13
Neuroscience Letters, 123 (1991) 13-16 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100076B NSL 07520
Growth hormone-releasing factor (GRF) suppresses the in vitro proliferation of mammotrophs from the adult rat Tadashi Shinkai 1,2, Hiroshi O o k a 2 and Tetsuo N o u m u r a 1 1Department of Regulation Biology, Faculty of Science, University of Saitama, Urawa (Japan) and ZDepartment of Cell Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received 13 August 1990; Revised version received 25 October 1990; Accepted 29 October 1990)
Key words: GRF; Mammotroph; Somatotroph; Culture; Rat In order to examine the hypothalamic control of acidophilic proliferation, anterior pituitary cells from adult female rats were cultured with or without rat growth hormone-releasing factor fragment 1-29 (GRF-29). Changes in the numbers of mammotrophs and somatotrophs during culture were measured by immunocytochemical staining. The addition of GRF suppressed the increase in the number of mammotrophs even at the very low concentration of 10- ~2M. The number of somatotrophs increased in the medium containing GRF. The increase in mammotroph number was blocked by cytosine arabinoside, a mitotic inhibitor. GRF had no effect on the in vitro proliferation of fibroblasts. These results indicate the important role of hypothalamic GRF !n the differential growth and secretion of the acidophils in vivo.
Physiological regulating mechanisms observed in the hypothalamus include a number of neuropeptides which control pituitary functions, and form the central part of the dominance of the nervous system over the endocrine system. The secretion of various hormones from the anterior pituitary is stimulated or suppressed by releasing or inhibitory hormones of the hypothalamus [6, 14, 18]. However, few works have reported on the hypothalamic control of the population of secretory cells. The proliferation of thyrotrophs, gonadotrophs and adrenocorticotrophs is regulated by negative feedback loops in which hormones secreted from the target organs suppress the proliferation of those cells directly, or indirectly through the inhibition of the secretion of releasing hormones. Brain control is supposed to be more important for the population of acidophils, which receive no clear feedback regulation. The homology in the molecular structures of prolactin (PRL) and growth hormone (GH) suggests a close relationship between their secretory cells. Some cells secrete both of the hormones as 'somatomammotrophs' [10]. For this reason, it is important to examine the effect of GRF, which stimulates the proliferation of somatotrophs [2], on the population of mammotrophs. It has recently been reported that the percentage of mammotrophs in the pituitary cell cultures Correspondence: T. Shinkai, Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo- 173, Japan.
from 5-day-old rats increases when the culture medium contains G R F [5, 7]. However, the anterior pituitary cells show active proliferation and differentiation in the early postnatal stages, and it is expected that their responses to hypothalamic peptides differ from those in adult animals. This presumption is confirmed by the fact that G R F conspicuously reduced the percentage of somatotrophs in the study on 5-day-old rats [5, 7]. Therefore, experiments on cells from adult rats are necessary to analyze the brain regulation of the secretory cell population in the pituitary of mature animals. In the present study we cultured mammotrophs, which proliferate when deprived of hypothalamic factors [1], and observed the effect of G R F on the change in cell number. For each experiment, cells were prepared from the anterior pituitaries of 4 female Wistar rats (8 months of age, obtained from the colony of the Tokyo Metropolitan Institute of Gerontology) at random stages of the estrous cycle. These rats were maintained in environmentally controlled animal facilities and had free access to rat chow and water. After decapitation their anterior pituitaries were removed and dispersed with 1.2 U/ml dispase (grade I, Boehringer Mannheim), 0.15% collagenase (Cooper Biochemicals) and 0.00001% DNase (Worthington Biochemical) at 37°C. The cell suspension was then filtered through a glass filter (no. 80, Ikemoto Rika) and centrifuged at 400 g for 5 min at 4°C. The cell pellet was resuspended in Eagle's minimum essential medium (no. 2, Nissui) without Phenol red, which con-
