Functional properties of porcine-thyroid follicles in suspension

Molecular and Cellular Endocrinology, 28 (1982) 99Elsevier Scientific Publishers Ireland, Ltd.

112

99

FUNCTIONAL PROPERTIES OF PORCINE-THYROID FOLLICLES IN SUSPENSION F.A. KARLSSON,

K. WESTERMARK

Department of Internal Medicine, University of Uppsala (Sweden) Received

18 March

1982; revision

University

received

and B. WESTERMARK Hospital,

and ’ the Wallenberg

17 May 1982; accepted

a Laboratory,

19 May 1982

Porcine thyroid follicle cells were isolated (about 10’ cells per gram of tissue) and cultured in small aggregates in agarose-coated culture dishes. The aggregates became arranged into follicle-like structures capable of iodide uptake and organification. In the presence of TSH (0.2 mu/ml), the aggregation of follicles was enhanced, and iodide uptake as well as TSH-stimulated organification of iodide was increased compared with that in the control. In culture, the active iodide metabolism was gradually lost over a ‘I-day period. This was not due to a disappearance of the TSH-adenylate cyclase system, since CAMP production was retained and stimulated by TSH (half-maximal effect at about 1 mu/ml). Acutely, TSH stimulated iodide efflux and iodide organification (half-maximal effect at about 20 pU/ml). The stimulatory effect on organification was transient: within an hour further organification proceeded as in the absence of hormone. The effects on efflux and organification were already maximal at low TSH concentrations, whereas CAMP production was stimulated with up to SO-fold higher TSH levels, i.e. the findings were typical of spare receptors. In the continued presence of epidermal growth factor, a, potent mitogen for thyroid cells, the follicles increased in size and contained one single large lumen. Their capability to take up and organify iodide was reduced. Keywords:

iodide factor.

uptake;

iodide

orgamfication;

thyrotropin;

epidermal

growth

Suspensions of rat-thyroid epithelial cells free from fibroblasts and endothelial cells can be kept as separate follicles for several days (Nitsch and Wollman, 1980) in agarose-coated culture dishes. The cells maintain an architecture resembling follicles in vivo, and respond morphologically to thyrotropin in vitro. In the absence of TSH, such follicles in culture undergo a morphological change and form multicellular vesicles composed of cells with reversed polarity. This inversion may be prevented by culture in collagen gels (Chambard et al., 1981). Previously, studies of thyroid cells in vitro have used cells in monolayer cultures (Lissitzky et 0303-7207/82/0000-0000/$02.75

0 Elsevier Scientific

Publishers

Ireland,

Ltd.

F.A. Karlsson, K. Wesrermnrk, B. Westermark

100

al., 1971; Rapoport, 1976), or ceils ‘follicularized’ by chronic treatment with thyrotropin or dibutyryl cyclic AMP (Fayet et al., 1970a, b). In this report, we take advantage of the improved technique for isolation of histologically differentiated follicle-like clusters of thyroid cells and describe a method for large-scale preparation and culture of porcine thyroid cells in suspension. The basic characteristics of iodide metabolism, and effects of thyrotropin and epidermal growth factor are presented.

MATERIAL

AND

METHODS

Bovine TSH (Actyron, 3 U/mg) was purchased from Ferring AB, Malmo (Sweden); epidermal growth factor (EGF) from Collaborative Research Inc., Waltham, MA; DNAase and collagenase from Sigma; 3-isobutyl- 1-methylxanthine (IBMX) from Aldrich; Dispase from Boehringer-Mannheim; Percoll solution from Pharmacia AB, Uppsala (Sweden); carrier-free Nalz51 from Amersham (Great Britain); a kit for CAMP determinations from Becton Dickinson, New York. All other reagents were the highest quality commercially available. Isolation of thyroid follicles Fresh porcine thyroid glands were obtained from a local abattoir. Large vessels, connective tissue and fat were removed, and the tissue was sliced into small pieces of a few cubic millimeters. The tissue was digested with collagenase, 1 mg/ml, with the addition on DNAase, 0.01 mg/ml, and Dispase, 5 mg/ml, in buffer containing 20 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), pH 7.4, 146 mM NaCl, 4 mM KCl, 5 mM CaCl, (5 ml per gram wet weight tissue) and swirled on a water-bath for 60 min at 37°C. The digest was passed through a steel net (860 pm pore diameter) and subjected to another 60 min of enzymatic treatment. The cell suspension was then washed once in PBS, (phosphate-buffered saline), resuspended in buffer (1 ml per gram tissue) and layered on top of 5 vol. of 5% Percoll. After about 30 min, follicles had sedimented through the Percoll solution, whereas single cells, erythrocytes and debris remained on top. Sedimented follicles were washed twice with PBS, then suspended to a ‘concentration of about 5 X IO5 cells/ml in F-10 medium with 1% FCS (fetal calf serum), 100 units of penicillin and 50 pg of streptomycin per ml, and cultured in, a humidified atmosphere with 5% CO, on petri dishes or in Falcon flasks precoated with a thin layer of 1% agarose. The yield by this procedure was usually about 10’ cells per gram of tissue.

