Primary Pituitary Cell Cultures Research Articles

Impact of gsp Oncogene on the mRNA Content for Somatostatin and Dopamine Receptors in Human Somatotropinomas

Introduction: It has been reported in some series that gsp+ somatotropinomas are more sensitive to somatostatin analogues (SA) and dopamine’s actions which may be related to their somatostatin receptor (SSTR) and dopamine receptor (DR) profile. No previous studies have been undertaken to evaluate the SSTR and DR profile related with the gsp status in somatotropinomas. Objectives: To determine if (1) gsp status is correlated with response to octreotide LAR (LAR) and tumor expression patterns of SSTR1-5 and DR1-5 and (2) cAMP level can directly modulate SSTR and DR mRNA levels. Methods: Response to SA was evaluated by GH and IGF-I percent reduction after 3 and 6 months of treatment with LAR. Conventional PCR and sequencing were used to identify gsp+ tumors. Quantitative real-time PCR was used to determine SSTR and DR tumor expression. Primary pituitary cell cultures of primates were used to study whether SSTR and DR expression is regulated by forskolin. Results: The response to LAR did not significantly differ between patients with gsp+ and gsp– tumors; however, gsp+ tumors expressed higher levels of SSTR1, SSTR2, DR2 and a lower level of SSTR3. Forskolin increased SSTR1, SSTR2, DR1 and DR2 expression in cell cultures. Conclusion: Elevated SSTR1, SSTR2, and DR2 tumor expression may help improve responsiveness to SA and DA therapy; however, this study may not have been appropriately powered to observe significant effects in the clinical response. Elevated cAMP levels could be directly responsible for the upregulation in SSTR1, SSTR2 and DR2 mRNA levels observed in gsp+ patients.

Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

BackgroundThe incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries.ResultsHarvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency.ConclusionDepending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

The systemic inhibition of nitric oxide production rapidly regulates TRH mRNA concentration in the paraventricular nucleus of the hypothalamus and serum TSH concentration. Studies in control and cold-stressed rats

Neurons of the paraventricular nuclei of the hypothalamus (PVN) that synthesize the peptide thyrotropin releasing hormone (TRH) control energy homeostasis. Identifying the circuits which regulate these neurons is critical to fully understand integration of metabolic information and the mechanisms that set thyroid hormone levels. We tested the hypothesis that nitric oxide (NO) acutely controls PVN TRH expression and thyrotropin (TSH) secretion by the anterior pituitary. The subcutaneous treatment of rats with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases, enhanced PVN TRH mRNA and medio-basal hypothalamic TRH levels, and reduced serum TSH concentration. Analysis of the effect of a NO donor in primary cultures of hypothalamic or anterior pituitary cells suggested that the effect of NO includes a direct action on hypothalamic neurons. The cold stress-induced increase in TSH release was inhibited by sc l-NAME. Therefore, production of NO may control the activity of the hypothalamus–pituitary–thyroid axis.

Involvement of protein kinase C and intracellular Ca 2+ in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca 2+ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca 2+] i) in goldfish somatotropes. gbSS-28 partially reduced [Ca 2+] i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca 2+] i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca 2+] i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca 2+] i and did not alter basal [Ca 2+] i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca 2+] i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca 2+] i signals. gbSS-28 reduces basal GH release also via reduction in [Ca 2+] i but PKC is not involved in this regard.

Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli

Two GnRH receptors (GnRH-R I and GnRH-R II) were obtained in protandrous black porgy (Acanthopagrus schlegeli). We investigated their tissue distribution, developmental/seasonal changes and regulation of expression using in vivo and in vitro (primary cultures of dispersed pituitary cells) approaches. The relative expressions of GnRH-Rs in the pituitary and gonad were as follows: pituitary: GnRH-R I > GnRH-R II; testicular tissue: GnRH-R I > GnRH-R II; ovarian tissue: GnRH-R I = GnRH-R II. GnRH-R I but not GnRH-R II expression was higher in the pituitary during the spawning period as compared to the prespawning. The expression profiles of both forms of GnRH-R were variable in the gonads according to the gonadal stage and season. In vivo, hCG stimulated GnRH-R I and GnRH-R II expression in testis and ovary. The LHRH analog also up-regulated both receptors in testis and but increased only GnRH-R II in the ovary. Sex steroids (estradiol, E2 and testosterone, T) increased the expression of both receptors in the testis and ovary. In the pituitary, sex steroids (E2 and T) increased the expression of GnRH-R I, but not GnRH-II, both in vivo and in vitro. The LHRH analog also specifically up-regulated the expression of GnRH-R I, but not GnRH-R II, by pituitary cells in vitro. All these data suggest that GnRH-R I rather than GnRH-R II may play a major physiological role in the pituitary. In contrast, both GnRH-R I and GnRH-R II may participate in the regulation of gonadal functions, including a possible role during sex change.

Sustained Gonadotropin-Releasing Hormone Stimulation Mobilizes the cAMP/PKA Pathway to Induce Nitric Oxide Synthase Type 1 Expression in Rat Pituitary Cells In Vitro and In Vivo at Proestrus1

Previous in vivo studies have established that pituitary nitric oxide synthase type 1 (NOS1) is regulated by gonadotropin-releasing hormone (GnRH). The aim of our study was to elucidate the mechanisms of NOS1 regulation by GnRH in rat pituitary cells. Using a perifused cell system, we demonstrated that NOS1 induction was sensitive to GnRH pulse frequency and was maximally induced under continuous GnRH stimulation. In primary cultures of rat pituitary cells, sustained stimulation with the GnRH agonist triptorelin (GnRHa) increased NOS1 protein levels, whereas NOS2 and NOS3 levels were unaffected. NOS1 up-regulation occurred in gonadotroph cells only, in a time-dependent and concentration-dependent manner (maximum increase, 2.5-fold; half-maximal concentration, 0.17 nM). GnRHa effect was mimicked by cAMP pathway activators and, most importantly, was blocked by disruption of the protein kinase A (PKA) pathway using pharmacological inhibitors such as Rp-cAMP or drug phosphatase technology-protein kinase inhibitor (DPT-PKI), a cell-permeant PKI peptide. In contrast, modulation of the PKC pathway and inhibition of the MAPK cascade were ineffective. Overall, these experiments demonstrated that GnRH-induced up-regulation of pituitary NOS1 is mediated notably by the cAMP/PKA pathway. Last, in vivo administration of a GnRH antagonist markedly inhibited the pituitary cAMP rise at proestrus in addition to suppressing NOS1 increase. Altogether, our data suggest that the cAMP/PKA signaling pathway is preferentially recruited under sustained GnRH stimulation in vivo during proestrus, allowing the expression of a specific set of PKA-regulated proteins, including NOS1, in gonadotroph cells.

Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach

Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylated-ghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormone-releasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT. Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.

Prostaglandin-D synthetase induces transcription of the LH beta subunit in the primary culture of chicken anterior pituitary cells via the PPAR signaling pathway

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken ( Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD 2 or PGJ 2, elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 μM) alone upregulated 15-deoxy-Δ 12,14-PGJ 2 (15-d-PGJ 2; derived from PGJ 2)/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 μM arachidonic acid additionally increased 15-d-PGJ 2/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r = 0.64; P < 0.05) between PGDS and the peroxisome proliferator-activated receptor A ( PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r = 0.65; P < 0.05) and between PGDS and PPARG (r = 0.67; P < 0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P < 0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P < 0.05); in addition, both antagonists attenuated arachidonic acid–induced LHB mRNA levels (P < 0.05) and PGDS-induced (in the presence of 1 μM arachidonic acid) LHB mRNA and LHB protein (P < 0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD 2-, PGJ 2-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.

