J. Anat. (1979), 129, 4, pp. 759-767 With 8 figures Printed in Great Britain
759
Fine structure of gonadotrophs and prolactin producing cells in the rat pars distalls in organ culture T. G. BAKER AND B. A. YOUNG*
Hormone Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh, 23 Chalmer's Street, Edinburgh EH3 9EW and * Department of Anatomy, Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL
(Accepted 11 December 1978) INTRODUCTION
The pioneer work on the maintenance of the pituitary gland in organ culture was done by Petrovic in 1959 (personal communication to B.A.Y.). Later work has been reviewed by Petrovic (1961, 1963), Pasteels (1961), Gaillard & Schaberg (1965) and Tixier-Vidal (1975). In recent years the technique has been successful in maintaining mammalian pituitary tissue for varying periods of time in such a state that it continues to show normal morphological appearances (Young & Baker, 1975; Tixier-Vidal, 1975), and continues to release gonadotrophic hormones into the culture medium, especially when gonadotrophin-releasing hormone (GnRH) is added to the culture (Martin & Klein, 1976; Baker, Sharpe & Fraser, in preparation). It would seem therefore that this technique might be of value in determining the mechanisms (especially the influence of GnRH steroid hormones) controlling the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The present results form part of a large series of experiments in which the morphology of the pituitary gland, and the amounts (determined by radioimmunoassay) of FSH and LH in the culture medium were studied in over 500 rat pituitary glands cultured for 8 days. The present report describes and compares the ultrastructure of organ-cultured and control pituitary glands derived from sexually mature male and female rats, to test the validity of the in vitro techniques employed. MATERIALS AND METHODS
A total of 26 Hooded Wistar rats aged 6-8 weeks were used in the study. Male and female rats were killed with chloroform vapour and the entire pituitary gland was cleanly and aseptically dissected into Eagle's minimal essential culture medium at 34 'C. The glands were then cut into explants measuring 1-2 mm3. Explants from one group of animals were immediately fixed for electron microscopy, while the remaining glands were cultured, with or without additions of GnRH, for 8 days. The techniques used for maintaining the explanted pituitary glands in organ culture have been described before (Baker & Neal, 1969; Baker & Young, 1973). All the explants from one pituitary were placed on lens tissue supported on a stainless steel grid in a sterile plastic Petri dish. Each dish received 5 ml of nutrient fluid consisting of Eagle's minimum essential medium (with Earle's salts and HEPES buffer) supplemented with donor calf serum (20 % v/v), glutamine (0-3 g/L), and 0021-8782/79/2828-6860 $02.00 © 1979 Anat. Soc. G.B. & I.
760
T. G. BAKER AND B. A. YOUNG
antibiotics (Gentamycin and Fungizone; all from Flow Laboratories, Irvine, U.K.). The level of the medium in each Petri dish was such that the explants were above the surface and received nutrients only by diffusion through the lens tissue. Some of the cultures were treated with GnRH (Hoechst Ltd, Hounslow, U.K.) at concentrations of 0 1 and 0 01 jug/ml of medium for male and female rat pituitaries respectively, doses which from other experiments appeared to be optimal in terms of the release of FSH and LH in culture. Racks containing four Petri dishes were placed in modified 'Kilner' preserving jars (United Glass Ltd, see Baker & Neal, 1969) which were gassed at 0 703 kg/cm2 above atmospheric pressure with a mixture containing 5 % C02, 57 % 02, and 38 % N2. The high gas pressure was maintained throughout the period of culture (apart