14
tained 0.15% NaHCO3 and 0.03% glutamine (Nissui). The dispersed cells were loaded onto a discontinuous Percoll (Pharmacia) gradient with densities of 1.045 and 1.065 g/ml and centrifuged at 400 g for 20 min at 4°C [19]. Fibroblasts condensed on top of the gradient, and mammotrophs condensed at the interface of the layers. The cell suspension was removed from the interface and washed twice in medium supplemented with 10% fetal bovine serum (HyClone). Approximately 2 x 103 cells were inoculated into tissue culture chambers (8 chambers/slide, no. 4818, Lab-Tek) and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. After 3 days, GRF (rat growth hormone-releasing factor fragment 1-29, Sigma) was added and incubation was continued for a further 6 days. The cells were then fixed in Bouin's solution for 12 h at room temperature. An immunocytochemical technique (PAP) using rabbit antirat PRL serum (HAC-RT26-01RBP85, Institute of Endocrinology, Gunma University) and GH serum (HAC-RT25-01-RBP85) was employed for light microscopy [16]. Mammotrophs and somatotrophs were counted using an ocular mesh micrometer. Data were analyzed using the Student's t-test. Fig. 1 shows the changes in the number of both mammotrophs and somatotrophs in the culture. The increase in mammotroph number during a culture period of 6 days was completely suppressed by the addition of GRF. The number of somatotrophs did not change over the culture period without GRF, but increased in the medium containing GRF. The mammotroph number increased linearly throughout the culture period (Fig. 2).
150
Control mm•
-I-
GRF
I-.T-
500
i ¢D
(J 300 I
I
I
3
6
9
Days of primary culture Fig. 2. Changes in mammotroph number during a culture period of 6 days (days 3-9 of culture). Each value represents the mean cell number of 4 culture wells _ S.E.M.
The suppressive effect of GRF on the increase in mammotroph number was observed even at the very low concentration of 10 -12 M, although there was no effect at a concentration of 10-14 M (Fig. 3). The fact that the minimum effective dose of GRF to suppress mammotrophs was so low, as was that to stimulate somatotrophs, suggests that the response of mammotrophs to GRF was not pharmacological but physiological.
Mammotrophs 200 -
*
150
.
100
400
100
-r
._
50
5O
i
Mammotrophs
Somatotrophs
Fig. 1. Changes in the number of mammotrophs and somatotrophs during a culture period of 6 days with or without GRF (10-6 M). Relative cell number represents the percentage of the number at the start of the culture period. Each bar represents the mean + S.E.M. of 5 separate experiments. *P < 0.01 compared to control.
0
10 "14 10 "12 10 "1° 10 "e 10 4 Concentration of GRF (M)
Fig. 3. Changes in cell number during a culture period of 6 days with various concentrations of GRF. Relative cell number represents the percentage of the number at the start of culture period. Each bar represents the mean cell number of four culture wells + S.E.M.
15
i
m
150
I-7 Control ~ GRF "1-
100 =. so
CA(-)
CA(+)
Fig. 4. Changes in the number of mammotrophs during a culture period of 6 days with 10-l° M GRF and 4 x 10 -7 M cytosine arabinoside (CA). Relative cell number represents the percentage of the number at the start of the culture period. Each bar represents the mean cell number of 4 culture wells + S.E.M.
