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Endocrinology Vol. 145, No. 12 5548-5559
Copyright © 2004 by The Endocrine Society

Evidence for a Novel Intrapituitary Autocrine/Paracrine Feedback Loop Regulating Growth Hormone Synthesis and Secretion in Grass Carp Pituitary Cells by Functional Interactions between Gonadotrophs and Somatotrophs

Hong Zhou, Xinyan Wang, Wendy K. W. Ko and Anderson O. L. Wong

Department of Zoology, University of Hong Kong, Hong Kong, People’s Republic of China

Address all correspondence and requests for reprints to: Dr. Anderson O. L. Wong, Associate Professor, Room 4S-12, Kadoorie Biological Sciences Building, Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China. E-mail: olwong{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Gonadotropin (GTH) and GH released from the pituitary are known to interact at multiple levels to modulate the functions of the gonadotrophic and somatotrophic axes. However, their interactions at the pituitary level have not been fully characterized. In this study, autocrine/paracrine regulation of GH synthesis and secretion by local interactions between gonadotrophs and somatotrophs was examined using grass carp pituitary cells as a cell model. Exogenous GTH and GH induced GH release and GH mRNA expression in carp pituitary cells. Removal of endogenous GTH and GH by immunoneutralization with GTH and GH antisera, respectively, suppressed GH release, GH production, and GH mRNA levels. GH antiserum also blocked the stimulatory effects of exogenous GTH on GH release and GH mRNA levels. In reciprocal experiments, GH release and GH mRNA expression induced by exogenous GH was significantly reduced by GTH antiserum. In addition, exogenous GH was found to be inhibitory to basal GTH release and treatment with GH antiserum elevated GTH secretion at low doses but suppressed GTH production at high doses. These results suggest that local interactions between gonadotrophs and somatotrophs may form an intrapituitary feedback loop to regulate GH release and synthesis. In this model, GTH released from gonadotrophs induces GH release and GH production in neighboring somatotrophs. GH secreted maintains somatotroph sensitivity to GTH stimulation, and at the same time, inhibits basal GTH release in gonadotrophs. This feedback loop may represent a novel mechanism regulating GH release and synthesis in lower vertebrates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
GONADOTROPIN (GTH) AND GH are the principal hormones released from the anterior pituitary regulating reproductive functions and body growth, respectively. These two hormones are known to interact at multiple levels to modulate the functions of the gonadotrophic and somatotrophic axes. GH receptors have been identified in the gonads as well as in other structures of the male and female reproductive systems (1). At the gonadal level, GH and FSH stimulate progesterone production in granulosa cells in a synergistic manner (2), and this potentiating effect can be related to LH receptor up-regulation caused by GH treatment (3). In Leydig cells, GH stimulates mRNA expression of steroidogenic acute regulatory protein (4), which controls mitochondrial uptake of cholesterol and constitutes the rate-limiting step in steroid synthesis (5). At the pituitary level, transcripts of GH receptors (6) and GH-binding sites (7) are located in several pituitary cell types including gonadotrophs, suggesting that endogenous GH may act in a paracrine manner regulating gonadotroph functions. This idea is supported by the findings that the stimulatory actions of GnRH on LH and/or FSH release can be inhibited by GH immunoneutralization, both in vivo (8) and in vitro (9). The effect of GH on GTH secretion, however, is controversial because stimulatory (9), inhibitory (8), and no effects (10) have been reported. The cause of these discrepancies is still unclear and may be related to species specificity, variations in physiological status, and/or difference in research methodology. It is also worth mentioning that a unique cell population with coexpression of GH and GTH (both at the transcript level and protein level) has been reported in the rat pituitary (11). This population of multihormonal cells increases rapidly from the metestrus to midcycle (12) and has been proposed be functional in providing a cocktail of hormones for optimal gonadotropic activity during the critical phase of the reproductive cycle (13).

In mammals, GH release is also under the influence of the gonadotrophic axis, especially via the release of sex steroids. In the rat, castration can reduce the number of somatotrophs in the anterior pituitary (14). This reduction in somatotrophs occurs with a drop in GH mRNA levels and a loss of the transcripts for Pit-1 (15), a pituitary-specific Pit-Oct-Unc transcription factor essential for GH, prolactin (PRL), and TSH gene expression (16). These inhibitory actions on somatotrophs, however, can be restored by testosterone replacement (15). Sex steroids are also known to modify GH pulsatility (17), probably by regulating the gene expression of GHRH and somatostatin (SRIF) in the arcuate (18) and periventricular nuclei (19), respectively. Besides the central actions, sex steroids can also exert their effects at the pituitary level by altering the responsiveness of pituitary cells to stimulation by SRIF and GHRH (20). Because a low level of LH receptor transcripts can be detected in the human pituitary (21), the possibility of a local action(s) of GTH in the pituitary cannot be excluded. Although intercellular communication among pituitary cells has received increasing attention as a novel mechanism regulating pituitary functions (22), the direct actions of GTH on GH synthesis and secretion at the pituitary level have not been previously examined.