Living-cell imaging of transgenic rat anterior pituitary cells in primary culture reveals novel characteristics of folliculo-stellate cells

Folliculo-stellate (FS) cells in the anterior pituitary gland appear to possess multifunctional properties. Recently, the development of transgenic rats (S100b-green fluorescent protein (GFP) rats) that express GFP specifically in FS cells in the anterior pituitary gland has allowed us to distinguish and observe living FS cells in other kinds of pituitary cells. We used S100b-GFP rats to investigate the topographic affinity of FS cells for other pituitary cells. We observed living FS cells in enzymatically dispersed anterior pituitary cells of S100b-GFP rats under a fluorescent microscope, and noted that FS cells markedly extended and contracted cytoplasmic processes and formed interconnections with neighboring FS cells. In addition, FS cells adhered to small clusters of GFP-negative cells, which were primarily hormone-producing cells, and these clusters further aggregated during the course of cytoplasmic contraction. In the presence of laminin, fibronectin, and varying types of collagen, FS cells showed marked changes in shape and specific proliferative activity; however, GFP-negative cells did not. On reverse transcription-PCR analysis and immunohistochemistry, FS cells were shown to express integrin subunits, which are the cell surface receptors for extracellular matrix (ECM). In the anterior pituitary gland, FS cells and the various types of hormone-producing cells generate a unique topography in the presence of basement membrane components and interstitial collagens. The novel characteristics of FS cells observed in the present study suggest that in the anterior pituitary gland, FS cells play important roles in determining and/or maintaining local cellular arrangement in the presence of ECM components.

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10 −8 M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10 −8 M) and GnRH (0.1 and 1 × 10 −9 M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10 −8 M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10 −8 M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10 −8 M), caused no action when used alone, its co-incubation with GnRH (1 × 10 −9 M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.

Time-resolved fluoroimmunoassay (TR-FIA) of gonadotropins.

Time-resolved fluoroimmunoassay (TR-FIA), which has recently been developed as a non-isotopic immunoassay, was intended for assay of gonadotropins (LH and FSH). Ovine LH or FSH was labeled with N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2, N3, N3-tetraacetic acid Eu-chelate (DTTA), and competitive binding assay was performed using a 96-weel titer plate previously coated with a second antibody, followed by measurement using time-resolved fluorometry. TR-FIA for standard ovine LH (NIDDK, LH-I-3) or FSH (NIDDK, FSH-I-1) had a sensitivity of about 25 pg/50 microliters sample. The assay system was applied to heterologous assay of porcine gonadotropins. Linearity was obtained by the dilution test using medium from primary culture of porcine anterior pituitary cells. Intra- and inter-assay coefficients of variation of LH and FSH determination in 31 different porcine samples were satisfactorily low, between 3.5 and 8.1% (intra-assay) and between 1.7 and 13.1% (inter-assay). Correlation coefficients between radioimmunoassay and TR-FIA were calculated to be 0.945 for LH and 0.978 for FSH. Stimulation of LH and FSH release with GnRH was observed by TR-FIA. This non-isotopic TR-FIA thus provides as good sensitivity, reproducibility and accuracy as conventional RIAs.

Drug effects on triiodothyronine uptake by rat anterior pituitary cells in vitro.