Fig. 4 shows the effect of a mitotic inhibitor, cytosine arabinoside (4 x 10 - 7 M), on the number of mammotrophs. No increase was observed in mammotroph number during a 6 day culture period in the presence of
I
|
~--6~A|
200
i
150
rj
cytosine arabinoside. The cell number in cultures containing G R F was not affected by the mitotic inhibitor. These results indicate that the increase in mammotroph number during a culture period is due to the proliferation of the cell, and that G R F inhibits the proliferation of mammotrophs. Fig. 5 shows the effect on the in vitro proliferation of rat pituitary fibroblasts obtained from the top of the Percoll gradient. G R F did not suppress the increase in the number of fibroblasts even at the high concentration of 10 - 6 M. This result indicates that G R F is not a general mitotic inhibitor, and that the inhibition of cell proliferation by G R F is a specific response of mammotrophs. The present study leads to the conclusion that G R F from the hypothalamus inhibits the proliferation of mammotrophs and suppresses the increase in their number in the anterior pituitary. Some recent studies have reported the stimulation of PRL secretion by shortterm G R F treatment [3, 4, 15]. However, chronic G R F supply is expected to lead to a decrease in PRL level as a consequence of the prevention of cell proliferation demonstrated by the present study. Dopamine secreted by the hypothalamus inhibits both the hormone secretion and the proliferation of mammotrophs [8, 11, 17]. It is noteworthy that GRF inhibits only the latter. In the present study, it was confirmed that the number of somatotrophs in vitro was increased by the presence of GRF. The majority of acidophils are somatotrophs in the pituitary of immature rats [7, 12]. The mammotrophs increase gradually during the course of maturation and aging, and they far outnumber the somatotrophs in aged rats [13]. Hypothalamic G R F may control the relative populations of acidophils through their inverse responses to GRF. The content of G R F in the median eminence of the rat decreases with aging [9]. The results of the present study emphasize the importance of hypothalamic regulation in the cell kinetics of the anterior pituitary.
.~ 100
|
We are grateful to Professor K. Wakabayashi and Dr. S. Tanaka of Gunma University for the supply of antibodies. This study was supported in part by a Grant-inAid 01740449 from the Ministry of Education, Science and Culture of Japan.
50
3days 6days Culture period Fig. 5. Changes in number of the fibroblasts during culture periods of 3 and 6 days with GRF. Relative cell number represents the percentage of the number at the start of culture period. Each bar represents the mean cell number of 4 culture wells __+S.E.M.
1 Baker, B.L., Reel, J.R., Van Dewark, S.D. and Yu, Y.Y., Persistence of cell types in monolayer cultures of dispersed cells from the pituitary pars distalis as revealed by immunohistochemistry, Anat. Rec., 179 (1974) 93-106. 2 Billestrup, N., Swanson, L.W. and 'Vale, W., Growth hormonereleasing factor stimulates proliferation of somatotrophs in vitro, Proc. Natl. Acad. Sci., 83 (1986) 6854-6857. 3 Casanueva, F.F., Brorras, C.G., Bruguera, B., Lima, L., Muruais, C., Tresguerres, J.A.F. and Devesa, J., Growth hormone and prolactin secretion after growth hormone-releasing hormone adminis-
16 tration, in anorexia nervosa patients, normal controls and tamoxifen-pretreated volunteers, Clin. Endocrinol., 27 (1987) 517-523. 4 Farmer, P.K., Tyler, J.M. and Stachura, M.E., Monensin influences basal and human growth hormone-releasing hormone-44 induced release of stored and new rat growth hormone and prolactin, Mol. Cell. Endocrinol., 62 (1989) 253-262. 5 Frawley, L.S. and Hoeffler, J.P., Hypothalamic peptides affect the ratios of GH and PRL cells: role of cell division, Peptides, 9 (1989) 825-828. 6 Guillemin, R., Peptides in the brain: the new endocrinology of the neuron, Science, 202 (1978) 390-402. 7 Hoeffler, J.P. and Frawley, L.S., Hypothalamic factors differentially affect the proportions of cells that secrete growth hormone or prolactin, Endocrinology, 120 (1987) 791-795. 8 Jahn, G.A., Machiavelli, G.A., Kalbermann, L.E., Szijan, I., Alonso, G.E. and Burdman, J.A., Relationships among release of prolactin, synthesis of DNA and growth of the anterior pituitary gland of the rat: effects of oestrogen and sulpiride, J. Endocrinol., 94 (1982) 1-10. 9 Morimoto, N., Kawakami, F., Makino, S., Chihara, K., Hasegawa, M. and Ibata, Y., Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus, Neuroendocrinology, 47 (1988) 459-464. 10 Papka, R.E. and Nikitovitch-Winer, M.B., Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labeling for pituitary hormones and neuropeptides, Am. J. Anat., 175 (1986) 289-306. 11 Perez, R.L., Machiavelli, G.A., Romano, M.I. and Burdman, J.A., Prolactin release, oestrogens and proliferation of prolactin-secret-
