DOMESTIC

ANIMAL

Vol. 9(3):209-218,1992

ENDOCRINOLOGY

ANDROGENS MODULATE GROWTH HORMONE-RELEASING FACTOR-INDUCED GH RELEASE FROM BOVINE ANTERIOR PITUITARY CELLS IN STATIC CULTURE H.A. Hassan,’ R.A. Merkel,1~2~3 W.J. Enright and H.A. Tucker’ Departments of Animal Science and Food Science and Human Nutrition Michigan State University, East Lansing, MI 48824 Received November 26, 1991

ABSTRACT Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10’ cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17P (E,, lo-” to 1O-8M), testosterone (T, 10m8to 10e5M), dihydrotestosterone (DHT, 10e9to 10. 6 M) or 5a-androstane-3a, 17P-diol (3a-diol, 10-l’ to lo-* M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P>O.O5) basal GH released at 24,48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF l-44NH, (lo-* M). In non-steroid treated wells, GRF increased (PO.O5) GRF-induced GH release; however, preincubation with T (10-j M) and DHT ( 10-9, 10e8and 10m7M) increased (PcO.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.

INTRODUCTION The endocrine system has a major impact on body growth and development. Principal among the hormones associated with growth is growth hormone (GH). Cattle (1,2), sheep (3), pigs (4) and rats (5) exhibit gender-associated dimorphic patterns of GH secretion. The GH secretory pattern into the circulation of males has a greater pulse amplitude and lower baseline than females (6). In addition, gonadally intact males of all domestic species grow faster, and have more muscle and less fat than females or castrates (7,8). Furthermore, endogenous (9) and exogenous (10) steroid hormones increase blood concentrations of GH, improve growth rate, feed utilization and feed efficiency of livestock (11). Thus, it has been suggested that these observations on growth are, at least in part, modified through sex steroids stimulation of GH secretion. The mechanism(s) by which gonadal steroids affect GH secretion is not clear. Whether sex steroids act directly on pituitary somatotropes, hypothalamic neurons or both to modify GH secretion, is not known. Therefore, the objective of the present study was to investigate the effect of estradiol-17P, testosterone and its metabolites (DHT and 3a-diol) on GH release from isolated bovine anterior pituitary cells in static culture.

MATERIALS AND METHODS Hormones and chemicals. Estradiol-17P (E,), testosterone (T), Sa-androstane-17P-ol3-one (DHT), W-androstane-3a,17P-diol(3a-diol), HEPES buffer, penicillin and streptomycin solution, and collagenase type 1A were purchased from Sigma Chemical Company Copyright 0 1992 Butterworth-Heinemann