from the short time needed for the daily changes of medium) since this was found virtually to eliminate the necrotic changes which otherwise characterize explanted tissues (see Baker & Neal, 1969; Baker & Young, 1973; Young & Baker, 1975). The culture fluid was completely replaced with fresh nutrient every 24 hours and, at the end of the period of culture (8 days), the explants were fixed for electron microscopy. Both the explants and the non-cultured control glands were fixed with Millonig's buffered formalin, post-fixed with 1 % osmium tetroxide, and embedded in Araldite. Ultrathin sections, cut on a Reichert EMU 2 microtome, were stained with uranyl acetate and lead hydroxide, and were examined with an AEI 801 electron microscope. RESULTS
The fine structure of the secretory cells in the organ-cultured pituitary glands was strikingly similar to that of corresponding cells in the non-cultured control glands. Some slight damage to plasma and other membrane systems was found in most of the control (Figs. 1, 2) and cultured material (Figs. 3-8). This damage was believed to have arisen during shipment from Edinburgh to Belfast of formalinfixed material in cacodylate buffer. However, the membrane damage was uniform in all the specimens examined, and hence it was decided that valid comparison of cultured and control explants was possible. The features of the various secretory cells were similar to those described by Farquhar (1971), although stellate (follicular, or supporting) cells were not observed in the cultured material (cf. rabbit adenohypophysis; Young & Baker, 1975). The following description is restricted to prolactin-secreting cells and gonadotrophs because of our special interest in these cells in relation to current work on hormone production (Baker, Sharpe & Fraser, in preparation). Cells which, on morphological grounds, were believed to be producing prolactin (see Tixier-Vidal, 1975; Costoff, 1973) were numerous in both the controls and the cultured glands (Figs. 1, 3). Such cells contained abundant endoplasmic reticulum, a Golgi zone of flattened saccules and small vesicles, the latter often containing electron-dense material. They also contained elongated mitochondria and electrondense secretory granules, which were irregular in shape, and measuring 400-500 nm in diameter as a rule, although larger dense bodies were encountered occasionally. Cells which, on morphological grounds, were believed to be gonadotrophs were often encountered. There was no evidence for distinct populations of FSH and LH gonadotrophs. The addition of GnRH to the cultured pituitaries produced some morphological changes in these cells (see below). The presumed gonadotrophs of the female controls were slightly elongated and
761
Organ culture of rat pituitary *.
.1:\
t
Fig. 1. Electron micrograph of a prolactin cell from a control male rat.
-K Fig.2. Electron micrograph of
: a
x 12600.
2
prolactin cell (P) and a gonadotroph (G) female rat. x 12600.
from a control
762
T. G. BAKER AND B. A. YOUNG
Fig. 3. Electron micrograph of a prolactin cell from an organ-cultured control male rat. x 12600.
Fig. 4. Electron micrograph of a gonadotroph from an organ-cultured control female rat. x 12 600.
Organ culture of rat pituitary
¾
.r
.
Fig. 5. Electron micrograph of a gonadotroph from an organ-cultured control female rat. x 12600.
Fig. 6. Electron micrograph of a gonadotroph from a GnRH-treated organ-cultured male rat. Golgi zone (G); membrane flow (t). x 12600.
763
764
T. G. BAKER AND B. A. YOUNG
Fig. 7. Electron micrograph from a GnRH-treated orgsn-cultured female rat. Gonadotroph packed with granules (G). x 12600.
~~
Fig. 8. Electron micrograph from
a
~
~
~
~
~
1
GnRH-treated organ-cultured female rat. Degranulated cell (c). x 12600.
