scholarly journals Ligand-Selective Signal Transduction by Two Endogenous GnRH Isoforms Involves Biased Activation of the Class I PI3K Catalytic Subunits p110β, p110γ, and p110δ in Pituitary Gonadotropes and Somatotropes

Endocrinology ◽  
2015 ◽  
Vol 156 (1) ◽  
pp. 218-230 ◽  
Author(s):  
Joshua G. Pemberton ◽  
James L. Stafford ◽  
John P. Chang
Keyword(s):  
Protein Complexes ◽  
Primary Cultures ◽  
Cell Types ◽  
Class I ◽  
Pituitary Cells ◽  
Catalytic Subunits ◽  
Transduction Mechanisms ◽  
Lh Release ◽  
Differential Involvement ◽  
Lh Secretion

Abstract In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110β, p110γ, and p110δ). Importantly, p110β and p110γ, but not p110α or p110δ, can be directly activated by the Gβγ heterodimer of Gαβγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110β, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110β only regulate basal LH secretion. However, p110β and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110β and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gβγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

Ionophore A23187 partially reverses LH secretory defect of pituitary cells from old rats

1987 ◽  
Vol 253 (3) ◽  
pp. E233-E237
Author(s):  
R. S. Chuknyiska ◽  
M. R. Blackman ◽  
G. S. Roth
Keyword(s):  
Primary Cultures ◽  
Extracellular Calcium ◽  
Base Line ◽  
Ionophore A23187 ◽  
Pituitary Cells ◽  
Lh Release ◽  
Old Rats ◽  
Lh Releasing Hormone ◽  
Lh Secretion

We measured in vitro release of luteinizing hormone (LH) in the presence of 1.5 mM extracellular calcium, with and without LH-releasing hormone (LHRH; 10(-10) to 10(-7) M) or the ionophore A23187 (10(-7) to 10(-4) M), in primary cultures of anterior pituitary cells from intact mature (6 mo) and old (24 mo) male and intact and ovariectomized mature and old female Wistar rats. Base-line as well as LHRH- and A23187-mediated LH secretion was decreased from cells of old rats. However, exposure to A23187 led to a nearly twofold greater augmentation of LH release from cells of old rats, thus decreasing the apparent age-related LH secretory deficit by approximately one-half. We then measured LHRH-mediated (10(-8) M) vs. A23187-mediated (10(-4) M) LH release with and without extracellular calcium (0.08-1.5 mM). For cells from both mature and old rats, there was a similar calcium dependency for A23187- and LHRH-mediated LH release, with optimal LH secretion at 1.0-1.5 mM extracellular calcium concentrations. Again, both LHRH- and A23187-stimulated LH release was significantly lower and exposure to A23187 led to a greater increase in LH release from cells of old rats. Taken together with similar findings in other systems, these data suggest that the in vitro LH secretory defect of pituitary cells from old rats results in part from one or more defects in calcium mobilization and that such alterations may be a widespread manifestation of aging.

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells

1992 ◽  
Vol 70 (7) ◽  
pp. 963-969 ◽  
Author(s):  
Gabriela T. Pérez ◽  
Marta E. Apfelbaum
Keyword(s):  
Steroid Hormones ◽  
Pituitary Cells ◽  
Short Term ◽  
Stimulatory Action ◽  
Rat Pituitary ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone ◽  
Rat Pituitary Cells ◽  
Lh Secretion

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17β (E2), progesterone (P), and 5α-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT. While the stimulatory effect of E2 was evident after both acute (133%) and chronic (119%) treatment, that of DHT appears to be exerted mainly after long-term priming (118%). These results suggest that the steroids modulate GnRH-induced LH secretion by acting on both synthesis and release of LH. On the other hand, total hormone content was not affected by P. The acute (5 h) effects of E2, P, and DHT on the GnRH response in E2-primed (24 h) cells during a short-term incubation, were also tested. Addition of P to the pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of E2 on GnRH-induced LH release and total content. Conversely, the augmentative E2 effect on pituitary responsiveness to GnRH was abolished by DHT. Taken together, these findings suggest that the physiological significance of the stimulatory action of progesterone could be to define the final magnitude of the LH preovulatory surge, while the inhibition by DHT could be required to limit the LH surge to that day of proestrus.Key words: luteinizing hormone, gonadotrophin-releasing hormone, steroid hormones, cultured pituitary cells.

