Growth hormone secretion in old female rats
hemolytic plaque
analyzed by the reverse
assay
Sumio Takahashi
Zoo/ogira/ Institute, Faculty of Science,
Hiroshima University, Kagamiyama,
Higashi-Hiroshima, Japan
Takahashi S. Growth hormone secretion in old female rats analyzed by the assay. Acta Endocrinol 1992;127:531-5. ISSN 0001-5598
reverse
hemolytic plaque
Growth hormone (GH) release was studied in young (3\p=n-\4-month-old) and old persistent diestrous (20\p=n-\ 21-month-old) female rats using the reverse hemolytic plaque assay. A bimodal distribution of reverse hemolytic plaque area was observed in both young and old female rats. The mean and median of the plaque area of GH cells from old females were smaller than those from young female rats. The percentage of plaque-forming GH cells in old female rats was lower than in young female rats. The percentage of large plaque-forming GH cells (plaque area, more than 8 \m=x\103 \g=m\m2) was lower in old female rats than in young female rats. GH-releasing hormone (GHRH) increased the mean and median of plaque areas in both young and old female rats. However, responsiveness to GHRH was reduced in old female rats. These results indicate that the amount of GH released from individual GH cells decreases with age in female rats, resulting in diminished GH secretion. Sumió Takahashi,
Japan
Department of Biology. Faculty of Science, Okayama University, Tsushima, Okayama 700,
Growth hormone (GH) secretion declines with age in rats (1, 2). The amplitudes of GH pulsatile release are smaller in old rats than in young rats (1,2). We found that each GH cell in old female rats has less GH and GH-mRNA content than those in young female rats (3). This decrease in GH synthesis at the gene transcription level in old rats probably causes the decrease in GH secretion. Pituitary responsiveness to GH-releasing hormone (GHRH) was reduced in old rats in the in vivo study (46). On the contrary, no age-related alterations were reported in either the in vivo study (7) or the in vitro study (4). One of the reasons for this discrepancy may be the difference in ages of the rats examined, since GH cell population changes with age (8). The GH cell population consisted of three morphologically different types of cells (8, 9). One type of GH cell contained large secretory granules, and another type small secretory granules. An intermediate type of GH cell contained both large and small secretory granules. The relative proportions of each subtype of GH cells changed with age (8). The physiological significance of these alterations in a GH cell population is not fully understood. As GH cell popula¬ tions change, as stated above, it is necessary to know the age-related functional changes of individual GH cells. The present study aimed at clarifying changes in the secretory capacity of each GH cell in old female rats. A reverse hemolytic plaque assay was used for this pur¬ pose, since this assay system enables detection of GH release at the individual cell level.
Material and methods Animals Female rats of the Wistar/Tw strain were used; they were housed in a temperature-controlled room with a 12-h light (06.00-18.00) and 12-h dark cycle, and given rat chow (CLEA Japan Inc., Tokyo) and water ad libitum. The mean life span and the age-related changes in the estrous cycle of these rats have been reported previously (10, 11). Young female rats (3-4 months of age) and old persistent diestrous female rats (20-21 months of age) were used in this study.
Pituitary cell dissociation and culture The rats were anesthetized and sacrificed by decapi¬ tation. Pituitaries were removed and the posterior and intermediate lobes were discarded. The anterior lobe was cut into small pieces with a sterile razor blade, and the fragments suspended in Hanks' solution containing HEPES (20 mmol/1) and bovine serum albumin (0.3% w/v, BSA) (HSH-BSA). After being rinsed twice in the same HSH-BSA solution, they were incubated with trypsin (0.5% w/v, Type III, 10,400 IU/mg, Sigma, St Louis, MO) in HSH-BSA at 37°C for 15 min. The fragments were incubated with DNase for 1 min and then with soybean trypsin inhibitor (0.1% w/v, Sigma, St Louis, MO) in HSH-BSA at 37°C for 15 min. After
532
Sumió Takahashi
rinsed in Ca2+-Mg2+ free-HSH-BSA, they were incubated in 2 mmol/1 EDTA-HSH solution and 1 mmol/ 1 EDTA-HSH solution for 5 and 15 min respectively, rinsed in Ca2 + -Mg2 + free-HSH-BSA twice, and triturated with a Pasteur pipette for 10 min. Dissociated pituitary cells were filtered with a nylon net and suspended in Dulbecco modified Eagle's medium containing 20 mmol/ 1 HEPES (DMEMH) supplemented with fetal calf serum (10% v/v, FCS). The cell viability was assessed by trypan blue exclusion at the end of dispersion. The cell viability was between 90 and 95% irrespective of the age groups. The dissociated cells were suspended at a density of 3 x 105 cell/ml in DMEMH with 10% FCS and were seeded in 35-mm plastic culture dishes and incubated at 37°C in a water-saturated atmosphere of 5% C02 and 9 5% air for two days, before the plaque assay, in order to allow the cell damage caused by the enzymatic cell dispersion to heal.
