- Email: [email protected]
[25] Isolation and functional aspects of free luteal cells
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scribed by Schrey et al. ~4The cell column is placed in a water bath and medium (usually KRB with 0.5% BSA and 0.02% glucose) is perfused at a rate of 0.4-0.6 ml/min. When using a Cytodex cell column, cells are seeded directly after dissociation, onto Cytodex beads (contained in bacteriological petri dishes) at a density of 1-2 × 105 cells/mg. Cytodex in Ham's F10 containing 10% fetal calf serum, penicillin, and streptomycin. On the third day, cells are washed with normal KRB solution (i.e., containing 2 mM Ca z÷ and 2 mM Mg 2+) containing 0.5% BSA and 0.02% glucose. The Cytodex beads are then packed into the perifusion apparatus. Usually a smaller column is used (i.e., approximately 20 mg Cytodex) and is perfused at a rate of 0.4-0.5 ml/min.
[25] I s o l a t i o n a n d F u n c t i o n a l A s p e c t s o f F r e e L u t e a l C e l l s By JUDITH L. LUBORSKYand HAROLD R. BEHRMAN
Introduction During each ovarian cycle, changes in the endocrine function of the ovary are dependent on differentiation of follicular cells and luteal cells. Differentiation of ovarian cells is expressed as changes in morphology, cellular metabolism, and their responsiveness to gonadotropins. Ovarian cell function has been studied in vitro with intact follicles and copora lutea, tissue slices, or more recently with isolated granulosa or luteal cells. Luteal cell function in particular has been studied in granulosa cells that have been allowed to luteinize in culture ~-3 or by direct isolation of luteal cells from lutenized tissue. 4-7 Since the function of the corpus luteum must be terminated for a subsequent ovulation to occur or extended for a pregnancy to be maintained, the functional state of corpus luteum is delicately balanced by both i G. L. K u m a r i and C. P. Channing, J. Steroid Biochem. 11, 781 (1979). 2 K. M. H e n d e r s o n and K. P. M c N a t t y , J. Endocrinol. 73, 71 (1977). 3 R. W. T u r e c k and J. F. Strauss, J. Clin. Endocrinol. Metab. 54, 367 (1982). 4 T-C. Liu and J. Gorski, Endocrinology 88, 419 (1971). 5 D. G o s p o d a r o w i c z and F. Gospodarowicz, Endocrinology 90, 1427 (1972). 6 D. H. W u , W. G. Wiest, and A. C. Enders, Endocrinology 98, 1378 (1976). 7 K. R. S i m m o n s , J. L. Caffrey, J. L. Phillips, J. H. Abel, and G. D. Niswender, Proc. Soc. Exp. Biol. Med. 152, 366 (1976).
METHODS IN ENZYMOLOGY,VOL. 109
Copyright © 1985by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2
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tropic and lytic endocrine regulators. Isolated luteal cell preparations were developed in order to examine cell regulation independent of variables present in vivo, such as changes in blood flow and endogenous hormone concentrations. Isolated cells can be uniformly and rapidly treated with drugs and hormones. Their use does not have the problems of sample variation and nonuniform exposure of cells that are inherent in isolated corpora lutea and luteal tissue slices. Luteal cells isolated from s h e e p , 7-9 cow, 5,1° pig, H rat, 6,n-14 guinea pig, 15 rabbit, 4 monkey, 16 and human corpora l u t e a 17-2° have been used to study aspects of their cellular morphology, biochemical responses and hormone receptor function. General Methods of Luteal Cell Isolation In general, methods of luteal cell isolation employ enzymatic treatment of minced or sliced luteal tissue with collagenase (to break down connective tissue) and deoxyribonuclease (DNase) (to break down released DNA that may cause cells to clump). Additional enzymes such as hyaluronidase or Pronase are also used in s o m e l a b o r a t o r i e s . 5,6,1°,1z Enzyme treatment is usually coupled with mechanical treatment to further disperse cells. A systematic study of conditions for rat luteal cell isolation examined duration of incubation, and combinations of variable concentrations of collagenase, trypsin, and hyaluronidase, as well as bovine serum albumin (BSA) and Ca 2+ concentrations necessary to maintain maximum viability, morphology, and steroidogenesis. A final combination of all three enzymes (2940 U trypsin, 2320 U collagenase, and 16,900 U hyaluronidase/ l0 ml of medium) plus 0.5% BSA and 3.3 mM Ca 2+ was chosen. 6 8 R. J. Rodgers and J. D. O'Shea, Aust. J. Biol. Sci. 35, 441 (1982). 9 C. E. Ahmed, H. R. Sawyer, and G. D. Niswender, Endocrinology 109, 1380 (1981). i0 j. Ursely and P. Leymarie, J. Endocrinol. 83, 303 (1979). II M. Lemon and M. Loir, J. Endocrinol. 72, 351 (1977). n R. F. Wilkinson, E. Anderson, and J. Aalberg, J. Ultrastruct. Res. 57, 168 (1976). 13 H. R. Behrman, S. L. Preston, and A. K. Hall, Endocrinology 107, 656 (1980). 14 j. L. Luborsky and H. R. Behrman, Mol. Cell. Endocrinol. 15, 61 (1979). 15 M. C. Richardson and M. J. Peddle, J. Reprod. Fertil. 66, 117 (1982). 16 R. L. Stouffer, W. E. Nixon, B. J. Gulyas, D. K. Johnson, and G. D. Hodgen, Steroids 27, 543 (1976). i7 M. T. Williams, M. S, Roth, J. M. Marsh, and W. J. LeMaire, J. Clin. Endocrinol. Metab. 48, 427 (1979). is M. C. Richardson and G. M. Masson, J. Endocrinol. 87, 247 (1980). 19 L. T. Goldsmith, M. Essig, P. Sarosi, P. Beck, and G. Weiss, J. Clin. Endocrinol. Metab. 53, 8980 (1981). 2o M. L. Polan, A. H. DeCherney, F. P. Haseltine, H. C. Mezer, and H. R. Behrman, J. Clin. Endocrinol. Metab. 56, 288 (1983).
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Luteinizing hormone (LH) receptor binding and function varies with different preparations of collagenase 21,22; these changes are thought to be due to variations in trypsin activity. However, trypsin improved separation of endothelial and large and small luteal cells of ovine corpus luteum, and the luteal cells retained their ability to respond to LH. a A systematic study of tryspin effects on primate luteal cells showed that a brief exposure (10 min) did not change human chorionic gonadotropin (hCG)-induced progesterone secretion, while a prolonged exposure (3 hr) significantly reduced both basal and hCG-stimulated progesterone secretion. 23 Variations in cell isolation with different collagenase preparations may be due to contamination with clostripain; collagenase free of clostripain was reported to produce uniform and consistent preparations of pancreatic cells. 24 After cell dispersion with enzyme(s), intercellular junctions are not completely separated. Frequently, some cells are well separated while the majority are still in small clumps or have attached debris from broken cells. Recently, additional treatment with trypsin and/or a divalent cation chelator was introduced to facilitate the separation of intercellular junctions. 8,14 Trypsin in low Ca 2+ medium containing ethyleneglycol bis(flaminoethylether) (EGTA) separates intercellular junctions. 8 A brief exposure to low Ca 2+ medium containing ethylenediaminetetraacetic acid (EDTA) also improves separation of luteal cells.14 Prolonged exposure (greater than 3-5 min) or high concentrations of EDTA (>2 mM) are deleterious to cell f u n c t i o n . 14 A sequential treatment with EDTA and hypotonic sucrose has been reported to enhance separation of gap junctions between granulosa cells. 25 Cell suspensions have been further enriched by separation on gradients of Ficoll, 8,1° Percoll, 13,26s u c r o s e , 9 o r B S A . 11,12 These gradients were used simply to remove membrane debris and dead c e l l s 13'26 o r to obtain cells of different sizes. 8,1°-12 Removal of membrane debris and broken cells is particularly important for morphological and receptor binding studies but does not appear to be essential for studies of cellular metabolism. Final yields of cells are about 1-10 × l0 7 cells/g of luteal tissue.4,5,7,8,10,16,26 2~ M. L. Dufau and K. J. Catt, this series, Vol. 37, p. 252. 22 j. L. Luborsky and H. R. Behrman, unpublished observations (1982). 23 B. J. Gulyas, L. C. Yuan, and G. D. Hodgen, Steroids 35, 43 (1980). 24 G. R. Gunther, G. S. Schultz, B. E. Hull, H. A. Alicea, and J. D. Jamieson, J. Cell Biol. 75, 368 (1979). 25 K. L. Campbell and A. R. Midgley, J. Cell Biol. 75, 246 (1977). 26 B. C. McNamara, C. E. G. Cranna, R. Booth, and D. A. Stansfield, Biochem. J. 192, 559 (1980).
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Morphological Aspects of Isolated Luteal Cells Isolated luteal cells retain the morphological characteristics of steroid secreting cells after isolation. They contain abundant mitochondria, variable amounts of lipid droplets, and an extensive smooth endoplasmic reticulum, similar to cells in vioo. 12'27'28 Two size ranges of isolated cells, of 15-25 and 30-50/zm in diameter, from monkey, rat, pig, sheep, and cow corpus luteum have been reported, s,1°-12,29 A comparison of cell types in vivo and in vitro suggests that the larger cells are extremely fragile and that there is a lower recovery after isolation than for the smaller cells.S, 10,12 Other cells, such as endothelial and blood cells, are found in isolated luteal cell preparations, and these can be reduced by further purification) It was suggested by several authors 8,11 that the small and large cell types originate from thecal and granulosa cells, respectively; it has also been suggested 12that these cells are part of a continuum of differentiating luteal cells. The actual source of the different size cells has not been established but it is clear that they differ morphologically and metabolically. The large cells contain more progesterone, are less responsive to LH, have more surface projections, and contain more mitochondria. 8,1°-n,z9 Isolated luteal cells have been maintained in culture for extended periods of time. Histochemical analysis of bovine luteal cells cultured for seven days showed lipid material that stained with Sudan Black and Oil Red, as well as 3fl-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase, characteristic of luteal cells. 3° Ultrastructural analysis indicates that cells retain their typical steroidogenic features, 12,29but small cells tend to lose their lipid droplets by extrusion during culture. J2 LH receptors have been localized on surfaces of rat 14 and monkey 3~ luteal cells with a ferritin-LH and a horseradish peroxidase-hCG conjugate, respectively. In both cases, receptors are located at intervals over the entire cell surface. Rate luteal cells in suspension do not appear to maintain their surface polarity to the same degree as found in vivo, with a basal smooth surface and an apical microvillus surface containing L H receptors. 28 Cultured monolayers of rat luteal cells 12may be more similar to cells in vivo but this has not been documented with respect to receptor localization. Primate luteal cells have been reported to lack morphological 27 A. K. Christensen and S. W. GiUim, in "The Gonads" (K. W. McKerns, ed.), p. 415. North-Holland Publ., Amsterdam, 1969. 28 W. Anderson, Y.-H. Kang, M. E. Perotti, T. A. Bramley, and R. J. Ryan, Biol. Reprod. 20, 362 (1979). 29 B. J. Gulyas, R. L. Stouffer, and G. D. Hodgen, Biol. Reprod. 20, 779 (1979). 3o D. Gospodarowicz and F. Gospodarowicz, Exp. Cell Res. 75, 353 (1972). 31 B. J. Gulyas, S. Matsuura, H.-C. Chen, L. C. Yuan, and G. D. Hodgen, Biol. Reprod. 25, 609 (1981).