Porcine ihyroid follicles in suspension

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Iodide uptake, efflux and organification Measurements of iodide uptake, iodide efflux and iodide organification in short-term experiments were carried out with cultured follicles washed once with medium and suspended to a concentration of about l-2 X IO6 cells per ml in F-10 medium with 1% FCS buffered with 20 mM Hepes, pH 7.4. The results were based on at least two experiments. In experiments of iodide uptake, propylthiouracil (PTU) was added to 10 PM to block organification. Suspensions (300 ~1) were then added to plastic tubes (Microfuge 1.5 ml) with medium (30 ~1) containing trace amounts of lz51 (2pCi/ml) and 10 PM potassium iodide. After incuba(Microfuge, 5 set, tion at 37”, uptake was stopped by centrifugation 10000 X g) and two washings of the cells with ice-cold PBS with 1 PM KI, and radioactivity of the cells was counted. Iodide efflx was determined by pre-incubating suspensions of cells for 30 min at 37O. with 10 PM PTU, 1 PM KI, and ‘251 (0.2 pCi/ml) Samples (300 ~1) of cells were then added to medium (30 ~1) with various concentrations of bovine TSH. The samples were mixed rapidly and further incubated for various periods of time. The incubations were stopped by centrifugation (Microfuge, 5 set), cell pellets were washed twice with ice-cold PBS with 1 PM KI, and the radioactivity was counted. Iodide organification was determined by incubating follicle suspensions in F-10 medium with 1% FCS with ‘25I (0.2 pCi/ml) at 37’ with or without additives for various periods of time: for 15-45-min experiments in plastic tubes (Microfuge) in medium supplemented with 20 mM Hepes, pH 7.4, in open air at 37”, for long-term experiments on agarosecoated Linbro dishes (25 mm) in regular medium in a humidified atmosphere containing 5% CO,. After incubationj the cells were washed as in the efflux experiments, the pellets were resuspended and radioactivity was precipitated in 10% trichloroacetic acid (TCA) with 0.5 mg bovine serum albumin as carrier. Other methods Production of CAMP in cell suspensions was determined after the incubation of cells (0.5 ml, about lo6 cells/ml) in F-10 medium supplemented with 20 mM Hepes, pH 7.4, and 0.5 mM IBMX with various concentrations of TSH for 20 min. The cells were centrifugated (Microfuge, 5 set) and extracted with 150 ~1 6% TCA. TCA was then removed by 4 extractions (1 ml each) with water-saturated ether. Cell counting. Attempts to obtain monocellular suspensions from freshly prepared or suspension-cultured thyroid follicles failed, and reliable estimates of cell numbers could not be obtained by cell counting. Cell numbers were therefore determined by comparing the DNA content of a

