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

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.

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Decreased in vitro secretion of LH, FSH, and free alpha-subunits by pituitary cells from old male rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1985.249.2.e145 ◽

1985 ◽

Vol 249 (2) ◽

pp. E145-E151

Author(s):

M. R. Blackman ◽

A. Mukherjee ◽

P. D. Tsitouras ◽

S. M. Harman

Keyword(s):

Repeated Measures ◽

Primary Cultures ◽

In Vitro Release ◽

Male Rats ◽

Pituitary Cells ◽

Cell Functions ◽

Alpha Subunits ◽

Old Rats ◽

Lh Releasing Hormone

Various in vivo and in vitro pituitary-Leydig cell functions were examined in mature (5-8 mo) vs. old (22-25 mo) male Wistar rats. Old rats exhibited decreased serum concentrations of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) (P less than 0.0025) but similar levels of prolactin (P less than 0.01) both before and 4 wk after castration. The in vitro release of LH, FSH, and free alpha-subunits was measured in primary cultures of dispersed anterior pituitary cells from both intact and castrated rats. During 48 h of incubation, basal secretion rates of the glycoproteins were decreased (P less than 0.001) from cells of both intact and castrated old rats. After stimulation with LH-releasing hormone (LRH) in single (10(-8) M) or multiple (10(-10) - 10(-7) M) doses, the total amounts of the glycoproteins secreted were also less from cells of both intact and castrated old rats. However, repeated measures analysis of variance revealed age-related hyporesponsivity to LRH stimulation only for LH secretion by cells from intact rats. The basal molar ratios of alpha/(LH + FSH) secreted by cells from intact but not castrated old rats were lower than those from cells of the corresponding mature rats. Moreover, after LRH stimulation (10(-10) - 10(-7) M), molar secretory ratios of alpha/(LH + FSH) decreased from cells of intact mature rats but increased from cells of intact old rats. These in vitro data suggest that the reduced serum LH and FSH levels in intact and castrated old male rats result in part from decreased secretion of these glycoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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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

Journal of Endocrinology ◽

10.1677/joe.0.1270149 ◽

1990 ◽

Vol 127 (1) ◽

pp. 149-159 ◽

Cited By ~ 11

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

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Inhibin-like activity in Sertoli cell culture media and testicular homogenates from rats of various ages

Journal of Endocrinology ◽

10.1677/joe.0.1130103 ◽

1987 ◽

Vol 113 (1) ◽

pp. 103-110 ◽

Cited By ~ 15

Author(s):

A. M. Ultee-van Gessel ◽

F. H. de Jong

Keyword(s):

Sertoli Cells ◽

Culture Media ◽

Reciprocal Relationship ◽

Pituitary Cells ◽

In Vitro Experiments ◽

Old Rats ◽

Lh Secretion

ABSTRACT The influence of age on testicular inhibin in untreated, neonatally hemicastrated and prenatally irradiated rats was studied using in-vivo and in-vitro experiments. In testicular cytosols prepared from 1-, 7-, 14-, 21-, 42- and 63-day-old rats concentrations of testicular inhibin could be measured with an in-vitro bioassay method using dispersed pituitary cells. Preparations of testicular cytosols caused a dose-dependent suppression of pituitary FSH secretion, whereas no effects were found on LH secretion. Testicular content of inhibin increased gradually with age, while after 14 days of age a relatively large increase of peripheral FSH concentrations occurred in all experimental groups. Neonatal hemicastration or prenatal irradiation resulted in decreased inhibin content of the testis and increased plasma FSH levels. The production of inhibin activity by Sertoli cells obtained from 7-, 14-, 21-, 42- and 63-day-old normal rats was measured during a 24-h incubation period on the third day of culture. The inhibin production per 106 plated Sertoli cells decreased rapidly after 14 days of age and the lowest production of inhibin was found in Sertoli cells from rats of 63 days of age. After preincubation with ovine FSH significantly larger amounts of inhibin activity were detected in spent media from 21-day-old rat testes. In contrast, suppression of inhibin production was found after preculture in the presence of testosterone at most of the ages studied. These data from in-vivo and in-vitro experiments indicate that a reciprocal relationship exists between pituitary FSH secretion and inhibin production before the age of 21 days. This relationship supports the concept that inhibin is a physiologically important modulator of FSH secretion before puberty, while the role of the large amount of testicular inhibin present at the older ages remains to be determined. J. Endocr. (1987) 113, 103–110