The bony fish, or teleosts, represent a unique model for the study of pituitary interactions between the gonadotrophs and somatotrophs. Unlike mammals with a random pattern of cell distribution in the pituitary, a distinct zonation of individual cell types can be identified in the pituitary of teleosts, e.g. grass carp (23). In this case, lactotrophs are located in the rostral pars distalis, whereas somatotrophs and gonadotrophs are restricted to the proximal pars distalis. In the proximal pars distalis, gonadotrophs always exhibit a patchy distribution with cell clusters embedding in a matrix of somatotrophs. The close proximity between the two cell types provides the anatomical substrate for local interactions between gonadotrophs and somatotrophs. In salmonids, e.g. rainbow trout, parallel increases in the population size of somatotrophs and gonadotrophs expressing GTH-II (or LH in fish) have been reported during sexual maturation (24). In Cyprinids, e.g. goldfish (25), GH secretion always increases with GTH-II levels during sexual recrudescence and spawning period. These observations are related to the permissive/facilitative role of GH in reproductive functions in fish, especially during oocyte maturation, spermatogenesis, and steroidogenesis (for review, see Ref. 26). In the goldfish, the preovulatory GTH-II surge is known to occur with a concurrent increase in GH release (27), which has been attributed to the stimulatory effects of GnRH on GH and GTH-II secretion at the pituitary level (28). GnRH-stimulated GH and GTH-II release have also been reported in other teleosts, including the rainbow trout (29) and tilapia (30). Apparently, the parallel increase in GH and GTH-II secretion in fish is particularly important for seasonal breeders with overlapping somatic growth and gonadal development during the spawning season. At present, it is unclear whether autocrine/paracrine interactions between somatotrophs and gonadotrophs in the pituitary also contribute to the coordinated release of GH and GTH-II in fish.

In this study, local interactions between gonadotrophs and somatotrophs in regulating GH synthesis and secretion were tested in primary cultures of grass carp pituitary cells. Using a static incubation approach, the direct actions of exogenous GTH and GH on GH secretion, cellular GH content, total GH production, and steady-state GH mRNA expression were examined at the pituitary cell level. The results of these studies were further confirmed by removing endogenously secreted GTH and GH using immunoneutralization. Using a column perifusion approach, the acute effects of GTH and GH on the kinetics of GH secretion in carp pituitary cells were also investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals
One-year-old (1+) Chinese grass carps (Ctenopharyngodon idellus) with body weight ranging from 1.5 to 2.0 kg were purchased from local wholesale markets and kept in a well-aerated 200-liter aquaria under 12-h light, 12-h dark photoperiod at 18 ± 2 C. Because the grass carp at this stage was prepubertal (gonadosomatic index ≤ 0.2%) and sexual dimorphism was not apparent, juvenile fish of mixed sexes were used for the preparation of pituitary cells. During the procedures of cell preparation, fish were killed by anesthesia in 0.05% MS222 (Sigma, St. Louis, MO) followed by spinosectomy according to the regulations of animal use at the University of Hong Kong.

Reagents and test substances
Porcine GH, human chorionic gonadotropin (HCG), soybean trypsin inhibitor, and Ca2+-free S-MEM were obtained from Sigma. Salmon GnRH (sGnRH) was purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA). MEM Eagle medium, fetal bovine serum (FBS), type II trypsin, and antibiotic-antimycotic solution were acquired from Gibco Life Technologies, Inc. (Rockville, MD). Porcine GH, HCG, and sGnRH were dissolved in double-distilled deionized water and stored frozen as concentrated stocks in small aliquots at –80 C. On the day of experiments, frozen stocks of test substances were diluted with prewarmed culture medium (28 C) to appropriate concentrations 30 min before adding to cell cultures. Other chemicals and materials used in this study were obtained from commercial sources and were of the highest quality available.

Preparation of grass carp pituitary cells
Carp pituitary cells were prepared by trypsin/DNase II digestion as described previously (23). Briefly, pituitaries were excised from 1-yr-old (1+) fish, diced into fragments using a McILwain tissue chopper (Brinkmann, Mississauga, ON, Canada), and digested in type II trypsin (4 mg/ml, Gibco) for 30 min at 28 C. After that, the reaction was terminated by adding trypsin inhibitor (2.5 mg/ml, Sigma), and pituitary fragments were dispersed in Ca2+-free MEM [S-MEM with 26 mM NaHCO3, 25 mM HEPES, 1% antibiotic-antimycotic, and 0.1% BSA (pH 7.7)] with DNase II (0.01 mg/ml, Sigma). Pituitary cells were harvested by filtration through a sterilized nylon mesh with 30 µm pore size and pelleted by centrifugation at 1000 rpm at 4 C for 10 min. The cell yield was estimated to be approximately 6 x 106 cells/pituitary with a cell viability of 96.5 ± 0.5% (n = 12). Pituitary cells were incubated overnight (>15 h) in carp MEM [MEM Eagle with 26 mM NaHCO3, 25 mM HEPES, and 1% antibiotic-antimycotic (pH 7.7)] with 5% FBS at 28 C under 5% CO2 and saturated humidity. On the following day, culture medium was replaced with serum-free carp MEM with 0.1% BSA, and drug treatment was initiated for the duration as indicated in individual experiments.