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polarity both in vivo 32 and in vitro31; this has not been correlated with studies of receptor localization. Responses of isolated luteal cells to hormones and growth factors involve a morphological component or shape change. In general, cells appear more spherical and polygonal when treated with LH (hCG) or dibutyryladenosine 3' : 5'-cyclic monophosphate [(Bu)2cAMP] and during enhanced steroidogenic activity. 3,33,34 Cell shape is probably determined by microtubules and microfilaments since shape changes are inhibited by colcemid, vinblastin, or cytochalasin B. 33 In the absence of hormone or after its removal, more flattened, oblong cells are observed. 3 It is possible that L H may control and maintain morphological differentiation. Glucocorticoids and testosterone inhibit cell growth but progesterone does n o t . 33 In the presence of fibroblast growth factor (FGF) and epidermal growth factor (EGF), cell growth is stimulated and cells become more flattened. 35 Cells in suspension culture have not been reported to undergo shape changes. Biochemical/Endocrine Function of Isolated Luteal Cells Isolated luteal cells have been used in numerous studies to examine the regulation of steroidogenesis by LH. The functional capacity of freshly isolated luteal cells has been reported to reflect their functional capacity in vivo with respect to progesterone production in response to L H . 16'26'36 Effects of L H on human luteal cells during the cycle 34 and primate luteal cells during pregnancy 36 parallel that seen in vioo. 36 LH stimulated progesterone production and increases in adenosine 3' : 5"-cyclic monophosphate (cAMP) levels and cAMP binding to protein kinase 37,38 are directly correlated, Aminoglutethimide inhibits cholesterol side chain cleavage and progesterone production from cholesterol esters 26,39 and compactin blocks cholesterol biosynthesis in vitro, z6 In the presence of aminoglutethimide but not compactin, rapid (short-term) responses of cells to L H were blocked, suggesting stored cholesterol but not cholesterol biosynthesis is required for acute responses to L H . 26 Also, 32 B. J. Gulyas, Am. J. Anat. 139, 95 (1974). 33 D. Gospodarowicz and F. Gospodarowicz, Endocrinology 75, 458 (1975). 34 K. Kamei, Nippon Sanka Fujinka Gakki Zasshi 34, 261 (1982). 35 D. Gospodarowicz, C. R. I11, and C. R. Birdwell, Endocrinology 100, 1121 (1977). 36 R. L. Stouffer, W. E. Nixon, B. J. Gulyas, and G. D. Hodgen, Endocrincology 100, 596 (1977). 37 G. B. Sala, M. L. Dufau, and K. J. Catt, J. Biol. Chem. 254, 2077 (1979). 38 W. Y. Ling, M. T. Williams, and J. M. Marsh, J. Endocrinol. 86, 45 (1980). 39 M. Lemon and P. Mauleon, J. Reprod. Fertil. 64, 315 (1982).
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inhibition of cholesterol ester cleavage by cannabinoids reduced progesterone production. 4° Ca 2÷ has been shown to augment LH-dependent steroidiogenesis. 41,42In isolated cells L H action is blocked by 2-bromopalmirate indicating involvement of fatty acid oxidation. 43 L H selectively stimulates incorporation of 32p into phosphatidic acid, phosphatidylinositol, and polyphosphoinositides. 44 A direct action of biogenic amines acting at separate receptors, on L H stimulated progesterone production was s h o w n . 4547 Biogenic amine action on cAMP accumulation in isolated luteal cells was shown to decrease with increasing corpus luteum age. 47 Danazol was reported to inhibit LH, cholera toxin, and (Bu)2cAMP-stimulated progesterone production but not corresponding increases in cAMP, suggesting it acts distal to the hormone receptor site. 48 Responsiveness of the two cell types to L H appears to differ. Large cells produce higher basal levels of progesterone but are less responsive to L H 8,1°,1~ and there are less L H receptors on large cells. 31 In addition, in a continuous perfusion system, experiments with large and small cells "in series" showed that small cells can increase the amount of progesterone produced by large cells but not the reverse. H During maintenance of primate and other luteal cells in long-term culture, progesterone and estrogen production gradually decline ~9,29and this is not prevented by addition of thyroxine, insulin, cortisol, or cholesterol. 49 Also, bovine serum was reported to decrease progesterone production in bovine luteal cells) ° Lipoproteins increase progesterone production in cultured rat, 5~ bovine, 5° and human 3 luteal cells. Luteal macrophages were reported to maintain progesterone production of mouse luteal cells. 52
40 S. Burstein, S. A. Hunter, and T. S. Shoupe, Res. Commun. Chem. Pathol. Pharmacol. 24, 413 (1979). 41 N. Kamiya, Nippon Naibunpi Gakkai Zasshi 58, 833 (1982). 42 L. J. Dorflinger, P. J. Albert, A. T. Williams, and H. R. Behrman, Endocrinology 114, 1208 (1984). 43 C. H. Tan and J. Robinson, Life Sci. 30, 1205 (1982). 44 j. S. Davis, R. V. Farese, and J. M. Marsh, Endocrinology 109, 469 (1981). 45 R. C. Rhodes and R. D. Randel, Comp. Biochem. Physiol. 72, 113 (1982). 46 A. W. Jordan, J. L. Caffrey, and G. D. Niswender, Endocrinology 103, 385 (1978). 47 E. Norjavaara, G. Selstam, and K. Ahren, Acta Endocrinol. (Copenhagen) 100, 613 (1982). 48 M. Menon, S. Azhar, and K. M. Menon, Am. J. Obstet. Gynecol. 136, 524 (1980). 49 B. J. Gulyas, L. C. Yuan, and G. D. Hodgen, Biol. Reprod. 23, 21 (1980). 50 j. L. Pate and W. A. Condon, Mol. Cell. Endocrinol. 28, 551 (1982). 51 S. Azhar and K. M. Menon, J. Steroid Biochem. 16, 175 (1982). 52 T. M. Kirsch, A. C. Friedman, R. L. Vogel, and G. L. Flickinger, Biol. Reprod. 25, 629 (1981).
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Prolactin (PRL) maintains LH receptor and steroidogenic function in rat luteal cells in v i v o . 53 In vitro, PRL maintains basal steroidogenesis in rat luteal cells without a specific stimulation of its o w n ) ,54 PRL also suppresses 20a-steroid dehydrogenase in the rat. 6,55 Its role in human luteal function is not as clear. In humans, PRL was reported to have a dual, age-dependent, luteolytic and luteotropic action, without an effect on responses to h C G ) 6 Only a minimal effect of PRL was seen on primate luteal cells) 7 Prostaglandin-F2~ (PGFz~) has been shown to be luteolytic in a variety of animals. In some studies the newly formed corpus luteum is refractory to PGFz~ ,z,~5,58but later, a marked inhibition of LH stimulated cAMP and progesterone production by PGF2~ in isolated luteal cells 59-62 occurs. PGFz,~ appears to have no effect on bovine luteal cells 63but is luteolytic in vivo. 64 In early luteal cells of humans, in the presence of no LH, or low levels of LH, PGFz~ stimulated progesterone production) 8 From these studies, PGF2~ acts directly and acutely at the LH receptor, as well as at other sites such as on steroidogenesis6°,62 and in vivo on delivery of hormone from blood. 64 Estrogen is thought to be involved in luteolysis of human, primate, and bovine corpus luteum. Direct inhibition by estrogen of LH and (Bu)zcAMP stimulated progesterone production but not LH-stimulated cAMP production suggests that estrogen does reduce bovine, 65 human, 66,67 and primate 68 luteal cell function, but at a point beyond LH 53 H. R. Behrman, D. L. Grinwich, M. Hichens, and G. D. Macdonald, Endocrinology 103, 349 (1978). 54 K. Shiota and W. G. Wiest, Adv. Exp. Med. Biol. 112, 169 (1979). 55 M. Lahav, S. A. Lamprecht, A. Amsterdam, and H. R. Lindner, Mol. Cell. Endocrinol. 6, 293 (1977). 56 T. Sawada, Nippon Sanka Fujinka Gakki Zasshi 34, 2212 (1982). 57 R. L. Stouffer, J. L. Coensgen, and G. D. Hodgen, Steroids 35, 523 (1980). 58 R. L. Stouffer, W. E. Nixon, and G. D. Hodgen, Biol. Reprod. 20, 897 (1979). 59 j. p. Thomas, L. J. Dorflinger, and H. R. Behrman, Proc. Natl. Acad. Sci. US.A. 75, 1334 (1978). 6o A. W. Jordan, Biol. Reprod. 25, 327 (1981). 61 A. K. Hall and J. Robinson, J. Endocrinol. 81, 157 (1979). 62 H. R. Behrman, J. L. Luborsky, C. Y. Pang, K. Wright, and L. J. Dorflinger, Adv. Exp. Biol. Med. 112, 557 (1979). 63 j. E. Hixon and W. Hansel, Adv. Exp. Med. Biol. 112, 613 (1979). 64 H. R. Behrman, Annu. Rev. Physiol. 41, 685 (1979). 65 M. T. Williams and J. M. Marsh, Endocrinology 103, 1611 (1978). 66 M. Thibier, N. el-Hassan, M. R. Clark, W. J. LeMaire, and J. M. Marsh, J. Clin. Endocrinol. Matab. 50, 590 (1980). 67 M. Richardson and G. M. Masson, J. Endocrinol. 91, 197 (1981). 68 R. L. Stouffer, L. A. Bennett, and G. D. Hodgen, Endocrinology 106, 519 (1980).