being
Reverse
hemolytic plaque assay
The cells were harvested from the culture dishes using a 0.05% trypsin solution and were dispersed into single cells (the cell viability on the day of assay was more than 95% in both age groups). These monodispersed cells were rinsed in DMEMH containing 0.1% BSA (DMEMHBSA) and then mixed with an equal volume of a 16% suspension of ovine red blood cells previously coupled to Protein A with chromium chloride hexahydrate (final concentration 2 x 105 cells/ml). The cell mixture was infused into a poly-L-lysine-coated Cunningham slide chamber and preincubated at 37°C, 95% air-5% C02, for 45 min. After preincubation, the chamber was rinsed in DMEMH-BSA and then filled with a mixture of DMEMHBSA and anti-rat growth hormone serum (final dilution of 1:20), GH-releasing hormone (GHRH, Peptide Inst., Osaka) and somatostatin (SRIF, Sigma, St Louis, MO) when needed. Incubation was carried out for 1.5 h. This incubation time was determined from the preliminary study in which the maximum percentage of plaqueforming GH cells had been achieved by 1.5 h. After incubation, the chamber was washed and filled with a mixture of DMEMH-BSA and guinea pig complement (ICN ImmunoBiologicals, Lisle, IL, final dilution 1:160) and secretagogues (GHRH and SRIF) when needed. The complement reaction was carried out for 30 min and terminated by infusion of 2% glutaraldehyde solution. The GH antiserum used was generated against rat GH. The specificity of the antiserum was checked using an enzyme-labelled immunoassay. The antiserum immunocytochemically stained pituitary cells (immunoreactive GH cells) at a dilution of 1:12,800. The diluted antiserum (1:6,400) was incubated with rat GH
(NIDDK, rGH-I-5)
or rat prolactin (NIDDK, rat concentrations of 0.1, 1, 2.5 and 5 yg at prolactin-I-5) rGH or rPRL/50 ul diluted antiserum, at 4°C for two days. Using these two antisera, immunocytochemical
acta endocrinologica 1992,127
staining was carried out. The antisera preabsorbed with
rGH (1 and 5 at all.
yg) did not show any immunoreactive cells
The specificity of the plaque assays for GH secretion checked as follows. Preincubation of a diluted antiserum (1:20) with highly purified rat GH (NIDDK, rGH-I-5, 10 yg rGH/100 ¿d of diluted antiserum) at 4°C for 16 h completely abolished plaque formation. Omis¬ sion of specific antiserum or guinea pig complement also abolished plaque formation. was
The observation
of the plaques
The pituitary cells were stained with Turuk solution to facilitate the observation of hemolytic plaques. Plaque diameters were measured with an ocular micrometer. Plaque areas were calculated from the diameter of the plaques, because those formed were usually circular.
Statistical
analyses
Statistical analysis was carried out by two-way analysis of variance with unequal but proportional subclass numbers (12). Percentages of plaque-forming cells and percentages of occurrence of small and large plaqueforming cells were arcsine-transformed and analyzed.