Static incubation of carp pituitary cells
Freshly dispersed pituitary cells were cultured in poly-D-lysine (0.1 µg/ml) precoated 24-well clustered plates (Costar, Corning Inc., Corning, NY) at a seeding density of approximately 2.5 x 106 cells/ml/ well in carp MEM with 5% FBS. After overnight incubation, culture medium was replaced with serum-free carp MEM containing 1% BSA with or without drug treatment. For time-course studies, culture medium was harvested at 2, 24, and 48 h after the administration of test substances. For dose-response studies, the duration of drug treatment was routinely fixed at 48 h unless stated otherwise. After incubation, culture medium was collected, and pituitary cells were lysed in double-distilled deionized water by three cycles of freezing and thawing. These samples, including culture medium and cell lysate, were stored frozen at –20 C, and their GH and GTH contents were later quantified by RIAs previously validated for carp GH (23) and carp GTH-II (31), respectively. The data obtained were used to evaluate the effects of drug treatment on basal secretion and cellular contents of GH and GTH-II in carp pituitary cells. In these experiments, total hormone production was defined as the sum of hormone released into culture medium and cellular hormone content.

Column perifusion of carp pituitary cells
Freshly dispersed pituitary cells were cultured in serum-free carp MEM with preswollen Cytodex2 beads (Pharmacia Biotech, Uppsala, Sweden) at 28 C for 3 h under 5% CO2 and saturated humidity. After cell attachment (≥90%), FBS was introduced to give a final dose of 5%. After overnight incubation, Cytodex2 beads with pituitary cells attached were harvested by centrifugation at 1000 rpm at 4 C for 10 min and transferred into 0.5 ml microcolumns (approximately 3 x 106 cells/ column). Pituitary cells were then perifused with prewarmed carp MEM (28 C) with 0.1% BSA at a flow rate of 15 ml/h in an ACUSYST-S perifusion system (Endotronics Inc., Minneapolis, MN). After 3 h of continuous perifusion, basal GH and GTH-II release from pituitary cells remained relatively stable in the absence of stimulation. Test substances, including serially dilutions of porcine GH and HCG, were added into the microcolumns via a three-way stopcock. Perifusate from individual columns was collected in 5-min fractions and stored frozen at –20 C. The GH or GTH-II contents in these samples were later quantified using RIAs.

Measurement of steady-state GH and GTH mRNA levels
After drug treatment, culture medium was aspirated and pituitary cells were dissolved in TRIZOL (Gibco). Phase separation was induced by adding bromochloropropane and total RNA in the aqueous phase was precipitated using isopropanol. RNA samples obtained were denatured at 70 C for 15 min in a denaturing solution [52% formamide, 6.7% formaldehyde, and 1x saline sodium citrate (SSC)] and vacuum blotted onto a positively charged nylon membrane using a Bio-Dot SF microfiltration unit (Bio-Rad Laboratories, Hercules, CA). The RNA-blotted membrane was then UV-cross-linked using a Stratalinker 2400 (Stratagene, La Jolla, CA), prehybridized for 3 h in a hybridization solution [5x SSC, 50% formamide, 0.02% sodium dodecyl sulfate (SDS), 0.1% N-lauroyl-sarcosine, and 1% blocking reagent (Roche, Mannheim, Germany)], and hybridized overnight at 42 C with digoxigenin (DIG)-labeled cDNA probes for grass carp GH mRNA, GTH-IIß mRNA, and GTH{alpha} mRNA, respectively. The probes were labeled with a PCR DIG probe synthesis kit (Roche) using primers flanking position 75–444 of grass carp GH cDNA (GenBank no. M27094), position 61–379 of grass carp GTH-IIß cDNA (GenBank no. X61051), and position 82–344 of grass carp GTH{alpha} cDNA (GenBank no. X61050), respectively. After hybridization, the membrane was subjected to two 5-min washes at room temperature in 2x SSC with 0.1% SDS. Clearance of DIG-labeled probe nonspecifically associated with the membrane was carried out by two 15-min washes at 65 C in 0.5x SSC containing 0.1% SDS. Hybridization signals for GH mRNA were visualized using a DIG luminescent detection kit (Roche) in an IC440 CF digital image station (Eastman Kodak, New Haven, CT). In these experiments, parallel probing of a duplicated membrane using a DIG-labeled probe for grass carp 18S RNA was used as an internal control.