Organ culture ofjrat pituitary 765 contained secretory granules of 200-250 nm diameter (Fig. 4). Their endoplasmic reticulum was not conspicuous, but was sometimes seen concentrated towards the plasma membrane. Occasionally the plane of section passed through a large Golgi zone (Fig. 5). A characteristic feature of these cells was that, when the plane of section did not include a profile of the nucleus, numerous mitochondria were seen in the middle of the cell. Gonadotrophs in the cultures treated with GnRH sometimes showed signs of stimulation. Thus in some cells the Golgi zone formed a complete ring of saccules containing secretory granules in process of formation. In others the endoplasmic reticulum scattered throughout the cytoplasm had dilated cisternae and sometimes appeared to be extending by 'membrane flow' from the outer nuclear membrane (Fig. 6). The cells were sometimes packed with granules (Figs. 6, 7), but occasionally a cell looked as if it might have lost its granules (Fig. 8). DISCUSSION
The fact that the morphologies of the gonadotrophs and prolactin-secreting cells respectively were strikingly similar in the treated and non-treated cultures and in the control group of pituitaries is a good indication that the techniques employed in the present study are adequate for studies of pituitary function for up to 8 days in organ culture. Indeed, we gained the impression, which needs to be confirmed using biochemical and immunological techniques, that the gonadotrophs were more 'active' (in secretory terms) in the organ-cultured explants (especially when treated with GnRH) than in the controls. The gonadotrophs were disappointing in that we had hoped to detect changes in the number of these cells, and/or in the number of secretory granules per cell, in explants treated with GnRH (see Tixier-Vidal, 1975). It is well known, however, that only fairly gross changes are likely to be detected with the electron microscope because of sampling error and thinness of section, Nevertheless, some evidence for active secretion following treatment with GnRH was obtained, and in any case the rate of secretion of gonadotrophs with the doses of GnRH used in the present study might well have been balanced by the rate of release of FSH and LH into the culture medium. Although the morphological activity of the gonadotrophs seemed to be greater in the GnRH-treated cultures than in either the non-treated cultures or the controls, it was not possible to distinguish between cells producing FSH and LH. The results of the present study are in good agreement with those of Tixier-Vidal (1975) and Farquhar, Skutelsky & Hopkins (1975) who studied organ cultures and dissociated pituitary cells respectively. The present technique for organ culture has the great advantage that cellular integrity and inter-relationships are maintained in the explants. With dissociated pituitary cells these vital functions are disrupted, and membrane-associated surface receptors are likely to be damaged by the enzyme dispersion techniques. The present method permits good maintenance of the explants for 8 days in culture, which is a significant improvement over that of Martin & Klein (1976) which is only suitable for 4 day cultures of pituitaries from neonatal rats: glands from rats aged 40 days consistently showed necrosis at the centre of the explant. The improvements obtained with the present technique probably stem from the use of explants rather than whole glands, from the addition to the nutrient medium of donor calf serum rather than bovine serum albumin, and from the use of a gas phase using less oxygen (56 %; cf. 95 % used by Martin & Klein, 1976).
T. G. BAKER AND B. A. YOUNG 766 High oxygen concentrations are known to be cytotoxic: increasing the pressure at which the gas is maintained in the culture vessel (to 0703 kg cm2 above atmospheric pressure) permits a drastic reduction in oxygen concentration (see Baker & Neal, 1969). The large number of prolactin-secreting cells observed during the present study confirm the observations of Tixier-Vidal (1975). It is noteworthy that our preliminary results using radioimmunoassay indicate that the level of prolactin in the daily changes of culture medium is very high (Baker, Sharpe & Fraser, unpublished results). The fact that stellate (so-called follicular) cells were not seen in the cultured pituitary glands accords with the view that such cells participate in a transport system for nutrients when the portal system is intact (Young, 1977): in the absence of the portal system in culture the cells seemingly