12
13
14 15
16 17
18 19
ing cells in the anterior pituitary gland of adult male rats, J. Endocrinol., 108 (1986) 399-403. Poole, M.C. and Kornegay, W.D., Cellular distribution within the rat adenohypophysis: a morphometric study, Anat. Rec., 204 (1982) 45-53. Putten, L.J.A. van, Zwieten, M.J. van, Mattheij, J.A.M. and Kemenade, J.A.M van, Studies on Prolactin-secreting cells in aging rats of different strains. I. Alterations in pituitary histology and serum prolactin levels as related to aging, Mech. Ageing Dev., 42 (1988) 75-90. Schally, A.V., Aspects of hypothalamic regulation of the pituitary gland, Science, 202 (1978) 18-28. Stachura, M.E., Tyler, J.M. and Farmer, P.K., Combined effects of human growth hormone (GH) releasing factor-44 (GRF) and somatostatin (SRIF) on post-SRIF rebound release of GH and prolactin: a model for GRF-SRIF modulation of secretion, Endocrinology, 123 (1988) 1476-1482. Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979, pp. 104-169. Takahashi, S. and Kawashima, S., Proliferation of prolactin cells in the rat: Effect of estrogen and bromocriptine, Zool. Sci., 4 (1987) 855-860. Vale, W., Rivier, C. and Brown, M., Regulatory peptides of the hypothalamus, Annu. Rev. Physiol., 39 (1977) 473-527. Velkeniers, B., Hooghe-Peter, E.L., Hooghe, R., Belayew, A., Smets, G., Claeys, A., Robberecht, P. and Vanhaelst, L., Prolactin cell subpopulations separated on discontinuous Percoll gradient: An immunocytochemical, biochemical, and physiological characterization, Endocrinology, 123 (1988) 1619-1630.
Neuroscience Letters, 123 (1991) 13-16 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100076B NSL 07520
Growth hormone-releasing factor (GRF) suppresses the in vitro proliferation of mammotrophs from the adult rat Tadashi Shinkai 1,2, Hiroshi O o k a 2 and Tetsuo N o u m u r a 1 1Department of Regulation Biology, Faculty of Science, University of Saitama, Urawa (Japan) and ZDepartment of Cell Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received 13 August 1990; Revised version received 25 October 1990; Accepted 29 October 1990)
Key words: GRF; Mammotroph; Somatotroph; Culture; Rat In order to examine the hypothalamic control of acidophilic proliferation, anterior pituitary cells from adult female rats were cultured with or without rat growth hormone-releasing factor fragment 1-29 (GRF-29). Changes in the numbers of mammotrophs and somatotrophs during culture were measured by immunocytochemical staining. The addition of GRF suppressed the increase in the number of mammotrophs even at the very low concentration of 10- ~2M. The number of somatotrophs increased in the medium containing GRF. The increase in mammotroph number was blocked by cytosine arabinoside, a mitotic inhibitor. GRF had no effect on the in vitro proliferation of fibroblasts. These results indicate the important role of hypothalamic GRF !n the differential growth and secretion of the acidophils in vivo.
Physiological regulating mechanisms observed in the hypothalamus include a number of neuropeptides which control pituitary functions, and form the central part of the dominance of the nervous system over the endocrine system. The secretion of various hormones from the anterior pituitary is stimulated or suppressed by releasing or inhibitory hormones of the hypothalamus [6, 14, 18]. However, few works have reported on the hypothalamic control of the population of secretory cells. The proliferation of thyrotrophs, gonadotrophs and adrenocorticotrophs is regulated by negative feedback loops in which hormones secreted from the target organs suppress the proliferation of those cells directly, or indirectly through the inhibition of the secretion of releasing hormones. Brain control is supposed to be more important for the population of acidophils, which receive no clear feedback regulation. The homology in the molecular structures of prolactin (PRL) and growth hormone (GH) suggests a close relationship between their secretory cells. Some cells secrete both of the hormones as 'somatomammotrophs' [10]. For this reason, it is important to examine the effect of GRF, which stimulates the proliferation of somatotrophs [2], on the population of mammotrophs. It has recently been reported that the percentage of mammotrophs in the pituitary cell cultures Correspondence: T. Shinkai, Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo- 173, Japan.