In nonthyroidal illness, numerous drugs such as glucocorticoids, dopamine, fenclofenac, furosemide and diphenylhydantoin may modify the close inverse-feedback relationship between circulating thyroid hormones and TSH. Such effects could involve altered hypothalamic TRH secretion, a direct effect on TSH production by the thyrotroph, alterations in circulating free thyroid hormone concentrations, or changes in thyroid hormone uptake by the thyrotroph. We therefore examined the effect of nonsteroidal antiinflammatory drugs (NSAID), diuretics, the synthetic flavonoid EMD 21388, and diphenylhydantoin, on [125I]T3 cellular uptake in rat pituitary primary cell cultures. Uptake of [125I]T3 (cell-associated counts of washed cells) was measured at 15 min after the addition of 50 pmol/L [125I]T3 in protein-free medium (37 degrees C, pH 7.4). Uptake of [125I]T3 by pituitary cells was 6.0 +/- 1.7% of total counts (mean +/- SD, n = 18). Unlabeled T3 (10 mumols/L) displaced 92% of total uptake. The IC50 of unlabeled T3 for the displacement of [125I]T3 was 1.2 mumols/L. T4 and rT3 were approximately 10% as effective as T3 itself in inhibiting [125I]T3 uptake, while triac did not affect cellular [125I]T3 uptake. Inhibition of [125I]T3 uptake at drug concentrations of 100 mumols/L was seen with the diuretics, furosemide (9%), bumetanide (14%), piretanide (12%) and ethacrynic acid (76%), the NSAID, meclofenamic acid (35%) and fenclofenac (52%), EMD 21388 (49%), and the anticonvulsant, diphenylhydantoin (23%). Aspirin, up to 500 mumols/L, had no effect on [125I]T3 uptake. Our results indicate that ethacrynic acid, meclofenamic acid, fenclofenac, EMD 21388 and diphenylhydantoin affect plasma membrane T3 uptake in the pituitary. This potential influence on TSH release will be contrary to the previously-demonstrated direct inhibitory effect of these drugs on TSH release.

Molecular cloning of pit-1 cDNA from porcine anterior pituitary and its involvement in pituitary stimulation by growth hormone-releasing factor.

Pit-1/GHF-1 (hereafter Pit-1) is a pituitary-specific transcription factor that participates in growth hormone (GH), prolactin and thyroid-stimulating hormone gene expression and in proliferation of the cells that produce these hormones. In this study, we determined the nucleotide sequence of porcine Pit-1 cDNA, which shows high homology (95%) with the known mammalian Pit-1s. Some differences in amino acid sequence were found in the amino terminal region, but the POU-specific and POU-homeo domains were well conserved. Porcine anterior pituitary cells in primary culture were treated for 24 h with GH-releasing factor (GRF), forskolin and phorbol ester (TPA). These agents stimulated progressive secretion of GH into the culture media. The levels of Pit-1, GH, cJun and cFos mRNAs were examined using the reverse transcription-polymerase chain reaction. Stimulation with GRF for 2 h increased the Pit-1 mRNA level 3-fold. This response was transient, as the level returned to the control level after 6 h. Forskolin and TPA evoked similar increases, but their effects appeared after 30 min and reached their maxima after 2 h. In contrast, GRF and forskolin, but not TPA, increased the GH mRNA level 2-fold after 24 h. The cJun mRNA level showed no significant change in response to these agents over 24 h and GRF and TPA increased the cFos mRNA level 1.4 and 2.3-fold, respectively, after 30 min. These results suggest that increasing Pit-1 mRNA level in response to GRF plays a role in the early event of the GH gene expression or with the assistance of other transcription factors that modulate GH gene. Furthermore, the GRF-induced increase in cFos mRNA level by GRF and TPA did not appear to be participated straightforward in the GH gene expression, suggesting that cFos alone is insufficient to the activation.

Brain gonadotropin releasing hormone3 expression variation during oogenesis and sexual behavior and its effect on pituitary hormonal expression in the blue gourami

To gain a better insight regarding the roles of gonadotropin releasing hormone3 (GnRH3) in the regulation of reproduction in the suborder Labyrinthici, GnRH3 expression was investigated during the reproduction cycle of the male and female blue gourami ( Trichogaster trichopterus). The full-length blue gourami brain GnRH3 gene was sequenced (EMBL acc. no. EU107388) and was found to be expressed in both the brain and pituitary of the blue gourami. High mRNA levels were detected only in the brain of females with oocytes in the maturation stage. Correspondingly, significantly greater mRNA levels of GnRH3 were detected in mature males than in immature ones. In primary cultures of dispersed pituitary cells, GnRH3 significantly increased βFSH and βLH subunit mRNA levels in cells from both females and males, whereas GH gene transcription was affected differently by GnRH3 in females, as compared to males. Thus, we propose that GnRH3 can act differentially in the blue gourami females and males. In females, GnRH3 may be involved in the final maturation stage of the oocyte and induces βFSH, βLH and GH gene expression; in males, it is engaged in sexual behavior and spermatogenesis regulation via βFSH and βLH stimulation and dowregulation of GH transcription.