do not survive as such (but cf. rabbit adenohypophysis, Young & Baker, 1975), although it is conceivable, but highly improbable, that they rapidly become transformed into granular cells in culture. A major problem with this experiment was the fact that organ culturing was carried out in Edinburgh before the material was transported in buffer to Belfast for post-fixation and subsequent processing. This was almost certainly the underlying cause of the membrane breakage in some specimens in both the cultured and non-cultured control group. Nevertheless, as is clear from the photomicrographs, the general standards of organ culture and fixation were very high, and thus the technical basis of our further experiments on the amounts of FSH and LH produced after treatment of explants with GnRH and various steroid hormones (oestradiol and testosterone) is validated (Baker, Sharpe & Fraser, in preparation). SUMMARY
A technique is described which permits the maintenance for up to 8 days in organ culture of explanted rat pituitary glands. Electron microscope studies showed that the cultured glands (whether treated with gonadotrophin-releasing hormone (GnRH) or not) were almost identical to controls in appearance, although the stellate (follicular) cells seemingly were eliminated in vitro. Gonadotrophs and prolactin-secreting cells in male and female pituitary glands were similar in appearance both in vivo and in vitro to comparable cells described by other authors. There was some evidence for an increase in the secretory activity of these cell types (particularly gonadotrophs) when the explants were treated with GnRH. The present technique thus provides a suitable and valid 'model' with which to study the effects of GnRH and steroid hormones on the release and synthesis of FSH and LH in vitro. The authors would like to thank Professor J. J. Pritchard for assistance given in preparing this paper. The expenses incurred in these studies were defrayed from a grant to T. G. B. by the Medical Research Council. We are grateful to Mrs June Hardiman, Mrs Ann Rae and Mr G. R. Dickson for expert technical assistance, and to Hoechst (U.K.) Ltd for the generous supply of the GnRH used in these experiments. Mrs Joan Hamilton kindly did the secretarial work.
Organ culture of rat pituitary
767
REFERENCES
BAKER, T. G. & NEAL, P. (1969). The effects of x-irradiation on mammalian oocytes in organ culture. Biophysik 6, 39-45. BAKER, T. G., SHARPE, R. & FRASER, H. M. (1979). Secretion of FSH and LH by male and female rat pituitary glands in organ culture. (In preparation.) BAKER, T. G. & YOUNG, B. A. (1973). Organ culture of the rat thyroid gland. Experimenta 29, 1548-1550. COSTOFF, A. (1973). Ultrastructure of Rat Adenohypophysis, Correlations with Functions. New York: Academic Press. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Memoirs of the Society for Endocrinology 19, 79-122. FARQUHAR, M. G., SKUTELSKY, E. H. & HOPKINS, C. R. (1975). Structure and function of the anterior pituitary and dispersed cells. In The Anterior Pituitary (ed. A. Tixier-Vidal and M. G. Farquhar), pp. 83-135. New York: Academic Press. GAILLARD, R. P. & SCHABERG, A. (1965). Endocrine glands. In Cells and Tissues in Culture (ed. E. N. Willmer), vol. 2, pp. 631-643. New York: Academic Press. MARTIN, J. E. & KLEIN, D. G. (1976). Long-term organ culture of rat anterior pituitary gland. Endocrinology 99, 1189-1198. PASTEELS, J. L. (1961). Premiers resultats de culture combinee in vitro d'hypophyse et d'hypothalamus dans le but d'en apprecier la s&retion de prolactine. Compte rendu hebdomadaire des seances de l'Academie des sciences 253, 3074-3075. PETROVIC, A. (1961). Recherches sur la prehypophyse en culture organotypique: Evolution structurale et action sur les organes effecteurs associes in vitro. Thesis, Imprimerie Alsatia, Colmar, Strasbourg. PETROVIC, A. (1963). Cytophysiologie de l'adenohypophyse des mammiferes en culture organotypique: Fonction gonadotrope, thyr6otrope et corticotrope. In Cytologie de l'Adenohypophyse (ed. J. Benoit and C. Da Lage), pp. 121-136. Paris: CNRS. TIXIER-VIDAL, A. (1975). Ultrastructure of anterior pituitary cells in culture. In The Anterior Pituitary (ed. A. Tixier-Vidal and M. G. Farquhar), pp. 181-230. New York: Academic Press. YOUNG, B. A. & BAKER, T. G. (1975). Ultrastructural observations on organ cultured rabbit adenohypophysis. LC.R.S. Journal of Medical Science 3, 597-598. YOUNG, B. A. (1977). Some observations on the ultrastructure of the stellate cells of the pars distalis of the guinea-pig. Journal of Anatomy 124, 153-156.
759
Fine structure of gonadotrophs and prolactin producing cells in the rat pars distalls in organ culture T. G. BAKER AND B. A. YOUNG*
Hormone Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh, 23 Chalmer's Street, Edinburgh EH3 9EW and * Department of Anatomy, Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL
(Accepted 11 December 1978) INTRODUCTION
The pioneer work on the maintenance of the pituitary gland in organ culture was done by Petrovic in 1959 (personal communication to B.A.Y.). Later work has been reviewed by Petrovic (1961, 1963), Pasteels (1961), Gaillard & Schaberg (1965) and Tixier-Vidal (1975). In recent years the technique has been successful in maintaining mammalian pituitary tissue for varying periods of time in such a state that it continues to show normal morphological appearances (Young & Baker, 1975; Tixier-Vidal, 1975), and continues to release gonadotrophic hormones into the culture medium, especially when gonadotrophin-releasing hormone (GnRH) is added to the culture (Martin & Klein, 1976; Baker, Sharpe & Fraser, in preparation). It would seem therefore that this technique might be of value in determining the mechanisms (especially the influence of GnRH steroid hormones) controlling the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The present results form part of a large series of experiments in which the morphology of the pituitary gland, and the amounts (determined by radioimmunoassay) of FSH and LH in the culture medium were studied in over 500 rat pituitary glands cultured for 8 days. The present report describes and compares the ultrastructure of organ-cultured and control pituitary glands derived from sexually mature male and female rats, to test the validity of the in vitro techniques employed. MATERIALS AND METHODS
A total of 26 Hooded Wistar rats aged 6-8 weeks were used in the study. Male and female rats were killed with chloroform vapour and the entire pituitary gland was cleanly and aseptically dissected into Eagle's minimal essential culture medium at 34 'C. The glands were then cut into explants measuring 1-2 mm3. Explants from one group of animals were immediately fixed for electron microscopy, while the remaining glands were cultured, with or without additions of GnRH, for 8 days. The techniques used for maintaining the explanted pituitary glands in organ culture have been described before (Baker & Neal, 1969; Baker & Young, 1973). All the explants from one pituitary were placed on lens tissue supported on a stainless steel grid in a sterile plastic Petri dish. Each dish received 5 ml of nutrient fluid consisting of Eagle's minimum essential medium (with Earle's salts and HEPES buffer) supplemented with donor calf serum (20 % v/v), glutamine (0-3 g/L), and 0021-8782/79/2828-6860 $02.00 © 1979 Anat. Soc. G.B. & I.
760
T. G. BAKER AND B. A. YOUNG
antibiotics (Gentamycin and Fungizone; all from Flow Laboratories, Irvine, U.K.). The level of the medium in each Petri dish was such that the explants were above the surface and received nutrients only by diffusion through the lens tissue. Some of the cultures were treated with GnRH (Hoechst Ltd, Hounslow, U.K.) at concentrations of 0 1 and 0 01 jug/ml of medium for male and female rat pituitaries respectively, doses which from other experiments appeared to be optimal in terms of the release of FSH and LH in culture. Racks containing four Petri dishes were placed in modified 'Kilner' preserving jars (United Glass Ltd, see Baker & Neal, 1969) which were gassed at 0 703 kg/cm2 above atmospheric pressure with a mixture containing 5 % C02, 57 % 02, and 38 % N2. The high gas pressure was maintained throughout the period of culture (apart from the short time needed for the daily changes of medium) since this was found virtually to eliminate the necrotic changes which otherwise characterize explanted tissues (see Baker & Neal, 1969; Baker & Young, 1973; Young & Baker, 1975). The culture fluid was completely replaced with fresh nutrient every 24 hours and, at the end of the period of culture (8 days), the explants were fixed for electron microscopy. Both the explants and the non-cultured control glands were fixed with Millonig's buffered formalin, post-fixed with 1 % osmium tetroxide, and embedded in Araldite. Ultrathin sections, cut on a Reichert EMU 2 microtome, were stained with uranyl acetate and lead hydroxide, and were examined with an AEI 801 electron microscope. RESULTS
The fine structure of the secretory cells in the organ-cultured pituitary glands was strikingly similar to that of corresponding cells in the non-cultured control glands. Some slight damage to plasma and other membrane systems was found in most of the control (Figs. 1, 2) and cultured material (Figs. 3-8). This damage was believed to have arisen during shipment from Edinburgh to Belfast of formalinfixed material in cacodylate buffer. However, the membrane damage was uniform in all the specimens examined, and hence it was decided that valid comparison of cultured and control explants was possible. The features of the various secretory cells were similar to those described by Farquhar (1971), although stellate (follicular, or supporting) cells were not observed in the cultured material (cf. rabbit adenohypophysis; Young & Baker, 1975). The following description is restricted to prolactin-secreting cells and gonadotrophs because of our special interest in these cells in relation to current work on hormone production (Baker, Sharpe & Fraser, in preparation). Cells which, on morphological grounds, were believed to be producing prolactin (see Tixier-Vidal, 1975; Costoff, 1973) were numerous in both the controls and the cultured glands (Figs. 1, 3). Such cells contained abundant endoplasmic reticulum, a Golgi zone of flattened saccules and small vesicles, the latter often containing electron-dense material. They also contained elongated mitochondria and electrondense secretory granules, which were irregular in shape, and measuring 400-500 nm in diameter as a rule, although larger dense bodies were encountered occasionally. Cells which, on morphological grounds, were believed to be gonadotrophs were often encountered. There was no evidence for distinct populations of FSH and LH gonadotrophs. The addition of GnRH to the cultured pituitaries produced some morphological changes in these cells (see below). The presumed gonadotrophs of the female controls were slightly elongated and
761
Organ culture of rat pituitary *.
.1:\
t
Fig. 1. Electron micrograph of a prolactin cell from a control male rat.
-K Fig.2. Electron micrograph of
: a
x 12600.
2
prolactin cell (P) and a gonadotroph (G) female rat. x 12600.
from a control
762
T. G. BAKER AND B. A. YOUNG
Fig. 3. Electron micrograph of a prolactin cell from an organ-cultured control male rat. x 12600.
Fig. 4. Electron micrograph of a gonadotroph from an organ-cultured control female rat. x 12 600.
Organ culture of rat pituitary
¾
.r
.
Fig. 5. Electron micrograph of a gonadotroph from an organ-cultured control female rat. x 12600.
Fig. 6. Electron micrograph of a gonadotroph from a GnRH-treated organ-cultured male rat. Golgi zone (G); membrane flow (t). x 12600.
763
764
T. G. BAKER AND B. A. YOUNG
Fig. 7. Electron micrograph from a GnRH-treated orgsn-cultured female rat. Gonadotroph packed with granules (G). x 12600.
~~
Fig. 8. Electron micrograph from
a
~
~
~
~
~
1
GnRH-treated organ-cultured female rat. Degranulated cell (c). x 12600.
Organ culture ofjrat pituitary 765 contained secretory granules of 200-250 nm diameter (Fig. 4). Their endoplasmic reticulum was not conspicuous, but was sometimes seen concentrated towards the plasma membrane. Occasionally the plane of section passed through a large Golgi zone (Fig. 5). A characteristic feature of these cells was that, when the plane of section did not include a profile of the nucleus, numerous mitochondria were seen in the middle of the cell. Gonadotrophs in the cultures treated with GnRH sometimes showed signs of stimulation. Thus in some cells the Golgi zone formed a complete ring of saccules containing secretory granules in process of formation. In others the endoplasmic reticulum scattered throughout the cytoplasm had dilated cisternae and sometimes appeared to be extending by 'membrane flow' from the outer nuclear membrane (Fig. 6). The cells were sometimes packed with granules (Figs. 6, 7), but occasionally a cell looked as if it might have lost its granules (Fig. 8). DISCUSSION
The fact that the morphologies of the gonadotrophs and prolactin-secreting cells respectively were strikingly similar in the treated and non-treated cultures and in the control group of pituitaries is a good indication that the techniques employed in the present study are adequate for studies of pituitary function for up to 8 days in organ culture. Indeed, we gained the impression, which needs to be confirmed using biochemical and immunological techniques, that the gonadotrophs were more 'active' (in secretory terms) in the organ-cultured explants (especially when treated with GnRH) than in the controls. The gonadotrophs were disappointing in that we had hoped to detect changes in the number of these cells, and/or in the number of secretory granules per cell, in explants treated with GnRH (see Tixier-Vidal, 1975). It is well known, however, that only fairly gross changes are likely to be detected with the electron microscope because of sampling error and thinness of section, Nevertheless, some evidence for active secretion following treatment with GnRH was obtained, and in any case the rate of secretion of gonadotrophs with the doses of GnRH used in the present study might well have been balanced by the rate of release of FSH and LH into the culture medium. Although the morphological activity of the gonadotrophs seemed to be greater in the GnRH-treated cultures than in either the non-treated cultures or the controls, it was not possible to distinguish between cells producing FSH and LH. The results of the present study are in good agreement with those of Tixier-Vidal (1975) and Farquhar, Skutelsky & Hopkins (1975) who studied organ cultures and dissociated pituitary cells respectively. The present technique for organ culture has the great advantage that cellular integrity and inter-relationships are maintained in the explants. With dissociated pituitary cells these vital functions are disrupted, and membrane-associated surface receptors are likely to be damaged by the enzyme dispersion techniques. The present method permits good maintenance of the explants for 8 days in culture, which is a significant improvement over that of Martin & Klein (1976) which is only suitable for 4 day cultures of pituitaries from neonatal rats: glands from rats aged 40 days consistently showed necrosis at the centre of the explant. The improvements obtained with the present technique probably stem from the use of explants rather than whole glands, from the addition to the nutrient medium of donor calf serum rather than bovine serum albumin, and from the use of a gas phase using less oxygen (56 %; cf. 95 % used by Martin & Klein, 1976).
T. G. BAKER AND B. A. YOUNG 766 High oxygen concentrations are known to be cytotoxic: increasing the pressure at which the gas is maintained in the culture vessel (to 0703 kg cm2 above atmospheric pressure) permits a drastic reduction in oxygen concentration (see Baker & Neal, 1969). The large number of prolactin-secreting cells observed during the present study confirm the observations of Tixier-Vidal (1975). It is noteworthy that our preliminary results using radioimmunoassay indicate that the level of prolactin in the daily changes of culture medium is very high (Baker, Sharpe & Fraser, unpublished results). The fact that stellate (so-called follicular) cells were not seen in the cultured pituitary glands accords with the view that such cells participate in a transport system for nutrients when the portal system is intact (Young, 1977): in the absence of the portal system in culture the cells seemingly do not survive as such (but cf. rabbit adenohypophysis, Young & Baker, 1975), although it is conceivable, but highly improbable, that they rapidly become transformed into granular cells in culture. A major problem with this experiment was the fact that organ culturing was carried out in Edinburgh before the material was transported in buffer to Belfast for post-fixation and subsequent processing. This was almost certainly the underlying cause of the membrane breakage in some specimens in both the cultured and non-cultured control group. Nevertheless, as is clear from the photomicrographs, the general standards of organ culture and fixation were very high, and thus the technical basis of our further experiments on the amounts of FSH and LH produced after treatment of explants with GnRH and various steroid hormones (oestradiol and testosterone) is validated (Baker, Sharpe & Fraser, in preparation). SUMMARY
A technique is described which permits the maintenance for up to 8 days in organ culture of explanted rat pituitary glands. Electron microscope studies showed that the cultured glands (whether treated with gonadotrophin-releasing hormone (GnRH) or not) were almost identical to controls in appearance, although the stellate (follicular) cells seemingly were eliminated in vitro. Gonadotrophs and prolactin-secreting cells in male and female pituitary glands were similar in appearance both in vivo and in vitro to comparable cells described by other authors. There was some evidence for an increase in the secretory activity of these cell types (particularly gonadotrophs) when the explants were treated with GnRH. The present technique thus provides a suitable and valid 'model' with which to study the effects of GnRH and steroid hormones on the release and synthesis of FSH and LH in vitro. The authors would like to thank Professor J. J. Pritchard for assistance given in preparing this paper. The expenses incurred in these studies were defrayed from a grant to T. G. B. by the Medical Research Council. We are grateful to Mrs June Hardiman, Mrs Ann Rae and Mr G. R. Dickson for expert technical assistance, and to Hoechst (U.K.) Ltd for the generous supply of the GnRH used in these experiments. Mrs Joan Hamilton kindly did the secretarial work.
Organ culture of rat pituitary
767
REFERENCES
BAKER, T. G. & NEAL, P. (1969). The effects of x-irradiation on mammalian oocytes in organ culture. Biophysik 6, 39-45. BAKER, T. G., SHARPE, R. & FRASER, H. M. (1979). Secretion of FSH and LH by male and female rat pituitary glands in organ culture. (In preparation.) BAKER, T. G. & YOUNG, B. A. (1973). Organ culture of the rat thyroid gland. Experimenta 29, 1548-1550. COSTOFF, A. (1973). Ultrastructure of Rat Adenohypophysis, Correlations with Functions. New York: Academic Press. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Memoirs of the Society for Endocrinology 19, 79-122. FARQUHAR, M. G., SKUTELSKY, E. H. & HOPKINS, C. R. (1975). Structure and function of the anterior pituitary and dispersed cells. In The Anterior Pituitary (ed. A. Tixier-Vidal and M. G. Farquhar), pp. 83-135. New York: Academic Press. GAILLARD, R. P. & SCHABERG, A. (1965). Endocrine glands. In Cells and Tissues in Culture (ed. E. N. Willmer), vol. 2, pp. 631-643. New York: Academic Press. MARTIN, J. E. & KLEIN, D. G. (1976). Long-term organ culture of rat anterior pituitary gland. Endocrinology 99, 1189-1198. PASTEELS, J. L. (1961). Premiers resultats de culture combinee in vitro d'hypophyse et d'hypothalamus dans le but d'en apprecier la s&retion de prolactine. Compte rendu hebdomadaire des seances de l'Academie des sciences 253, 3074-3075. PETROVIC, A. (1961). Recherches sur la prehypophyse en culture organotypique: Evolution structurale et action sur les organes effecteurs associes in vitro. Thesis, Imprimerie Alsatia, Colmar, Strasbourg. PETROVIC, A. (1963). Cytophysiologie de l'adenohypophyse des mammiferes en culture organotypique: Fonction gonadotrope, thyr6otrope et corticotrope. In Cytologie de l'Adenohypophyse (ed. J. Benoit and C. Da Lage), pp. 121-136. Paris: CNRS. TIXIER-VIDAL, A. (1975). Ultrastructure of anterior pituitary cells in culture. In The Anterior Pituitary (ed. A. Tixier-Vidal and M. G. Farquhar), pp. 181-230. New York: Academic Press. YOUNG, B. A. & BAKER, T. G. (1975). Ultrastructural observations on organ cultured rabbit adenohypophysis. LC.R.S. Journal of Medical Science 3, 597-598. YOUNG, B. A. (1977). Some observations on the ultrastructure of the stellate cells of the pars distalis of the guinea-pig. Journal of Anatomy 124, 153-156.
