Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
12 (1978) 177-187 Scientific Publishers, Ltd.
177
DISSOCIATION OF THE PORCINE ANTERIOR PITUITARY: THE KINETICS OF LUTEINIZING HORMONE RELEASE IN RESPONSE TO LUTEINIZING HORMONE-RELEASING HORMONE Ameae M. WALKER * and Colin R. HOPKINS Department of Histology and Cell Biology (Medical), The Medical School, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K.
Received 21 April 1978; accepted 4 August 1978
A method has been developed for disaggregating porcine anterior pituitary tissue and for providing dissociated preparations which have good, stable viability in culture. The secretory capacity for these preparations in terms of their ability to release luteinizing hormone in response to luteinizing hormone-releasing hormone has been documented in detail. In following the short-term kinetics of LH secretion a biphasic pattern of release has been demonstrated in which a sharp initial peak, maximal at 2 min, is followed by a second, more prolonged phase of release reaching a maximum between 10 and 20 min. Keywords:
adenohypophysis; tion coupling.
gonadotrophin
secretion;
isolated cells; stimulus-secre-
In recent years several methods for disaggregating rat anterior pituitary tissue have been developed (Portanova et al., 1970; Vale et al., 1972; Hopkins and Farquhar, 1973) and it has been established that with culture the dissociated cells are able to provide a stable and responsive preparation which reacts to a variety of specific secretagogues in a manner consistent with observations made in the intact gland in vivo (Vale et al., 1972). The advantage of these preparations which makes them particularly useful for studies on the regulation of the secretory process derives from the fact that in suspension cells are thoroughly randomized and thus, unlike intact tissue, they provide multiple representative aliquots for bioassay. Despite the usefulness of the available preparations, however, there remains a need for a dissociated preparation which is both relatively unconstrained by the availability of tissue and well characterized in terms of secretory capacity. In this report we describe a preparation derived from porcine glands which fulfils these specific requirements. The gland in this species is relatively large (approx. 200 mg) and since we have been able to obtain viable preparations from material obtained at abattoir slaughter, * Present address: Section for Cell Biology, Yale Medical School, New Haven, Corm. 06510, U.S.A.
A.M. Walkerand CR. Hopkins
178
copious amounts of starting tissue are available. Moreover, since the chemistry and physiology of the gonadotrophins and the major principle concerned in the control of gonadotrophin secretion are well documented in this species (Matsuo et al., 1971; Maghuin-Rogister and Hennen, 1973; Maghuin-Rogister et al., 1973; Pomerantz et al., 1974), it has been possible to characterize the secretory capacity of the preparation in terms of its ability to release a specific secretory product, luteinizing hormone (LH), in response to a specific secretagogue, luteinizing hormone-releasing hormone (LH-RH). Here we describe the dissociation procedure, document the secretion of LH in response to LH-RH and demonstrate that the pattern of LH release in response to this secretagogue is biphasic.
MATERIALS
AND METHODS
Materials
Reagents were obtained from the following sources: soybean trypsin inhibitor (SBTI) from the Sigma Chemical Co., London, U.K.; bovine serum albumin (BSA) (fraction V) from the Armour Pharmaceutical Co., Eastbourne, U.K.; trypsin (Difco, 1 : 250, controls 553214 and 595505) from Difco Laboratories, Detroit, Mich., U.S.A.; collagenase (CLS 45D006, CLS 44A193, CLS 44B231) from the Worthington Biochemical Corporation, Freehold, N.J., U.S.A.; tissue culture media and sera from Bio Cult Laboratories Ltd., Paisley, U.K; and penicillin and streptomycin (Crystamycin) from Glaxo, Greenford, U.K. Bordetella pertissis vaccine was kindly provided by Lilly Research Centre Ltd., Windlesham, UK.; “‘1 for protein iodination was obtained from the Radiochemical Centre, Amersham, UK., precipitating serum from Wellcome Reagents Ltd., Beckenham, UK., and fluorescein-labelled goat anti-rabbit y-globulin from Miles Laboratories, Stoke Poges, U.K. Purified porcine LH for immunization (LER-778-4) and for use as standard in the assay (LER-786-3) was generously provided by Dr. Leo Reichert, Jr., Emory University, Atlanta, Ga., U.S.A. Purified porcine TSH was kindly provided by Dr. Georges Hennen, Departement Clinique et de Semieologie Medicales, Hopital Universitaire de Baviere, Liege, Belgium. Bovine LH (NIH-LH-B9) and porcine FSH (NIH-FSHP2) were provided by the Hormone Distribution Program, NIAMDD, Bethesda, Md., U.S.A. Animals The donor animals were Landrace hogs (castrates) and gilts which were 7 months of age and on average 206 lb when brought to slaughter. The pituitary glands were provided free of charge by the Walls Meat Co., Godley, Hyde, Cheshire, U.K. Incubation
media
The incubation medium used throughout the dissociation procedure was a Krebs-Ringer bicarbonate solution (Krebs, 1950) with calcium and magnesium
Dissociated cells from porcine pituitary
119
reduced to 0.1 and 1.2 mM, respectively, containing a complete amino acid supplement with 2.5 mg/ml glucose, 0.25 mg/ml bovine serum albumin and penicillin and streptomycin at 30 III/ml (KRB complete). The medium was maintained at pH 7.4 by gassing with 95% 02 : 5% CO*. For ‘calcium- and magnesium-free’ media (KRB - Ca2+ and Mg2+) these ions were simply omitted. All glassware used for handling cells was siliconized. Preparation of poly-L-lysine (pLys)-coated dishes Plastic petri dishes (5 cm diam.; Sterilin Ltd., Richmond, Surrey, U.K.) were coated under sterile conditions with poly-L-lysine (type l-B, mol. wt. 2-4000, Sigma, London) by rinsing them sequentially with 1 mg/ml aqueous pLys, then water, and allowing them to dry. Immunocytochemistry Dissociated cells cultured for 48 h on plys-coated coverslips were fixed in 2% formaldehyde, rinsed in phosphate-buffered saline (PBS) and incubated for 60 min at 20°C in the rabbit anti-porcine LH raised for radioimmunoassay (diluted 1 : 10 with PBS). These preparations were then thoroughly washed (2 h) with PBS before being incubated with goat anti-rabbit y-globulin conjugated to fluorescein isothiocyanate (diluted 1 : 10 with PBS) for 60 min at 20°C. They were finally washed again in PBS before being mounted in PBS/glycerine. Preparations were examined in a Zeiss Ultraphot II fitted with accessories for epifluorescence. Control preparations incubated with non-immune serum instead of rabbit anti-porcine LH showed negligible fluorescence. Deoxyribonucleic acid (DNA) was assayed according to the method of Burton (1956). Preparation of material for microscopy Cells were fixed either in suspension or in situ, attached to petri dishes, with dilute Kamovsky’s fixative (Kamovsky, 1965) for 15 min at room temperature. Thereafter they were processed for microscopy as described previously (Hopkins and Farquhar, 1973). Radioimmunoassay Antisera against LER-7784 were raised according to the procedure described by Vaitukaitis et al. (1971). The antiserum chosen for radioimmunoassay and immunocytochemistry has less than 1% cross-reactivity with porcine FSH and less than 2% cross-reactivity with TSH. The sensitivity of the assay is better than 0.5 ng/ml: the coefficient of intra-assay variation is 11.4% and that of inter-assay variation 17%. Media collected for LH radioimmunoassay were centrifuged at 3000g for 15 min, decanted and stored at -2O’C until assayed. To estimate cell content of LH, 1 ml of 1 mM EGTA/O.25% egg albumin was added to the attached cells and they were then freeze-thawed 3 times before centrifugation (3000 g X 15 min) and the removal of the supernatant for assay.
180
A.M. Walkerand C.R. Hopkins
RESULTS Dissociation procedure
In the present study, crude enzyme preparations have been employed and it has been found that once the efficacy of a given batch of enzyme has been established, consistent and reproducible results are obtained (i.e. variation within a preparation is uncommon). In particular, we evaluated the efficacy of sequential incubations in crude collagenase and crude trypsin. These enzymes were selected because, although preliminary studies showed that the preparations and procedures published for rat tissues (Vale et al., 1972; Hopkins and Farquhar, 1973) were ineffective with porcine material, cell viability within the treated tissue remained high even after prolonged incubation. The sequence, concentration, length of incubation and temperature dependence of the enzyme incubations were assessed in detail and optimized on the basis of the yield of viable, single cells. On this basis the most efficient procedure is as follows. The glands are removed from the animals within 30 min of death and placed in KRB + Ca2+ and Mg’+ containing 300 IU/ml penicillin and streptomycin. The posterior lobes are removed and the anterior lobes cleanly chopped in approx. 1 mm cubes. The freshly chopped cubes are washed in KRB f Ca*+ and Mg*” containing 30 W/ml penicillin and streptomycin and then incubated with constant shaking in 40 ml 0.075% collagenase in KRB t Ca*’ and Mg*+ at 37°C for 60 min. The collagenase medium is decanted and the blocks washed 3 X 5 min in KRB Ca*+ and Mg*+. The blocks are next incubated, again with constant shaking, in 40 ml 0.5% trypsin in KRB - Ca*+ and Mg*+ at 20°C for 60 mm. At the end of this incubation the tissue which remains as intact blocks is carefully transferred to 15 ml conical centrifuge tubes and allowed to settle out. The trypsin medium is decanted off and replaced by a fresh KRB - Ca*+ and Mg*+ in which the blocks are dispe&ed by gently pipetting with a flame-polished Pasteur pipette. At intervals, as the medium becomes milky, the fragmented blocks are allowed to settle out and the free cells in suspension are decanted into ‘KRB complete’. The pipetting is continued until no further dispersion occurs. Residual tryptic activity is inhibited by the addition of 1 ml of 1 mg/ml SBTI to each 10 ml of suspension. Other enzymes present in the crude collagenase and crude trypsin preparations are presumably inactivated both by dilution during subsequent processing of the cells and by the addition of serum during cell culture. Following a 5-min incubation with SBTI, the suspension is centrifuged at 250g for 5 min to pellet the cells and the milky supernatant containing fine debris is decanted off. The pelleted cells are then gently resuspended in KRB complete and this suspension layered over 4.5 ml of 4% BSA in the same medium in a 15 ml conical centrifuge tube. Centrifugation at 250 g for 3 min effectively removes red blood cells and remaining debris from the pelleted cells. The final pellet is resuspended in Dulbecco’s minimum essential medium (DMEM) and ready for use. The acute suspension produced by this procedure contains predominantly single
Dismcia~ed cells from porcine pituitary
181
secretory cells, with some aggregates of follicular cells and endothelial components (fig. la). On the basis of trypan blue staining the cells are better than 98% viable and the cell yield is between 15 and 20 X lo6 cells/gland. This represents a yield of approximately 15% on a DNA basis. The tine structural organization of the dissociated cells is good (fig. lb) and the LH/DNA ratio indicates gonadotrophin cells in the acute suspension is proportionally the initial tissue.
that the number of the same as that of
Cell culture
Preliminary studies showed that in the acutely dissociated cells, as in preparations derived from the rat pituitary (Hopes and Farquhar, 1973), hormone secretion was impaired and indicated that in order for them to recover their responsiveness to LH*RH stimulation it would be necessary to establish the cells in culture. When .aliquotted into petri dishes in DMEM t 10% fetal calf serum, the dissociated cells remain viable for at least 3 weeks. Within 24 h most of them attach lightly to the dish surface, but in order for them to adhere firmly enough to withstand repeated changes of medium they need to be incubated for at least 48 h. Use of LH antiserum to identify LH immunocytochemic~ly within the attached cells shows that the LH cells are randomly distributed throughout the preparation. Counts of a total of 1000 immunofluorescent cells indicate that LH cells comprise 30 + 4% of the total population in both hog and gilt preparations.
After 4 days in culture and in response to LH-RH the attached cells release LH in a highly reproducible dose-related manner. The magnitude of the response in preparations derived from hog glands is about twice that of preparations derived from gilts, although the shape of the log dose-response curve from the two preparations is otherwise identical. As shown in fig. 2, the curve is sigmoidal, halfmaximal at 4 X 10-r’ M and maximal at 10-s M. In hog cell preparations incubated over a 2-h interval there is less than 5% difference in response between replicate dishes; in gilt preparations the variation is greater but not more than 10%. During a 2-h incubation period with maximal LH-RH stimulation the cells of both hog and gilt preparations release approximately 70% of their total LH content. In order to document the recovery of the secretory response within the first few days of culture, that is, before the cells become firmly adherent, acutely dissociated cells were aliquotted onto poly-L-lysinecoated dishes. This coating induces the cells to attach immediately and does not appear to affect their secretory response (Hopkins, 1977). Using gilt cells attached in this way it could be shown that acutely dissociated cells do not respond to LH-RH and that even after 24 h in culture their response in terms of experimental~control remains poor (fig. 3). This is primarily because at this time the level of LH release in the unstimulated controls is high. At 48 h and up to 96 h, however, the basal levels of LH release are reduced and consequently the response to LH-RH expressed as experimental/control improves.
182
A.M. Walker and C.R. Hopkins
Fig. 1. (a) Light micrograph snowmg cells from an acutely dissociated suspension. Granulecontaining secretory cells, clusters of follicular cells and some endothelial elements are shown (X360). (b) Transmission electron micrograph showing cells from the acute dissociated suspension. The fine structural organization of the different cell types is well preserved and similar in all respects to that observed in intact tissue fragments. At the upper right is a non-secretory follicular cell and lower right a gonadotrophin cell. (X5760).
183
Dissociated cells f?om porcine pituitary
10-11
10-10 M LHRH 10-g
10-s
10-7
Fig. 2. Dose-response
curve for LH-RH-stimulated LH release obtained using gilt cells cultured for 4 days. Dishes containing 2 X lo6 cells were rinsed free of serum with 3 X 5 mm changes in DMEM containing 1 mg/ml gelatin. They were then incubated in replicates of 5 at 37’C for 2 h in this medium with varying concentrations of LH-RH. At the end of the incubation media were collected, centrifuged to remove any free cells or cell debris and freeze stored at -20°C cells from hog pituitaries; until radioimmunoassay. Each bar represents tSEM. A-, lu, cells from gilt pituitaries. Arrow indicates half-maximal dose level.
The half-maximal response over the period 48-96 h remains unchanged at 4 X lo-” M, but the magnitude of the response expressed in terms of ng LH released/ lo6 cells decreases by approximately 40%. Fig. 4 shows the form and distribution of the LH cells at 48 h.
A.M. Walker and C.R. Hopkins
184
N-1I#
lo-I'M
lo-'M
lo-'M
Fig. 3. Dose-response curves for LH-RH-stimulated for 24 h (o -), 72 h (ad) 48 h ( l-), SEMs are omitted for clarity. Variations between preparations incubated 48-96 h.
1F7M LHRH LH response obtained and 96 h (Areplicate dishes did
using cells cultured ) post-dissociation. not exceed 10% in
Fig. 4. Light micrograph showing gonadotrophin cells identified using specific immunofluorescence for porcine LH. The cells were dissociated, plated onto poly-L-lysine-coated petri dishes and incubated 48 h before treatment with the rabbit anti-porcine LH serum characterized by radioimmunoassay. The LH cells in this preparation comprise approximately 30% of the total preparation. (Phase contrast/fluorescence optics, X400.)
Dissociated cells from porcine pituitary
Fig. 5. Kinetics
of LH release in response to continuous stimulation 1-min intervals. Gilt cells (10 X lo6 on 2.5 cm poly-L-lysme-coated 2 days before being rinsed free of serum and subjected to repeated gelatin containing LH-RH as indicated. The cells remained tightly procedure. The profiles were derived from 5 replicate dishes; bar pattern .-,
185
by LH-RH followed at dishes) were cultured for 1-min changes of DMEM/ adherent throughout this variations are +SEM. The
of LH release is clearly biphasic with an initial peak at 2 min and a second 1O-8 M LH-RH; o------o, 5 X 10-l’ LH-RH.
at 20 min.
Based on these preliminary studies, preparations in which cells attached to pLyscoated dishes and incubated for 48 h were chosen for a detailed analysis of the pattern of LH release. When the kinetics of LH release are followed at l-min intervals (fig. 5) it is clear that within the first 10 min there is an initial phase of release which is maximal at 2 min. After this initial peak the rate of LH release sharply declines until at 6 min it almost reaches prestimulation levels. Between 10 and 30 min there is a second increase in the rate of LH release which leads to the maximum indicated in fig. 5 at about 20 min. This pattern of release is seen in both hog and gilt preparations.
DISCUSSION The method of dissociation outlined in this report has been used sucessfully to provide an experimental preparation in more than 150 separate experiments, and
186
A.M. Walker and C.R. Hopkins
although the pituitaries are removed between 20 and 30 min after the animals are killed, viability in the final preparation is usually better than 98%. The magnitude of the LH-RH response has varied from S- to 25-fold but the sensitivity in terms of the half-maximal response is remarkably consistent. Compared with the reported sensitivity of rat preparations (Vale et al., 1972; Hopkins and Farquhar, 1973) this response is almost 2-fold better. At all dose levels the amount of LH released by LH-RH in preparations derived from hog pituitaries is greater than that released from gilt preparations. This difference is probably due to the fact that the hogs are castrates (Purves, 1961), although the immunocytochemical observations suggest that the increased amount of LH released cannot be due simply to an increase in gonadotroph number. The most consistent responses (i.e. least variation between replicates) are also observed in preparations derived from hog pituitaries, and perhaps this too is related to the endocrine environment in castrate animals. Interestingly, in response to a maximal dose of LH-RH, porcine cells in culture release a proportionally larger amount of their LH content (approx. 70%) than do rat cells cultured under similar conditions (lo-15%) (Hopkins, 1974). Moreover, if LH released from gilt cells in culture is multiplied to correct for the cell yield during dissociation, giving LH released/pituitary gland, and the amor& of LH released in vivo corrected for total blood volume, the performance of the dissociated cells compares favourably with that reported from the gland in vivo (Parvizi et al., 1976). The rapidity with which LH-RH stimulates an initial increase in the rate of LH release correlates well with an early peak at 6 min found in the rat in vivo following a single tail vein injection of LH-RH (Johnson and Mallampati, 1975) and the overall magnitude of the response occurring during the first 30 min of stimulation is similar to that reported from studies on the rat in which perfused intact tissue fragments were used (Edwardson and Gilbert, 1976; Kilpatrick et al., 1976; Williams, 1976). In a recent study using dissociated pituitary cells in the rat a biphasic response to LH-RH stimulation was also observed (Hopkins, 1977) and more recently it has been reported (Schrey et al., 1977) that in a similar preparation thyrotropin-releasing hormone may also provoke a biphasic pattern of thyrotropin release. In this context it is of special interest that in vivo the pattern of LH release has been shown to be pulsatile (Midgley and Jaffe, 1971; Gay and Sheth, 1972). In conclusion, the present report described a stable in vitro preparation which is especially useful for the study of LH-RH-LH stimulus-secretion coupling. As a starting preparation for the subfractionation of specific cell types and for subcellular fractionation studies it has the same potential advantage as the other similar preparations. In addition, it provides a substantially increased amount of starting material.
Dissociated cells from porcine pituitary
187
ACKNOWLEDGEMENT This work was carried out during the tenure of an MRC postgraduate studentship and was supported by an MRC project grant (G975/517/C) awarded to C.R.H.
REFERENCES Burton, K. (1956) Biochem. J. 62, 315-323. Edwardson, J.A. and Gilbert, D. (1976) 3. Endocrinol. 68, 197-207. Gay, V.L. and Sheth, N.S. (1972) Endocrinology 90, 158-161. Greenwood, F.C., IIunter, W.M. and Glover, J.S. (1963) Biochem. J. 89,114-123. Hopkins, C.R. (1974) J. Cell Biol. 63,143a. Hopkins, CR. (1977) J. Cell Biol. 73,685-695. Hopkins, C.R. and Farquhar, M.G. (1973) J. Cell Biol. 59,276-303. Johnson, D.C. and Mallampati, R.S. (1975) J. Endocrinol. 66, 13-20. Karnovsky, M.J. (196.5) J. Cell Biol. 27, 137A-138A (abstr.). Kilpatrick, M.J., Collins, W.P. and Newton, J.R. (1976) J. Reprod. Fertil. 46,25-30. Krebs, H.A. (1950) Biochim. Biophys. Acta 4,249. Maghuin-Rogister, G. and Hennen, G. (1973) Eur. J. Biochem. 39,235-253. Maghuin-Rogister, G., Combarnous, Y. and Hennen, G. (1973) Eur. J. Biochem. 39,255-263. Matsuo, H., Baba, Y., Nair, R.M.G., Arimura, A. and Schally, A.V. (1971) Biochem. Biophys. Res. Commun. 43,1334-1339. Midgley, A.R. and Jaffe, R.B. (1971) J. Clin. Endocrinol Metab. 33,962-969. Parvizi, N., Elsaesser, F., Smidt, D. and Ellendorf, F. (1976) J. Endocrinol. 69,193-203. Pomerantz, D.K., Ellendorff, F., Elazaesser, F., Konig, A. and Smidt, D. (1974) Endocrinology 94,330-335. Portanova, R., Smith, 0. and Sayers, G. (1970) Proc. Sot. Exp. Biol. Med. 133,573-576. Purves, H.D. (1961) In: Sex and Internal Secretion, Ed.: W.C. Young (Williams and Wilkins Co., Baltimore) pp. 161-238. Schrey, M.P., Brown, B.L. and Ekins, R.P. (1977) Mol. Cell. Endocrinol. 8, 271-282. Vaitukaitis, J., Robbins, J.B., Neischlag, E. and Ross, G.T. (1971) J. Clin. Endocrinol. 33,988991. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guillemin, R. (1972) Endocrinology 91, 562-572. Williams, J.A. (1976) J. Physiol. (London) 260, 105-I 16.