Data transformation and statistics
For perifusion studies, GH/GTH-II data (in nanograms per milliliter) from individual microcolumns were expressed as a percentage of the mean hormone contents in the first six fractions collected at the beginning of individual experiments before drug treatment (referred to as percent basal). This transformation was conducted to allow pooling of data from separate columns without distorting the profile of hormone release during the course of perifusion. In static incubation experiments, GH/GTH-II data for hormone release and hormone content were expressed as micrograms per milliliter and nanograms per 106 cells, respectively, and these data were used for subsequent calculation of total hormone production for individual samples. Expression levels of GH/GTH mRNA were measured in terms of arbitrary density unit and normalized against the amount of 18S RNA in the same sample to adjust for potential variations in cell number between wells and to check for the absence of RNA degradation. Because the 18S RNA levels did not exhibit significant changes in these experiments, normalized GH/GTH mRNA data were simply transformed into percent control for statistical analysis. Data presented in this study were the pooled results of at least three independent experiments and were analyzed by Student’s t test or ANOVA followed by Fisher’s least significance difference (LSD) test. Differences were considered significant at P < 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
In mammals, GH release from the anterior pituitary is under the control of hypothalamic regulators (e.g. GHRH and SRIF), hormone release from target organs (e.g. IGF), feedback regulation by GH itself, and autocrine/paracrine factors (e.g. cytokines) acting locally within the pituitary (32). However, functional interactions among pituitary cells in GH regulation through the release of classic pituitary hormones have not been fully characterized. In this study, using grass carp pituitary cells as a cell model, we examined the functional role of pituitary hormones, namely GTH and GH, in regulating GH synthesis and secretion via local interactions between gonadotrophs and somatotrophs. To test whether exogenous GTH has a direct effect on GH release and production at the pituitary level, a time-course study was conducted by static incubation of pituitary cells with 50 U/ml HCG for 2, 24, and 48 h, respectively (Fig. 1AGo). HCG was used in this study because it is commercially available, is more stable in solution, and has a higher binding affinity for LH receptors (33). More importantly, it is biologically active in fish, e.g. in goldfish (34), yellow perch (35), and channel catfish (36). Recently it has been shown that HCG has no cross-reactivity with fish FSH (or GTH-I) receptors (37). Within the first 2 h of incubation, HCG did not affect GH secretion, cellular GH content, and total GH production. When compared with the time-matched controls, HCG induced a time-dependent increase in GH release and GH production from 24 to 48 h. During the same period, a concurrent drop in cellular GH content (~14%) was also observed. HCG-induced GH secretion was further confirmed by subsequent dose-response studies. In this case, pituitary cells were incubated for 48 h with increasing levels of HCG (10–50 U/ml), and GH release was elevated in a dose-related fashion (Fig. 1BGo), whereas GH contents were reduced with a concurrent rise in GH production. The stimulatory effect of HCG on GH production was dose dependent and constituted mainly by GH released into the culture medium (>90%). These results are similar to those of the time-course studies and indicate that the amount of GH secretion far exceeded GH content residing in grass carp pituitary cells during the 48-h incubation.



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  		FIG. 1. Effects of exogenous GTH on GH secretion and synthesis in grass carp pituitary cells. A, Time course of HCG treatment on GH release (upper panel), GH content (middle panel), and GH production (lower panel). Pituitary cells were incubated with and without HCG (50 U/ml) for 2, 24, and 48 h, respectively. B, Dose dependence of HCG on GH secretion (upper panel), GH content (middle panel), and GH production (lower panel). Pituitary cells were exposed to increasing levels of HCG (10–50 U/ml) for 48 h under static incubation. In these experiments, culture medium was harvested for the measurement of GH release, whereas cell lysate was prepared for the determination of cellular GH content. Total GH production was defined as the sum of GH release and GH content. Data presented are expressed as mean ± SEM (n = 8). Groups denoted by different letters represent significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 
To test the acute effect of exogenous GTH on GH release at the pituitary level, a column perifusion approach was used to study the kinetics of GH secretion in carp pituitary cells. In these experiments, increasing doses of HCG (30–50 U/ml) was applied as 15-min pulses, and GH release was increased in a dose-dependent manner (Fig. 2Go). The GH responses observed were rapid with the peak GH release occurred within 15 min after the initiation of drug treatment. After that, GH release decreased gradually but remained elevated above basal levels, especially in the cases of high doses of HCG treatment (40 and 50 U/ml). In the same experiment, sGnRH was used as a positive control because it is known to stimulate GH and GTH secretion in fish models (38), and a 15-min pulse of sGnRH (1 µM) consistently increased GH release from grass carp pituitary cells. Owing to the flow-through nature of the perifusion system, accumulation of secreted products is rather unlikely (39). Therefore, the possibility of secondary actions of HCG on GH release via local secretion of growth factors/hormones can be minimized. Furthermore, the rapidity of the GH responses observed in this study also suggests that HCG may act directly on somatotrophs to trigger GH exocytosis.



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  		FIG. 2. Effects of exogenous GTH on the kinetic of GH release from grass carp pituitary cells. Pituitary cells were cultured in 0.5 ml microcolumns and perifused with 15-min pulses of increasing doses of HCG (30–50 U/ml). Samples of perfusate were collected at 5 min per faction for the measurement of GH release. Parallel treatment with a 15-min pulse of sGnRH (1 µM) was used as a positive control. GH data were transformed into percent basal as defined in Materials and Methods (mean basal = 28.4 ± 1.9 ng GH/ml) and are expressed as mean ± SEM (n = 6).