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(St. Louis, MO). Hank’s balanced salt solution (HBSS), calcium magnesium-free HBSS (CMF-HBSS), Dulbecco’s Modified Eagle Medium (DMEM), Fungizone, pancreatin 4X N.F., trypan blue stain (0.4 %), essential and non-essential amino acids, and newborn calf serum were purchased from GIBCO Laboratories (Grand Island, NY). Growth hormonereleasing hormone l-44-NH, (GRF) was generously donated by The Upjohn Company, Kalamazoo, MI. Cell dissociation and culture. Pituitary glands were aseptically removed at slaughter from either steers (14-20 mo of age), heifers (14-16 mo of age), cows (> 3 yr of age) or prepubertal male calves (13 mo of age) and kept in cold sterile oxygenated HBSS until transferred to the laboratory. The steers and heifers used for isolation of AP cells had not been implanted with any anabolic agent. The HBSS was supplemented with 25 mM HEPES, 10 U/ml penicillin, 10 pgJm1 streptomycin and 2.5 pg/ml Fungizone. All subsequent procedures were performed under sterile conditions using a horizontal laminar flow hood. Anterior pituitary (AP) cells were prepared as described by Vale et al. (12) and Padmanabhan et al. (13) as modified in our laboratory. Briefly, slices (1 mm thick, obtained using a Stadie Riggs Microtome) of the AP were incubated at 37 C with CMF-HBSS containing 0.3% collagenase and 3% BSA for 45-60 min. This was followed by incubation with 0.25% pancreatin in CMF-HBSS for 7-10 min. Cells were pelleted by centrifugation (400 x g for 5 min), washed 4 times and suspended (5 X lo5 cells/ml) in plating media. Total cell yield was more than 12 X lo6 cells per pituitary gland. Cell viability determined by trypan blue exclusion was greater than 90%. Cells (1 ml of cell suspension) were plated in Coming 24-well dishes and maintained at 37 C in a humidified (95%) atmosphere of 5% CO,. Plating medium (pH = 7.4) was DMEM supplemented with 10% newborn calf serum (Lot #4705), 1% essential and 1% non-essential amino acids, 25 mM HEPES, 10 U/ml penicillin, 10 cls/ml streptomycin and 2.5 @ml Fungizone which was added for the first 48 hr and omitted thereafter. Media were changed 48 hr after plating, and then at 24-hr intervals thereafter. All treatments were imposed 72 hr after plating, which allowed the AP cells to become confluent, aggregate and attain intercellular communication which has been reported to potentiate GH release (14). Media were collected every 24 hr following treatment (unless otherwise stated) and stored at -20 C until assayed for GH concentrations. Details of individual treatment protocols are described in Results. RIA of GH. GH concentrations in culture media were measured by a double antibody radioimmunoassay method developed in our laboratory (15,16). Bovine GH (NIH-b-18) was used as the reference standard. All samples from each experiment were measured in a single assay. The intra-assay coefficient of variation was less than 10%. Statistical analyses. Results were analyzed by split plot design with repeat measurements (17), using the M-stat software package (1985), followed by student t-test to compare individual groups; PcO.05 was considered significant. All data are presented as means + SE. RESULTS

Experimental conditions. Phenol red (PhR), a pH indicator, which is a component of plating media, exhibits estrogenic activity (18). To determine the effect of PhR on GH release, media were replaced on confluent cells with DMEM containing various concentrations of E, (O,lO-I*, lo-IO, 10e8 M) in the presence or absence of PhR (45 @I). This concentration of PhR is normally present in culture media. Media were collected every 24 hr for 72 hr from 6 wells/treatment combination. This experiment was repeated once. Concentrations of GH did not differ (bO.05) between cells incubated with or without PhR (data not shown). Similarly, no interactions were observed (bO.05) between PhR and different concentrations of E,. Thus, PhR was included in the media for all subsequent experiments. Fungizone (Fz), an antifungal agent, reduced GH secretion from cultured GH, cells (19); therefore, its effect was studied in our culture system. Cells were maintained in DMEM con-

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CELL SOURCE Figure 1. Basal growth hormone concentrations in the media from anterior pituitary cells obtained from calves, heifers, or cows at 72, 96 and 120 hr after plating (See Material and Methods). Cells isolated from the anterior pituitaries of prepubertal calves, 5 heifers, 5 steers or 5 cows were pooled by gender and age group. The pooled cells for each constitute a dispersion and the dispersion of each group was repeated once. Each bar represents the mean of 24 wells

steers 12-15 group f SE.