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induced increases in cAMP. 65,66 Isolated primate luteal cells also synthesize and secrete estrogen and respond to LH with an increase in estrogen production; neither estrogen production nor the estrogen/progesterone ratio increases with the age of the corpus luteum. 68 Testosterone has been reported to inhibit LH action in porcine 69 but not human 67 luteal cells. Recently, a direct inhibitory action of luteinizing hormone releasing hormone (LHRH) on rat luteal cells was shown. L H R H binds to specific receptors on luteal cells and inhibits LH-induced increases in cAMP accumulation and progesterone secretion. 7°-73L H R H stimulates incorporation of 32p into phospholipid. TM The antigonadotropic action of L H R H is similar to that for PGF2~ and these agents appear to increase intracellular C a 2+ levels. 42 At this time, the physiological significance of L H R H receptors on ovarian cells is not established. L H R H or an LHRH-like peptide may be one of several intraovarian factors that regulate ovarian function. 75 In this regard follicular fluid injected into monkeys reduced progesterone production and responsiveness to L H in subsequently isolated luteal cells .76 The corpus luteum has been identified as the source of the peptide hormone, relaxin. Long-term cultures of isolated rat 77 and human ~9luteal cells have been used to examine the factors regulating relaxin secretion. Its release in rats is stimulated by various combinations, but not singly, of LH, PRL, progesterone, epinephrine, and estrogen; (Bu)2cAMP did not stimulate relaxin secretion. The role of microfilaments and microtubules in steroidogenesis has been investigated in isolated luteal cells. Cytochalasin B and D decreased progesterone secretion in response to LH, (Bu)zcAMP, cholera toxin and 8-bromo-cAMP in rat luteal cells without a reduction of cAMP levels. 78 Cytochalasin B also decreased L H and (Bu)zcAMP stimulated progesterone secretion in bovine luteal cells. 79 Microtubule inhibitors had little effect on steroidogenesis when applied directly to luteal cells TM but if injected in vivo, progesterone production in subsequently isolated cells 69 M. G. Hunter, J. Reprod. Fertil. 63, 471 (1981). 70 A. K. Hall and H. R. Behrman, J. Endocrinol. 88, 27 (1981). 7~ H. R. Behrman, S. L. Preston, and A. K. Hall, Endocrinology 107, 656 (1980). 72 j. Massicotte, J. P. Borgus, R. Lachance, and F. Labile, J. Steroid Biochem. 14, 239 (1981). 73 R. N. Clayton, J. P. Harwood, and K. J. Catt, Nature (London) 282, 90 (1979). 74 C. K. Leung, V. Raymond, and F. Labile, Endocrinology 112, 1138 (1983). 75 A. T. Williams and H. R. Behrman, Semin. Reprod. Endocrinol 1, 269 (1983). 76 R. L. Stouffer and G. D. Hodgen, J. Clin. Endocrinol. Metab. 51, 669 (1980). 77 L. T. Goldsmith, H. S. Grob, and G. Weiss, Ann. N.Y. Acad. Sci. 380, 60 (1982). 78 S. Azhar and K. M. Menon, Biochem. J. 194, 19 (1981). 79 M. T. Williams and J. M. Marsh, Adv. Exp. Biol. Med. 112, 549 (1979).
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was decreased 8° without morphological evidence of microtubule disruption. Thus, cellular microfilaments were suggested to be involved in hormone regulated steroidogenesis. Hormone Receptor Function of Isolated Luteal Cells L H receptor binding studies were employed to quantitate specific properties of hormone-receptor interaction in relation to cellular function. Binding of [125I]hCG to bovine luteal cells and membranes was compared and it was concluded that the enzymatic treatment used to isolate cells did not change L H receptor binding kinetics or affinity. Binding to bovine cells was saturable, rapid and temperature dependent with an estimated binding capacity of 5 x 10 4 receptors/cell and an equilibrium binding constant (KD) of 5.3 × 10-l° M. 8j Prostaglandin El (PGEI) binding to bovine luteal cells was rapid, saturable, reversible, and specific with a KD = 2.4 nM and 1.8 × 105 receptors/cell, with most properties similar to receptors in isolated membranes. 82 Binding of [125I]hCG to cells from rats desensitized with hCG was compared to cells from untreated rats. The desensitized or refractory state was accompanied by a significant loss of hCG binding concomitant with decreased cAMP and progesterone production in response to LH. 83 This reduced steroid production was also due to an additional lesion in steroid biosynthesis, rather than to a simple loss of LH receptors. In studying recovery from desensitization, it was found that a full complement of L H receptors was required for maximum sensitivity (i.e., an L H response is obtained, but more hormone is needed to obtain a maximum response). Similarly, cells isolated from primate corpus luteum after hCG exposure were refractory to hCG. 84 Rat luteal cells increased their sensitivity to L H (cAMP) without a change in L H receptor binding during prolonged culture. 7° This increased sensitivity was impaired by early exposure to LH, PGFE~, LHRH, or an L H R H analog. Since there was no change in LH receptor binding, it was suggested that receptor desensitization may precede receptor loss and may be due to rapid uncoupling of the receptor and adenylate cyclase. A similar inhibition of L H action by PGF2~ was seen in in vivo studies with PGF2~ ,53,62 but this may be due to additional action of PGF2~ in vivo. 80 S. Azhar and E. Reaven, Am. J. Physiol. 243, E380 (1982). 81 S. Papaionannou and D. Gospodarowicz, Endocrinology 97, 114 (1975). 82 M. T. Lin and C. V. Rao, Mol. Cell. Endocrinol. 9, 311 (1978). 83 j. p. Harwood, M. Conti, P. M. Conn, M. L. Dufau, and K. J. Catt, Mol. Cell. Endocrinol. 11, 121 (1978). 84 R. L. Stouffer, W. E. Nixon, and G. D Hodgen, Biol. Reprod. 18, 858 (1978).
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Mechanisms for changes in the L H receptor content of cells have been studied. Loss of receptors from the ovine luteal cell surface occurred with a hi2 of 9.6 hr and about 85% was lost within 24 hr. Cell-associated radioactivity increased up to 4 hr, was maintained, and then decreased during 12-24 hr. This time course is similar to that seen in vivo during down-regulation. 6z Receptor loss was three times faster than membrane protein turnover. A majority of bound [125I]hCG was internalized and degraded. 85 Inhibitors of transglutaminase (inhibits receptor clustering) did not inhibit internalization of [125I]hCG86 but did produce an intracellular accumulation of [lzSI]hCG and an inhibition of hCG degradation. HCG binding may result in an increase in L H receptor aggregation, but LH receptors are not necessarily internalized as large clusters in coated pits. 14 Furthermore, inhibition of protein synthesis did not decrease the total number of L H receptors per cell, although it did decrease LH receptor degradation and the number of receptors on the cell surface. Thus, synthesis of new receptors is not required for continued binding and internalization, and it was suggested that receptors are recycled. 85 The effects of EGF and FGF and their binding to cells is similar to that in vivo; bovine luteal cells lose their proliferative response to EGF but retain their responsiveness to FGF. 87 Analysis of EGF binding showed that both granulosa cells and luteal cells bind EGF, but in luteal cells there is no mitogenic respnse. In fact, EGF receptors increased with luteinization (0.2 to 1 x 105). Down-regulation, or loss of EGF receptors in response to EGF, occurred in both granulosa and luteal cells. Low concentrations of EGF produced a greater loss of receptors than those that were occupied. 87 Preparation of Free Luteal Cells Animals. Ovaries from gonadotropin primed rats, given 50 IU pregnant mares serum (Gestyl, Organon) and 25 IU hCG (A.P.L., Ayerst) 64 hr later, were removed 4-8 days after hCG injection. Enzymatic Treatment. Ovaries are rapidly trimmed in medium A (GIBCO No. 320-1380 containing 0.1% BSA), weighed, and minced with a clean, sharp razor. The fragments are washed to remove excess blood cells. The fragments are added to a 25-ml Erlenmeyer flask containing 2000 IU collagenase (Worthington Biochemical Corporation) and 3000 IU DNase (Worthington Biochemical Corporation) per gram of tissue in 5 ml of medium A. An additional 5 ml of medium A is added and the solution is s5 D. E. Suter and G. D. Niswender, Endocrinology 112, 838 (1983). 86 C. E. Ahmed and G. D. Niswender, Endocrinology 109, 1388 (1981). 87 I. Vlodavsky, K. D. Brown, and G. Gospodarowicz, J. Biol. Chem. 253, 3744 (1978).
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gently mixed and aerated with 95% 02/5% CO2 for 1 min. The tissue is incubated for 1 hr at 37° with agitation (Dubnoff incubator, I00 cycles/ min). During the incubation the fragments are pipetted five or six times with a large bore, Pasteur pipet with a flame polished tip. At the end of incubation the tissue is centrifuged (100 g, 5 min) in a 15-ml round bottom centrifuge tube and the supernatant discarded. Low Ca 2÷ Treatment. After enzyme treatment, the majority of cells are in small clumps. Luteal cells are more difficult to separate than granulosa cells since they develop extensive intercellular junctions during luteinization.12 To improve separation of these junctions, cells are resuspended in 10 ml of medium A containing 1 mM EDTA. The solution is pipetted gently about six times to resuspend the pellet, with a Pasteur pipet with a flame polished tip, incubated for 2 min (22°), centrifuged (100 g, 5 min), and the supernatant discarded. The cells are extremely fragile in low Ca 2÷ conditions; additional time or increased concentrations of EDTA will damage cells. Mechanical Dispersion. The pellet is resuspended in 3 ml of medium A. Cells are mechanically dispersed by repeated pipetting with a flame polished Pasteur pipet, allowing clumps to settle (about 0.5 min) and removing the supernatant containing isolated cells. Cells are filtered through nylon mesh (Nyten, Tetko) supported by a disposable plastic funnel into a clean 15-ml polypropylene centrifuge tube. The pellet is resuspended in l ml of medium A and the procedure repeated until only a few small clumps of cells and connective tissue remain. Pooled cells are centrifuged (100 g, 5 min). This process minimizes mechanical manipulation of isolated cells. Cells may be used without further processing at this point; cells are resuspended in medium B (GIBCO No. 380-2360 containing 0.1% BSA) and preincubated 60 min before use. Alternatively, the cells are resuspended in 2 ml of medium A and further enriched on a Percoll column to remove membrane debris, some red blood cells, and nonfunctional luteal cells. Enrichment o f Luteal Cell Preparation. A discontinuous Percoll gradient is prepared by layering 3 ml each of 40, 30, 20, and 10% (bottom to top) Percoll solutions in a 15 ml conical polypropylene centrifuge tube. A Percoll stock solution is prepared from 9 ml of Percoll plus 1 ml of medium C (GIBCO No. 199, 10×) and is diluted with medium A. The cell suspension is layered on top and centrifuged through the gradient (100 g, 30 min). '?he excess medium is removed and cells at the 10-20% and 2030% interfaces are combined. Cells are rinsed with medium B (100 g, 10 min) and resuspended to about 2 to 4 x 10 6 cells/ml in medium B. One column will separate cells from 14 ovaries.