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Differential effects of melatonin on the stimulated release of LH from dispersed pituitary cells of the prepubertal female rat

Journal of Endocrinology ◽

10.1677/joe.0.1070107 ◽

1985 ◽

Vol 107 (1) ◽

pp. 107-112 ◽

Cited By ~ 5

Author(s):

A. M. Symons ◽

J. Arendt ◽

S. J. Pryde

Keyword(s):

Pubertal Development ◽

Specific Interaction ◽

Calcium Ionophore ◽

Significant Inhibition ◽

Pituitary Cells ◽

Female Rat ◽

Lh Release ◽

Rat Pituitary Cells ◽

Lh Releasing Hormone

ABSTRACT The effect of melatonin on the stimulated release of LH from prepubertal female rat pituitary cells in vitro was investigated. Significant inhibition of LH-releasing hormone and calcium ionophore-induced LH release was seen but not of potassium-induced release. These results suggest a specific interaction between melatonin and the endogenous events leading to LH release, and may implicate melatonin as an important neuroendocrine component of pubertal development in this species. J. Endocr. (1985) 107, 107–112

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cAMP augmentation of secretagogue-induced luteinizing hormone secretion

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1986.250.1.e62 ◽

1986 ◽

Vol 250 (1) ◽

pp. E62-E68 ◽

Cited By ~ 1

Author(s):

J. L. Turgeon ◽

D. W. Waring

Keyword(s):

Luteinizing Hormone ◽

Hormone Secretion ◽

Base Line ◽

Luteinizing Hormone Secretion ◽

Secretory Rate ◽

Camp Analogue ◽

Lh Release ◽

Lh Secretion ◽

Camp Elevation

Whether adenosine 3',5'-cyclic monophosphate (cAMP) acts as a mediator for luteinizing hormone-releasing hormone (LHRH) in either its immediate LH release action or in its self-priming action was investigated. Pituitary pieces from either proestrous or estrous rats were superfused in vitro in the presence of dibutyryl cAMP [(Bu)2cAMP], 8-bromo-cAMP (8BrcAMP), forskolin, or control for 2-3 h. For proestrous but not estrous pituitary pieces, a slight increase in base-line LH secretion rate occurred at approximately 70 min of exposure to elevated cAMP; in the same system LHRH caused an increase in LH secretory rate within 2 min in either proestrous or estrous tissue. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a severalfold augmentation of both LHRH- and elevated K+-induced LH secretion from proestrous but not estrous pituitary pieces; for these experiments, superfusion with a cAMP analogue or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation was evident after 30 min of cAMP elevation; longer exposures were coincident with greater potentiation. Cycloheximide prevented (Bu)2cAMP augmentation of LHRH-induced secretion. These data show that cAMP does not mediate the immediate LH release action of LHRH, but cAMP does augment LHRH- or K+-induced LH secretion with characteristics in common with the self-priming action of LHRH.

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

Acta Veterinaria Hungarica ◽

10.1556/avet.50.2002.1.10 ◽

2002 ◽

Vol 50 (1) ◽

pp. 79-92 ◽

Cited By ~ 3

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.

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Steroid effects on the in vitro LH secretion from pituitary cells of sexually immature carp (Cyprinus carpio L.) under the influence of naltrexone and morphine

Czech Journal of Animal Science ◽

10.17221/131/2009-cjas ◽

2010 ◽

Vol 55 (No. 6) ◽

pp. 250-257

Author(s):

M. Sokolowska-Mikolajczyk ◽

M. Socha ◽

P. Epler

Keyword(s):

Cyprinus Carpio ◽

Sex Steroids ◽

Pituitary Cells ◽

Control Medium ◽

Opioid Agonist ◽

Carp Cyprinus Carpio ◽

Lh Release ◽

Lh Secretion

Dispersed cells from sexually immature carp (footlings) pituitaries were exposed to estradiol (E2) or testosterone (T) (both at 3 × 10–8 M) in the presence of opioid receptor antagonist naltrexone (10–8 or 10–6 M) and/or agonist – morphine (10–8 or 10–6 M). Naltrexone alone at 10–6 M increased the LH level as compared with the control. Morphine (10–8 or 10–6 M), T and E2 had no influence on LH levels. The combination of T with naltrexone (10–8 M) stimulated LH release if compared with the control or with T alone. Morphine (both concentrations) with T caused significantly higher LH secretion than the control medium and T alone. Estradiol with naltrexone (10–8 and 10–6 M) had no influence on LH concentration. In media with E2 and morphine (10–8 M) LH levels were higher than in the control and estradiol alone. The results show that in common carp sex steroids affect the response of pituitary cells to opioid agonist or antagonist giving an evidence on the role of steroids in LH release mediated by the opioid system.