104

F.A. Karlsson, K. Westermark,

B. Westermark

follicle suspension with that of a known number of thyroid epithelial cells dispersed from monolayer cultures by trypsin treatment as described by Westermark and Westermark (1982). Follicles were spun down and washed twice in PBS (Microfuge 5 set), and the pellet was resuspended in 0.1% sodium dodecylsulfate. The DNA content was determined by the spectrofluorimetric method described by Hinegardner (197 1). Human lymphocytes and porcine thyroid epithelial cells were used as standards. Protein was measured according to Lowry et al. (195 1) with bovine serum albumin as standard. Follicle cultures were viewed in a Leitz Diavert inverted microscope. Micrographs were taken through a 10 X objective. RESULTS Morphology The freshly prepared suspension of thyroid cells contained aggregates of different sizes. In addition, single nucleated cells and erythrocytes were present. When cultured, thyroid follicles of more uniform size formed within the first 24 h. By removing the suspension and washing by low-speed centrifugation (100 X g, 3 mm), a pellet of thyroid follicles with high purity was obtained. From 1 g of tissue about 10’ cells were obtained. After thyroid cells had been isolated from monolayer cultures, DNA and protein contents were determined: they were about 6.5 ug and 0.25 mg per. lo6 cells, respectively. Thyroid follicles cultured in F-10 medium with 1% FCS responded to thyrotropin with a marked change in morphology: large follicular aggregates with a finely vacuolated texture were formed. This response was visible after a few hours and was fully developed within 24 h (Fig. 1). The cells were remarkably sensitive to thyrotropin, a morphological change being detected with only l-10 $l of TSH per ml. When follicles were cultured in the presence of epidermal growth factor (10 ng/ml) a different morphology developed. The follicles did not aggregate but expanded in size and contained a single large lumen. The gradual changes of follicle morphology as a result of hormonal treatment are illustrated in Fig. 1. Iodide uptake The thyroid follicles actively concentrated iodide. Initially, there was a rapid rise in iodide uptake. After about 30 min of incubation, however, the curve leveled off (Fig. 2). Acutely, addition of TSH reduced iodide net uptake owing to the flux phenomenon (see below). EGF (0.1-100 ng/ml), on the other hand, had no acute influence on iodide uptake (data not shown).

Porcine thyroid follicles in suspension

0

40

20

105

60

(mid

Fig. 2. Iodide uptake in porcine thyroid cells. Cells cultured for 1 day in F-10 medium with 1% FCS were incubated in duplicate and uptake was measured (mean-range) as described under Methods. 37°C (0), 0” (X).

Iodide organification Iodide uptake measured in the absence of PTU displayed a curvilinear pattern, with an initial rapid phase of accumulation of iodide within the cells (Fig. 3a), reflecting mainly iodide transport (cf. Fig. 2), followed by a more gradual and linear accumulation of radioactivity reflecting iodide organification. A linear increase in TCA-precipitable 125I occurred during 4-6 h of incubation. When TSH was added, this organification of iodide occurred with different kinetics. A rapid and concentration-dependent increase of TCA-precipitable material took place during the first 15-60 min (Fig. 3, b and c), whereafter a linear increase in organified material continued at a rate similar to that in control cultures (Fig. 3a). The transitory stimulation by TSH of organification might indicate a desensitization at the receptor-cyclase level or a consumption of substrate for iodination. The lower amount of total iodide that accumulated in the presence of TSH compared with the control reflects a stimulation of iodide efflux (see below). Iodide ejflux A notable phenomenon of stimulation of thyroid cells, in vitro, pre-incubated with radioactive iodide, is a rapid efflux of up to 90% of the iodide (Wilson et al., 1968; Fayet and Hovsepian, 1977). The efflux phenomenon seems related to an increase of the intracellular CAMP content. The thyroid follicles cultured in the present study showed a high sensitivity to TSH with respect to iodide efflux (Fig. 4a). A stimulation was detected with as little as l- 10 PU of TSH per ml, and the half-maxi-

F.A. Karlsson, K. Westermark,

B. Westermark

lTSH(ZmU/ml)

1

2 (hours)

3

4

TSH$J/ml:

15

45 (mln)

02

20 TSH

2000

(pU/ml)

Fig. 3. Iodide organification in porcine-thyroid cells cultured for 1 day in F-10 medium with 1% FCS. Cells were incubated in duplicate in medium (1 ml, 2 X IO6 cells) in the absence and presence of 2 mU bovine TSH per ml (a), in duplicate in medium with 20 mM Hepes, pH 7.4 (0.3 ml, 2X 106/ml), and various concentrations of bovine TSH for 15 and 45 min (b). Effect of TSH on organification during the 15-min incubation, shown as a concentration-response curve (c).

ma1 effect occurred at around 20 @/ml, i.e. a hormone concentration within the physiological range. The time-dependency of efflux is shown in Fig. 4b. CAMP production TSH stimulated CAMP production in the cell suspensions with halfmaximal effect at about 1 mU of TSH per ml (Fig. 5). When studied on the day of preparation, sometimes a low and insensitive CAMP response to TSH was observed (data not shown), probably as a result of enzymatic

