FSHß gene expression. Salmon gonadotrophin releasing hormone (sGnRH) increased LHß and FSHß mRNA levels in tilapia pituitary but these stimulatory actions were not either enhanced by treatment with irisin or blocked by irisin antiserum. Furthermore, the stimulation on LHß and FSHß mRNA expression was coincident with the enhancement of LHß and FSHß mRNA stability after irisin treatment. These results provide evidence that irisin may serve as a novel intrapituitary factor maintaining gonadotropins gene expression in tilapia pituitary.