from 5-day-old rats increases when the culture medium contains G R F [5, 7]. However, the anterior pituitary cells show active proliferation and differentiation in the early postnatal stages, and it is expected that their responses to hypothalamic peptides differ from those in adult animals. This presumption is confirmed by the fact that G R F conspicuously reduced the percentage of somatotrophs in the study on 5-day-old rats [5, 7]. Therefore, experiments on cells from adult rats are necessary to analyze the brain regulation of the secretory cell population in the pituitary of mature animals. In the present study we cultured mammotrophs, which proliferate when deprived of hypothalamic factors [1], and observed the effect of G R F on the change in cell number. For each experiment, cells were prepared from the anterior pituitaries of 4 female Wistar rats (8 months of age, obtained from the colony of the Tokyo Metropolitan Institute of Gerontology) at random stages of the estrous cycle. These rats were maintained in environmentally controlled animal facilities and had free access to rat chow and water. After decapitation their anterior pituitaries were removed and dispersed with 1.2 U/ml dispase (grade I, Boehringer Mannheim), 0.15% collagenase (Cooper Biochemicals) and 0.00001% DNase (Worthington Biochemical) at 37°C. The cell suspension was then filtered through a glass filter (no. 80, Ikemoto Rika) and centrifuged at 400 g for 5 min at 4°C. The cell pellet was resuspended in Eagle's minimum essential medium (no. 2, Nissui) without Phenol red, which con-
14
tained 0.15% NaHCO3 and 0.03% glutamine (Nissui). The dispersed cells were loaded onto a discontinuous Percoll (Pharmacia) gradient with densities of 1.045 and 1.065 g/ml and centrifuged at 400 g for 20 min at 4°C [19]. Fibroblasts condensed on top of the gradient, and mammotrophs condensed at the interface of the layers. The cell suspension was removed from the interface and washed twice in medium supplemented with 10% fetal bovine serum (HyClone). Approximately 2 x 103 cells were inoculated into tissue culture chambers (8 chambers/slide, no. 4818, Lab-Tek) and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. After 3 days, GRF (rat growth hormone-releasing factor fragment 1-29, Sigma) was added and incubation was continued for a further 6 days. The cells were then fixed in Bouin's solution for 12 h at room temperature. An immunocytochemical technique (PAP) using rabbit antirat PRL serum (HAC-RT26-01RBP85, Institute of Endocrinology, Gunma University) and GH serum (HAC-RT25-01-RBP85) was employed for light microscopy [16]. Mammotrophs and somatotrophs were counted using an ocular mesh micrometer. Data were analyzed using the Student's t-test. Fig. 1 shows the changes in the number of both mammotrophs and somatotrophs in the culture. The increase in mammotroph number during a culture period of 6 days was completely suppressed by the addition of GRF. The number of somatotrophs did not change over the culture period without GRF, but increased in the medium containing GRF. The mammotroph number increased linearly throughout the culture period (Fig. 2).
150
Control mm•
-I-
GRF
I-.T-
500
i ¢D
(J 300 I
I
I
3
6
9
Days of primary culture Fig. 2. Changes in mammotroph number during a culture period of 6 days (days 3-9 of culture). Each value represents the mean cell number of 4 culture wells _ S.E.M.
The suppressive effect of GRF on the increase in mammotroph number was observed even at the very low concentration of 10 -12 M, although there was no effect at a concentration of 10-14 M (Fig. 3). The fact that the minimum effective dose of GRF to suppress mammotrophs was so low, as was that to stimulate somatotrophs, suggests that the response of mammotrophs to GRF was not pharmacological but physiological.
Mammotrophs 200 -
*
150
.
100
400
100
-r
._
50
5O
i
Mammotrophs
Somatotrophs
Fig. 1. Changes in the number of mammotrophs and somatotrophs during a culture period of 6 days with or without GRF (10-6 M). Relative cell number represents the percentage of the number at the start of the culture period. Each bar represents the mean + S.E.M. of 5 separate experiments. *P < 0.01 compared to control.
0
10 "14 10 "12 10 "1° 10 "e 10 4 Concentration of GRF (M)
Fig. 3. Changes in cell number during a culture period of 6 days with various concentrations of GRF. Relative cell number represents the percentage of the number at the start of culture period. Each bar represents the mean cell number of four culture wells + S.E.M.
15
i
m
150
I-7 Control ~ GRF "1-
100 =. so
CA(-)
CA(+)
Fig. 4. Changes in the number of mammotrophs during a culture period of 6 days with 10-l° M GRF and 4 x 10 -7 M cytosine arabinoside (CA). Relative cell number represents the percentage of the number at the start of the culture period. Each bar represents the mean cell number of 4 culture wells + S.E.M.
Fig. 4 shows the effect of a mitotic inhibitor, cytosine arabinoside (4 x 10 - 7 M), on the number of mammotrophs. No increase was observed in mammotroph number during a 6 day culture period in the presence of
I
|
~--6~A|
200
i
150
rj
cytosine arabinoside. The cell number in cultures containing G R F was not affected by the mitotic inhibitor. These results indicate that the increase in mammotroph number during a culture period is due to the proliferation of the cell, and that G R F inhibits the proliferation of mammotrophs. Fig. 5 shows the effect on the in vitro proliferation of rat pituitary fibroblasts obtained from the top of the Percoll gradient. G R F did not suppress the increase in the number of fibroblasts even at the high concentration of 10 - 6 M. This result indicates that G R F is not a general mitotic inhibitor, and that the inhibition of cell proliferation by G R F is a specific response of mammotrophs. The present study leads to the conclusion that G R F from the hypothalamus inhibits the proliferation of mammotrophs and suppresses the increase in their number in the anterior pituitary. Some recent studies have reported the stimulation of PRL secretion by shortterm G R F treatment [3, 4, 15]. However, chronic G R F supply is expected to lead to a decrease in PRL level as a consequence of the prevention of cell proliferation demonstrated by the present study. Dopamine secreted by the hypothalamus inhibits both the hormone secretion and the proliferation of mammotrophs [8, 11, 17]. It is noteworthy that GRF inhibits only the latter. In the present study, it was confirmed that the number of somatotrophs in vitro was increased by the presence of GRF. The majority of acidophils are somatotrophs in the pituitary of immature rats [7, 12]. The mammotrophs increase gradually during the course of maturation and aging, and they far outnumber the somatotrophs in aged rats [13]. Hypothalamic G R F may control the relative populations of acidophils through their inverse responses to GRF. The content of G R F in the median eminence of the rat decreases with aging [9]. The results of the present study emphasize the importance of hypothalamic regulation in the cell kinetics of the anterior pituitary.
.~ 100
|
We are grateful to Professor K. Wakabayashi and Dr. S. Tanaka of Gunma University for the supply of antibodies. This study was supported in part by a Grant-inAid 01740449 from the Ministry of Education, Science and Culture of Japan.
50
3days 6days Culture period Fig. 5. Changes in number of the fibroblasts during culture periods of 3 and 6 days with GRF. Relative cell number represents the percentage of the number at the start of culture period. Each bar represents the mean cell number of 4 culture wells __+S.E.M.