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Cell Yield and Viability. The cells are counted in a hemocytometer. Generally this method yields about 1-2 x 10 6 cells/ovary. Before Percoll enrichment the preparation contains about 50-60% luteal cells and after PercoU enrichment it contains about 70-80% luteal cells. Cell viability is about 90% when tested for the ability of cells to exclude trypan blue. 88 Similar but more quantitative results are obtained when the LDH content (LDH kit, Statzyme, Worthington) of medium is tested before and after 90 min of incubation at 37°. Total protein synthesis, as measured by incorporation of [14C]amino acids into perchloric acid precipitable proteins, 89 remains constant over 24 hr. Methods of Incubation and Preparation of Samples for Analysis. For short-term experiments luteal cells are incubated in 12 × 75 glass tubes in medium B at 105 to 10 6 cells/ml/tube. The incubation is terminated by immersion of the tubes in a boiling water bath for 10 min. Samples are then frozen until analysis by radioimmunoassay for cAMP 9° or progesterone. 91 For hormone binding experiments 2 ml of ice cold medium B is added, the cells are centrifuged (1000 g, 15 min), the supernatant aspirated, and the radioactivity in the cell pellet counted. For longer term experiments, cells are cultured for up to 48 hr in disposable plastic tissue culture trays (CoStar) with l06 cells/well/0.5 ml in medium B containing 50 IU/ml myocostatin, 100 IU/ml penicillinstreptomycin, and 10% fetal calf serum (GIBCO). The cells are cultured on individual nitrocellulose filters (Millipore, HAWG 01300) soaked in medium 2 for 16 hr at 4o.70 The presoaked filters are held in place by narrow plastic rings. For measurement of secreted cAMP, the medium is transferred to test tubes with 1 mM theophylline and frozen until they are analyzed. To measure intracellular cAMP, the filter with attached cells is placed in 1 ml of medium B containing 1 mM theophylline and heated in a boiling water bath for 10 min. For measurement of [J25I]hCG binding, [~25I]hCG of high specific activity 9~ is incubated with cells on filters and the medium removed, the filters rinsed, and placed in test tubes and the radioactivity counted. as H. J. Phillips, in "Tissue Culture: Methods and Applications" (P. F. Kruse and M. K. Patterson, eds.), p. 406. Academic Press, New York, 1973. 89 R. J. Mans and E. D. Novelli, Biochem. Biophys. Res. Commun. 3, 540 (1960). ~ A. L. Steiner, in "Methods of Hormone Radioimmunoassay" (B. M. Jaffee and H. R. Behrman, eds.), p. 3. Academic Press, New York, 1979. 91 G. P. Orczyk, M. Hichens, G. Arth, and H. R. Behrman, in "Methods of Hormone Radioimmunoassay" (B. M. Jaffee and H. R. Behrman, eds.), p. 701. Academic Press, New York, 1979. 9~ M. Hichens, D. L. Grinwich, and H. R. Behrman, Prostaglandins 7, 449 (1979).
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In experiments where adenosine triphosphate (ATP) production was measured, incubations are carried out in 0.5 ml of medium B with 1-3 x 105 cells/tube. Reactions are stopped by the addition of 0.1 ml of 50% trichloroacetic acid (TCA) and samples are stored at 4° until they are analyzed. The TCA treated cells are extracted four times with 3 ml of diethyl ether, the pH adjusted to 7 with 0.02 N NaOH, and ATP assayed by the luciferin-luciferase assay (Sigma, L0663). 93 For ultrastructural analysis~4 the cells are rinsed gently with medium B and after centrifugation (100 g, 5 min), 1 ml of 2% glutaraldehyde, in 0.067 M cacodylate buffer at pH 7.4 containing 0.5% tannic acid (350 mOsM) is added to the pellet for 20-30 min at 23°. With a large bore plastic pipet the cell pellet is transferred to a 1.5-ml microfuge tube, spun briefly (>3 sec) in a Beckman microfuge B and rinsed with 0.1 M cacodylate buffer containing sucrose (3 l0 mOsM). One-half milliliter of 2% OsO4 is added for 1 hr at 22°. Buffer containing sucrose is then added and cells are centrifuged (<3 sec). The pellet is rinsed again. During this procedure the cells are not handled except to "lift" the pellet with a small flat wooden spatula. The tips of each microfuge tube (5 mm length) are cut with a sharp clean razor and the pellet scooped onto filter paper tied over the end of a polyethylene test tube (1.0-1.5 cm opening) open at both ends. The pellet is covered with a second piece of filter paper, which is tied with surgical thread. The tubes are carried through routine dehydration (5 min each in an ethanol series, 2 hr in 50 : 50 propylene oxide : resin, overnight with two changes in 100% resin) and embedded without handling the cells. After infiltration, the upper layer of filter paper is removed and the cell pellet lifted (scooped) into a drop of resin in the bottom of an embedding capsule which is then filled with resin. For Epon 812 (Fullam) or Embed-812 (Polysciences), capsules were polymerized overnight at 60°. Sections were examined in a Hitachi 12 electron microscope. Applications of Dispersed Luteal Cells The dispersed rat luteal cell preparation contains greater than 80% luteal cells with the remainder endothelial and blood cells. Based on light microscopic (Fig. 1) and electron microscopic (Fig. 2) observation there are two sizes of luteal cells. Their average diameters are about 15 and 25 ~m. Both types of cells contain abundant smooth endoplasmic reticulum, mitochondria, and lipid droplets similar to cells in vivo. a7 The larger cells appear to contain less lipid droplets, more smooth endoplasmic reticulum, and relatively little smooth surface area (Figs. 4 and 5). 93 T. J. Brennan, R. Ohkawa, S. D. Gore, and H. R. Behrman, Endocrinology U ] , 449 (1983).
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FIG. 1. Isolated rat luteal cells after dissociation of corpora lutea. This preparation contains about 60% luteal cells (arrows) and debris which is evident as particulate matter. Nomarski optics, x 120. FIG. 2. The same preparation of isolated rat luteal cells after enrichment with a discontinuous Percoll density gradient. Substantial membrane debris has been removed as well as large clumps of cells and some blood cells. This preparation contains about 80% luteal cells (arrows). Nomarski optics. × 120. R a t l u t e a l c e l l s b i n d [125I]hCG s i m i l a r to i s o l a t e d m e m b r a n e s 92 w i t h a KD o f 0.5 × 101° M - 1 . 94 T h e y r e s p o n d to L H w i t h a d o s e - d e p e n d e n t i n c r e a s e in c A M P a n d p r o g e s t e r o n e p r o d u c t i o n . 13,59 R e s p o n s i v e n e s s to L H is i n v e r s e l y r e l a t e d to t h e a g e o f t h e c o r p u s l u t e u m . 2°,95 LH receptors, localized with a ferritin-LH conjugate, are distributed at i r r e g u l a r i n t e r v a l s a l o n g t h e l u t e a l cell s u r f a c e . B o t h specific i n t e r n a l ization of bound ferritin-LH by small vesicles, and nonspecific bulk upt a k e w e r e o b s e r v e d a n d a r e r a p i d p r o c e s s e s . 14 B i n d i n g a n d m i c r o a g g r e g a t i o n o f r e c e p t o r s a p p e a r to b e r e l a t e d to f e r r i t i n - L H c o n c e n t r a t i o n . 96 L H 94 j. L. Luborsky, L. J. Dorflinger, K. Wright, and H. R. Behrman, Endocrinology 115, in press (1984). 95 H. R. Behrman, A. K. Hall, S. L. Preston, and S. D. Gore, Endocrinology 110, 38 (1982). 96 j. L. Luborsky and H. R. Behrman, Endocrinology 115, in press (1984).
312
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FIG. 3. A higher magnification view of the luteal cells shown in Fig. 2, to show larger luteal cells (LC), smaller luteal cells (lc), and probable endothelial cells (E) which are also present. Luteal cells typically have a more granular cytoplasm than endothelial cells. Nomarski optics. × 1200.
receptor aggregation decreased in the presence of PGF2~ and increased in the presence of (Bu)2cAMP96 suggesting that L H receptor aggregation is also related to cell function. With this preparation of luteal cells we showed a direct action of PGF2~ on L H stimulated cAMP and progesterone production. PGF2~ rapidly inhibits the acute action of L H by inhibition of cAMP production. 59,62 Luteal cells have specific receptors for PGF2a. 97'98 L H R H and D-Trp 6 L H R H also inhibit LH stimulated cAMP and progesterone production. 7° The rapid inhibition by PGF2~ and L H R H was not due to a decrease in LH binding. However, following the initial rapid inhibition, PGF2~ was shown to reduce the subsequent equilibrium binding level of [~25I]hCG (i.e., after 2 to 3 hr) by about 10%. This decrease was not due to a change in the initial association or dissociation rate but resulted in a decreased binding capacity for LH. 94 Upon initial binding, LH increases the number of 97 j. L. Luborsky, K. Wright, and H. R. Behrman, Adv. Exp. Biol. Med. 112, 633 (1979). 98 K. Wright, J. L. Luborsky, and H. R. Behrman, Mol. Cell. Endocrinol. 13, 25 (1979).
[25]
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~
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6
~.~
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available receptors by a similar small increment (about 10%) and this is blocked by PGF2~ .94 Thus, inhibition of LH action by PGF2~ occurs at the level of the coupling mechanism between the L H receptor and adenylate cyclase. In long-term cultures, LH-dependent increases in cAMP and progesterone production are inhibited by a prior pulse (24 hr earlier) of LH, PGF2~, L H R H , o r D - T r p 6 L H R H . An associated loss of L H receptors, similar to that seen in vivo, was not observed. 7° In this same study, untreated cells exhibited an increased (100x) sensitivity to L H at 24 hr. We also reported with luteal cells the first evidence that adenosine amplified the action of a polypeptide hormone. 2°,99 In rat luteal cells, adenosine amplifies L H action partly by an intracellular action (about 80%) and partly by interaction with a cell surface catalytic site. 99 Leydig cells do not respond to adenosine with an amplification of LH. 99 Adenosine also amplifies F S H action in human and rat granulosa cells and LH action in human luteal cells. 2° In addition, adenosine attenuates the inhibition of L H by PGF2,,, but this action is not associated with a change in L H receptor binding or prostaglandin synthesis. 95 Adenosine is less active in rat and human luteal cells from older corpora lutea. 2°,95 The basis for this purine amplification of LH action in luteal cells appears to be due to a rapid increase in ATP levels which follows a similar time course to the increase in cAMP production in response to L H . 93 Neither L H nor PGF2~, however, changes ATP levels. Thus, the ability of the cell to produce cAMP appears to be dependent on ATP levels and adenosine increases this capacity. The mechanism of action of antigonadotropins such as PGF2~ and L H R H was further investigated with respect to their possible action on transmembrane ion flux cations. Ouabain and monensin, inhibitors of Na + flux, inhibited LH-dependent increases in cAMP and progesterone production in intact cells but did not inhibit cAMP production in isolated membranes.l°° Prior treatment with L H or removal of extracellular Ca 2÷ prevented subsequent inhibition by ouabain and monensin. Thus, Ca 2+ appeared to regulate inhibition of L H action. This was confirmed in another study with PGF2~ and LHRH. 42 LH-stimulated cAMP production is enhanced in the absence of extracellular Ca 2+, the calcium ionophore A23187 inhibited L H stimulated cAMP, and LH-stimulated adenylate cyclase is directly inhibited by C a 2+ in isolated membranes. Verapamil, which blocks Ca 2+ channels, did not prevent inhibition of LH action by PGF2~ or L H R H . Thus acute increases in intracellular C a 2+, rather than an influx of extracellular Ca 2+, appear to inhibit activation of adenylate 99 A. K. Hall, S. L. Preston, and H. R. Behrman, J. Biol. Chem. 256, 10390 (1981). 100 S. D. Gore and H. R. Behrman, Endocrinology 114, 2020 (1984).