0 Elsevier/North-Holland
12 (1978) 177-187 Scientific Publishers, Ltd.
177
DISSOCIATION OF THE PORCINE ANTERIOR PITUITARY: THE KINETICS OF LUTEINIZING HORMONE RELEASE IN RESPONSE TO LUTEINIZING HORMONE-RELEASING HORMONE Ameae M. WALKER * and Colin R. HOPKINS Department of Histology and Cell Biology (Medical), The Medical School, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K.
Received 21 April 1978; accepted 4 August 1978
A method has been developed for disaggregating porcine anterior pituitary tissue and for providing dissociated preparations which have good, stable viability in culture. The secretory capacity for these preparations in terms of their ability to release luteinizing hormone in response to luteinizing hormone-releasing hormone has been documented in detail. In following the short-term kinetics of LH secretion a biphasic pattern of release has been demonstrated in which a sharp initial peak, maximal at 2 min, is followed by a second, more prolonged phase of release reaching a maximum between 10 and 20 min. Keywords:
adenohypophysis; tion coupling.
gonadotrophin
secretion;
isolated cells; stimulus-secre-
In recent years several methods for disaggregating rat anterior pituitary tissue have been developed (Portanova et al., 1970; Vale et al., 1972; Hopkins and Farquhar, 1973) and it has been established that with culture the dissociated cells are able to provide a stable and responsive preparation which reacts to a variety of specific secretagogues in a manner consistent with observations made in the intact gland in vivo (Vale et al., 1972). The advantage of these preparations which makes them particularly useful for studies on the regulation of the secretory process derives from the fact that in suspension cells are thoroughly randomized and thus, unlike intact tissue, they provide multiple representative aliquots for bioassay. Despite the usefulness of the available preparations, however, there remains a need for a dissociated preparation which is both relatively unconstrained by the availability of tissue and well characterized in terms of secretory capacity. In this report we describe a preparation derived from porcine glands which fulfils these specific requirements. The gland in this species is relatively large (approx. 200 mg) and since we have been able to obtain viable preparations from material obtained at abattoir slaughter, * Present address: Section for Cell Biology, Yale Medical School, New Haven, Corm. 06510, U.S.A.
A.M. Walkerand CR. Hopkins
178
copious amounts of starting tissue are available. Moreover, since the chemistry and physiology of the gonadotrophins and the major principle concerned in the control of gonadotrophin secretion are well documented in this species (Matsuo et al., 1971; Maghuin-Rogister and Hennen, 1973; Maghuin-Rogister et al., 1973; Pomerantz et al., 1974), it has been possible to characterize the secretory capacity of the preparation in terms of its ability to release a specific secretory product, luteinizing hormone (LH), in response to a specific secretagogue, luteinizing hormone-releasing hormone (LH-RH). Here we describe the dissociation procedure, document the secretion of LH in response to LH-RH and demonstrate that the pattern of LH release in response to this secretagogue is biphasic.
MATERIALS
AND METHODS
Materials
Reagents were obtained from the following sources: soybean trypsin inhibitor (SBTI) from the Sigma Chemical Co., London, U.K.; bovine serum albumin (BSA) (fraction V) from the Armour Pharmaceutical Co., Eastbourne, U.K.; trypsin (Difco, 1 : 250, controls 553214 and 595505) from Difco Laboratories, Detroit, Mich., U.S.A.; collagenase (CLS 45D006, CLS 44A193, CLS 44B231) from the Worthington Biochemical Corporation, Freehold, N.J., U.S.A.; tissue culture media and sera from Bio Cult Laboratories Ltd., Paisley, U.K; and penicillin and streptomycin (Crystamycin) from Glaxo, Greenford, U.K. Bordetella pertissis vaccine was kindly provided by Lilly Research Centre Ltd., Windlesham, UK.; “‘1 for protein iodination was obtained from the Radiochemical Centre, Amersham, UK., precipitating serum from Wellcome Reagents Ltd., Beckenham, UK., and fluorescein-labelled goat anti-rabbit y-globulin from Miles Laboratories, Stoke Poges, U.K. Purified porcine LH for immunization (LER-778-4) and for use as standard in the assay (LER-786-3) was generously provided by Dr. Leo Reichert, Jr., Emory University, Atlanta, Ga., U.S.A. Purified porcine TSH was kindly provided by Dr. Georges Hennen, Departement Clinique et de Semieologie Medicales, Hopital Universitaire de Baviere, Liege, Belgium. Bovine LH (NIH-LH-B9) and porcine FSH (NIH-FSHP2) were provided by the Hormone Distribution Program, NIAMDD, Bethesda, Md., U.S.A. Animals The donor animals were Landrace hogs (castrates) and gilts which were 7 months of age and on average 206 lb when brought to slaughter. The pituitary glands were provided free of charge by the Walls Meat Co., Godley, Hyde, Cheshire, U.K. Incubation
media
The incubation medium used throughout the dissociation procedure was a Krebs-Ringer bicarbonate solution (Krebs, 1950) with calcium and magnesium
Dissociated cells from porcine pituitary
119
reduced to 0.1 and 1.2 mM, respectively, containing a complete amino acid supplement with 2.5 mg/ml glucose, 0.25 mg/ml bovine serum albumin and penicillin and streptomycin at 30 III/ml (KRB complete). The medium was maintained at pH 7.4 by gassing with 95% 02 : 5% CO*. For ‘calcium- and magnesium-free’ media (KRB - Ca2+ and Mg2+) these ions were simply omitted. All glassware used for handling cells was siliconized. Preparation of poly-L-lysine (pLys)-coated dishes Plastic petri dishes (5 cm diam.; Sterilin Ltd., Richmond, Surrey, U.K.) were coated under sterile conditions with poly-L-lysine (type l-B, mol. wt. 2-4000, Sigma, London) by rinsing them sequentially with 1 mg/ml aqueous pLys, then water, and allowing them to dry. Immunocytochemistry Dissociated cells cultured for 48 h on plys-coated coverslips were fixed in 2% formaldehyde, rinsed in phosphate-buffered saline (PBS) and incubated for 60 min at 20°C in the rabbit anti-porcine LH raised for radioimmunoassay (diluted 1 : 10 with PBS). These preparations were then thoroughly washed (2 h) with PBS before being incubated with goat anti-rabbit y-globulin conjugated to fluorescein isothiocyanate (diluted 1 : 10 with PBS) for 60 min at 20°C. They were finally washed again in PBS before being mounted in PBS/glycerine. Preparations were examined in a Zeiss Ultraphot II fitted with accessories for epifluorescence. Control preparations incubated with non-immune serum instead of rabbit anti-porcine LH showed negligible fluorescence. Deoxyribonucleic acid (DNA) was assayed according to the method of Burton (1956). Preparation of material for microscopy Cells were fixed either in suspension or in situ, attached to petri dishes, with dilute Kamovsky’s fixative (Kamovsky, 1965) for 15 min at room temperature. Thereafter they were processed for microscopy as described previously (Hopkins and Farquhar, 1973). Radioimmunoassay Antisera against LER-7784 were raised according to the procedure described by Vaitukaitis et al. (1971). The antiserum chosen for radioimmunoassay and immunocytochemistry has less than 1% cross-reactivity with porcine FSH and less than 2% cross-reactivity with TSH. The sensitivity of the assay is better than 0.5 ng/ml: the coefficient of intra-assay variation is 11.4% and that of inter-assay variation 17%. Media collected for LH radioimmunoassay were centrifuged at 3000g for 15 min, decanted and stored at -2O’C until assayed. To estimate cell content of LH, 1 ml of 1 mM EGTA/O.25% egg albumin was added to the attached cells and they were then freeze-thawed 3 times before centrifugation (3000 g X 15 min) and the removal of the supernatant for assay.
180
A.M. Walkerand C.R. Hopkins
RESULTS Dissociation procedure
In the present study, crude enzyme preparations have been employed and it has been found that once the efficacy of a given batch of enzyme has been established, consistent and reproducible results are obtained (i.e. variation within a preparation is uncommon). In particular, we evaluated the efficacy of sequential incubations in crude collagenase and crude trypsin. These enzymes were selected because, although preliminary studies showed that the preparations and procedures published for rat tissues (Vale et al., 1972; Hopkins and Farquhar, 1973) were ineffective with porcine material, cell viability within the treated tissue remained high even after prolonged incubation. The sequence, concentration, length of incubation and temperature dependence of the enzyme incubations were assessed in detail and optimized on the basis of the yield of viable, single cells. On this basis the most efficient procedure is as follows. The glands are removed from the animals within 30 min of death and placed in KRB + Ca2+ and Mg’+ containing 300 IU/ml penicillin and streptomycin. The posterior lobes are removed and the anterior lobes cleanly chopped in approx. 1 mm cubes. The freshly chopped cubes are washed in KRB f Ca*+ and Mg*” containing 30 W/ml penicillin and streptomycin and then incubated with constant shaking in 40 ml 0.075% collagenase in KRB t Ca*’ and Mg*+ at 37°C for 60 min. The collagenase medium is decanted and the blocks washed 3 X 5 min in KRB Ca*+ and Mg*+. The blocks are next incubated, again with constant shaking, in 40 ml 0.5% trypsin in KRB - Ca*+ and Mg*+ at 20°C for 60 mm. At the end of this incubation the tissue which remains as intact blocks is carefully transferred to 15 ml conical centrifuge tubes and allowed to settle out. The trypsin medium is decanted off and replaced by a fresh KRB - Ca*+ and Mg*+ in which the blocks are dispe&ed by gently pipetting with a flame-polished Pasteur pipette. At intervals, as the medium becomes milky, the fragmented blocks are allowed to settle out and the free cells in suspension are decanted into ‘KRB complete’. The pipetting is continued until no further dispersion occurs. Residual tryptic activity is inhibited by the addition of 1 ml of 1 mg/ml SBTI to each 10 ml of suspension. Other enzymes present in the crude collagenase and crude trypsin preparations are presumably inactivated both by dilution during subsequent processing of the cells and by the addition of serum during cell culture. Following a 5-min incubation with SBTI, the suspension is centrifuged at 250g for 5 min to pellet the cells and the milky supernatant containing fine debris is decanted off. The pelleted cells are then gently resuspended in KRB complete and this suspension layered over 4.5 ml of 4% BSA in the same medium in a 15 ml conical centrifuge tube. Centrifugation at 250 g for 3 min effectively removes red blood cells and remaining debris from the pelleted cells. The final pellet is resuspended in Dulbecco’s minimum essential medium (DMEM) and ready for use. The acute suspension produced by this procedure contains predominantly single
Dismcia~ed cells from porcine pituitary
181
secretory cells, with some aggregates of follicular cells and endothelial components (fig. la). On the basis of trypan blue staining the cells are better than 98% viable and the cell yield is between 15 and 20 X lo6 cells/gland. This represents a yield of approximately 15% on a DNA basis. The tine structural organization of the dissociated cells is good (fig. lb) and the LH/DNA ratio indicates gonadotrophin cells in the acute suspension is proportionally the initial tissue.
that the number of the same as that of
Cell culture
Preliminary studies showed that in the acutely dissociated cells, as in preparations derived from the rat pituitary (Hopes and Farquhar, 1973), hormone secretion was impaired and indicated that in order for them to recover their responsiveness to LH*RH stimulation it would be necessary to establish the cells in culture. When .aliquotted into petri dishes in DMEM t 10% fetal calf serum, the dissociated cells remain viable for at least 3 weeks. Within 24 h most of them attach lightly to the dish surface, but in order for them to adhere firmly enough to withstand repeated changes of medium they need to be incubated for at least 48 h. Use of LH antiserum to identify LH immunocytochemic~ly within the attached cells shows that the LH cells are randomly distributed throughout the preparation. Counts of a total of 1000 immunofluorescent cells indicate that LH cells comprise 30 + 4% of the total population in both hog and gilt preparations.
After 4 days in culture and in response to LH-RH the attached cells release LH in a highly reproducible dose-related manner. The magnitude of the response in preparations derived from hog glands is about twice that of preparations derived from gilts, although the shape of the log dose-response curve from the two preparations is otherwise identical. As shown in fig. 2, the curve is sigmoidal, halfmaximal at 4 X 10-r’ M and maximal at 10-s M. In hog cell preparations incubated over a 2-h interval there is less than 5% difference in response between replicate dishes; in gilt preparations the variation is greater but not more than 10%. During a 2-h incubation period with maximal LH-RH stimulation the cells of both hog and gilt preparations release approximately 70% of their total LH content. In order to document the recovery of the secretory response within the first few days of culture, that is, before the cells become firmly adherent, acutely dissociated cells were aliquotted onto poly-L-lysinecoated dishes. This coating induces the cells to attach immediately and does not appear to affect their secretory response (Hopkins, 1977). Using gilt cells attached in this way it could be shown that acutely dissociated cells do not respond to LH-RH and that even after 24 h in culture their response in terms of experimental~control remains poor (fig. 3). This is primarily because at this time the level of LH release in the unstimulated controls is high. At 48 h and up to 96 h, however, the basal levels of LH release are reduced and consequently the response to LH-RH expressed as experimental/control improves.
182
A.M. Walker and C.R. Hopkins
Fig. 1. (a) Light micrograph snowmg cells from an acutely dissociated suspension. Granulecontaining secretory cells, clusters of follicular cells and some endothelial elements are shown (X360). (b) Transmission electron micrograph showing cells from the acute dissociated suspension. The fine structural organization of the different cell types is well preserved and similar in all respects to that observed in intact tissue fragments. At the upper right is a non-secretory follicular cell and lower right a gonadotrophin cell. (X5760).
183
Dissociated cells f?om porcine pituitary
10-11
10-10 M LHRH 10-g
10-s
10-7
Fig. 2. Dose-response
curve for LH-RH-stimulated LH release obtained using gilt cells cultured for 4 days. Dishes containing 2 X lo6 cells were rinsed free of serum with 3 X 5 mm changes in DMEM containing 1 mg/ml gelatin. They were then incubated in replicates of 5 at 37’C for 2 h in this medium with varying concentrations of LH-RH. At the end of the incubation media were collected, centrifuged to remove any free cells or cell debris and freeze stored at -20°C cells from hog pituitaries; until radioimmunoassay. Each bar represents tSEM. A-, lu, cells from gilt pituitaries. Arrow indicates half-maximal dose level.
The half-maximal response over the period 48-96 h remains unchanged at 4 X lo-” M, but the magnitude of the response expressed in terms of ng LH released/ lo6 cells decreases by approximately 40%. Fig. 4 shows the form and distribution of the LH cells at 48 h.
A.M. Walker and C.R. Hopkins
184
N-1I#
lo-I'M
lo-'M
lo-'M
Fig. 3. Dose-response curves for LH-RH-stimulated for 24 h (o -), 72 h (ad) 48 h ( l-), SEMs are omitted for clarity. Variations between preparations incubated 48-96 h.
1F7M LHRH LH response obtained and 96 h (Areplicate dishes did
using cells cultured ) post-dissociation. not exceed 10% in
Fig. 4. Light micrograph showing gonadotrophin cells identified using specific immunofluorescence for porcine LH. The cells were dissociated, plated onto poly-L-lysine-coated petri dishes and incubated 48 h before treatment with the rabbit anti-porcine LH serum characterized by radioimmunoassay. The LH cells in this preparation comprise approximately 30% of the total preparation. (Phase contrast/fluorescence optics, X400.)
Dissociated cells from porcine pituitary
Fig. 5. Kinetics
of LH release in response to continuous stimulation 1-min intervals. Gilt cells (10 X lo6 on 2.5 cm poly-L-lysme-coated 2 days before being rinsed free of serum and subjected to repeated gelatin containing LH-RH as indicated. The cells remained tightly procedure. The profiles were derived from 5 replicate dishes; bar pattern .-,
185
by LH-RH followed at dishes) were cultured for 1-min changes of DMEM/ adherent throughout this variations are +SEM. The
of LH release is clearly biphasic with an initial peak at 2 min and a second 1O-8 M LH-RH; o------o, 5 X 10-l’ LH-RH.
at 20 min.
Based on these preliminary studies, preparations in which cells attached to pLyscoated dishes and incubated for 48 h were chosen for a detailed analysis of the pattern of LH release. When the kinetics of LH release are followed at l-min intervals (fig. 5) it is clear that within the first 10 min there is an initial phase of release which is maximal at 2 min. After this initial peak the rate of LH release sharply declines until at 6 min it almost reaches prestimulation levels. Between 10 and 30 min there is a second increase in the rate of LH release which leads to the maximum indicated in fig. 5 at about 20 min. This pattern of release is seen in both hog and gilt preparations.
DISCUSSION The method of dissociation outlined in this report has been used sucessfully to provide an experimental preparation in more than 150 separate experiments, and
186
A.M. Walker and C.R. Hopkins
although the pituitaries are removed between 20 and 30 min after the animals are killed, viability in the final preparation is usually better than 98%. The magnitude of the LH-RH response has varied from S- to 25-fold but the sensitivity in terms of the half-maximal response is remarkably consistent. Compared with the reported sensitivity of rat preparations (Vale et al., 1972; Hopkins and Farquhar, 1973) this response is almost 2-fold better. At all dose levels the amount of LH released by LH-RH in preparations derived from hog pituitaries is greater than that released from gilt preparations. This difference is probably due to the fact that the hogs are castrates (Purves, 1961), although the immunocytochemical observations suggest that the increased amount of LH released cannot be due simply to an increase in gonadotroph number. The most consistent responses (i.e. least variation between replicates) are also observed in preparations derived from hog pituitaries, and perhaps this too is related to the endocrine environment in castrate animals. Interestingly, in response to a maximal dose of LH-RH, porcine cells in culture release a proportionally larger amount of their LH content (approx. 70%) than do rat cells cultured under similar conditions (lo-15%) (Hopkins, 1974). Moreover, if LH released from gilt cells in culture is multiplied to correct for the cell yield during dissociation, giving LH released/pituitary gland, and the amor& of LH released in vivo corrected for total blood volume, the performance of the dissociated cells compares favourably with that reported from the gland in vivo (Parvizi et al., 1976). The rapidity with which LH-RH stimulates an initial increase in the rate of LH release correlates well with an early peak at 6 min found in the rat in vivo following a single tail vein injection of LH-RH (Johnson and Mallampati, 1975) and the overall magnitude of the response occurring during the first 30 min of stimulation is similar to that reported from studies on the rat in which perfused intact tissue fragments were used (Edwardson and Gilbert, 1976; Kilpatrick et al., 1976; Williams, 1976). In a recent study using dissociated pituitary cells in the rat a biphasic response to LH-RH stimulation was also observed (Hopkins, 1977) and more recently it has been reported (Schrey et al., 1977) that in a similar preparation thyrotropin-releasing hormone may also provoke a biphasic pattern of thyrotropin release. In this context it is of special interest that in vivo the pattern of LH release has been shown to be pulsatile (Midgley and Jaffe, 1971; Gay and Sheth, 1972). In conclusion, the present report described a stable in vitro preparation which is especially useful for the study of LH-RH-LH stimulus-secretion coupling. As a starting preparation for the subfractionation of specific cell types and for subcellular fractionation studies it has the same potential advantage as the other similar preparations. In addition, it provides a substantially increased amount of starting material.
Dissociated cells from porcine pituitary
187
ACKNOWLEDGEMENT This work was carried out during the tenure of an MRC postgraduate studentship and was supported by an MRC project grant (G975/517/C) awarded to C.R.H.
REFERENCES Burton, K. (1956) Biochem. J. 62, 315-323. Edwardson, J.A. and Gilbert, D. (1976) 3. Endocrinol. 68, 197-207. Gay, V.L. and Sheth, N.S. (1972) Endocrinology 90, 158-161. Greenwood, F.C., IIunter, W.M. and Glover, J.S. (1963) Biochem. J. 89,114-123. Hopkins, C.R. (1974) J. Cell Biol. 63,143a. Hopkins, CR. (1977) J. Cell Biol. 73,685-695. Hopkins, C.R. and Farquhar, M.G. (1973) J. Cell Biol. 59,276-303. Johnson, D.C. and Mallampati, R.S. (1975) J. Endocrinol. 66, 13-20. Karnovsky, M.J. (196.5) J. Cell Biol. 27, 137A-138A (abstr.). Kilpatrick, M.J., Collins, W.P. and Newton, J.R. (1976) J. Reprod. Fertil. 46,25-30. Krebs, H.A. (1950) Biochim. Biophys. Acta 4,249. Maghuin-Rogister, G. and Hennen, G. (1973) Eur. J. Biochem. 39,235-253. Maghuin-Rogister, G., Combarnous, Y. and Hennen, G. (1973) Eur. J. Biochem. 39,255-263. Matsuo, H., Baba, Y., Nair, R.M.G., Arimura, A. and Schally, A.V. (1971) Biochem. Biophys. Res. Commun. 43,1334-1339. Midgley, A.R. and Jaffe, R.B. (1971) J. Clin. Endocrinol Metab. 33,962-969. Parvizi, N., Elsaesser, F., Smidt, D. and Ellendorf, F. (1976) J. Endocrinol. 69,193-203. Pomerantz, D.K., Ellendorff, F., Elazaesser, F., Konig, A. and Smidt, D. (1974) Endocrinology 94,330-335. Portanova, R., Smith, 0. and Sayers, G. (1970) Proc. Sot. Exp. Biol. Med. 133,573-576. Purves, H.D. (1961) In: Sex and Internal Secretion, Ed.: W.C. Young (Williams and Wilkins Co., Baltimore) pp. 161-238. Schrey, M.P., Brown, B.L. and Ekins, R.P. (1977) Mol. Cell. Endocrinol. 8, 271-282. Vaitukaitis, J., Robbins, J.B., Neischlag, E. and Ross, G.T. (1971) J. Clin. Endocrinol. 33,988991. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guillemin, R. (1972) Endocrinology 91, 562-572. Williams, J.A. (1976) J. Physiol. (London) 260, 105-I 16.