Sensitivity of rat adenohypophyseal cells to estradiol and LHRH during long-term culture

1981 ◽  
Vol 240 (6) ◽  
pp. E602-E608
Author(s):  
L. Lagace ◽  
F. Labrie ◽  
T. Antakly ◽  
G. Pelletier
Keyword(s):  
Luteinizing Hormone ◽  
Cell Types ◽  
Thyroid Stimulating Hormone ◽  
Time Interval ◽  
Pituitary Cells ◽  
Luteinizing Hormone Releasing Hormone ◽  
Lh Release ◽  
Lh Rh ◽  
Stimulating Hormone

To determine possible effects of the time in culture on the responsiveness of the different pituitary cell types to estrogens, rat anterior pituitary cells were incubated up to 20 days in the presence or absence of 10 nM 17 beta-estradiol. Whereas spontaneous luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) release decreased by 85-90%, follicle-stimulating hormone (FSH) and prolactin accumulation in medium were only 50% decreased after 20 days in culture, thus suggesting that the secretion of FSH and prolactin is less dependent on extrinsic stimulatory factors. Estradiol increased spontaneous LH release and its responsiveness to luteinizing hormone-releasing hormone (LH-RH) up to day 16 in culture, whereas the stimulatory effect of the estrogen on FSH secretion was significant only up to day 6. The stimulatory effect of estradiol on basal TSH release was seen up to day 8 in culture, whereas that on spontaneous prolactin release increased progressively after day 8 in culture up to the last time interval studied (20 days). As revealed by immunocytochemistry, the stimulatory effect of estradiol was not due to changes of cell growth.

Effects of crude and highly purified bovine inhibin (Mr 32 000 form) on gonadotrophin production by ovine pituitary cells in vitro: inhibin enhances gonadotrophin-releasing hormone-induced release of LH

1990 ◽  
Vol 127 (1) ◽  
pp. 149-159 ◽  
Author(s):  
S. Muttukrishna ◽  
P. G. Knight
Keyword(s):  
Primary Cultures ◽  
Suppressive Effect ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified Mr 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated. As in rats, both crude and highly purified inhibin preparations promoted a dose (P < 0·001)- and time (P < 0·001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P< 0·001) increased by the presence of either bFF (+ 75%) or highly-purified inhibin (+ 64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 μl specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17β (1 pmol/l–10 nmol/l) nor monomeric α-subunit of bovine inhibin (2·5–40 ng/ml) significantly affected basal or GnRH-induced release of LH. These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat. Journal of Endocrinology (1990) 127, 149–159

scholarly journals Cell Type-Specific Metabolism of Peptidylglycineα -Amidating Monooxygenase in Anterior Pituitary*

Endocrinology ◽  
2000 ◽  
Vol 141 (8) ◽  
pp. 3020-3034 ◽  
Author(s):  
Rajaa El Meskini ◽  
Richard E. Mains ◽  
Betty A. Eipper
Keyword(s):  
Anterior Pituitary ◽  
Secretory Granules ◽  
Primary Cultures ◽  
Cell Types ◽  
Pituitary Cell ◽  
Male Rat ◽  
Pituitary Cells ◽  
Cell Type ◽  
Cell Content ◽  
Monooxygenase Activity

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.

scholarly journals Short term kinetics of luteinizing hormone secretion studied in dissociated pituitary cells attached to manipulable substrates.

1977 ◽  
Vol 73 (3) ◽  
pp. 685-695 ◽  
Author(s):  
C R Hopkins
Keyword(s):  
Luteinizing Hormone ◽  
Hormone Secretion ◽  
Pituitary Cells ◽  
Short Term ◽  
Luteinizing Hormone Secretion ◽  
Luteinizing Hormone Releasing Hormone ◽  
Dissociated Cells ◽  
Lh Release ◽  
Lh Secretion ◽  
Kinetics Of

With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.

scholarly journals Identification of estradiol/ERα-regulated genes in the mouse pituitary

2011 ◽  
Vol 210 (3) ◽  
pp. 309-321 ◽  
Author(s):  
Hyun Joon Kim ◽  
Mary C Gieske ◽  
Kourtney L Trudgen ◽  
Susan Hudgins-Spivey ◽  
Beob Gyun Kim ◽  
...  
Keyword(s):  
Estrogen Receptor Α ◽  
Pituitary Cells ◽  
Gene Expression Microarray ◽  
Intracellular Vesicle ◽  
17Β Estradiol ◽  
Lh Release ◽  
Mouse Pituitary ◽  
Cellular Components ◽  
Lh Secretion

Estrogen acts to prime the pituitary prior to the GnRH-induced LH surge by undiscovered mechanisms. This study aimed to identify the key components that mediate estrogen action in priming the pituitary. RNA extracted from the pituitaries of metestrous (low estrogen) and proestrus (high estrogen) stage mice, as well as from ovariectomized wild-type and estrogen receptor α (ERα) knockout mice treated with 17β-estradiol (E2) or vehicle, was used for gene expression microarray. Microarray data were then aggregated, built into a functional electronic database, and used for further characterization of E2/ERα-regulated genes. These data were used to compile a list of genes representing diverse biological pathways that are regulated by E2via an ERα-mediated pathway in the pituitary. This approach substantiates ERα regulation of membrane potential regulators and intracellular vesicle transporters, among others, but not the basic components of secretory machinery. Subsequent characterization of six selected genes (Cacna1a, Cacna1g, Cited1, Abep1, Opn3, andKcne2) confirmed not only ERα dependency for their pituitary expression but also the significance of their expression in regulating GnRH-induced LH secretion. In conclusion, findings from this study suggest that estrogen primes the pituitary via ERα by equipping pituitary cells with critical cellular components that potentiate LH release on subsequent GnRH stimulations.

scholarly journals Sex dependent action of Aroclor 1254 on basal and sGnRHa-stimulated secretion of LH from the pituitary cells of common carp, Cyprinus carpio L.