Results Fig. 1 shows the typical patterns of plaque sizes of GHRH (10"7 mol/1), SRIF (10-7 mol/1) and vehicle-treated pituitary cells of young female rats. GHRH increased the
plaque area and SRIF decreased it compared with that of vehicle-treated controls. This distribution pattern revealed a bimodal distribution of plaque area: small plaques and large plaques. Small plaques were defined as those whose areas were less than 8 x 103 yra2; large plaques were defined as those whose areas were greater than 8 x 103 /mi2. GHRH increased the relative propor¬ tion of large plaques and SRIF decreased it compared with that of controls. A dose-response to GHRH and SRIF was studied, but only the highest dose (10~7 mol/1) of GHRH and SRIF induced significant changes (data not shown). These results clearly indicate that hemolytic plaques were formed by GH cells. Fig. 2 shows typical patterns of plaque size of GH cells in young and old female rats with or without GHRH. The mean and median of plaque areas from old females were smaller than those of young female rats. The large plaque-forming cells were less frequently observed in old female rats than in young females. GHRH increased the frequences of large plaques in both young and old females. Table 1 gives the effects of GHRH on the plaque sizes of GH cells in young and old females. Percentages of plaque-forming cells significantly differed between young and old rats (p
hemolytic plaque
analyzed by the reverse
assay
Sumio Takahashi
Zoo/ogira/ Institute, Faculty of Science,
Hiroshima University, Kagamiyama,
Higashi-Hiroshima, Japan
Takahashi S. Growth hormone secretion in old female rats analyzed by the assay. Acta Endocrinol 1992;127:531-5. ISSN 0001-5598
reverse
hemolytic plaque
Growth hormone (GH) release was studied in young (3\p=n-\4-month-old) and old persistent diestrous (20\p=n-\ 21-month-old) female rats using the reverse hemolytic plaque assay. A bimodal distribution of reverse hemolytic plaque area was observed in both young and old female rats. The mean and median of the plaque area of GH cells from old females were smaller than those from young female rats. The percentage of plaque-forming GH cells in old female rats was lower than in young female rats. The percentage of large plaque-forming GH cells (plaque area, more than 8 \m=x\103 \g=m\m2) was lower in old female rats than in young female rats. GH-releasing hormone (GHRH) increased the mean and median of plaque areas in both young and old female rats. However, responsiveness to GHRH was reduced in old female rats. These results indicate that the amount of GH released from individual GH cells decreases with age in female rats, resulting in diminished GH secretion. Sumió Takahashi,
Japan
Department of Biology. Faculty of Science, Okayama University, Tsushima, Okayama 700,
Growth hormone (GH) secretion declines with age in rats (1, 2). The amplitudes of GH pulsatile release are smaller in old rats than in young rats (1,2). We found that each GH cell in old female rats has less GH and GH-mRNA content than those in young female rats (3). This decrease in GH synthesis at the gene transcription level in old rats probably causes the decrease in GH secretion. Pituitary responsiveness to GH-releasing hormone (GHRH) was reduced in old rats in the in vivo study (46). On the contrary, no age-related alterations were reported in either the in vivo study (7) or the in vitro study (4). One of the reasons for this discrepancy may be the difference in ages of the rats examined, since GH cell population changes with age (8). The GH cell population consisted of three morphologically different types of cells (8, 9). One type of GH cell contained large secretory granules, and another type small secretory granules. An intermediate type of GH cell contained both large and small secretory granules. The relative proportions of each subtype of GH cells changed with age (8). The physiological significance of these alterations in a GH cell population is not fully understood. As GH cell popula¬ tions change, as stated above, it is necessary to know the age-related functional changes of individual GH cells. The present study aimed at clarifying changes in the secretory capacity of each GH cell in old female rats. A reverse hemolytic plaque assay was used for this pur¬ pose, since this assay system enables detection of GH release at the individual cell level.
Material and methods Animals Female rats of the Wistar/Tw strain were used; they were housed in a temperature-controlled room with a 12-h light (06.00-18.00) and 12-h dark cycle, and given rat chow (CLEA Japan Inc., Tokyo) and water ad libitum. The mean life span and the age-related changes in the estrous cycle of these rats have been reported previously (10, 11). Young female rats (3-4 months of age) and old persistent diestrous female rats (20-21 months of age) were used in this study.
Pituitary cell dissociation and culture The rats were anesthetized and sacrificed by decapi¬ tation. Pituitaries were removed and the posterior and intermediate lobes were discarded. The anterior lobe was cut into small pieces with a sterile razor blade, and the fragments suspended in Hanks' solution containing HEPES (20 mmol/1) and bovine serum albumin (0.3% w/v, BSA) (HSH-BSA). After being rinsed twice in the same HSH-BSA solution, they were incubated with trypsin (0.5% w/v, Type III, 10,400 IU/mg, Sigma, St Louis, MO) in HSH-BSA at 37°C for 15 min. The fragments were incubated with DNase for 1 min and then with soybean trypsin inhibitor (0.1% w/v, Sigma, St Louis, MO) in HSH-BSA at 37°C for 15 min. After
532
Sumió Takahashi
rinsed in Ca2+-Mg2+ free-HSH-BSA, they were incubated in 2 mmol/1 EDTA-HSH solution and 1 mmol/ 1 EDTA-HSH solution for 5 and 15 min respectively, rinsed in Ca2 + -Mg2 + free-HSH-BSA twice, and triturated with a Pasteur pipette for 10 min. Dissociated pituitary cells were filtered with a nylon net and suspended in Dulbecco modified Eagle's medium containing 20 mmol/ 1 HEPES (DMEMH) supplemented with fetal calf serum (10% v/v, FCS). The cell viability was assessed by trypan blue exclusion at the end of dispersion. The cell viability was between 90 and 95% irrespective of the age groups. The dissociated cells were suspended at a density of 3 x 105 cell/ml in DMEMH with 10% FCS and were seeded in 35-mm plastic culture dishes and incubated at 37°C in a water-saturated atmosphere of 5% C02 and 9 5% air for two days, before the plaque assay, in order to allow the cell damage caused by the enzymatic cell dispersion to heal.