Porcine lhyroid follicles in suspension

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Buffer

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20pUlmlTSH A

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TSH(pU/ml)

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Fig.4. Iodide efflux of porcine-thyroid cells. Concentration-response curve of TSH on efflux (meantrange) from porcine-thyroid cells incubated for 30 min (a), and time-dependence of efflux (mean of duplicates) in the presence of a high and a low dose of TSH (b).

digestion of follicle cell surface. During the following days the concentration response curve and the amount of CAMP produced in general was not changed (Fig. 6). In some experiments, though, a small decrease in the absolute amounts of CAMP was noted after 7 days of culture. Long-term During

effects of TSH and EGF on iodide uptake and organification

culture

a gradual

decrease

I ; s a I_ .-c” :: E z 0 ;;‘ n 3

80-

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on CAMP formation (mean*SEM, 3 Expts.) F-10 medium with 1% FCS. Incubations were comparison, a dotted dose-response curve of 3c) is included.

108

F.A. Karlsson, K. Westermark,

Day

&YO

6’ 0.02

4 0.2

I

I

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2

20

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Fig. 6. CAMP production in porcine-thyroid cells kept in long-term culture in F-10 medium with 1% FCS. Suspensions of cells were incubated in duplicate with various doses of bovine TSH and CAMP accumulation determined as described under Methods.

TSH 0.2 mu/ml

1

3

7

1 DAYS

3

7

EGF 1Ongh

1

3

7

IN CULTURE

Fig. 7. Iodide organification and accumulation in porcine-thyroid cells in long-term culture in the presence of TSH (0.2 mu/ml) and EGF (10 ng/ml); mean+range. Suspensions (1 ml, about IO6 cells) were cultured in duplicate in F-10 medium with 1% FCS; iodide accumulation (dotted bars) and organification (hatched bars) were determined after 30 min incubations as described under methods.

Porcine thyroid folkles

in suspension

109

fication took place. The capacity to organify iodide was retained at a higher level by culture of the follicles in 0.2 mU TSH per ml. Culture in medium with EGF (10 ng/ml), on the other hand, reduced the extent of organification (Fig. 7). The decrease of iodide metabolism started as the cultures were initiated. After 7 days the cells, irrespective of culture in medium supplemented with TSH, were low-functioning.

DISCUSSION In the present study we used a technique for preparation and culture of thyroid aggregates based on the findings reported by Mauchamp and Fayet (1974) and Nitsch and Wollman (1980). By culturing dense suspensions of porcine-thyroid cells on culture dishes not treated for tissue culture the cells were found not to adhere to the plastic but to form aggregates of follicle-like structures (Mauchamp et al., 1979). In the presence of TSH or dibutyryl CAMP (dcAMP) the aggregation was enhanced, and the cells expressed active iodide metabolism: iodide uptake and organification was qualitatively observable and present up to 5 days in culture. Nitsch and Wollman (1980) reported culture of rat-thyroid aggregates obtained after enzymatic treatment of minced glands and filtration of the digest through a 60-p net. Such aggregates, when kept in suspension on agarose-treated culture dishes, formed histotypically differentiated follicles, which responded morphologically to TSH in an in-vivo-like manner. At low serum concentration the follicles could be cultured for up to 12 days with the characteristic morphology unchanged, although with time an increase in the number of follicles composed of cells with reversed polarity was noticed. The reversal of the normal polarity of the thyroid cells was much enhanced in high serum concentrations. In a recent study, Chambard et al. (1981) found that collagen stabilizes thyroid follicles in suspension to maintain normal polarity. In the present study, we adopted the findings described above to be able to culture porcine-thyroid cells in large amounts for further biochemical studies of thyroid function. For dissociation of the digest we used a net with a porosity of 860~. In our hands, nets with smaller pore size gave a lower yield and a larger proportion of single cells, without increasing the uniformity of the aggregates. A considerable further purification of the suspensions of aggregates was achieved by sedimentation through a Percoll solution. It is well known from earlier studies that thyroid cells in monolayer culture rapidly lose the capacity for iodide uptake and organification as the cultures are established. As described by Fayet and co-workers (1970a, b) thyroid cells, when cultured in the presence of TSH or dcAMP