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cyclase. This suggests that an increase in intracellular Ca 2÷, independent of an extracellular source, mediates the action of PGF2~ and LHRH. In conclusion, we have used isolated luteal cells to show a relationship between L H receptor topography and cell function, PGF2~ and L H R H inhibition of L H action, activation of adenylate cyclase by LH is regulated by the intracellular Ca 2÷ concentration, and that adenosine amplifies LH and FSH action by an increase in ATP levels. Acknowledgments Supported by NIH Grants HD 10718 (HRB) and HD 14098 (JLL).
[26] C u l t u r e a n d C h a r a c t e r i s t i c s o f H o r m o n e - R e s p o n s i v e N e u r o b l a s t o m a × G l i o m a H y b r i d Cells
By BERND HAMPRECHT, THOMAS GLASER, GEORG RElSER, ERNST BAYER, and FRIEDRICH PROPST
Due to the complexity of the mammalian nervous system, results from biochemical or pharmacological experiments with pieces or homogenates of nervous tissue are difficult to interpret. Especially it is hard to assign a certain effect observed to a certain cell type. The most desirable situation would be to have at one's disposal the numerous cell types as homogenous cell populations for studying their individual differentiated functions and their mechanisms of intercellular communication. Only recently a few cases have been reported of apparently relatively homogeneous populations of certain cell types from nervous tissue. Therefore, the hope to be able to study molecular mechanisms of nervous tissue functions has been resting completely on model systems derived from tumors of the nervous system. Initially, Sato's laboratory developed clonal rat glioma ~ and mouse neuroblastoma 2 cell lines that still expressed differentiated functions known to occur in glial cells and neurons, respectively. Other groups i p. Benda, J. Lightbody, G. Sato, L. Levine, and W. Sweet, Science 161, 370 (1968). 2 G. Augusti-Tocco and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 64, 311 (1969).
METHODS IN ENZYMOLOGY, VOL. 109
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2
HORMONALLY RESPONSIVE CELLS
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scribed by Schrey et al. ~4The cell column is placed in a water bath and medium (usually KRB with 0.5% BSA and 0.02% glucose) is perfused at a rate of 0.4-0.6 ml/min. When using a Cytodex cell column, cells are seeded directly after dissociation, onto Cytodex beads (contained in bacteriological petri dishes) at a density of 1-2 × 105 cells/mg. Cytodex in Ham's F10 containing 10% fetal calf serum, penicillin, and streptomycin. On the third day, cells are washed with normal KRB solution (i.e., containing 2 mM Ca z÷ and 2 mM Mg 2+) containing 0.5% BSA and 0.02% glucose. The Cytodex beads are then packed into the perifusion apparatus. Usually a smaller column is used (i.e., approximately 20 mg Cytodex) and is perfused at a rate of 0.4-0.5 ml/min.
[25] I s o l a t i o n a n d F u n c t i o n a l A s p e c t s o f F r e e L u t e a l C e l l s By JUDITH L. LUBORSKYand HAROLD R. BEHRMAN
Introduction During each ovarian cycle, changes in the endocrine function of the ovary are dependent on differentiation of follicular cells and luteal cells. Differentiation of ovarian cells is expressed as changes in morphology, cellular metabolism, and their responsiveness to gonadotropins. Ovarian cell function has been studied in vitro with intact follicles and copora lutea, tissue slices, or more recently with isolated granulosa or luteal cells. Luteal cell function in particular has been studied in granulosa cells that have been allowed to luteinize in culture ~-3 or by direct isolation of luteal cells from lutenized tissue. 4-7 Since the function of the corpus luteum must be terminated for a subsequent ovulation to occur or extended for a pregnancy to be maintained, the functional state of corpus luteum is delicately balanced by both i G. L. K u m a r i and C. P. Channing, J. Steroid Biochem. 11, 781 (1979). 2 K. M. H e n d e r s o n and K. P. M c N a t t y , J. Endocrinol. 73, 71 (1977). 3 R. W. T u r e c k and J. F. Strauss, J. Clin. Endocrinol. Metab. 54, 367 (1982). 4 T-C. Liu and J. Gorski, Endocrinology 88, 419 (1971). 5 D. G o s p o d a r o w i c z and F. Gospodarowicz, Endocrinology 90, 1427 (1972). 6 D. H. W u , W. G. Wiest, and A. C. Enders, Endocrinology 98, 1378 (1976). 7 K. R. S i m m o n s , J. L. Caffrey, J. L. Phillips, J. H. Abel, and G. D. Niswender, Proc. Soc. Exp. Biol. Med. 152, 366 (1976).
METHODS IN ENZYMOLOGY,VOL. 109
Copyright © 1985by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2
[251
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tropic and lytic endocrine regulators. Isolated luteal cell preparations were developed in order to examine cell regulation independent of variables present in vivo, such as changes in blood flow and endogenous hormone concentrations. Isolated cells can be uniformly and rapidly treated with drugs and hormones. Their use does not have the problems of sample variation and nonuniform exposure of cells that are inherent in isolated corpora lutea and luteal tissue slices. Luteal cells isolated from s h e e p , 7-9 cow, 5,1° pig, H rat, 6,n-14 guinea pig, 15 rabbit, 4 monkey, 16 and human corpora l u t e a 17-2° have been used to study aspects of their cellular morphology, biochemical responses and hormone receptor function. General Methods of Luteal Cell Isolation In general, methods of luteal cell isolation employ enzymatic treatment of minced or sliced luteal tissue with collagenase (to break down connective tissue) and deoxyribonuclease (DNase) (to break down released DNA that may cause cells to clump). Additional enzymes such as hyaluronidase or Pronase are also used in s o m e l a b o r a t o r i e s . 5,6,1°,1z Enzyme treatment is usually coupled with mechanical treatment to further disperse cells. A systematic study of conditions for rat luteal cell isolation examined duration of incubation, and combinations of variable concentrations of collagenase, trypsin, and hyaluronidase, as well as bovine serum albumin (BSA) and Ca 2+ concentrations necessary to maintain maximum viability, morphology, and steroidogenesis. A final combination of all three enzymes (2940 U trypsin, 2320 U collagenase, and 16,900 U hyaluronidase/ l0 ml of medium) plus 0.5% BSA and 3.3 mM Ca 2+ was chosen. 6 8 R. J. Rodgers and J. D. O'Shea, Aust. J. Biol. Sci. 35, 441 (1982). 9 C. E. Ahmed, H. R. Sawyer, and G. D. Niswender, Endocrinology 109, 1380 (1981). i0 j. Ursely and P. Leymarie, J. Endocrinol. 83, 303 (1979). II M. Lemon and M. Loir, J. Endocrinol. 72, 351 (1977). n R. F. Wilkinson, E. Anderson, and J. Aalberg, J. Ultrastruct. Res. 57, 168 (1976). 13 H. R. Behrman, S. L. Preston, and A. K. Hall, Endocrinology 107, 656 (1980). 14 j. L. Luborsky and H. R. Behrman, Mol. Cell. Endocrinol. 15, 61 (1979). 15 M. C. Richardson and M. J. Peddle, J. Reprod. Fertil. 66, 117 (1982). 16 R. L. Stouffer, W. E. Nixon, B. J. Gulyas, D. K. Johnson, and G. D. Hodgen, Steroids 27, 543 (1976). i7 M. T. Williams, M. S, Roth, J. M. Marsh, and W. J. LeMaire, J. Clin. Endocrinol. Metab. 48, 427 (1979). is M. C. Richardson and G. M. Masson, J. Endocrinol. 87, 247 (1980). 19 L. T. Goldsmith, M. Essig, P. Sarosi, P. Beck, and G. Weiss, J. Clin. Endocrinol. Metab. 53, 8980 (1981). 2o M. L. Polan, A. H. DeCherney, F. P. Haseltine, H. C. Mezer, and H. R. Behrman, J. Clin. Endocrinol. Metab. 56, 288 (1983).
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Luteinizing hormone (LH) receptor binding and function varies with different preparations of collagenase 21,22; these changes are thought to be due to variations in trypsin activity. However, trypsin improved separation of endothelial and large and small luteal cells of ovine corpus luteum, and the luteal cells retained their ability to respond to LH. a A systematic study of tryspin effects on primate luteal cells showed that a brief exposure (10 min) did not change human chorionic gonadotropin (hCG)-induced progesterone secretion, while a prolonged exposure (3 hr) significantly reduced both basal and hCG-stimulated progesterone secretion. 23 Variations in cell isolation with different collagenase preparations may be due to contamination with clostripain; collagenase free of clostripain was reported to produce uniform and consistent preparations of pancreatic cells. 24 After cell dispersion with enzyme(s), intercellular junctions are not completely separated. Frequently, some cells are well separated while the majority are still in small clumps or have attached debris from broken cells. Recently, additional treatment with trypsin and/or a divalent cation chelator was introduced to facilitate the separation of intercellular junctions. 8,14 Trypsin in low Ca 2+ medium containing ethyleneglycol bis(flaminoethylether) (EGTA) separates intercellular junctions. 8 A brief exposure to low Ca 2+ medium containing ethylenediaminetetraacetic acid (EDTA) also improves separation of luteal cells.14 Prolonged exposure (greater than 3-5 min) or high concentrations of EDTA (>2 mM) are deleterious to cell f u n c t i o n . 14 A sequential treatment with EDTA and hypotonic sucrose has been reported to enhance separation of gap junctions between granulosa cells. 25 Cell suspensions have been further enriched by separation on gradients of Ficoll, 8,1° Percoll, 13,26s u c r o s e , 9 o r B S A . 11,12 These gradients were used simply to remove membrane debris and dead c e l l s 13'26 o r to obtain cells of different sizes. 8,1°-12 Removal of membrane debris and broken cells is particularly important for morphological and receptor binding studies but does not appear to be essential for studies of cellular metabolism. Final yields of cells are about 1-10 × l0 7 cells/g of luteal tissue.4,5,7,8,10,16,26 2~ M. L. Dufau and K. J. Catt, this series, Vol. 37, p. 252. 22 j. L. Luborsky and H. R. Behrman, unpublished observations (1982). 23 B. J. Gulyas, L. C. Yuan, and G. D. Hodgen, Steroids 35, 43 (1980). 24 G. R. Gunther, G. S. Schultz, B. E. Hull, H. A. Alicea, and J. D. Jamieson, J. Cell Biol. 75, 368 (1979). 25 K. L. Campbell and A. R. Midgley, J. Cell Biol. 75, 246 (1977). 26 B. C. McNamara, C. E. G. Cranna, R. Booth, and D. A. Stansfield, Biochem. J. 192, 559 (1980).