taining Fz for 48 hr after plating. Then wells were assigned to Fz treatment (0,2.5 @ml) for 72 hr. Media were collected every 24 hr from 36 wells/treatment. This experiment was repeated twice. Fz reduced (PcO.05) GH concentration in the media by 38% after 72 hr of incubation (425 vs 263 @ml). Thus, Fz was added only during the first 48 hr of incubation. Source of anterior pituitary glands. In preliminary studies we observed that gender and age of animal from which AP cells were isolated affected basal GH concentration in media during incubation in static culture. To confirm this observation, AP from a number of animals (either 5 steers, 5 heifers, 5 cows or 12-15 calves) were pooled within each gender and age group for each dispersion. The number of dispersions (replications) for each gender and age group as well as the number of wells/treatment is stated for each experiment. Media were collected from confluent cells every 24 hr for an additional 72 hr from 12 wells/gender and age group to measure basal GH concentrations. All experiments were performed with independent pools of pituitary cells collected from each gender and age group. This experiment was repeated once. Concentrations of GH after 24,48 and 72 hr were greatest when calf or heifer AP were used (Figure 1). Consequently, all subsequent data were collected using confluent AP cells from calves. Dose of GRF for challenge. Bovine GRF ( 1@12to 10e8M) stimulates GH release from bovine AP in a dose-dependent manner (20). The effect of GRF concentrations on GH release was tested in our culture system. Confluent cells were washed 4 times and incubated for 1 hr in serum-free media with GRF (0 and 10m9 to 10m6 M); there were 12 wells/treatment and the experiment was repeated once. Increasing GRF concentrations, up to lo-* M, linearly increased (PcO.01) GH concentrations in the media (Figure 2). Since a near maximum GH response was observed with 1W8M GRF, this concentration was used in subsequent experiments.

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400-

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Figure 2. Effect of GRF concentration (0 or IO’ to 10 6 M) on growth hormone concentrations in the media from calf an pituita ry cells incubated for 1hr. The cells isolated from anterior pituitaries obtained from 12-15 prepubettal male CdWS were pooled I for each of the two replications of this experiment. Each bar represents the mean of 24 wells f SE.

Duration of GRF challenge. Loss of GRF bioactivity in serum-containing media has been reported and is attributed to the presence of the proteolytic enzyme, dipeptidyl amino peptidase (DPP-IV), which cleaves GRF at several sites yielding inactive peptide fragments (21). This enzyme has been found to be widely distributed in mammalian cells (22) and has been observed in extracts of bovine AP (23). Additionally, it has been reported recently that proteolytic degradation of GRF occurred when incubated with bovine AP cells in vitro (24). Thus, inactivation of GRF by DPP-IV may occur in serum-free media. The temporal effectiveness of GRF to stimulate GH release in serum-free media was studied over a 6-hr period. Confluent cells were washed (4 times) and incubated in serum-free media for 1 hr, media collected and basal GH concentration of each individual well was determined. Cells were then treated with GRF (0, 10.’ M) in serum-free media for 1, 2, 3, 4, or 6 hr. At the end of each period, media were collected from 12 wells per treatment, and then those wells were terminated. This experiment was repeated once. Rate of basal GH secretion decreased with time (Figure 3); GRF induced a 270, 177, 174, 169 and 152% increase in GH concentration over non-GRF-treated wells at 1, 2,3,4 and 6 hr, respectively. These results are consistent with those of Fukata et al. (25) and Brazeau et al. (26) who also observed the maximum GRF-stimulated GH release during the first hr and a diminished response thereafter. Thus, we challenged AP cells with 10m8MGRF for 1 hr only in all subsequent experiments. Incubation with steroid hormones. Confluent cells were incubated with media containing various concentrations of either E, (0 and 10.” to 10e8M), T (0 and lOmEto 10.’ M), DHT (0 and 10e9to 10m6M) or 3a-diol(0 and 10-l’ to lo-* M) for 24,48 or 72 hr. Media were collected every 24 hr with 24 wells for each steroid concentration. Each experiment was repeated three times. Incubations with E,, T, DHT or 3a-diol had no effect (bO.05) on GH concentrations in the media at any sampling time (Figure 4). While incubation with the steriods did not effect GH concentrations at 24,48, or 72 hr, overall GH concentrations were reduced at 48 and 72 hr compared to those at 24 hr.

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TIME (Hr) Figure 3. Effect of duration of GRF challenge (I to 6 hr) on growth hormone concentrations released into the media. The cells isolated from anterior pituitaries obtained from 12-15 prepubertal male calves were pooled for each of the two replications of this experiment. Each bar represents the mean of 24 wells f SE.