 
To further evaluate the role of endogenous GTH in GH synthesis and secretion at the pituitary level, immunoneutralization using a GTH antiserum (gift from Dr. R. E. Peter, University of Alberta, Canada) was conducted in carp pituitary cells under static incubation. This antiserum was previously validated by immunostaining to be specific for carp GTH-II and has no cross-reactivity with GH and PRL in carp species (23). In this study, immunoneutralization using GTH antiserum (1:2500) induced a drop in GH release (Fig. 3AGo). This inhibitory action was noted within 2 h of incubation with GTH antiserum, and GH levels in the culture medium remained inhibited up to 48 h. In parallel experiments, pituitary cells were exposed for 48 h with increasing concentrations of GTH antiserum (1:10,000 to 1:1,000). GH levels in the culture medium were suppressed in a dose-dependent manner (Fig. 3BGo) with a concurrent rise in cellular GH contents (Fig. 3CGo). In addition, GH production, calculated as the sum of GH release and GH contents, was inhibited gradually with increasing levels of GTH antiserum (Fig. 3DGo). In these experiments, treatment with normal rabbit serum (NRS) was used as a negative control, and NRS (1:1000) did not affect basal GH release, GH content, and GH production (Fig. 3Go, C and D, insets), confirming that the actions of GTH antiserum were not due to nonspecific effects of serum treatment. These results, as a whole, suggest that endogenous GTH-II secreted by gonadotrophs can act in a paracrine manner to stimulate GH synthesis and secretion in somatotrophs. To our knowledge, a direct stimulatory action of GTH on GH synthesis and secretion at the pituitary level has not been previously reported.



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  		FIG. 3. Effects of GTH immunoneutralization on GH secretion and synthesis in grass carp pituitary cells. Time course of GTH immunoneutralization on GH release (A). Pituitary cells were incubated with and without GTH antiserum (GTH AS, 1:2500) for 2, 24, and 48 h, respectively. Dose dependence of GTH antiserum on GH release (B), GH content (C), and GH production (D) are shown. Pituitary cells were exposed to increasing levels of GTH antiserum (1:10,000 to 1:1,000) for 48 h under static incubation. In these experiments, parallel treatment with NRS (1:1000) was used as a negative control (insets). Data presented are expressed as mean ± SEM (n = 8). Groups denoted by different letters represent significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 
In mammals, GH can exert ultrashort feedback at the pituitary cell level to inhibit its own secretion, and this phenomenon is known to be species specific. For example, treatment with GH reduces GH release in bovine pituitary cells (40) but has no effects on GH secretion in rat pituitary cells (41) or purified somatotrophs (42). Because the pituitary actions of GH on GH gene expression have not been examined, it is unclear whether GH ultrashort feedback also plays a role in GH synthesis. To test whether GH can exert a direct action at the pituitary level to modulate GH secretion in fish, static incubation of grass carp pituitary cells was performed for 24 h with increasing levels of porcine GH (10–1000 ng/ml). Porcine GH was used in this study because it is commercially available, does not cross-react with our RIA for carp GH, and was previously shown to be biologically active in inducing IGF-I and -II expression in carp species (43). In these experiments, GH levels released into the culture medium were elevated in a dose-dependent manner with increasing concentrations of porcine GH (Fig. 4AGo). This stimulatory action, however, was not observed using a perifusion approach (Fig. 5Go), and porcine GH (10–1000 ng/ml) administered as 15-min pulses was not effective in altering GH secretion in pituitary cells under column perifusion. In contrast, sGnRH (1 µM), the positive control in this study, consistently increased GH secretion, suggesting that the responsiveness of somatotrophs to stimulation by GH-releasing factors was still intact.



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  		FIG. 4. Functional role of GH in GH secretion and synthesis in grass carp pituitary cells. Dose dependence of GH- induced GH release (A). Pituitary cells were incubated for 24 h with increasing doses of porcine GH (10–1000 ng/ml). Effects of GH immunoneutralization on GH release (B), GH content (C), and GH production (D) are shown. Pituitary cells were exposed to increasing levels of GH antiserum (1:10,000 to 1:1,000) for 48 h under static incubation. In these experiments, parallel treatment with an antiserum (1:1000) for carp prolactin (PRL AS) was used as a negative control (insets). Data presented are expressed as mean ± SEM (n = 8) and groups denoted by different letters represent significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 


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  		FIG. 5. Effects of exogenous GH on the kinetic of GH release from grass carp pituitary cells. Pituitary cells were cultured in 0.5-ml microcolumns and perifused with 15-min pulses of increasing doses of porcine GH (10–1000 ng/ml). In this study, parallel treatment with a 15-min pulse of sGnRH (1 µM) was used as a positive control. GH data were transformed into percent basal (mean basal = 23.5 ± 1.6 ng GH/ml) and are expressed as mean ± SEM (n = 6).

 
These results indicate that GH-induced GH release is not an acute effect but requires a prolonged incubation for the manifestation of GH responses. A similar observation has also been reported in IGF action at the pituitary level. In the rat, IGF inhibition on GH release is known to have a slow onset (3–24 h) in pituitary cells under static incubation (44) and cannot be readily demonstrated by column perifusion (45). This phenomenon may be related to the need of de novo protein synthesis for IGF actions (46). Whether a similar mechanism also exists in GH-induced GH release in grass carp is still unknown. However, we do not exclude the possibility that GH might stimulate GH release in pituitary cells via local release of autocrine/paracrine factors. If this were the case, a prolonged incubation would be needed for the accumulation of local growth factors/hormones.