being
Reverse
hemolytic plaque assay
The cells were harvested from the culture dishes using a 0.05% trypsin solution and were dispersed into single cells (the cell viability on the day of assay was more than 95% in both age groups). These monodispersed cells were rinsed in DMEMH containing 0.1% BSA (DMEMHBSA) and then mixed with an equal volume of a 16% suspension of ovine red blood cells previously coupled to Protein A with chromium chloride hexahydrate (final concentration 2 x 105 cells/ml). The cell mixture was infused into a poly-L-lysine-coated Cunningham slide chamber and preincubated at 37°C, 95% air-5% C02, for 45 min. After preincubation, the chamber was rinsed in DMEMH-BSA and then filled with a mixture of DMEMHBSA and anti-rat growth hormone serum (final dilution of 1:20), GH-releasing hormone (GHRH, Peptide Inst., Osaka) and somatostatin (SRIF, Sigma, St Louis, MO) when needed. Incubation was carried out for 1.5 h. This incubation time was determined from the preliminary study in which the maximum percentage of plaqueforming GH cells had been achieved by 1.5 h. After incubation, the chamber was washed and filled with a mixture of DMEMH-BSA and guinea pig complement (ICN ImmunoBiologicals, Lisle, IL, final dilution 1:160) and secretagogues (GHRH and SRIF) when needed. The complement reaction was carried out for 30 min and terminated by infusion of 2% glutaraldehyde solution. The GH antiserum used was generated against rat GH. The specificity of the antiserum was checked using an enzyme-labelled immunoassay. The antiserum immunocytochemically stained pituitary cells (immunoreactive GH cells) at a dilution of 1:12,800. The diluted antiserum (1:6,400) was incubated with rat GH
(NIDDK, rGH-I-5)
or rat prolactin (NIDDK, rat concentrations of 0.1, 1, 2.5 and 5 yg at prolactin-I-5) rGH or rPRL/50 ul diluted antiserum, at 4°C for two days. Using these two antisera, immunocytochemical
acta endocrinologica 1992,127
staining was carried out. The antisera preabsorbed with
rGH (1 and 5 at all.
yg) did not show any immunoreactive cells
The specificity of the plaque assays for GH secretion checked as follows. Preincubation of a diluted antiserum (1:20) with highly purified rat GH (NIDDK, rGH-I-5, 10 yg rGH/100 ¿d of diluted antiserum) at 4°C for 16 h completely abolished plaque formation. Omis¬ sion of specific antiserum or guinea pig complement also abolished plaque formation. was
The observation
of the plaques
The pituitary cells were stained with Turuk solution to facilitate the observation of hemolytic plaques. Plaque diameters were measured with an ocular micrometer. Plaque areas were calculated from the diameter of the plaques, because those formed were usually circular.
Statistical
analyses
Statistical analysis was carried out by two-way analysis of variance with unequal but proportional subclass numbers (12). Percentages of plaque-forming cells and percentages of occurrence of small and large plaqueforming cells were arcsine-transformed and analyzed.
Results Fig. 1 shows the typical patterns of plaque sizes of GHRH (10"7 mol/1), SRIF (10-7 mol/1) and vehicle-treated pituitary cells of young female rats. GHRH increased the
plaque area and SRIF decreased it compared with that of vehicle-treated controls. This distribution pattern revealed a bimodal distribution of plaque area: small plaques and large plaques. Small plaques were defined as those whose areas were less than 8 x 103 yra2; large plaques were defined as those whose areas were greater than 8 x 103 /mi2. GHRH increased the relative propor¬ tion of large plaques and SRIF decreased it compared with that of controls. A dose-response to GHRH and SRIF was studied, but only the highest dose (10~7 mol/1) of GHRH and SRIF induced significant changes (data not shown). These results clearly indicate that hemolytic plaques were formed by GH cells. Fig. 2 shows typical patterns of plaque size of GH cells in young and old female rats with or without GHRH. The mean and median of plaque areas from old females were smaller than those of young female rats. The large plaque-forming cells were less frequently observed in old female rats than in young females. GHRH increased the frequences of large plaques in both young and old females. Table 1 gives the effects of GHRH on the plaque sizes of GH cells in young and old females. Percentages of plaque-forming cells significantly differed between young and old rats (p