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In vitro effect of leptin on anterior pituitary cells LH secretory activity during early pregnancy in pig

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2017-0010 ◽

2017 ◽

Vol 20 (1) ◽

pp. 67-76 ◽

Cited By ~ 1

Author(s):

G. Siawrys ◽

A. Gajewska

Keyword(s):

Early Pregnancy ◽

Anterior Pituitary ◽

Leptin Receptor ◽

Full Range ◽

Primary Cultures ◽

Pituitary Cells ◽

Leptin Sensitivity ◽

Vitro Effect ◽

Lh Secretion

Abstract Leptin modulates reproductive activity but its potential influence on LH secretion from anterior pituitary (AP) cells during implantation period in pigs (days 14-16 of pregnancy) remained unexplored. This study focused on determination whether leptin affects basal and GnRH-induced LH secretion and intracellular accumulation and whether leptin receptor (OB-Rb) mRNA is expressed in the AP gland during implantation in pigs. Four individual AP glands were developed into separate primary cultures. 2×105 cells/ml were preincubated (72 h) and next, for 3.5 h, experimentally treated with GnRH (100 ng/ml), leptin (10-11, 10-9, 10-7, 10-6 M) alone, or given in respective combinations with GnRH. In the AP gland, OB-Rb mRNA expression was determined by real-time PCR method. Leptin activated LH secretion and its concentration-dependent effect was observed as stimulation shown in a full range tested (culture 1) and exhibited only at 10-6 M (culture 2). A pooled data analysis revealed that basal LH secretion increased at 10-9, 10-7 and 10-6 M, but GnRH-induced LH release decreased at 10-6 M. Leptin down-regulated GnRH-induced LH secretion in all cultures, but only culture 3 exhibited sensitivity for all concentrations tested. Basal LH accumulation was activated in culture 1 (at 10-11 M) and inhibited in culture 4 (at 10-9 M). In the presence of GnRH leptin up-regulated LH accumulation with individual culture leptin-sensitivity (culture 1-3), while down-regulated LH accumulation in culture 4. Obtained data indicate that OB-Rb mRNA is expressed in the AP gland and leptin alone and in combination with GnRH specifically modulates LH activity during early pregnancy in pigs.

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Gonadotrophin surge-attenuating factor attenuates in-vitro LH secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season

Journal of Endocrinology ◽

10.1677/joe.0.1350221 ◽

1992 ◽

Vol 135 (2) ◽

pp. 221-227 ◽

Cited By ~ 6

Author(s):

P. A. Fowler ◽

C. Townsend ◽

I. E. Messinis ◽

P. Cunningham ◽

A. Templeton

Keyword(s):

Breeding Season ◽

Follicular Fluid ◽

Primary Cultures ◽

Pituitary Cells ◽

Partial Reduction ◽

Human Follicular Fluid ◽

Gonadotrophin Releasing Hormone ◽

Lh Secretion ◽

Stimulation Of

ABSTRACT Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophinreleasing hormone (GnRH) was markedly reduced (43·5 ± 5·2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71·3± 13·2 to 117·8±11·2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 μmol/l), progesterone (1 pmol/l to 1 μmol/l) or oestradiol and progesterone combined (1 pmol/l to 1 μmol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83·4±7·6 to 110·6±5·0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10–30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season. In addition, neither oestradiol nor progesterone singly or in combination caused the observed attenuation of GnRH-induced LH secretion that is ascribed to GnSAF bioactivity. Journal of Endocrinology (1992) 135, 221–227

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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 ◽

10.1210/en.2014-1640 ◽

2015 ◽

Vol 156 (1) ◽

pp. 218-230 ◽

Cited By ~ 5

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.

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