To further examine the role of endogenous GH in regulating GH synthesis and secretion, pituitary cells were subjected to immunoneutralization for 48 h with increasing doses of GH antiserum (1:10,000 to 1:1,000 dilution). This antiserum was raised against carp GH (gift from Dr. R. E. Peter, University of Alberta, Canada) and was previously shown to have no cross-reactivity with GTH and PRL in carp species (23). In this study, GH immunoneutralization induced a dose-dependent decrease in GH release (Fig. 4BGo) and GH production (Fig. 4DGo). During the same period, a rise in GH contents was noted in the treatment groups exposed to higher levels of GH antiserum (1:5000 to 1:1000; Fig. 4CGo). As a negative control to confirm the specificity of GH immunoneutralization, pituitary cells were also exposed to an antiserum raised against carp PRL (1:1000; Fig. 4Go, B–D, insets). In this case, removal of endogenous PRL by antiserum treatment did not affect GH secretion, GH content, and total GH production. As a whole, we have shown that treatment with exogenous GH induced GH release, whereas the removal of endogenous GH inhibited GH release and GH production in grass carp pituitary cells. These findings indicate that GH may serve as a novel autocrine/paracrine factor in the pituitary to maintain GH synthesis and secretion. In contrast to mammals, the results of these experiments do not support the presence of a GH ultrashort feedback in the carp species. Using laser capture microdissection coupled to RT-PCR, we have shown that GH receptor transcripts are expressed in immunoidentified somatotrophs isolated from grass carp pituitary cells (47). In our recent study (48), we also reported that porcine GH was effective in elevating intracellular Ca2+ levels in enriched carp somatotrophs. These findings strongly suggest that somatotrophs are the target cells for endogenous GH in the carp model. Nevertheless, as mentioned in the preceding section, GH may also exert its actions through the release of paracrine factors in the pituitary via indirect actions on other pituitary cell types.

Because GTH and GH can maintain GH production and secretion in carp pituitary cells, it raises the possibility that their stimulatory actions on GH synthesis may involve modification of GH gene expression. Furthermore, it is conceivable that the stimulatory actions of GTH, at least partly, are mediated via the release of endogenous GH. To test these hypotheses, the direct action of HCG on GH mRNA expression in grass carp pituitary cells were examined in the presence of GH antiserum using a static incubation approach (Fig. 6AGo, left panel). As a reciprocal experiment, the effect of porcine GH on GH mRNA expression was also tested in the presence of GTH antiserum (Fig. 6BGo, left panel). In these studies, treatment of pituitary cells with HCG (40 U/ml) and porcine GH (100 ng/ml) for 48 h resulted in an increase in steady-state GH mRNA levels. Incubation with GTH antiserum (1:2500) or GH antiserum alone (1:2500), however, suppressed basal expression of GH mRNA. The stimulatory action of HCG on GH mRNA levels was abolished by treatment with GH antiserum. Interestingly, GTH antiserum was also effective in blocking GH-induced GH mRNA expression. In these experiments, samples of culture medium were also collected to study the effects of immunoneutralization on HCG- and GH-induced GH secretion. In this case, HCG and porcine GH consistently increased GH release. Treatment with the antisera for GH and GTH, in contrast, reduced basal GH secretion and blocked the GH-releasing effects of HCG and porcine GH, respectively. Unlike antiserum treatment, NRS (1:2500), the negative control of these studies, was not effective in altering both basal and stimulated GH release or GH mRNA levels (Fig. 6Go, A and B, right panels). These results indicate that both GTH and GH are essential for the maintenance of basal levels of GH gene expression in grass carp pituitary cells. Furthermore, GTH-induced GH mRNA expression is dependent on endogenous GH. Interestingly enough, endogenous GTH is also required for GH-induced GH gene expression. Parallel measurement of GH release also reveals that endogenous GH and GTH are required for the GH-releasing effects of HCG and porcine GH, respectively. Because gonadotrophs and somatotrophs are colocalized in the proximal pars distalis of the carp pituitary (23), these findings may suggest that these two hormones by acting in an autocrine/paracrine manner are essential to maintain the sensitivity of somatotrophs to stimulation by GTH and GH.



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  		FIG. 6. Interactions of GTH and GH in regulating GH gene expression and GH secretion in grass carp pituitary cells. Effects of immunoneutralization on HCG- (A) and porcine GH-induced (B) GH mRNA expression (upper panels) and GH release (lower panels). Pituitary cells were incubated for 48 h with either HCG (40 U/ml) or porcine GH (100 ng/ml) with simultaneous treatment of GH antiserum (GH AS; 1:2500) or GTH antiserum (GTH AS; 1: 2500), respectively. After drug treatment, medium was collected for the measurement of GH release. Total RNA was isolated from pituitary cells for the measurement of steady-state GH mRNA. In this study, parallel treatment with NRS (1:2500) was used as a negative control. Data presented are expressed as mean ± SEM (n = 8). Groups denoted by different letters represent significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 
It is worth mentioning that a reciprocal relationship between GH release and GH content was consistently observed in pituitary cells exposed to GTH/GH immunoneutralization. The cause of this phenomenon is unclear, but we speculate that it may be related to the dynamic balance between GH stores and GH exocytosis. Because the majority of GH produced in carp pituitary cells was released into the culture medium, it would be expected that activation of GH exocytosis for a prolonged period could deplete intracellular GH stores, presumably by emptying the readily releasable pool of secretory vesicles. On the contrary, a prolonged inhibition on GH release might have increased GH accumulation in intracellular GH stores, which may account for the increase in GH contents caused by immunoneutralization. In the rat, a similar reciprocal relationship between GH release and GH content has been reported after anterolateral deafferentation of medial basal hypothalamus (49). Under static incubation, bromocriptine, a dopamine agonist, is known to suppress basal GH release but increase GH contents in rat pituitary cells (50). Similar observations in fish may suggest that there is an intricate balance among GH production, GH stores, and GH exocytosis in somatotrophs throughout vertebrate evolution.