PACAP stimulation of maturational gonadotropin secretion in goldfish involves extracellular signal-regulated kinase, but not nitric oxide or guanylate cyclase, signaling

In goldfish, nitric oxide synthase (NOS) immunoreactivity is present in gonadotropes and extracellular signal-regulated protein kinase (ERK) mediates GnRH stimulation of gonadotropin release and synthesis. In this study, we tested the possible involvement of nitric oxide (NO) and ERK in mediating PACAP-stimulated maturational gonadotropin (GTH-II) release from primary cultures of dispersed goldfish pituitary cells. In static incubation experiments, PACAP-induced GTH-II release was unaffected by two inhibitors of NOS synthase, AGH and 1400W; whereas addition of a NO donor, SNAP, elevated GTH-II secretion. In perifusion experiments, neither NOS inhibitors (AGH, 1400W and 7-Ni) nor NO scavengers (PTIO and rutin hydrate) attenuated the GTH-II response to pulse applications of PACAP. In addition, the GTH-II responses to PACAP and the NO donor SNP were additive while PTIO blocked SNP action. Although dibutyryl cGMP increased GTH-II secretion in static incubation, inhibition of guanylate cyclase (GC), a known down-stream target for NO signaling, did not reduce the GTH-II response to pulse application of PACAP. On the other hand, GTH-II responses to PACAP in perifusion were attenuated in the presence of two inhibitors of ERK kinase (MEK), U 0126 and PD 98059. These results suggest that although increased availability of NO and cGMP can lead to increased GTH-II secretion, MEK/ERK signaling, rather than NOS/NO/GC activation, mediates PACAP action on GTH-II release in goldfish.

Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)

To ascertain the neuroendocrine function of the kisspeptin/GPR54 system in non-mammalian species, full-length cDNAs encoding for Kiss1 and Kiss2 as well as their putative cognate receptors GPR54a and GPR54b, were isolated from goldfish (Carassius auratus). The deduced protein sequences between Kiss1 and Kiss2 in goldfish share very low similarity, but their putative mature peptides (kisspeptin-10) are relatively conserved. RT-PCR analysis demonstrated that the goldfish kiss1 gene (gfkiss1) is highly expressed in the optic tectum-thalamus, intestine, kidney, and testis, while the goldfish kiss2 gene (gfkiss2) is mainly detected in the hypothalamus, telencephalon, optic tectum thalamus, adipose tissue, kidney, heart, and gonads. The two receptor genes (gfgpr54a and gfgpr54b) are highly expressed in the brain regions including telencephalon, optic tectum thalamus, and hypothalamus. Both mature goldfish kisspeptin-10 peptides (gfKiss1-10 and gfKiss2-10) are biologically active as they could functionally interact with the two goldfish receptors expressed in cultured eukaryotic cells to trigger the downstream signaling pathways with different potencies. The actions of gfKiss1-10 and gfKiss2-10 on LH secretion were further investigated in vitro and in vivo. Intraperitoneal administration of gfKiss1-10 to sexually mature female goldfish could increase the serum LH levels. However, this peptide does not significantly influence LH release from goldfish pituitary cells in primary culture, indicating that the peptide does not exert its actions at the pituitary level. On the other hand, gfKiss2-10 appears to be a much less potent peptide as it exhibits no significant in vivo bioactivity and is also inactive on the primary pituitary cells.

Pituitary primary cell culture of Common carp (Cyprinus Carpio) and evaluation of its secretion effect on endocrine activity of incubated ovarian follicles

Summary In this study, five carp pituitary glands were collected and dispersed enzymatically and mechanically. Then, the cells were cultivated as monolayer in MEM (minimum essential medium Eagle). The culture media were collected after 72 h and frozen at -20°C. Carp ovarian follicles also were separated mechanically and incubated in BSS (basic salt solution) Cortland medium in 24-well microplates for 48 h at 20°C. Then, they were divided into two groups: control group which were incubated in BSS medium and experimental group which subdivided into three subgroups according to treatment with different concentration of collected pituitary secretion (50, 100 and 200 µl/ml). Follicles culture media were collected 24 h later and were analyzed for 17-β-oestradiol (E2) and 17-α-hydroxy progesterone (P4) content by radioimmunoassay (RIA). The results showed that adding low concentration (50 µl/ml) of collected pituitary secretion (CPS) increased steroid hormones (E2 and P4) secretion of incubated ovarian follicles significantly (P 0.05).

Estrogens exert a rapid apoptotic action in anterior pituitary cells

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.

Differential regulation of gonadotropins (FSH and LH) and growth hormone (GH) by neuroendocrine, endocrine, and paracrine factors in the zebrafish—An in vitro approach

Recently, zebrafish has quickly risen as a model species for functional analysis of the brain–pituitary–gonad axis. However, one of the hurdles for such work in this popular model organism is the small size of its pituitary gland, which makes it difficult to investigate the regulation of pituitary hormone expression and secretion in vitro. To provide a solution to this problem and demonstrate the value of zebrafish in reproductive endocrinology, the present study was undertaken to establish a primary pituitary cell culture followed by investigating the regulation of FSHβ ( fshb), LHβ ( lhb), and GH ( gh) expression by a variety of neuroendocrine, endocrine, and paracrine factors. All the factors examined influenced the expression of fshb, lhb, and gh in vitro except epidermal growth factor (EGF) despite the expression of its receptor egfr in the pituitary. Acting in a similar manner, gonadal steroids (estradiol and testosterone) stimulated both fshb and lhb, but had no effect on gh. In contrast, all other factors tested (gonadotropin-releasing hormone, GnRH; pituitary adenylate cyclase-activating polypeptide, PACAP; activin/follistatin, and insulin-like growth factor I, IGF-I) exhibited distinct effects on the expression of the three target genes studied, suggesting roles for these factors in the differential regulation of two gonadotropins and growth hormone and therefore the gonadotrophic and somatotrophic axes.

Functional expression of CXCR4 in somatotrophs: CXCL12 activates GH gene, GH production and secretion, and cellular proliferation

The interaction of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR4 may play an important role in the regulation of anterior pituitary function. In this study, we investigated the expression of CXCL12 and CXCR4 and their role in normal rat pituitary and GH-producing GH3 tumor cell line. RT-PCR analysis and immunohistochemistry revealed that CXCR4 was expressed in normal rat anterior pituitary and GH3 tumor cells. Double immunofluorescent staining showed the complete colocalization of CXCR4 with GH in rat pituitary, indicating that CXCR4 is specifically expressed in rat somatotrophs. Using rat primary pituitary cell cultures and GH releasing hormone receptor expressing stable GH3 cells (GH3-GHRHR), we evaluated the function of CXCL12 compared with GHRH. CXCL12 stimulated GH gene activation in both primary rat anterior pituitary cells and GH3-GHRHR cells. CXCL12 also stimulated GH secretion from primary rat pituitary cells in a dose-dependant manner. BrdU incorporation was increased in response to CXCL12 addition in GH3 cell culture, indicating CXCL12-induced cell proliferation. CXCL12-dependent phosphorylation of ERK1/2 was also confirmed by western blot analysis, supporting the evidence that MAPK is an intracellular mediator of CXCL12/CXCR4 interaction in GH3 cell proliferation. In conclusion, these results indicate that CXCL12/CXCR4 interaction plays an important role in GH production, secretion, and the proliferation of somatotrophs.