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DHT 3a-DIOL STEROID (-Log CONCENTRATION) Figure 4. Effect of incubation (72 hr) with various concentrations of sex steroids on basal growth hormone concentrations in the media from calf anterior pituitary cells. Anterior pituitary cells from 12-15 calves were pooled for each of three replications of this experiment. Each bar represents the mean of 72 wells f SE. E = estradiol-17P (o or IO-” to lo-* M), T = testosterone (0 or IO-’ to IO’ M), DHT = dihydrotestosterone (0 or lO-9 to 10~bM), 3a- diol = 5a-androstane-3a,17P-diol (0 or 10 ” to IO-y M).

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DHT 3wDIOL E STEROID (-Log CONCENTRATION) Figure 5. Effect of GRF l-U-NH, (0 or 10~’M) treatment for 1 hr on growth hormone concentrations in the media from anterior pituitary cells incubated for 24 hr with different concentrations of the sex steroids. Anterior pituitary cells from 15 calves were pooled for each of three replications of this experiment. Each bar represents the mean of 72 wells f SE. estradioLl7P (0 or lo-” to 10’ M), T = testosterone (0 or lo-” to 10.’ M), DHT = dihyrotestosterone (0 or 10’ to 10’ 3a-diol = 5a-androstane-3a,l7p-diol (0 or 10” to 10’ M).

calf 12. E= M),

GRF challenge after incubation with sex steroids. Confluent cells were incubated for 24 hr with the aforementioned sex steroids at different concentrations, then washed 4 times and challenged in serum-free media with GRF (0, 10e8M). Media were collected after 1 hr challenge from 12 wells; each study was repeated 3 times. GRF challenge increased (PcO.05) average GH concentrations from 58 f 3 to 134 k 5 ng/ml in control wells. Neither E, nor 3a-diol affected (P>O.O5) GH release after GRF challenge (Figure 5). In contrast, incubation with DHT ( 10m9to 10m7M) or T (lo-* M) increased (PcO.05) GRF-induced GH release (235 + 10, 190 * 8, 185 f 6 and 195 + 7 ng/ml, respectively). DISCUSSION The gender-associated dimorphic pattern of GH secretion in many mammalian species implies that the gonadal steroids play a role in the secretion of GH; however, the mechanism of this action has not been elucidated. Previous studies on the effect of sex steroids on GH production from the AP have been contradictory (27,28,29). This prompted the present study to investigate the effect of gonadal steroids on GH release from bovine AP cells in static cultures. Estrogens stimulate GH secretion in vivo (11). Several attempts to investigate the mechanism of E, action on GH secretion have provided conflicting results. For instance, it has been reported that E,, when compared with T, increased serum GH in rats with renal autotransplanted pituitaries (30). Lack of T action may be explained by low Sa-reductase activity, which occurs for up to 14 d after grafting (3 l), and a consequent decline in the conversion of T to DHT which appears to be the active metabolite eliciting androgenic activity. Also, Simmard et al. (29) observed an increase in basal and GRF-stimulated GH release