To further elucidate the functional interactions between GH and GTH at the pituitary level, the direct actions of GH on GTH release from carp pituitary cells were examined using column perifusion. Increasing levels of porcine GH (10–1000 ng/ml) were applied as 15-min pulses and induced a dose-dependent inhibition on basal GTH-II release (Fig. 7Go). This inhibitory action was rapid, and a drop in GTH-II release was noted 10 min after the initiation of drug treatment. The maximal inhibition on GTH-II release was observed within 20–25 min after GH perifusion. After that, GTH-II release gradually increased and returned to basal levels for low doses of GH treatment (10–100 ng/ml). In the case of a high dose of GH treatment (1000 ng/ml), GTH-II release remained suppressed at levels well below the prepulse basal (~58%). In these experiments, sGnRH was used as a positive control. A 15-min pulse of sGnRH (1 µM) consistently increased GTH-II release in carp pituitary cells under column perifusion. In parallel experiments using 24- and 48-h static incubation, increasing doses of porcine GH (10–1000 ng/ml) did not alter basal GTH-II secretion (Fig. 8AGo). A drop in GTH-II release, however, was observed with high doses of GH (100–1000 ng/ml) when the duration of drug treatment was reduced to 2 h. Apparently the sensitivity to GH inhibition was lost as a result of prolonged incubation. To establish the functional role of endogenous GH in GTH release, pituitary cells were subjected to 48-h static incubation with increasing concentrations of GH antiserum (1:10,000 to 1:1,000). Treatment with low levels of GH antiserum (1:10,000 to 1:5,000) increased GTH-II secretion (Fig. 8BGo) but suppressed GTH-II contents (Fig. 8CGo), whereas there was no change in GTH-II production (Fig. 8DGo). When higher levels of GH antiserum (1:2500 to 1:1000) were used, GTH-II release was inhibited and a partial recovery of the inhibition on GTH-II contents was noted. During this period, GTH-II production was significantly suppressed. In these experiments, treatment with NRS (1:1000), the negative control for GH immunoneutralization, was not effective in altering GTH-II secretion, GTH-II content, and GTH-II production (Fig. 8Go, B–D, insets).



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  		FIG. 7. Effects of exogenous GH on the kinetic of GTH-II release from carp pituitary cells. Pituitary cells were cultured in 0.5-ml microcolumns and perifused with 15-min pulses of increasing doses of porcine GH (10–1000 ng/ml). In these experiments, parallel treatment with a 15-min pulse of sGnRH (1 µM) was used as a positive control. GTH-II data were transformed into percent basal (mean basal = 1.44 ± 0.09 ng GTH/ml) and are expressed as mean ± SEM (n = 6).

 


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  		FIG. 8. Functional role of GH in GTH secretion and synthesis in grass carp pituitary cells. Dose dependence of GH treatment on GTH-II release (A). Pituitary cells were incubated for 2, 24, and 48 h with increasing doses of porcine GH (10–1000 ng/ml). An asterisk (*) represents a significant difference (P < 0.05), compared with the time-matched control without drug treatment (Student’s t test). Effects of GH immunoneutralization on GTH-II release (B), GTH-II content (C), and GTH-II production (D) are shown. Pituitary cells were exposed to increasing levels of GH antiserum (1:10,000 to 1:1,000) for 48 h under static incubation. In these experiments, parallel treatment with NRS (1:1000) was used as a negative control (insets). Data presented are expressed as mean ± SEM (n = 8) and groups denoted by different letters represent a significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 
To test whether GH can modify GTH gene expression, GTH-IIß mRNA levels were monitored in pituitary cells exposed to increasing doses of porcine GH (10–300 ng/ml). In this case, basal levels of GTH-IIß mRNA were increased in a dose-dependent manner in response to GH stimulation (Fig. 9AGo). Parallel treatment with GH antiserum (1:2500) also revealed that removal of endogenous GH by immunoneutralization markedly suppressed basal expression of GTH-IIß mRNA (Fig. 9BGo). A similar treatment of GH antiserum, however, did not affect basal levels of GTH{alpha} mRNA, which encodes the glycoprotein {alpha}-subunit shared commonly by GTH-I/FSH, GTH-II/LH, HCG, and TSH (Fig. 9BGo, inset). Again, the inhibitory action of GH antiserum was not mimicked by NRS (1:2500), confirming that the response of GTH-IIß mRNA was specific to GH immunoneutralization.



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  		FIG. 9. Functional role of GH in GTH gene expression in grass carp pituitary cells. Effects of exogenous GH on GTH-II ß mRNA expression (A) are shown. Pituitary cells were incubated for 48 h with increasing concentrations of porcine GH (10–300 ng/ml). Effects of GH immunoneutralization on GTH-IIß mRNA expression (B) are shown. Pituitary cells were incubated for 48 h with GH antiserum (GH AS; 1:2500). Treatment with NRS (1:2500) was used as a negative control. In this study, parallel measurement of GTH{alpha} mRNA in pituitary cells exposed to GH antiserum (1:2500) was also conducted (inset). Data presented are expressed as mean ± SEM (n = 8), and groups denoted by different letters represent a significant difference at P < 0.05 (ANOVA followed by Fisher’s LSD test).

 
These results, taken together, suggest that endogenous GH inhibits GTH-II secretion but at the same time maintains GTH synthesis by activating GTH-IIß gene expression. Apparently, the expression of GTH{alpha} subunit is not under the control of GH. In this study, GTH-II release was also suppressed by treatment with high doses of GH antiserum. This phenomenon is suspected to be the result of a drop in GTH synthesis, which might have reduced/depleted the readily releasable pool of secretory vesicles in gonadotrophs. Because only high doses of GH antiserum was effective in inhibiting GTH-II production, it would be logical to assume that a low level of endogenous GH is good enough to maintain GTH synthesis in grass carp pituitary cells. Probably, gonadotrophs have a high sensitivity but a low saturation threshold for GH-induced GTH synthesis. It is also noted that increasing doses of porcine GH were unable to suppress GTH-II secretion after prolonged incubation (≥24 h).

These results are at variance with those in 2-h incubation or perifusion experiments and, presumably, are due to the saturating effect of endogenous GH accumulated during the period of prolonged incubation. In mammals, the role of GH in LH and/or FSH secretion is still controversial. In general, the stimulatory action of GH on GTH release is more readily observed in transgenic models, e.g. in transgenic mice with human GH (9) or GH-deficient Ames dwarf mice (51). In the case of the inhibitory action, only in vivo studies have been reported. For example, GH treatment inhibits plasma LH levels in the rat, and the opposite effect can be obtained by infusion with GH antiserum (8). In amenorrheic women, GH treatment does not alter LH pulse frequency but reduces the pulse amplitude as well as integrated LH plasma levels (52). In these in vivo studies, the site of action for GH has not been determined, and to our knowledge, a direct action of GH at the pituitary level to inhibit LH and/or FSH release has not been previously reported.

In the grass carp, a direct inhibitory action of GH on GTH-II release from pituitary cells forms an important component for the functional interactions between somatotrophs and gonadotrophs (Fig. 10Go). In this case, GTH-II secreted from carp gonadotrophs exerts paracrine stimulation on neighboring somatotrophs to induce GH exocytosis. The combined actions of GH (autocrine effect) and GTH-II (paracrine effect) on carp somatotrophs can maintain GH gene expression, GH production, and sensitivity of somatotrophs to stimulation by GTH and GH at the pituitary level. In addition, endogenously secreted GH exerts a feedback control (paracrine effect) on neighboring gonadotrophs to inhibit GTH-II secretion. In this feedback loop, a low level of GH is required for the maintenance of GTH synthesis, and GH may also induce GH release indirectly by acting on other pituitary cells to trigger the release of stimulatory paracrine factors. This intrapituitary feedback loop may represent a novel mechanism operating at the pituitary level to maintain GH synthesis and secretion in lower vertebrates. Whether a similar system also exists in the pituitary of mammals is unclear, and future investigations are clearly warranted. The local interactions between GTH and GH in regulating GH synthesis and secretion in grass carp may provide a functional account for the close anatomical relationship between gonadotrophs and somatotrophs in the pituitary of bony fish. The local interactions between gonadotrophs and somatotrophs at the pituitary level also provide new insights on the mechanisms causing the parallel increases in GTH-II and GH release observed in fish during sexual recrudescence and spawning seasons.



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  		FIG. 10. Working model of an intrapituitary feedback loop formed by local interactions between gonadotrophs and somatotrophs (see Discussion for details).

 


   Acknowledgments
 
Special thanks are given to Drs. R. E. Peter and J. P. Chang (University of Alberta, Canada) for the supply of antisera and hormone standards for GH and GTH-II RIA. We are also indebted to Dr. W. K. K. Ho (Chinese University of Hong Kong) for his support to set up the assay system for GH mRNA measurement.


   Footnotes
 
This work was supported by RGC (HK) and CRCG (HKU) grants (to A.O.L.W.). Financial support from the Department of Zoology, Hong Kong University (to H.Z. and X.W.) in the form of postgraduate studentship is also acknowledged.

Abbreviations: DIG, Digoxigenin; FBS, fetal bovine serum; GTH, gonadotropin; HCG, human chorionic gonadotropin; LSD, least significance difference; NRS, normal rabbit serum; PRL, prolactin; SDS, sodium dodecyl sulfate; sGnRH, salmon GnRH; SRIF, somatostatin; SSC, saline sodium citrate.

Received March 19, 2004.

Accepted for publication August 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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