Influence of lactotroph cell in rats H. A.
density on prolactin secretion
Pasolli, A. I. Torres and A. Aoki
Centro de Microscopía Electrónica, Facultad de Ciencias Médicas Universidad Nacional de Córdoba, Casilla Postal 362, 5000 Córdoba, Argentina (Requests for offprints should be addressed to A. Aoki) received
25 November 1991
ABSTRACT
The relationships between the stimulation of prolactin secretion and proliferation of lactotrophs was studied from a multidisciplinary standpoint in three experimental models. Administration of both oestrogen and sulpiride resulted in a significant increase in prolactin secretion and in the lactotroph population. A single injection of 10 \g=m\goestradiol benzoate (OB) induced a twofold increase in the proliferation of lactotrophs
(morphometrically as volume density), which increased further (2\m=.\5-fold) after three OB injections. Parallel changes were observed in the net counts made on lactotrophs sectioned through the nucleus to avoid possible distortions in volume density caused by hypertrophic cytoplasms. Comparable results were
obtained with the mitotic index in the same groups of rats exposed to treatment with colchicine. The effect also
sulpiride proliferation lactotrophs significant ( 1 7-fold) but less pronounced than in rats
of
of
on
was
\m=.\
treated with oestrogens. The treatments with oestrogen and sulpiride did not stimulate lactotrophic activity in a similar way, as judged by the levels of serum prolactin and the storage patterns of small and big
prolactin
in
pituitary glands.
Serum
prolactin
(mean \m=+-\s.e.m.) in control ovariectomized rats
4\m=.\0\m=+-\0\m=.\9
\g=m\g/l and
one
and three
injections
was
of OB
INTRODUCTION The different endocrine cells in the pituitary gland do not occur in a fixed proportion but undergo extensive changes in response to physiological and experimental stimuli (Takahashi & Kawashima, 1982; Haggi, Torres, Maldonado & Aoki, 1986; Phelps, 1986;
Chen, 1987).
In lactating rats, there is a remarkable proliferation of lactotrophs, the number of which comprises up to
to 14\m=.\4\m=+-\5\m=.\0and 28\m=.\8\m=+-\4\m=.\6 \g=m\g/l highest levels of serum prolactin were seen in sulpiride-treated rats (467\m=.\2\m=+-\28\m=.\7 \g=m\g/l). Striking differences occurred in the pituitary contents of big prolactin, the control values increasing from 5\m=.\3\m=+-\0\m=.\5to 10\m=.\2\m=+-\1\m=.\3\g=m\g/mgafter one OB injection and to 14\m=.\7 \m=+-\0\m=.\7\g=m\g/mg after three OB injections. In contrast, the concentration of big prolactin in sulpiride-treated rats was very low (1\m=.\85\m=+-\0\m=.\2\g=m\g/mg), 2\m=.\8-timessmaller than the controls. Other changes were also found in the small prolactin content in pituitary tissue with higher values in all the experimental models. These differences could only be detected after differential extraction of big and small molecular forms of prolactin.
raised these levels
respectively.