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Morphological Aspects of Isolated Luteal Cells Isolated luteal cells retain the morphological characteristics of steroid secreting cells after isolation. They contain abundant mitochondria, variable amounts of lipid droplets, and an extensive smooth endoplasmic reticulum, similar to cells in vioo. 12'27'28 Two size ranges of isolated cells, of 15-25 and 30-50/zm in diameter, from monkey, rat, pig, sheep, and cow corpus luteum have been reported, s,1°-12,29 A comparison of cell types in vivo and in vitro suggests that the larger cells are extremely fragile and that there is a lower recovery after isolation than for the smaller cells.S, 10,12 Other cells, such as endothelial and blood cells, are found in isolated luteal cell preparations, and these can be reduced by further purification) It was suggested by several authors 8,11 that the small and large cell types originate from thecal and granulosa cells, respectively; it has also been suggested 12that these cells are part of a continuum of differentiating luteal cells. The actual source of the different size cells has not been established but it is clear that they differ morphologically and metabolically. The large cells contain more progesterone, are less responsive to LH, have more surface projections, and contain more mitochondria. 8,1°-n,z9 Isolated luteal cells have been maintained in culture for extended periods of time. Histochemical analysis of bovine luteal cells cultured for seven days showed lipid material that stained with Sudan Black and Oil Red, as well as 3fl-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase, characteristic of luteal cells. 3° Ultrastructural analysis indicates that cells retain their typical steroidogenic features, 12,29but small cells tend to lose their lipid droplets by extrusion during culture. J2 LH receptors have been localized on surfaces of rat 14 and monkey 3~ luteal cells with a ferritin-LH and a horseradish peroxidase-hCG conjugate, respectively. In both cases, receptors are located at intervals over the entire cell surface. Rate luteal cells in suspension do not appear to maintain their surface polarity to the same degree as found in vivo, with a basal smooth surface and an apical microvillus surface containing L H receptors. 28 Cultured monolayers of rat luteal cells 12may be more similar to cells in vivo but this has not been documented with respect to receptor localization. Primate luteal cells have been reported to lack morphological 27 A. K. Christensen and S. W. GiUim, in "The Gonads" (K. W. McKerns, ed.), p. 415. North-Holland Publ., Amsterdam, 1969. 28 W. Anderson, Y.-H. Kang, M. E. Perotti, T. A. Bramley, and R. J. Ryan, Biol. Reprod. 20, 362 (1979). 29 B. J. Gulyas, R. L. Stouffer, and G. D. Hodgen, Biol. Reprod. 20, 779 (1979). 3o D. Gospodarowicz and F. Gospodarowicz, Exp. Cell Res. 75, 353 (1972). 31 B. J. Gulyas, S. Matsuura, H.-C. Chen, L. C. Yuan, and G. D. Hodgen, Biol. Reprod. 25, 609 (1981).
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polarity both in vivo 32 and in vitro31; this has not been correlated with studies of receptor localization. Responses of isolated luteal cells to hormones and growth factors involve a morphological component or shape change. In general, cells appear more spherical and polygonal when treated with LH (hCG) or dibutyryladenosine 3' : 5'-cyclic monophosphate [(Bu)2cAMP] and during enhanced steroidogenic activity. 3,33,34 Cell shape is probably determined by microtubules and microfilaments since shape changes are inhibited by colcemid, vinblastin, or cytochalasin B. 33 In the absence of hormone or after its removal, more flattened, oblong cells are observed. 3 It is possible that L H may control and maintain morphological differentiation. Glucocorticoids and testosterone inhibit cell growth but progesterone does n o t . 33 In the presence of fibroblast growth factor (FGF) and epidermal growth factor (EGF), cell growth is stimulated and cells become more flattened. 35 Cells in suspension culture have not been reported to undergo shape changes. Biochemical/Endocrine Function of Isolated Luteal Cells Isolated luteal cells have been used in numerous studies to examine the regulation of steroidogenesis by LH. The functional capacity of freshly isolated luteal cells has been reported to reflect their functional capacity in vivo with respect to progesterone production in response to L H . 16'26'36 Effects of L H on human luteal cells during the cycle 34 and primate luteal cells during pregnancy 36 parallel that seen in vioo. 36 LH stimulated progesterone production and increases in adenosine 3' : 5"-cyclic monophosphate (cAMP) levels and cAMP binding to protein kinase 37,38 are directly correlated, Aminoglutethimide inhibits cholesterol side chain cleavage and progesterone production from cholesterol esters 26,39 and compactin blocks cholesterol biosynthesis in vitro, z6 In the presence of aminoglutethimide but not compactin, rapid (short-term) responses of cells to L H were blocked, suggesting stored cholesterol but not cholesterol biosynthesis is required for acute responses to L H . 26 Also, 32 B. J. Gulyas, Am. J. Anat. 139, 95 (1974). 33 D. Gospodarowicz and F. Gospodarowicz, Endocrinology 75, 458 (1975). 34 K. Kamei, Nippon Sanka Fujinka Gakki Zasshi 34, 261 (1982). 35 D. Gospodarowicz, C. R. I11, and C. R. Birdwell, Endocrinology 100, 1121 (1977). 36 R. L. Stouffer, W. E. Nixon, B. J. Gulyas, and G. D. Hodgen, Endocrincology 100, 596 (1977). 37 G. B. Sala, M. L. Dufau, and K. J. Catt, J. Biol. Chem. 254, 2077 (1979). 38 W. Y. Ling, M. T. Williams, and J. M. Marsh, J. Endocrinol. 86, 45 (1980). 39 M. Lemon and P. Mauleon, J. Reprod. Fertil. 64, 315 (1982).
[25]
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inhibition of cholesterol ester cleavage by cannabinoids reduced progesterone production. 4° Ca 2÷ has been shown to augment LH-dependent steroidiogenesis. 41,42In isolated cells L H action is blocked by 2-bromopalmirate indicating involvement of fatty acid oxidation. 43 L H selectively stimulates incorporation of 32p into phosphatidic acid, phosphatidylinositol, and polyphosphoinositides. 44 A direct action of biogenic amines acting at separate receptors, on L H stimulated progesterone production was s h o w n . 4547 Biogenic amine action on cAMP accumulation in isolated luteal cells was shown to decrease with increasing corpus luteum age. 47 Danazol was reported to inhibit LH, cholera toxin, and (Bu)2cAMP-stimulated progesterone production but not corresponding increases in cAMP, suggesting it acts distal to the hormone receptor site. 48 Responsiveness of the two cell types to L H appears to differ. Large cells produce higher basal levels of progesterone but are less responsive to L H 8,1°,1~ and there are less L H receptors on large cells. 31 In addition, in a continuous perfusion system, experiments with large and small cells "in series" showed that small cells can increase the amount of progesterone produced by large cells but not the reverse. H During maintenance of primate and other luteal cells in long-term culture, progesterone and estrogen production gradually decline ~9,29and this is not prevented by addition of thyroxine, insulin, cortisol, or cholesterol. 49 Also, bovine serum was reported to decrease progesterone production in bovine luteal cells) ° Lipoproteins increase progesterone production in cultured rat, 5~ bovine, 5° and human 3 luteal cells. Luteal macrophages were reported to maintain progesterone production of mouse luteal cells. 52
40 S. Burstein, S. A. Hunter, and T. S. Shoupe, Res. Commun. Chem. Pathol. Pharmacol. 24, 413 (1979). 41 N. Kamiya, Nippon Naibunpi Gakkai Zasshi 58, 833 (1982). 42 L. J. Dorflinger, P. J. Albert, A. T. Williams, and H. R. Behrman, Endocrinology 114, 1208 (1984). 43 C. H. Tan and J. Robinson, Life Sci. 30, 1205 (1982). 44 j. S. Davis, R. V. Farese, and J. M. Marsh, Endocrinology 109, 469 (1981). 45 R. C. Rhodes and R. D. Randel, Comp. Biochem. Physiol. 72, 113 (1982). 46 A. W. Jordan, J. L. Caffrey, and G. D. Niswender, Endocrinology 103, 385 (1978). 47 E. Norjavaara, G. Selstam, and K. Ahren, Acta Endocrinol. (Copenhagen) 100, 613 (1982). 48 M. Menon, S. Azhar, and K. M. Menon, Am. J. Obstet. Gynecol. 136, 524 (1980). 49 B. J. Gulyas, L. C. Yuan, and G. D. Hodgen, Biol. Reprod. 23, 21 (1980). 50 j. L. Pate and W. A. Condon, Mol. Cell. Endocrinol. 28, 551 (1982). 51 S. Azhar and K. M. Menon, J. Steroid Biochem. 16, 175 (1982). 52 T. M. Kirsch, A. C. Friedman, R. L. Vogel, and G. L. Flickinger, Biol. Reprod. 25, 629 (1981).
304
HORMONALLY RESPONSIVE CELLS
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Prolactin (PRL) maintains LH receptor and steroidogenic function in rat luteal cells in v i v o . 53 In vitro, PRL maintains basal steroidogenesis in rat luteal cells without a specific stimulation of its o w n ) ,54 PRL also suppresses 20a-steroid dehydrogenase in the rat. 6,55 Its role in human luteal function is not as clear. In humans, PRL was reported to have a dual, age-dependent, luteolytic and luteotropic action, without an effect on responses to h C G ) 6 Only a minimal effect of PRL was seen on primate luteal cells) 7 Prostaglandin-F2~ (PGFz~) has been shown to be luteolytic in a variety of animals. In some studies the newly formed corpus luteum is refractory to PGFz~ ,z,~5,58but later, a marked inhibition of LH stimulated cAMP and progesterone production by PGF2~ in isolated luteal cells 59-62 occurs. PGFz,~ appears to have no effect on bovine luteal cells 63but is luteolytic in vivo. 64 In early luteal cells of humans, in the presence of no LH, or low levels of LH, PGFz~ stimulated progesterone production) 8 From these studies, PGF2~ acts directly and acutely at the LH receptor, as well as at other sites such as on steroidogenesis6°,62 and in vivo on delivery of hormone from blood. 64 Estrogen is thought to be involved in luteolysis of human, primate, and bovine corpus luteum. Direct inhibition by estrogen of LH and (Bu)zcAMP stimulated progesterone production but not LH-stimulated cAMP production suggests that estrogen does reduce bovine, 65 human, 66,67 and primate 68 luteal cell function, but at a point beyond LH 53 H. R. Behrman, D. L. Grinwich, M. Hichens, and G. D. Macdonald, Endocrinology 103, 349 (1978). 54 K. Shiota and W. G. Wiest, Adv. Exp. Med. Biol. 112, 169 (1979). 55 M. Lahav, S. A. Lamprecht, A. Amsterdam, and H. R. Lindner, Mol. Cell. Endocrinol. 6, 293 (1977). 56 T. Sawada, Nippon Sanka Fujinka Gakki Zasshi 34, 2212 (1982). 57 R. L. Stouffer, J. L. Coensgen, and G. D. Hodgen, Steroids 35, 523 (1980). 58 R. L. Stouffer, W. E. Nixon, and G. D. Hodgen, Biol. Reprod. 20, 897 (1979). 59 j. p. Thomas, L. J. Dorflinger, and H. R. Behrman, Proc. Natl. Acad. Sci. US.A. 75, 1334 (1978). 6o A. W. Jordan, Biol. Reprod. 25, 327 (1981). 61 A. K. Hall and J. Robinson, J. Endocrinol. 81, 157 (1979). 62 H. R. Behrman, J. L. Luborsky, C. Y. Pang, K. Wright, and L. J. Dorflinger, Adv. Exp. Biol. Med. 112, 557 (1979). 63 j. E. Hixon and W. Hansel, Adv. Exp. Med. Biol. 112, 613 (1979). 64 H. R. Behrman, Annu. Rev. Physiol. 41, 685 (1979). 65 M. T. Williams and J. M. Marsh, Endocrinology 103, 1611 (1978). 66 M. Thibier, N. el-Hassan, M. R. Clark, W. J. LeMaire, and J. M. Marsh, J. Clin. Endocrinol. Matab. 50, 590 (1980). 67 M. Richardson and G. M. Masson, J. Endocrinol. 91, 197 (1981). 68 R. L. Stouffer, L. A. Bennett, and G. D. Hodgen, Endocrinology 106, 519 (1980).
[25]
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induced increases in cAMP. 65,66 Isolated primate luteal cells also synthesize and secrete estrogen and respond to LH with an increase in estrogen production; neither estrogen production nor the estrogen/progesterone ratio increases with the age of the corpus luteum. 68 Testosterone has been reported to inhibit LH action in porcine 69 but not human 67 luteal cells. Recently, a direct inhibitory action of luteinizing hormone releasing hormone (LHRH) on rat luteal cells was shown. L H R H binds to specific receptors on luteal cells and inhibits LH-induced increases in cAMP accumulation and progesterone secretion. 7°-73L H R H stimulates incorporation of 32p into phospholipid. TM The antigonadotropic action of L H R H is similar to that for PGF2~ and these agents appear to increase intracellular C a 2+ levels. 42 At this time, the physiological significance of L H R H receptors on ovarian cells is not established. L H R H or an LHRH-like peptide may be one of several intraovarian factors that regulate ovarian function. 75 In this regard follicular fluid injected into monkeys reduced progesterone production and responsiveness to L H in subsequently isolated luteal cells .76 The corpus luteum has been identified as the source of the peptide hormone, relaxin. Long-term cultures of isolated rat 77 and human ~9luteal cells have been used to examine the factors regulating relaxin secretion. Its release in rats is stimulated by various combinations, but not singly, of LH, PRL, progesterone, epinephrine, and estrogen; (Bu)2cAMP did not stimulate relaxin secretion. The role of microfilaments and microtubules in steroidogenesis has been investigated in isolated luteal cells. Cytochalasin B and D decreased progesterone secretion in response to LH, (Bu)zcAMP, cholera toxin and 8-bromo-cAMP in rat luteal cells without a reduction of cAMP levels. 78 Cytochalasin B also decreased L H and (Bu)zcAMP stimulated progesterone secretion in bovine luteal cells. 79 Microtubule inhibitors had little effect on steroidogenesis when applied directly to luteal cells TM but if injected in vivo, progesterone production in subsequently isolated cells 69 M. G. Hunter, J. Reprod. Fertil. 63, 471 (1981). 70 A. K. Hall and H. R. Behrman, J. Endocrinol. 88, 27 (1981). 7~ H. R. Behrman, S. L. Preston, and A. K. Hall, Endocrinology 107, 656 (1980). 72 j. Massicotte, J. P. Borgus, R. Lachance, and F. Labile, J. Steroid Biochem. 14, 239 (1981). 73 R. N. Clayton, J. P. Harwood, and K. J. Catt, Nature (London) 282, 90 (1979). 74 C. K. Leung, V. Raymond, and F. Labile, Endocrinology 112, 1138 (1983). 75 A. T. Williams and H. R. Behrman, Semin. Reprod. Endocrinol 1, 269 (1983). 76 R. L. Stouffer and G. D. Hodgen, J. Clin. Endocrinol. Metab. 51, 669 (1980). 77 L. T. Goldsmith, H. S. Grob, and G. Weiss, Ann. N.Y. Acad. Sci. 380, 60 (1982). 78 S. Azhar and K. M. Menon, Biochem. J. 194, 19 (1981). 79 M. T. Williams and J. M. Marsh, Adv. Exp. Biol. Med. 112, 549 (1979).
306
HORMONALLY RESPONSIVE CELLS
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was decreased 8° without morphological evidence of microtubule disruption. Thus, cellular microfilaments were suggested to be involved in hormone regulated steroidogenesis. Hormone Receptor Function of Isolated Luteal Cells L H receptor binding studies were employed to quantitate specific properties of hormone-receptor interaction in relation to cellular function. Binding of [125I]hCG to bovine luteal cells and membranes was compared and it was concluded that the enzymatic treatment used to isolate cells did not change L H receptor binding kinetics or affinity. Binding to bovine cells was saturable, rapid and temperature dependent with an estimated binding capacity of 5 x 10 4 receptors/cell and an equilibrium binding constant (KD) of 5.3 × 10-l° M. 8j Prostaglandin El (PGEI) binding to bovine luteal cells was rapid, saturable, reversible, and specific with a KD = 2.4 nM and 1.8 × 105 receptors/cell, with most properties similar to receptors in isolated membranes. 82 Binding of [125I]hCG to cells from rats desensitized with hCG was compared to cells from untreated rats. The desensitized or refractory state was accompanied by a significant loss of hCG binding concomitant with decreased cAMP and progesterone production in response to LH. 83 This reduced steroid production was also due to an additional lesion in steroid biosynthesis, rather than to a simple loss of LH receptors. In studying recovery from desensitization, it was found that a full complement of L H receptors was required for maximum sensitivity (i.e., an L H response is obtained, but more hormone is needed to obtain a maximum response). Similarly, cells isolated from primate corpus luteum after hCG exposure were refractory to hCG. 84 Rat luteal cells increased their sensitivity to L H (cAMP) without a change in L H receptor binding during prolonged culture. 7° This increased sensitivity was impaired by early exposure to LH, PGFE~, LHRH, or an L H R H analog. Since there was no change in LH receptor binding, it was suggested that receptor desensitization may precede receptor loss and may be due to rapid uncoupling of the receptor and adenylate cyclase. A similar inhibition of L H action by PGF2~ was seen in in vivo studies with PGF2~ ,53,62 but this may be due to additional action of PGF2~ in vivo. 80 S. Azhar and E. Reaven, Am. J. Physiol. 243, E380 (1982). 81 S. Papaionannou and D. Gospodarowicz, Endocrinology 97, 114 (1975). 82 M. T. Lin and C. V. Rao, Mol. Cell. Endocrinol. 9, 311 (1978). 83 j. p. Harwood, M. Conti, P. M. Conn, M. L. Dufau, and K. J. Catt, Mol. Cell. Endocrinol. 11, 121 (1978). 84 R. L. Stouffer, W. E. Nixon, and G. D Hodgen, Biol. Reprod. 18, 858 (1978).
[25]
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307
Mechanisms for changes in the L H receptor content of cells have been studied. Loss of receptors from the ovine luteal cell surface occurred with a hi2 of 9.6 hr and about 85% was lost within 24 hr. Cell-associated radioactivity increased up to 4 hr, was maintained, and then decreased during 12-24 hr. This time course is similar to that seen in vivo during down-regulation. 6z Receptor loss was three times faster than membrane protein turnover. A majority of bound [125I]hCG was internalized and degraded. 85 Inhibitors of transglutaminase (inhibits receptor clustering) did not inhibit internalization of [125I]hCG86 but did produce an intracellular accumulation of [lzSI]hCG and an inhibition of hCG degradation. HCG binding may result in an increase in L H receptor aggregation, but LH receptors are not necessarily internalized as large clusters in coated pits. 14 Furthermore, inhibition of protein synthesis did not decrease the total number of L H receptors per cell, although it did decrease LH receptor degradation and the number of receptors on the cell surface. Thus, synthesis of new receptors is not required for continued binding and internalization, and it was suggested that receptors are recycled. 85 The effects of EGF and FGF and their binding to cells is similar to that in vivo; bovine luteal cells lose their proliferative response to EGF but retain their responsiveness to FGF. 87 Analysis of EGF binding showed that both granulosa cells and luteal cells bind EGF, but in luteal cells there is no mitogenic respnse. In fact, EGF receptors increased with luteinization (0.2 to 1 x 105). Down-regulation, or loss of EGF receptors in response to EGF, occurred in both granulosa and luteal cells. Low concentrations of EGF produced a greater loss of receptors than those that were occupied. 87 Preparation of Free Luteal Cells Animals. Ovaries from gonadotropin primed rats, given 50 IU pregnant mares serum (Gestyl, Organon) and 25 IU hCG (A.P.L., Ayerst) 64 hr later, were removed 4-8 days after hCG injection. Enzymatic Treatment. Ovaries are rapidly trimmed in medium A (GIBCO No. 320-1380 containing 0.1% BSA), weighed, and minced with a clean, sharp razor. The fragments are washed to remove excess blood cells. The fragments are added to a 25-ml Erlenmeyer flask containing 2000 IU collagenase (Worthington Biochemical Corporation) and 3000 IU DNase (Worthington Biochemical Corporation) per gram of tissue in 5 ml of medium A. An additional 5 ml of medium A is added and the solution is s5 D. E. Suter and G. D. Niswender, Endocrinology 112, 838 (1983). 86 C. E. Ahmed and G. D. Niswender, Endocrinology 109, 1388 (1981). 87 I. Vlodavsky, K. D. Brown, and G. Gospodarowicz, J. Biol. Chem. 253, 3744 (1978).