110

F.A. Karlsson, K. We&v-mark, il. Watermark

and 10% serum, form two-dimensional follicle-like structures. Iodide uptake and organification develop after a few days in culture, with maximal capacity in these respects after about 7 and 4 days, respectively (Lissitsky et al., 197 1); at later times active iodide metabolism disappears. Fayet and Hovsepain (1977) using a system similar to that of Lissitzky and co-workers, reported that follicularized porcine thyroid cells cultured in 20% serum and 0.4 mM dcAMP developed active iodide uptake with a maximum at around day 3. However, iodide organification was not observed at any time during culture. The thyroid-follicle preparations in the present study maintained active iodide transport and organification at initiation of culture, without the need for a conditioning period with TSH or dcAMP added. This culture technique constitutes an advantage compared with the earlier methods described. However, in culture a successive loss of active iodide organification and iodide uptake took place. In the absence of TSH the follicles gradually lost the ability to accumulate iodide over a 7-day period. When cultured with 0.2 mU of TSH per ml, active iodide metabolism was temporarily increased and retained for a prolonged period. EGF, on the other hand, influenced the cells to a reduced iodide uptake and organification: active iodide metabolism disappeared after 3 days in culture. In tissue culture, when freshly isolated thyroid cells start to grow in monolayer the organification capacity is rapidly lost (Kerkof et al., 1964; Lissitzky et al., 1971). Such findings have led to the assumption that polarization of the thyroid cells is of critical importance for iodination of protein to occur. However, because cells kept in culture in follicle suspensions seem to maintain a polarized architecture for periods up to 12 days (Nitsch and Wollman, 1980), provided the serum concentration is low, the loss of active iodide metabolism must be due to other factors as well Possibly the loss of organification capacity reflects a decrease in the peroxide-generating system, as suggested by Rapoport et al. (1977). The decay of organification does not seem to be due to some limiting component of the culture medium as TSH was able to prolong the organification capacity in culture. EGF, in contrast, induced a rapid loss of protein iodination. The subsequent loss of active iodide uptake in culture is likewise not understood. As argued above, cons~tuents of the medium do not seem to be limiting. Further, the loss of active iodide metabolism in the thyroid follicles appears unrelated to the TSH receptor and adenylate-cyclase system, as CAMP production and TSH sensitivity of the cyclase remained during up to 10 days of culture. A TSH-sensitive cyclase is present in thyroid monolayer culture (Rapoport, 1976). Stimulation of iodide efflux and organi~cation was exquisitely sensi-

Porcine

follicles

in suspension

111

tive to TSH, half-maximal activation occurring at about 20 @J/ml, findings that make this system useful for bioassays of TSH and other thyroid-stimulating agents. Because half-maximal stimulation of total CAMP production occurred at about 1 mu/ml, the TSH-receptor adenylate-cyclase system in these cells displays characteristics typical of spare receptors, i.e. full biological response with only fractional occupancy of the receptors. The differences in doses of TSH needed for maximal bioresponse and CAMP production can be explained by a saturation of the CAMP-dependent protein kinase at low levels of CAMP production. Schumacher and Hilz (1978), in studies of isolated bovine thyrocytes, reported half-maximal activation of the CAMP-dependent protein kinase at 0.2 mU of TSH per ml and half-maximal stimulation of total CAMP production at 2 mu/ml. In culture, the morphology of the follicles was modulated in separate ways by the peptides TSH and EGF. The marked and sensitive changes in morphology of the thyroid cells when cultured with TSH is thought to reflect an increase of membrane activity and a stimulation of phagocytic events leading to formation of large intracellular lumina. This response was rapid and is probably the counterpart to the exo- and endocytotic events known to occur early, in viva, after TSH stimulation (Dumont, 1971). In the continued presence of TSH, the isolated follicles tended to form larger aggregates, possibly due to synthesis of an aggregation-promoting factor as described by Giraud et al. (1974) in studies of cells grown on plates. Inoue et al. (1980) reported culture of single thyroid cells in a rotary shaker in the presence and absence of TSH. Without TSH the cells became arranged in small aggregates with reversed polarity - pseudofollicles. With TSH, much larger aggregates of thyroid cells with polarity and morphology of in-vivo-like character were formed. EGF is a potent mitogen for thyroid cells both in monolayer (Westermark and Westermark, 1982) and in suspension cultures of porcine follicles (unpublished observations). The peptide altered the morphology in a way distinct from that induced by TSH. Large follicles were formed with flat cells, suggestive of low functional activity, an interpretation lending support to the reduction of active iodide metabolism noted with EGF-treated cultures. These cultures with large multicellular spheres most likely consisted of follicles with inverse cellular polarity. As illustrated in a report by Mauchamp et al. (1979), vesicles of such appearance are formed in long-term cultures of thyroid follicles in suspension in the absence of CAMP-stimulatory agents. As opposed to the cultures with TSH, formation of large multifollicular aggregates was not produced, indicating that EGF alters the properties of the basal surface of the cells or inhibits the production of an unidentified aggregation factor as discussed above.