The
In ovariectomized rats, treatment with several doses of oestrogen enhanced the proliferation observed in the lactotroph population and increased the number of mitoses. In turn, morphological data could be closely related to the higher levels of prolactin in serum and pituitary glands. A sustained stimulation of lactotrophic secretion was always followed by proliferation of lactotrophs, the number of which fluctuated in parallel with the degree of stimulation. Journal of Endocrinology (1992) 134, 241\p=n-\246
50% of the total cell population of the pituitary gland (Goluboff & Ezrin, 1969). Interruption of lactation is followed by a massive degeneration of redundant prolactin cells (Haggi et al. 1986). Cell proliferation has also been recorded for other pituitary cell types when submitted to hyperstimulation, as in the case of gonadotrophs after castration (Smith & Keefer, 1982 ; Sakuma, Shirasawa & Yoshimura, 1984; Ibrahim, Moussa & Childs, 1986), corticotrophs after chronic administration of corticotrophin-releasing factor
(Gertz, Contreras, McComb et al. 1987), somatotrophs and lactotrophs after injection with hypothal¬ amic factors (Hoeffler & Frawley, 1987) and thyrotrophs in experimentally hypothyroid rats (Surks & De Fesi, 1977). Undoubtedly, there is a close correlation between the degree of stimulation and the proliferation of specific cells in the pituitary gland; however, the role played by the density of endocrine
and frozen until
cells in hormone secretion has yet to be assessed. In the present study, we have investigated possible interrelations between the numerical density of lacto¬ trophs and the secretion of the different molecular forms of prolactin from pituitary glands when sub¬ jected to different experimental conditions. New data gathered from this multidisciplinary approach suggest a novel interpretation of the role played by the size of the lactotroph population on prolactin secretion.
Hemipituitaries were fixed by immersion in 2% (v/v) glutaraldehyde and 4% (w/v) formaldehyde in phos¬ phate buffer (pH 7-3) at room temperature for 5-6 h. The fixed tissue was dehydrated in a series of increasing concentrations of ethanol and embedded in Lowicryl K4M (Chemische Weke Lowi, Waldkraiburg, Germany). Preparation of the immunogold complexes and details of immunocytochemical techniques are described by Maldonado & Aoki (1986). The pituitary gland was divided into three zones : anterior, mid and posterior and one large sec¬ tion (1 pm thick) from each zone was cut, mounted onto a glass slide and stained for prolactin with the immunogold-silver technique. The sections were incu¬
MATERIALS AND METHODS
phosphate-buffered
Adult female rats of the Wistar strain and aged 3 to 5 months were housed in air-conditioned quarters with a fixed schedule of light (14 h) and darkness ( 10 h) and provided with free access to tap water and rodent chow (Nutrie, Cordoba, Argentina). The rats were ovariectomized under ether anaesthesia and used 5 weeks later. They were divided into four groups of three rats each and submitted to the following experi¬ mental treatments. Group 1 animals (control) were injected s.c. with 0-1 ml sunflower oil. Group 2 rats were injected s.c. with 10 pg oestradiol benzoate (OB) dissolved in 0-1 ml sunflower oil at noon and killed 48 h later. Group 3 received a s.c. injection of OB daily for 3 days at noon and were killed 48 h after the last injection. Group 4 were injected with 5 mg sulpiride daily for 4 days at 09.00 h and killed 2 h after the last injection. In addition, rats treated similarly to groups 1,2 and 3 were used to study the mitotic index in pituitary glands removed 4 h after an s.c. injection of colchicine (1 mg/kg body weight) diluted in saline solution. The maintenance and treatment of animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Experimental protocols All the procedures have
been
published previously et al. 1986).
(Torres & Aoki, 1985, 1987; Haggi
The adenohypophyses were divided into two halves, hemihypophysis was fixed for immunocytochemistry and the other was blotted dry, weighed one
performed.
radioimmunoassay (RIA)
Immunocytochemistry
and
was
morphometry
bated with 1% normal goat serum, diluted in saline (PBS) pH 7-3, for 15 min. The excess serum was removed and the sections were incubated with specific anti-rat prolactin serum
(NIDDK-NIH Pituitary Hormone Program, Bethesda, MD, U.S.A.) diluted at 1:1000 in 0 1% bovine serum albumin (BSA) PBS, for 48 h at 4 °C. After being washed in PBS, the sections were treated with 1% normal goat serum for 15 min followed by incubation in a drop of goat anti-rabbit IgG-gold complex (stock solution diluted 1 :3) for 2 h at room temperature. At the end of this incubation, the slides
washed in PBS and distilled water, allowed to 1 h and then exposed to the developer of Danscher & Ryter Norgaard (1983), for 30 min in total darkness. To validate the specificity of the immunostaining the following controls were performed : ( 1 ) adsorption of the antibodies with highly purified prolactin; (2) application of a non-immune serum followed by the gold complex; (3) replacement of primary antiserum with PBS-1% BSA; (4) substitution of IgG-gold com¬ plex with PBS-1% polyethyleneglycol. The lactotroph population was assessed by point counting, according to Weibel's technique (Weibel, 1969), Sections were photographed in a Zeiss photomicroscope III and printed at a final magnification of 185. A grid with 600 intersection points was super¬ imposed on the micrographs and analysed under a stereoscopic microscope. Points falling on immunore¬ active cells were taken as positive. The volume density (Vv) of lactotrophs was calculated as the number of points on immunopositive cells divided by the total number of intersection points of the grid. In addition, in the same sections all the lactotrophs cut through the nucleus were counted to evaluate the changes
were
dry at 37 °C for
occurring
in the net number of
cells.
prolactin-producing
For morphometry of mitosis, the anterior pituitary fixed by immersion in 4% (v/v) glutaraldehyde, 4% (v/v) formaldehyde in cacodylate buffer (01 mol/1). Each hemipituitary was diced into four blocks and treated with 1% osmium tetroxide for 3 h, dehyd¬ rated in a series of cold graded acetones and embedded in Araldite. One large semithin section was cut from each block, mounted on clean glass slides, and stained with 1% toluidine blue/1% borax. The total number of cells and cells in mitosis per section were counted in a light microscope at 1000. Between 2500 to 3000 cells were counted per pituitary gland. After all the measure¬ ments were completed for each animal, the average value was calculated and treated as an independent observation to determine the group mean. For statist¬ ical analysis of the morphometric data the non-para¬ metric one-way Kruskal-Wallis test was adopted. In the event of significance, two group comparisons were made in order to detect where significant differences appeared. For these two-group comparisons the Mann-Whitney test was used. For electron microscopic controls, thin sections were obtained from the same blocks, stained with uranyl acetate and lead citrate and studied in Siemens' Elmiskop 101 and Zeiss 109 electron
determined
new
test.
< 0-05.
applying Duncan's Significance is reported at
multiple
range
was
microscopes.
Radioimmunoassay The frozen hemipituitaries were homogenized in saline solution (015 mol NaCl/1), using a PotterElvehjem tissue grinder and equal aliquots were incu¬ bated for 3 h at 4 °C in the following extraction media: (a) 0-1 mol Tris-HCl/1, pH 7-3 for determina¬ tion of monomeric (small) prolactin content (buffer extract) and (b) 01 mol Tris-HCl/1, pH 9-0 with the addition of urea (final dilution 2-5 mol/1) for total prolactin contents. The difference in prolactin con¬ tents between urea and buffer extracts represents the amount of big (polymeric) prolactin stored in pitui¬
tary tissue. These procedures have been described in detail (Haggi & Aoki, 1981 ; Torres, Haggi & Aoki, 1984; Torres, Prada & Aoki, 1990). Prolactin was quantified by RIA applying a doubleantibody technique at two dose levels and the results are expressed in terms of rat prolactin-RP-3 (biologi¬ cal potency equivalent to 20-0 IU/mg) with reagents donated by NIDDK-NIH, Bethesda, MD, U.S.A. All samples were processed simultaneously to avoid interassay variations. The intra-assay coefficient of variation was less than 10%. The data on serum and pituitary prolactin were analysed by one-way analysis of variance, and the differences between means were
RESULTS
Morphometry The results of morphometry of lactotroph Vv and the number of lactotrophs are summarized in Table 1. Oestrogen treatment of ovariectomized rats induced a significant (P-
g
X °
g
"
SB
(m .s G
,
c
O X 'S
V.
U
2O C* * -
O o
2-
O O
& S w
ce
o .a C S
do
S a
c
.
.5 -a
S t,
o
Sí 3 W
ce b »
-g
B
ce 100
density on prolactin secretion
Pasolli, A. I. Torres and A. Aoki
Centro de Microscopía Electrónica, Facultad de Ciencias Médicas Universidad Nacional de Córdoba, Casilla Postal 362, 5000 Córdoba, Argentina (Requests for offprints should be addressed to A. Aoki) received
25 November 1991
ABSTRACT
The relationships between the stimulation of prolactin secretion and proliferation of lactotrophs was studied from a multidisciplinary standpoint in three experimental models. Administration of both oestrogen and sulpiride resulted in a significant increase in prolactin secretion and in the lactotroph population. A single injection of 10 \g=m\goestradiol benzoate (OB) induced a twofold increase in the proliferation of lactotrophs
(morphometrically as volume density), which increased further (2\m=.\5-fold) after three OB injections. Parallel changes were observed in the net counts made on lactotrophs sectioned through the nucleus to avoid possible distortions in volume density caused by hypertrophic cytoplasms. Comparable results were
obtained with the mitotic index in the same groups of rats exposed to treatment with colchicine. The effect also
sulpiride proliferation lactotrophs significant ( 1 7-fold) but less pronounced than in rats
of
of
on
was
\m=.\
treated with oestrogens. The treatments with oestrogen and sulpiride did not stimulate lactotrophic activity in a similar way, as judged by the levels of serum prolactin and the storage patterns of small and big
prolactin
in
pituitary glands.
Serum
prolactin
(mean \m=+-\s.e.m.) in control ovariectomized rats
4\m=.\0\m=+-\0\m=.\9
\g=m\g/l and
one
and three
injections
was
of OB
INTRODUCTION The different endocrine cells in the pituitary gland do not occur in a fixed proportion but undergo extensive changes in response to physiological and experimental stimuli (Takahashi & Kawashima, 1982; Haggi, Torres, Maldonado & Aoki, 1986; Phelps, 1986;
Chen, 1987).
In lactating rats, there is a remarkable proliferation of lactotrophs, the number of which comprises up to
to 14\m=.\4\m=+-\5\m=.\0and 28\m=.\8\m=+-\4\m=.\6 \g=m\g/l highest levels of serum prolactin were seen in sulpiride-treated rats (467\m=.\2\m=+-\28\m=.\7 \g=m\g/l). Striking differences occurred in the pituitary contents of big prolactin, the control values increasing from 5\m=.\3\m=+-\0\m=.\5to 10\m=.\2\m=+-\1\m=.\3\g=m\g/mgafter one OB injection and to 14\m=.\7 \m=+-\0\m=.\7\g=m\g/mg after three OB injections. In contrast, the concentration of big prolactin in sulpiride-treated rats was very low (1\m=.\85\m=+-\0\m=.\2\g=m\g/mg), 2\m=.\8-timessmaller than the controls. Other changes were also found in the small prolactin content in pituitary tissue with higher values in all the experimental models. These differences could only be detected after differential extraction of big and small molecular forms of prolactin.
raised these levels
respectively.
The
In ovariectomized rats, treatment with several doses of oestrogen enhanced the proliferation observed in the lactotroph population and increased the number of mitoses. In turn, morphological data could be closely related to the higher levels of prolactin in serum and pituitary glands. A sustained stimulation of lactotrophic secretion was always followed by proliferation of lactotrophs, the number of which fluctuated in parallel with the degree of stimulation. Journal of Endocrinology (1992) 134, 241\p=n-\246
50% of the total cell population of the pituitary gland (Goluboff & Ezrin, 1969). Interruption of lactation is followed by a massive degeneration of redundant prolactin cells (Haggi et al. 1986). Cell proliferation has also been recorded for other pituitary cell types when submitted to hyperstimulation, as in the case of gonadotrophs after castration (Smith & Keefer, 1982 ; Sakuma, Shirasawa & Yoshimura, 1984; Ibrahim, Moussa & Childs, 1986), corticotrophs after chronic administration of corticotrophin-releasing factor
(Gertz, Contreras, McComb et al. 1987), somatotrophs and lactotrophs after injection with hypothal¬ amic factors (Hoeffler & Frawley, 1987) and thyrotrophs in experimentally hypothyroid rats (Surks & De Fesi, 1977). Undoubtedly, there is a close correlation between the degree of stimulation and the proliferation of specific cells in the pituitary gland; however, the role played by the density of endocrine
and frozen until
cells in hormone secretion has yet to be assessed. In the present study, we have investigated possible interrelations between the numerical density of lacto¬ trophs and the secretion of the different molecular forms of prolactin from pituitary glands when sub¬ jected to different experimental conditions. New data gathered from this multidisciplinary approach suggest a novel interpretation of the role played by the size of the lactotroph population on prolactin secretion.
Hemipituitaries were fixed by immersion in 2% (v/v) glutaraldehyde and 4% (w/v) formaldehyde in phos¬ phate buffer (pH 7-3) at room temperature for 5-6 h. The fixed tissue was dehydrated in a series of increasing concentrations of ethanol and embedded in Lowicryl K4M (Chemische Weke Lowi, Waldkraiburg, Germany). Preparation of the immunogold complexes and details of immunocytochemical techniques are described by Maldonado & Aoki (1986). The pituitary gland was divided into three zones : anterior, mid and posterior and one large sec¬ tion (1 pm thick) from each zone was cut, mounted onto a glass slide and stained for prolactin with the immunogold-silver technique. The sections were incu¬
MATERIALS AND METHODS
phosphate-buffered
Adult female rats of the Wistar strain and aged 3 to 5 months were housed in air-conditioned quarters with a fixed schedule of light (14 h) and darkness ( 10 h) and provided with free access to tap water and rodent chow (Nutrie, Cordoba, Argentina). The rats were ovariectomized under ether anaesthesia and used 5 weeks later. They were divided into four groups of three rats each and submitted to the following experi¬ mental treatments. Group 1 animals (control) were injected s.c. with 0-1 ml sunflower oil. Group 2 rats were injected s.c. with 10 pg oestradiol benzoate (OB) dissolved in 0-1 ml sunflower oil at noon and killed 48 h later. Group 3 received a s.c. injection of OB daily for 3 days at noon and were killed 48 h after the last injection. Group 4 were injected with 5 mg sulpiride daily for 4 days at 09.00 h and killed 2 h after the last injection. In addition, rats treated similarly to groups 1,2 and 3 were used to study the mitotic index in pituitary glands removed 4 h after an s.c. injection of colchicine (1 mg/kg body weight) diluted in saline solution. The maintenance and treatment of animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Experimental protocols All the procedures have
been
published previously et al. 1986).
(Torres & Aoki, 1985, 1987; Haggi
The adenohypophyses were divided into two halves, hemihypophysis was fixed for immunocytochemistry and the other was blotted dry, weighed one
performed.
radioimmunoassay (RIA)
Immunocytochemistry
and
was
morphometry
bated with 1% normal goat serum, diluted in saline (PBS) pH 7-3, for 15 min. The excess serum was removed and the sections were incubated with specific anti-rat prolactin serum
(NIDDK-NIH Pituitary Hormone Program, Bethesda, MD, U.S.A.) diluted at 1:1000 in 0 1% bovine serum albumin (BSA) PBS, for 48 h at 4 °C. After being washed in PBS, the sections were treated with 1% normal goat serum for 15 min followed by incubation in a drop of goat anti-rabbit IgG-gold complex (stock solution diluted 1 :3) for 2 h at room temperature. At the end of this incubation, the slides
washed in PBS and distilled water, allowed to 1 h and then exposed to the developer of Danscher & Ryter Norgaard (1983), for 30 min in total darkness. To validate the specificity of the immunostaining the following controls were performed : ( 1 ) adsorption of the antibodies with highly purified prolactin; (2) application of a non-immune serum followed by the gold complex; (3) replacement of primary antiserum with PBS-1% BSA; (4) substitution of IgG-gold com¬ plex with PBS-1% polyethyleneglycol. The lactotroph population was assessed by point counting, according to Weibel's technique (Weibel, 1969), Sections were photographed in a Zeiss photomicroscope III and printed at a final magnification of 185. A grid with 600 intersection points was super¬ imposed on the micrographs and analysed under a stereoscopic microscope. Points falling on immunore¬ active cells were taken as positive. The volume density (Vv) of lactotrophs was calculated as the number of points on immunopositive cells divided by the total number of intersection points of the grid. In addition, in the same sections all the lactotrophs cut through the nucleus were counted to evaluate the changes
were
dry at 37 °C for
occurring
in the net number of
cells.
prolactin-producing
For morphometry of mitosis, the anterior pituitary fixed by immersion in 4% (v/v) glutaraldehyde, 4% (v/v) formaldehyde in cacodylate buffer (01 mol/1). Each hemipituitary was diced into four blocks and treated with 1% osmium tetroxide for 3 h, dehyd¬ rated in a series of cold graded acetones and embedded in Araldite. One large semithin section was cut from each block, mounted on clean glass slides, and stained with 1% toluidine blue/1% borax. The total number of cells and cells in mitosis per section were counted in a light microscope at 1000. Between 2500 to 3000 cells were counted per pituitary gland. After all the measure¬ ments were completed for each animal, the average value was calculated and treated as an independent observation to determine the group mean. For statist¬ ical analysis of the morphometric data the non-para¬ metric one-way Kruskal-Wallis test was adopted. In the event of significance, two group comparisons were made in order to detect where significant differences appeared. For these two-group comparisons the Mann-Whitney test was used. For electron microscopic controls, thin sections were obtained from the same blocks, stained with uranyl acetate and lead citrate and studied in Siemens' Elmiskop 101 and Zeiss 109 electron
determined
new
test.
< 0-05.
applying Duncan's Significance is reported at
multiple
range
was
microscopes.
Radioimmunoassay The frozen hemipituitaries were homogenized in saline solution (015 mol NaCl/1), using a PotterElvehjem tissue grinder and equal aliquots were incu¬ bated for 3 h at 4 °C in the following extraction media: (a) 0-1 mol Tris-HCl/1, pH 7-3 for determina¬ tion of monomeric (small) prolactin content (buffer extract) and (b) 01 mol Tris-HCl/1, pH 9-0 with the addition of urea (final dilution 2-5 mol/1) for total prolactin contents. The difference in prolactin con¬ tents between urea and buffer extracts represents the amount of big (polymeric) prolactin stored in pitui¬
tary tissue. These procedures have been described in detail (Haggi & Aoki, 1981 ; Torres, Haggi & Aoki, 1984; Torres, Prada & Aoki, 1990). Prolactin was quantified by RIA applying a doubleantibody technique at two dose levels and the results are expressed in terms of rat prolactin-RP-3 (biologi¬ cal potency equivalent to 20-0 IU/mg) with reagents donated by NIDDK-NIH, Bethesda, MD, U.S.A. All samples were processed simultaneously to avoid interassay variations. The intra-assay coefficient of variation was less than 10%. The data on serum and pituitary prolactin were analysed by one-way analysis of variance, and the differences between means were
RESULTS
Morphometry The results of morphometry of lactotroph Vv and the number of lactotrophs are summarized in Table 1. Oestrogen treatment of ovariectomized rats induced a significant (P-
g
X °
g
"
SB
(m .s G
,
c
O X 'S
V.
U
2O C* * -
O o
2-
O O
& S w
ce
o .a C S
do
S a
c
.
.5 -a
S t,
o
Sí 3 W
ce b »
-g
B
ce 100