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when rat AP cells were incubated with E,. In contrast, Fukata and Martin (28) and Wehrenberg et al. (27) reported no effect of E, on basal or GRF-stimulated GH release using rat AP cells. The latter data are consistent with the observations in our study. The stimulatory effect of E, on GH secretion in vivo may be due to other mechanisms. For instance, E, may act at the hypothalamic level to alter GRF and(or) somatostatin secretion. It is also possible that E, acts at extrahypothalamic sites to alter neurotransmitters such as acetylcholine and(or) serotonin (32) which may influence hypothalamic GRF or somatostatin secretion (33). In addition, E, may alter insulin-like growth factor(s) which influences serum GH concentrations in cattle (34,35). Also, the effect of E, on metabolic clearance rate of GH cannot be ignored because clearance rates in male rats have been reported to be faster than in females (36). Androgens play an important role in modulating the pituitary GH response to GRF in vivo (27). AP cells, especially gonadotropes, possess So-reductase which converts T to DHT (37). They also appear to possess the 3a- and 3P-hydroxysteroid oxidoreductases that convert DHT to 3a- and 3/%diol, respectively. The effect of these metabolites as intracellular mediators of T action on bovine AP cells with respect to GH release has not been reported. In the present study, neither T nor its metabolites (DHT or 3a-diol) had a direct effect on basal GH release. This lack of effect of T is consistent with reported observations (27,28). In addition, the current study showed that incubation with T and DHT increased GRF-induced GH release; however, T was a weaker promoter than DHT. Furthermore, the GH response to GRF increased with increased dose of T. In contrast, the GH response to GRF decreased with increasing dose of DHT. A possible explanation for the observed T action is that high concentrations of T ( 10e7M): 1) stimulate Sa-reductase activity as has been observed in human fibroblasts (38), and presumably favor the conversion of T to DHT, 2) display binding affinity to androgen receptors similar to what DHT exhibits at low concentrations (39,40). We speculate that these mechanisms, yet to be demonstrated, are functional in our culture system and may have contributed to the results obtained. Results obtained from the present study suggest that E, acts at sites other than the AP to alter GH concentrations. In addition, the differences in the pattern of circulating GH between male and female may be due, at least in part, to enhancement of somatotrope responsiveness to GRF by androgens. This may partially explain the role that androgens play in the characteristic pattern of high amplitude GH peaks in males. The mechanism whereby androgens induce periods of low baseline GH concentrations between peaks remains to be elucidated, but is likely to involve somatostatin. ACKNOWLEDGMENTS/FOOTNOTES This study was partially supported by the Michigan Agricultural Experiment Station. The authors gratefully acknowledge the generous gift of the growth hormone standard and the growth hormone-releasing factor from The Upjohn Company, Kalamazoo, MI. ‘Department of Animal Science. *Department of Human Nutrition. ‘Corresponding author: R.A. Merkel, Departments of Animal Science, and Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824. %esent address: Teagasc, Grange Research Centre, Dunsany, Co. Meath, Ireland.

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3 1. Martini L. The Sa-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implication. Endocr Rev 3: l-25, 1982. 32. Akuzawa K, Wakabayashi K. A serum-free culture of neurons in the septal, preoptic, and hypothalamic region. Effect of triiodothyronine and estradiol. Endocrinol Jpn 32: 163-173, 1985. 33. Armstrong JD, Spears JW. An endogenous opioid peptide agonist elevates concentrations of growth hormone in ruminants. J Anim Sci 66 [Suppl 1]:391 (Abstract), 1988. 34. Breier BH, Gluckman, PD, Bass JJ. Influence of nutritional status and estradiol-17B on plasma growth hormone, insulin-like growth factor-1 and -II and the response to exogenous growth hormone in young steers. J Endocrinol 118:243-250, 1988. 35. Emight WJ, Quirke JF, Gluckman PD, Breier BH, Kennedy LG, Hart IC, Roche JF, Coert A, Allen P. Effect of long-term administration of pituitary-derived bovine growth hormone and estradiol on growth in steers, J Anim Sci 68:2345-2356, 1990. 36. Badger TM, Millard WJ, Owens SM. LaRovere J, O’Sullivan D. Effects of gonadal steroids on clearance of growth hormone at steady state in the rat. Endocrinology 128: 1065-1072, 1991. 37. Cohen H, Loras B, Rocle B, Bourcet P. Testosterone metabolism in three-cloned pituitary cell lines. J Steroid Biochem 12:361-365, 1980. 38. Price P, Wass JAH, Griffin JE, Leshin M, Savage MO, Large DM, Bu’Lock DE, Anderson DC, Wilson JD, Besser GM. High dose androgen therapy in male pseudohermaphroditism due to So-reductase deficiency and disorders of the androgen receptor. J Clin Invest 74: 1496-1501, 1984. 39. Wilbert DM, Griffin JE, Wilson JD. Characterization of the cytosol androgen receptor of the human prostate. J Clin Endocrinol Metab 56:113-120, 1983. 40. Grino PB, Griffin JE, Wilson JD. Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 126: 1165-I 172, 1990.