112

F.A. Karlsson, K. Westermark,

B. Westermark

In conclusion, in the present report a procedure for isolation and culture of functioning porcine thyroid follicles in suspension has been described, a system that should prove valuabIe for future investigations of the regulation and growth of thyroid cells. In particular, the high sensitivity of these cells to both TSH and EGF makes them useful for studies aimed at clarifying the separate roles of and the interactions of these peptides with respect to thyroid growth and function.

ACKNOWLEDGEMENTS We thank Majstin Wik-Lundberg and Marianne Kastemar for excellent assistance. This work was supported by the Swedish medical Research Council (Project No. 4996) and the Swedish Cancer Society (Project No. 55).

REFERENCES Chambard, M., Gabrion, J., and Macuhamp, J. (1981) J. Cell Biol. 91, 157-166. Dumont, J.E. (1971) in: Vitamins and Hormones, Vol. 29, Eds. R.S. Harris, P.H. Munson, E. Dinfahrsy and J. GIover (Academic Press, New York) pp. 287-412. Fayet, G., and Hovsepain, S. (1977) Mol. Cell. Endocrinof. 7, 67-78. Fayet, G., and Lissitaky, S. (1970) FEBS Lett. 11, 185-188. Fayet, G., Pacheco, H., and Tixier, R. (1970) Bull. Sot. Chim.-Biol. 52,299-306. Giraud, A., Fayet, G., and Lissitsky, S. (1974) Exp. Cell Res. 87, 359-364. Hinegardner, R.T. ( 1971) Anal. Biochem. 39, 197-201. Inoue, K., Koriuchi, R., and Kondo, Y. (1980) Endocrinology 107, 1162-I 168. Karlsson, F.A., Dahlberg, P.A., and WEtlinder, 0. (1981) Acta Endocrinol. 97,60-66. Kerkof, P,k,, Long, P.J., and Chaikoff, I.L. (1964) Endocrinology 74, 170-179. Lissitzky, S., Fayet, G., Giraud, A., Verrier, B., and Torresani, J. (1971) Eur. .I. B&hem. 24, 88-99. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Mauchamp, J., and Fayet, G. (1974) Endocrinol. Exp. 8, 170. Mauchamp, J., Charrier, B., Takasu, N., Margotat, A., Chambard, M., and Dumas, D. (1979a) in: Hormones and Cell Regulation. Eds.: J. Dumont and J. Nunez (North-Holland, Amsterdam) pp. 5 I-68. Mauchamp, J., Margotat, A., Chambard, M., Charrier, B., Remy, L.. and Miche~-B~het, M. (1979b) Cell Tissue Res. 204, 417-430. Nits&, L., and WoIiman, S.H. (1980a) Proc. Natl. Acad. Sci. (U.S.A.) 77, 472-476. Rapoport, B. ( 1976) Endocrinology 98, 1189-I 197. Rapoport, B., Adams, R.J., and Rose, M. (1977) Endocrinology 100, 755-764. Schumacher, M., and Kilz, H. (1978) B&hem. Biophys. Res. Commun. SO, 5 11-518. Westermark, K,, and Westermark, B. (1982) Exp. Cell Res. 138, 47-55. Westermark, B., Karlsson, F.A., and W&tinder, 0. (1979) Proc. Nati. Acad. Sci. (U.S.A.) 76, 2022-2026. Wilson, B., Raghupathy, E., Tonoue, T., and Tong, W. (1968) Endocrinology 83, 877-884.