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Magdalena Socha ◽  
Mirosława Sokołowska-Mikołajczyk ◽  
Jarosław Chyb ◽  
Ewa Drąg-Kozak ◽  
Ewa Łuszczek-Trojnar
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Hormonal Regulation ◽  
Pituitary Cells ◽  
Aroclor 1254 ◽  
Cell Incubation ◽  
The Media ◽  
Lh Release ◽  
Sites Of Action ◽  
Lh Secretion

AbstractPolychlorinated biphenyls (PCBs) affect the hypothalamic-pituitary-gonadal axis in many vertebrates, changing the hormonal regulation of reproduction. To identify one of the possible sites of action of PCBs on gonadotropin release in common carp, the direct effects of Aroclor 1254 on luteinizing hormone (LH) secretion from dispersed pituitary cells were investigated. Pituitary cells were obtained from sexually mature male and female common carp (Cyprinus carpio L.) at the time of natural spawning. The cells were incubated with different concentrations of Aroclor 1254 (5, 10, 50 and 100 ng mL−1 medium) and/or salmon gonadotropin-releasing hormone analogue (sGnRHa) at a concentration of 10−8 M. LH levels were measured in the cultured medium by the ELISA method after 10 hours of cell incubation. Incubation of male pituitary cells in the presence of tested concentrations of Aroclor did not change the basal LH secretion to the media. In the female pituitary cell incubations Aroclor (5, 10 and 100 ng mL−1 medium) caused a significant increase in LH concentrations in comparison to control incubations. In the case of sGnRHa-stimulated LH secretion in incubations of cells of both sexes, all the concentrations of Aroclor significantly stimulated LH release and potentiated stimulatory effects of sGnRH analogue. These results indicate that endocrine disrupters, such as Aroclor 1254, may affect reproduction in fish, acting also directly on gonadotrophs at the level of the pituitary gland, changing LH secretion.

Effect of GnRH and its antagonist (Antarelix) on LH release from cultured bovine anterior pituitary cells

2002 ◽  
Vol 50 (1) ◽  
pp. 79-92 ◽  
Author(s):  
Annett Bellmann ◽  
F. Schneider ◽  
W. Kanitz ◽  
Keyword(s):  
Anterior Pituitary ◽  
Culture System ◽  
Gnrh Antagonist ◽  
Pituitary Cells ◽  
Maximal Effect ◽  
Static Culture ◽  
Lh Release ◽  
Lh Secretion

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10-5 or 10-4 M Antarelix followed by 10-6 M GnRH coincubation for a further 6h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10-6 M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10-4 and 10-5 M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10-4 M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.

scholarly journals Differential expression and regulation of progesterone receptor isoforms in rat and mouse pituitary cells and LβT2 gonadotropes

2006 ◽  
Vol 190 (3) ◽  
pp. 837-846 ◽  
Author(s):  
Judith L Turgeon ◽  
Dennis W Waring
Keyword(s):  
Progesterone Receptor ◽  
Mrna Level ◽  
Primary Cultures ◽  
Ovariectomized Rats ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Mouse Cells ◽  
Isoform Expression ◽  
Mouse Pituitary ◽  
Lh Secretion

Manipulation of endogenous progesterone receptor (PR) does not produce equivalent physiological effects in mouse and rat pituitary cells. To test whether this may be due in part to difference in PR isoform expression, we examined hormonally regulated pituitary PR-A and PR-B mRNA levels using quantitative real-time PCR. The LβT2 mouse gonadotrope line or pituitary cells from adult, ovariectomized rats or mice were cultured with or without 0.2 nM 17β-estradiol (E2) for 3 days. PR-A was the predominant form expressed for all groups. For mouse cells, E2 led to an increase in both isoforms without a change in the A:B ratio; for rat cells, the PR-B response to E2 was more robust resulting in a decrease in the A:B ratio. Exposure of E2-treated pituitary cells to 200 nM progesterone for 6 h decreased both PR-A and PR-B levels in rat cells, but had no effect on PR isoform expression in mouse cells even when exposure was extended to 12 h. The low level of PR expression found in LβT2 gonadotropes was unaffected by E2, alone or with progesterone. The weak PR expression and lack of responsiveness of LβT2 cells cannot be explained by a male phenotype as was shown by the more than tenfold higher PR mRNA level in primary cultures of male mouse pituitary cells, which responded to E2 stimulation with a proportional increase in PR isoforms similar to female cells. Functionally, E2-stimulated changes in PR mRNA isoform ratios in rat, mouse or LβT2 cells correlated with the degree of progesterone augmentation of GnRH-stimulated LH secretion in these models. These results are consistent with the hypothesis that robust GnRH priming and progesterone augmentation of LH secretion in the rat compared to these events in the mouse are a consequence, in part, of differences in the E2-modulated ratio of PR isoforms.

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