308
HORMONALLY RESPONSIVE CELLS
[25]
gently mixed and aerated with 95% 02/5% CO2 for 1 min. The tissue is incubated for 1 hr at 37° with agitation (Dubnoff incubator, I00 cycles/ min). During the incubation the fragments are pipetted five or six times with a large bore, Pasteur pipet with a flame polished tip. At the end of incubation the tissue is centrifuged (100 g, 5 min) in a 15-ml round bottom centrifuge tube and the supernatant discarded. Low Ca 2÷ Treatment. After enzyme treatment, the majority of cells are in small clumps. Luteal cells are more difficult to separate than granulosa cells since they develop extensive intercellular junctions during luteinization.12 To improve separation of these junctions, cells are resuspended in 10 ml of medium A containing 1 mM EDTA. The solution is pipetted gently about six times to resuspend the pellet, with a Pasteur pipet with a flame polished tip, incubated for 2 min (22°), centrifuged (100 g, 5 min), and the supernatant discarded. The cells are extremely fragile in low Ca 2÷ conditions; additional time or increased concentrations of EDTA will damage cells. Mechanical Dispersion. The pellet is resuspended in 3 ml of medium A. Cells are mechanically dispersed by repeated pipetting with a flame polished Pasteur pipet, allowing clumps to settle (about 0.5 min) and removing the supernatant containing isolated cells. Cells are filtered through nylon mesh (Nyten, Tetko) supported by a disposable plastic funnel into a clean 15-ml polypropylene centrifuge tube. The pellet is resuspended in l ml of medium A and the procedure repeated until only a few small clumps of cells and connective tissue remain. Pooled cells are centrifuged (100 g, 5 min). This process minimizes mechanical manipulation of isolated cells. Cells may be used without further processing at this point; cells are resuspended in medium B (GIBCO No. 380-2360 containing 0.1% BSA) and preincubated 60 min before use. Alternatively, the cells are resuspended in 2 ml of medium A and further enriched on a Percoll column to remove membrane debris, some red blood cells, and nonfunctional luteal cells. Enrichment o f Luteal Cell Preparation. A discontinuous Percoll gradient is prepared by layering 3 ml each of 40, 30, 20, and 10% (bottom to top) Percoll solutions in a 15 ml conical polypropylene centrifuge tube. A Percoll stock solution is prepared from 9 ml of Percoll plus 1 ml of medium C (GIBCO No. 199, 10×) and is diluted with medium A. The cell suspension is layered on top and centrifuged through the gradient (100 g, 30 min). '?he excess medium is removed and cells at the 10-20% and 2030% interfaces are combined. Cells are rinsed with medium B (100 g, 10 min) and resuspended to about 2 to 4 x 10 6 cells/ml in medium B. One column will separate cells from 14 ovaries.
[25]
ISOLATED LUTEAL CELLS
309
Cell Yield and Viability. The cells are counted in a hemocytometer. Generally this method yields about 1-2 x 10 6 cells/ovary. Before Percoll enrichment the preparation contains about 50-60% luteal cells and after PercoU enrichment it contains about 70-80% luteal cells. Cell viability is about 90% when tested for the ability of cells to exclude trypan blue. 88 Similar but more quantitative results are obtained when the LDH content (LDH kit, Statzyme, Worthington) of medium is tested before and after 90 min of incubation at 37°. Total protein synthesis, as measured by incorporation of [14C]amino acids into perchloric acid precipitable proteins, 89 remains constant over 24 hr. Methods of Incubation and Preparation of Samples for Analysis. For short-term experiments luteal cells are incubated in 12 × 75 glass tubes in medium B at 105 to 10 6 cells/ml/tube. The incubation is terminated by immersion of the tubes in a boiling water bath for 10 min. Samples are then frozen until analysis by radioimmunoassay for cAMP 9° or progesterone. 91 For hormone binding experiments 2 ml of ice cold medium B is added, the cells are centrifuged (1000 g, 15 min), the supernatant aspirated, and the radioactivity in the cell pellet counted. For longer term experiments, cells are cultured for up to 48 hr in disposable plastic tissue culture trays (CoStar) with l06 cells/well/0.5 ml in medium B containing 50 IU/ml myocostatin, 100 IU/ml penicillinstreptomycin, and 10% fetal calf serum (GIBCO). The cells are cultured on individual nitrocellulose filters (Millipore, HAWG 01300) soaked in medium 2 for 16 hr at 4o.70 The presoaked filters are held in place by narrow plastic rings. For measurement of secreted cAMP, the medium is transferred to test tubes with 1 mM theophylline and frozen until they are analyzed. To measure intracellular cAMP, the filter with attached cells is placed in 1 ml of medium B containing 1 mM theophylline and heated in a boiling water bath for 10 min. For measurement of [J25I]hCG binding, [~25I]hCG of high specific activity 9~ is incubated with cells on filters and the medium removed, the filters rinsed, and placed in test tubes and the radioactivity counted. as H. J. Phillips, in "Tissue Culture: Methods and Applications" (P. F. Kruse and M. K. Patterson, eds.), p. 406. Academic Press, New York, 1973. 89 R. J. Mans and E. D. Novelli, Biochem. Biophys. Res. Commun. 3, 540 (1960). ~ A. L. Steiner, in "Methods of Hormone Radioimmunoassay" (B. M. Jaffee and H. R. Behrman, eds.), p. 3. Academic Press, New York, 1979. 91 G. P. Orczyk, M. Hichens, G. Arth, and H. R. Behrman, in "Methods of Hormone Radioimmunoassay" (B. M. Jaffee and H. R. Behrman, eds.), p. 701. Academic Press, New York, 1979. 9~ M. Hichens, D. L. Grinwich, and H. R. Behrman, Prostaglandins 7, 449 (1979).
310
HORMONALLYRESPONSIVECELLS
[25]
In experiments where adenosine triphosphate (ATP) production was measured, incubations are carried out in 0.5 ml of medium B with 1-3 x 105 cells/tube. Reactions are stopped by the addition of 0.1 ml of 50% trichloroacetic acid (TCA) and samples are stored at 4° until they are analyzed. The TCA treated cells are extracted four times with 3 ml of diethyl ether, the pH adjusted to 7 with 0.02 N NaOH, and ATP assayed by the luciferin-luciferase assay (Sigma, L0663). 93 For ultrastructural analysis~4 the cells are rinsed gently with medium B and after centrifugation (100 g, 5 min), 1 ml of 2% glutaraldehyde, in 0.067 M cacodylate buffer at pH 7.4 containing 0.5% tannic acid (350 mOsM) is added to the pellet for 20-30 min at 23°. With a large bore plastic pipet the cell pellet is transferred to a 1.5-ml microfuge tube, spun briefly (>3 sec) in a Beckman microfuge B and rinsed with 0.1 M cacodylate buffer containing sucrose (3 l0 mOsM). One-half milliliter of 2% OsO4 is added for 1 hr at 22°. Buffer containing sucrose is then added and cells are centrifuged (<3 sec). The pellet is rinsed again. During this procedure the cells are not handled except to "lift" the pellet with a small flat wooden spatula. The tips of each microfuge tube (5 mm length) are cut with a sharp clean razor and the pellet scooped onto filter paper tied over the end of a polyethylene test tube (1.0-1.5 cm opening) open at both ends. The pellet is covered with a second piece of filter paper, which is tied with surgical thread. The tubes are carried through routine dehydration (5 min each in an ethanol series, 2 hr in 50 : 50 propylene oxide : resin, overnight with two changes in 100% resin) and embedded without handling the cells. After infiltration, the upper layer of filter paper is removed and the cell pellet lifted (scooped) into a drop of resin in the bottom of an embedding capsule which is then filled with resin. For Epon 812 (Fullam) or Embed-812 (Polysciences), capsules were polymerized overnight at 60°. Sections were examined in a Hitachi 12 electron microscope. Applications of Dispersed Luteal Cells The dispersed rat luteal cell preparation contains greater than 80% luteal cells with the remainder endothelial and blood cells. Based on light microscopic (Fig. 1) and electron microscopic (Fig. 2) observation there are two sizes of luteal cells. Their average diameters are about 15 and 25 ~m. Both types of cells contain abundant smooth endoplasmic reticulum, mitochondria, and lipid droplets similar to cells in vivo. a7 The larger cells appear to contain less lipid droplets, more smooth endoplasmic reticulum, and relatively little smooth surface area (Figs. 4 and 5). 93 T. J. Brennan, R. Ohkawa, S. D. Gore, and H. R. Behrman, Endocrinology U ] , 449 (1983).
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ISOLATED LUTEAL CELLS
311
FIG. 1. Isolated rat luteal cells after dissociation of corpora lutea. This preparation contains about 60% luteal cells (arrows) and debris which is evident as particulate matter. Nomarski optics, x 120. FIG. 2. The same preparation of isolated rat luteal cells after enrichment with a discontinuous Percoll density gradient. Substantial membrane debris has been removed as well as large clumps of cells and some blood cells. This preparation contains about 80% luteal cells (arrows). Nomarski optics. × 120. R a t l u t e a l c e l l s b i n d [125I]hCG s i m i l a r to i s o l a t e d m e m b r a n e s 92 w i t h a KD o f 0.5 × 101° M - 1 . 94 T h e y r e s p o n d to L H w i t h a d o s e - d e p e n d e n t i n c r e a s e in c A M P a n d p r o g e s t e r o n e p r o d u c t i o n . 13,59 R e s p o n s i v e n e s s to L H is i n v e r s e l y r e l a t e d to t h e a g e o f t h e c o r p u s l u t e u m . 2°,95 LH receptors, localized with a ferritin-LH conjugate, are distributed at i r r e g u l a r i n t e r v a l s a l o n g t h e l u t e a l cell s u r f a c e . B o t h specific i n t e r n a l ization of bound ferritin-LH by small vesicles, and nonspecific bulk upt a k e w e r e o b s e r v e d a n d a r e r a p i d p r o c e s s e s . 14 B i n d i n g a n d m i c r o a g g r e g a t i o n o f r e c e p t o r s a p p e a r to b e r e l a t e d to f e r r i t i n - L H c o n c e n t r a t i o n . 96 L H 94 j. L. Luborsky, L. J. Dorflinger, K. Wright, and H. R. