Cell Tissue Res. 199, 539-543 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Short Communication
Cytological Changes in the Pars distalis of the Female Rat Hypophysis Grafted Under the Kidney Capsule* L.I. Aguado, S. Rodriguez, A. Bilbao, and E.M. Rodriguez Instituto de Histologiay Patologia, UniversidadAustral de Chile, Valdivia, Chile
Summary. Intact female rats received transplants of two hypophysial partes distales under the kidney capsule. The plasma levels of prolactin were determined, and the ultrastructure of the grafted gland was studied 15, 45 and 90 days after the operation. Although prolactin levels in the three experimental groups were significantly higher than those in control rats, a decrease in prolactin level was detected in the 45-day samples. Parallel ultrastructural changes suggest that between the 45th and 90th postoperative day a process is initiated leading to hyperplasia and hypertrophy of prolactotrophs.
Key words: Pars distalis - Graft - Ultrastructure - Prolactin - Rat. Evidence indicating that the rat pars distalis transplanted distant from the hypothalamus is capable of secreting prolactin was first presented by Desclin (1950). Other authors confirmed and extended these observations using different methodological approaches (Everett, 1954, 1956; Sanders and Rennels, 1959; Chen et al., 1970; MiJhlbock and Boot, 1959). The hyperprolactinemia induced by the transplantation of the pars distalis under the kidney capsule, both in male and female rats, has been used to investigate its effects on target organs (Piacsek and Hostetter, 1971 ; Bartke et al., 1977; Negro-Vilar, 1977) or the direct effects of drugs on the prolactin cells (Donoso et al., 1974; Grfif et al., 1977). Despite the fact that pituitary transplantation has been widely used, little attention has been paid to the morphological events occurring in the grafted tissue itself. Santolaya and Rodriguez (1977) studied the ultrastructure of the hypophysis of the male rat chronically grafted under the kidney capsule of intact males. To our knowledge a parallel study of the hormonal levels and the ultrastructural morphology of the transplanted pars distalis at different postoperative intervals has not been undertaken. Dr. L.I.Aguado, Instituto de HistologJay Patologia,UniversidadAustral de Chile, Valdivia,Chile * Supported by Grants from PLAMIRH (93.173.2-77) and from the Direcci6n de Investigaciones, Universidad Austral (S-77-28)
Send offprint requests to:
0302-766X/79/0199/0539/$01.00
540
L.I. Aguado et al.
Materials and Methods Five-day cyclic female rats derived from a Holtzman stock, weighing about 150g, were used in the present study. The rats were divided into two groups. GroupL' 9 intact rats without any treatment. Group 11:24 intact rats with two partes distales grafted under the kidney capsule. All the animals were maintained under controlled photoperiods and temperature. Food and water were provided ad libitum. From each control rat only one blood sample was obtained, whereas from the experimental rats samples were collected 15, 45 and 90 days after the operation. All blood samples were obtained at diestrus by jugular puncture and after keeping the rats during 20min under light ether anesthesia (Chi and Shin, 1978). Plasma prolactin levels were determined by the double antibody radioimmunoassay (The kits where kindly provided by Dr. H.F. Parlow, NIAMDD-NIH, Bethesda, Maryland, USA). At each postoperative interval (15, 45 and 90 days) 4 rats were killed by decapitation, the grafted glands were dissected out and processed for electron microscopy (Rodriguez, 1969).
Results and Discussion Plasma Prolactin Levels. The experimental animals in all the postoperative intervals studied showed plasma prolactin levels that were significantly higher than those o f control rats at diestrus (Fig. 1 A). N o significant differences between the prolactin levels o f the blood samples collected 15, 45 and 90 days after the operation were found. A tendency for lower h o r m o n a l levels in the 45-day g r o u p was noted (Fig. 1 A). W h e n the prolactin levels at day 15 and 45 o f each experimental rat were compared, a substantial decline in the h o r m o n a l level at day 45 was noted in 71% o f the rats. Electron Microscopy. Fifteen days after the operation the grafted gland possessed all the cell types f o u n d in the gland in situ. With the exception o f p r o l a c t o t r o p h s , the remaining types o f secretory cells showed the same ultrastructural appearance as the corresponding cells o f the n o r m a l pars distalis. Prolactotrophs, although scarce, displayed a hypertrophied r o u g h endoplasmic reticulum and several Golgi complexes (Fig. 1 B). A few p o l y m o r p h o u s secretory granules were confined to the Golgi region. A l t h o u g h blood vessels were not numerous, their basement m e m b r a n e possessed long processes establishing wide surface contacts with the secretory cells. In the periphery o f the grafted tissue a large n u m b e r o f l y m p h o i d cells were found. Forty-five days after the operation the m o s t relevant and consistent feature in all animals studied was the penetration into the grafted gland o f n u m e r o u s l y m p h o i d cells o f various types (Fig. 1 C). In the 1 lxm thick sections studied light microscopically, the graft showed the appearance o f a lymphatic tissue. The n u m b e r o f secretory cells appeared to be reduced.
Fig. 1.A Plasma prolactin levelsin female rats at diestrus anesthetized with ether for 20 min. White bar, control rats (C); black bars, rats with two grafted partes distales. Vertical lines indicate + SD. P < 0.005 for all experimental groups compared with the control group. B Hypertrophied prolactotroph cell from a 15-day graft. Arrowheadsindicate processes of the perivascular basement membrane. The arrowpoints to an exocytotic figure, x 7500. C 45-day graft showing a large number of lymphoid cells intermingled with the secretory cells (arrows). x 2200. D 90-day graft. Several hypertrophied prolactotrophs (asterisks) are present, x 3800
Cytology of Grafted Hypophysis, Pars distalis
541
542
L.I. Aguado et al.
Ninety days after the operation, the graft was free of lymphoid cells and was highly vascularized. Although secretory cells other than prolactotrophs were present, the latter were the most abundant cell type (Fig. 1 D). When compared with the other two experimental groups, the number of prolactotrophs in the 90-day group increased significantly. The decrease in the level of plasma prolactin occurring in most of the operated rats of the 45-day group could be explained on the basis of the morphological findings. In fact, 45 days after transplantation the number of invading lymphoid cells largely exceeded that of the secretory cells. This immune response of the recipient animals might account for the drastic reduction in the population of secretory cells and for the decrease in the prolactin levels. Furthermore, the fact that this transitory hormonal fall also appears in ovariectomized-hypophysectomized female rats bearing pituitary grafts (Chen et al., 1970) suggests that hormonal factors are not involved in such a decrease. Intact male rats bearing grafts of the pars distalis also show a decline in the plasma levels of prolactin during the second postoperative month (Bartke et al., 1977). The substantial morphological differences between the gland grafted for 45 days and that transplanted for 90 days indicate that between these two postoperative intervals events take place leading to a hyperplasia and a hypertrophy of the prolactotrophs. This phenomenon might be related to a supression of the immune response, as suggested by the disappearance from the grafted tissue of the lymphoid cells. However, in the 90-day group plasma prolactin levels are not as high as expected from the morphological findings. We have no explantation for this apparent discrepancy. From the present results it becomes clear that the cytological changes in the grafted hypophysis follow certain time-dependent steps which should be taken into consideration when using pituitary transplantation as an experimental model.
References
Bartke, A., Smith, M.S., Michael, S.D., Peron, F.G., Dalterio, S.: Effectsof experimentally-induced chronic hyperprolactinemia on testosterone and gonadotropin levels in male rats and mice. Endocrinology 100, 182-186 (1977) Chen, C.L., Amenomori, Y., Lu, K.H., Voogt, J.L., Meites, J.: Serum prolactin levels in rats with pituitary transplants or hypothalamiclesions. Neuroendocrinology6, 220-227 (1970) Chi, H.L, Shin, S.H.: The effect of exposure to ether on prolactin secretion and the half-life of endogenous prolactin in normal and castrated male rats. Neuroendocrinology26, 193-201 (1978) Desclin, L.: Apropos du mrcanismed'action des oestrogrnessur le lobe antrrieur de l'hypophysechezle Rat. Ann. Endocrinol. (Paris) 11, 656-659 (1950) Donoso, A.O., Banz~n, A.M., Barcaglioni,J.C.: Further evidenceon the direct action of L-dopa on prolactin release. Neuroendocrinology15, 236-239 (1974) Everett,J.W.: Luteotrophic function of autografts of the rat hypophysis. Endocrinology54, 685-690 (1954) Everett, J.W.: Functional corpora lutea maintained for months by autografts of rat hypophyses. Endocrinology58, 786-796 (1956) Gr~if,K.I., Horowski,R., E1Eltreby,M.F.: Effectofprolactin inhibitoryagents on the ectopicanterior pituitary and the mammary gland in rats. Acta Endocrinol. (Kbh.) 85, 267-278 (1977)
Cytology of Grafted Hypophysis, Pars distalis
543
Miihlbock, O., Boot, L.M.: Induction of mammary cancer in mice without the mammary tumor agent by isografts of hypophyses. Cancer Res. 19, 402-412 (1959) Negro-Vilar, A., Saad, W.A., McCann, S.M.: Evidence for a role of prolactin in prostate and seminal vesicle growth in immature male rats. Endocrinology 1110, 729-737 (1977) Piacsek, B.E., Hostetter, M.W.: Functional capacity of transplanted pituitaries in superovulated rats. Biol. Reprod. 5, 282-290 (1971) Rodriguez, E.M.: Fixation of the central nervous system by ventricular perfusion with a threefold aldehyde mixture. Brain Res. 15, 395-412 (1969) Sanders, A.E., Rennels, E.G.: Evidence on the cellular source ofluteotrophin derived from a study of rat pituitary autografts. Z. Zellforsch. 49, 263-274 (1959) Santolaya, R.C., Rodriguez, E.M.: Ultrastructure of the male rat hypophysis chronically grafted under the kidney capsule. Cell Tissue Res. 179, 271-284 (1977)
Accepted April 7, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
Short Communication
Cytological Changes in the Pars distalis of the Female Rat Hypophysis Grafted Under the Kidney Capsule* L.I. Aguado, S. Rodriguez, A. Bilbao, and E.M. Rodriguez Instituto de Histologiay Patologia, UniversidadAustral de Chile, Valdivia, Chile
Summary. Intact female rats received transplants of two hypophysial partes distales under the kidney capsule. The plasma levels of prolactin were determined, and the ultrastructure of the grafted gland was studied 15, 45 and 90 days after the operation. Although prolactin levels in the three experimental groups were significantly higher than those in control rats, a decrease in prolactin level was detected in the 45-day samples. Parallel ultrastructural changes suggest that between the 45th and 90th postoperative day a process is initiated leading to hyperplasia and hypertrophy of prolactotrophs.
Key words: Pars distalis - Graft - Ultrastructure - Prolactin - Rat. Evidence indicating that the rat pars distalis transplanted distant from the hypothalamus is capable of secreting prolactin was first presented by Desclin (1950). Other authors confirmed and extended these observations using different methodological approaches (Everett, 1954, 1956; Sanders and Rennels, 1959; Chen et al., 1970; MiJhlbock and Boot, 1959). The hyperprolactinemia induced by the transplantation of the pars distalis under the kidney capsule, both in male and female rats, has been used to investigate its effects on target organs (Piacsek and Hostetter, 1971 ; Bartke et al., 1977; Negro-Vilar, 1977) or the direct effects of drugs on the prolactin cells (Donoso et al., 1974; Grfif et al., 1977). Despite the fact that pituitary transplantation has been widely used, little attention has been paid to the morphological events occurring in the grafted tissue itself. Santolaya and Rodriguez (1977) studied the ultrastructure of the hypophysis of the male rat chronically grafted under the kidney capsule of intact males. To our knowledge a parallel study of the hormonal levels and the ultrastructural morphology of the transplanted pars distalis at different postoperative intervals has not been undertaken. Dr. L.I.Aguado, Instituto de HistologJay Patologia,UniversidadAustral de Chile, Valdivia,Chile * Supported by Grants from PLAMIRH (93.173.2-77) and from the Direcci6n de Investigaciones, Universidad Austral (S-77-28)
Send offprint requests to:
0302-766X/79/0199/0539/$01.00
540
L.I. Aguado et al.
Materials and Methods Five-day cyclic female rats derived from a Holtzman stock, weighing about 150g, were used in the present study. The rats were divided into two groups. GroupL' 9 intact rats without any treatment. Group 11:24 intact rats with two partes distales grafted under the kidney capsule. All the animals were maintained under controlled photoperiods and temperature. Food and water were provided ad libitum. From each control rat only one blood sample was obtained, whereas from the experimental rats samples were collected 15, 45 and 90 days after the operation. All blood samples were obtained at diestrus by jugular puncture and after keeping the rats during 20min under light ether anesthesia (Chi and Shin, 1978). Plasma prolactin levels were determined by the double antibody radioimmunoassay (The kits where kindly provided by Dr. H.F. Parlow, NIAMDD-NIH, Bethesda, Maryland, USA). At each postoperative interval (15, 45 and 90 days) 4 rats were killed by decapitation, the grafted glands were dissected out and processed for electron microscopy (Rodriguez, 1969).
Results and Discussion Plasma Prolactin Levels. The experimental animals in all the postoperative intervals studied showed plasma prolactin levels that were significantly higher than those o f control rats at diestrus (Fig. 1 A). N o significant differences between the prolactin levels o f the blood samples collected 15, 45 and 90 days after the operation were found. A tendency for lower h o r m o n a l levels in the 45-day g r o u p was noted (Fig. 1 A). W h e n the prolactin levels at day 15 and 45 o f each experimental rat were compared, a substantial decline in the h o r m o n a l level at day 45 was noted in 71% o f the rats. Electron Microscopy. Fifteen days after the operation the grafted gland possessed all the cell types f o u n d in the gland in situ. With the exception o f p r o l a c t o t r o p h s , the remaining types o f secretory cells showed the same ultrastructural appearance as the corresponding cells o f the n o r m a l pars distalis. Prolactotrophs, although scarce, displayed a hypertrophied r o u g h endoplasmic reticulum and several Golgi complexes (Fig. 1 B). A few p o l y m o r p h o u s secretory granules were confined to the Golgi region. A l t h o u g h blood vessels were not numerous, their basement m e m b r a n e possessed long processes establishing wide surface contacts with the secretory cells. In the periphery o f the grafted tissue a large n u m b e r o f l y m p h o i d cells were found. Forty-five days after the operation the m o s t relevant and consistent feature in all animals studied was the penetration into the grafted gland o f n u m e r o u s l y m p h o i d cells o f various types (Fig. 1 C). In the 1 lxm thick sections studied light microscopically, the graft showed the appearance o f a lymphatic tissue. The n u m b e r o f secretory cells appeared to be reduced.
Fig. 1.A Plasma prolactin levelsin female rats at diestrus anesthetized with ether for 20 min. White bar, control rats (C); black bars, rats with two grafted partes distales. Vertical lines indicate + SD. P < 0.005 for all experimental groups compared with the control group. B Hypertrophied prolactotroph cell from a 15-day graft. Arrowheadsindicate processes of the perivascular basement membrane. The arrowpoints to an exocytotic figure, x 7500. C 45-day graft showing a large number of lymphoid cells intermingled with the secretory cells (arrows). x 2200. D 90-day graft. Several hypertrophied prolactotrophs (asterisks) are present, x 3800
Cytology of Grafted Hypophysis, Pars distalis
541
542
L.I. Aguado et al.
Ninety days after the operation, the graft was free of lymphoid cells and was highly vascularized. Although secretory cells other than prolactotrophs were present, the latter were the most abundant cell type (Fig. 1 D). When compared with the other two experimental groups, the number of prolactotrophs in the 90-day group increased significantly. The decrease in the level of plasma prolactin occurring in most of the operated rats of the 45-day group could be explained on the basis of the morphological findings. In fact, 45 days after transplantation the number of invading lymphoid cells largely exceeded that of the secretory cells. This immune response of the recipient animals might account for the drastic reduction in the population of secretory cells and for the decrease in the prolactin levels. Furthermore, the fact that this transitory hormonal fall also appears in ovariectomized-hypophysectomized female rats bearing pituitary grafts (Chen et al., 1970) suggests that hormonal factors are not involved in such a decrease. Intact male rats bearing grafts of the pars distalis also show a decline in the plasma levels of prolactin during the second postoperative month (Bartke et al., 1977). The substantial morphological differences between the gland grafted for 45 days and that transplanted for 90 days indicate that between these two postoperative intervals events take place leading to a hyperplasia and a hypertrophy of the prolactotrophs. This phenomenon might be related to a supression of the immune response, as suggested by the disappearance from the grafted tissue of the lymphoid cells. However, in the 90-day group plasma prolactin levels are not as high as expected from the morphological findings. We have no explantation for this apparent discrepancy. From the present results it becomes clear that the cytological changes in the grafted hypophysis follow certain time-dependent steps which should be taken into consideration when using pituitary transplantation as an experimental model.
References
Bartke, A., Smith, M.S., Michael, S.D., Peron, F.G., Dalterio, S.: Effectsof experimentally-induced chronic hyperprolactinemia on testosterone and gonadotropin levels in male rats and mice. Endocrinology 100, 182-186 (1977) Chen, C.L., Amenomori, Y., Lu, K.H., Voogt, J.L., Meites, J.: Serum prolactin levels in rats with pituitary transplants or hypothalamiclesions. Neuroendocrinology6, 220-227 (1970) Chi, H.L, Shin, S.H.: The effect of exposure to ether on prolactin secretion and the half-life of endogenous prolactin in normal and castrated male rats. Neuroendocrinology26, 193-201 (1978) Desclin, L.: Apropos du mrcanismed'action des oestrogrnessur le lobe antrrieur de l'hypophysechezle Rat. Ann. Endocrinol. (Paris) 11, 656-659 (1950) Donoso, A.O., Banz~n, A.M., Barcaglioni,J.C.: Further evidenceon the direct action of L-dopa on prolactin release. Neuroendocrinology15, 236-239 (1974) Everett,J.W.: Luteotrophic function of autografts of the rat hypophysis. Endocrinology54, 685-690 (1954) Everett, J.W.: Functional corpora lutea maintained for months by autografts of rat hypophyses. Endocrinology58, 786-796 (1956) Gr~if,K.I., Horowski,R., E1Eltreby,M.F.: Effectofprolactin inhibitoryagents on the ectopicanterior pituitary and the mammary gland in rats. Acta Endocrinol. (Kbh.) 85, 267-278 (1977)
Cytology of Grafted Hypophysis, Pars distalis
543
Miihlbock, O., Boot, L.M.: Induction of mammary cancer in mice without the mammary tumor agent by isografts of hypophyses. Cancer Res. 19, 402-412 (1959) Negro-Vilar, A., Saad, W.A., McCann, S.M.: Evidence for a role of prolactin in prostate and seminal vesicle growth in immature male rats. Endocrinology 1110, 729-737 (1977) Piacsek, B.E., Hostetter, M.W.: Functional capacity of transplanted pituitaries in superovulated rats. Biol. Reprod. 5, 282-290 (1971) Rodriguez, E.M.: Fixation of the central nervous system by ventricular perfusion with a threefold aldehyde mixture. Brain Res. 15, 395-412 (1969) Sanders, A.E., Rennels, E.G.: Evidence on the cellular source ofluteotrophin derived from a study of rat pituitary autografts. Z. Zellforsch. 49, 263-274 (1959) Santolaya, R.C., Rodriguez, E.M.: Ultrastructure of the male rat hypophysis chronically grafted under the kidney capsule. Cell Tissue Res. 179, 271-284 (1977)
Accepted April 7, 1979