1 Baker, B.L., Reel, J.R., Van Dewark, S.D. and Yu, Y.Y., Persistence of cell types in monolayer cultures of dispersed cells from the pituitary pars distalis as revealed by immunohistochemistry, Anat. Rec., 179 (1974) 93-106. 2 Billestrup, N., Swanson, L.W. and 'Vale, W., Growth hormonereleasing factor stimulates proliferation of somatotrophs in vitro, Proc. Natl. Acad. Sci., 83 (1986) 6854-6857. 3 Casanueva, F.F., Brorras, C.G., Bruguera, B., Lima, L., Muruais, C., Tresguerres, J.A.F. and Devesa, J., Growth hormone and prolactin secretion after growth hormone-releasing hormone adminis-
16 tration, in anorexia nervosa patients, normal controls and tamoxifen-pretreated volunteers, Clin. Endocrinol., 27 (1987) 517-523. 4 Farmer, P.K., Tyler, J.M. and Stachura, M.E., Monensin influences basal and human growth hormone-releasing hormone-44 induced release of stored and new rat growth hormone and prolactin, Mol. Cell. Endocrinol., 62 (1989) 253-262. 5 Frawley, L.S. and Hoeffler, J.P., Hypothalamic peptides affect the ratios of GH and PRL cells: role of cell division, Peptides, 9 (1989) 825-828. 6 Guillemin, R., Peptides in the brain: the new endocrinology of the neuron, Science, 202 (1978) 390-402. 7 Hoeffler, J.P. and Frawley, L.S., Hypothalamic factors differentially affect the proportions of cells that secrete growth hormone or prolactin, Endocrinology, 120 (1987) 791-795. 8 Jahn, G.A., Machiavelli, G.A., Kalbermann, L.E., Szijan, I., Alonso, G.E. and Burdman, J.A., Relationships among release of prolactin, synthesis of DNA and growth of the anterior pituitary gland of the rat: effects of oestrogen and sulpiride, J. Endocrinol., 94 (1982) 1-10. 9 Morimoto, N., Kawakami, F., Makino, S., Chihara, K., Hasegawa, M. and Ibata, Y., Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus, Neuroendocrinology, 47 (1988) 459-464. 10 Papka, R.E. and Nikitovitch-Winer, M.B., Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labeling for pituitary hormones and neuropeptides, Am. J. Anat., 175 (1986) 289-306. 11 Perez, R.L., Machiavelli, G.A., Romano, M.I. and Burdman, J.A., Prolactin release, oestrogens and proliferation of prolactin-secret-
12
13
14 15
16 17
18 19
ing cells in the anterior pituitary gland of adult male rats, J. Endocrinol., 108 (1986) 399-403. Poole, M.C. and Kornegay, W.D., Cellular distribution within the rat adenohypophysis: a morphometric study, Anat. Rec., 204 (1982) 45-53. Putten, L.J.A. van, Zwieten, M.J. van, Mattheij, J.A.M. and Kemenade, J.A.M van, Studies on Prolactin-secreting cells in aging rats of different strains. I. Alterations in pituitary histology and serum prolactin levels as related to aging, Mech. Ageing Dev., 42 (1988) 75-90. Schally, A.V., Aspects of hypothalamic regulation of the pituitary gland, Science, 202 (1978) 18-28. Stachura, M.E., Tyler, J.M. and Farmer, P.K., Combined effects of human growth hormone (GH) releasing factor-44 (GRF) and somatostatin (SRIF) on post-SRIF rebound release of GH and prolactin: a model for GRF-SRIF modulation of secretion, Endocrinology, 123 (1988) 1476-1482. Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979, pp. 104-169. Takahashi, S. and Kawashima, S., Proliferation of prolactin cells in the rat: Effect of estrogen and bromocriptine, Zool. Sci., 4 (1987) 855-860. Vale, W., Rivier, C. and Brown, M., Regulatory peptides of the hypothalamus, Annu. Rev. Physiol., 39 (1977) 473-527. Velkeniers, B., Hooghe-Peter, E.L., Hooghe, R., Belayew, A., Smets, G., Claeys, A., Robberecht, P. and Vanhaelst, L., Prolactin cell subpopulations separated on discontinuous Percoll gradient: An immunocytochemical, biochemical, and physiological characterization, Endocrinology, 123 (1988) 1619-1630.
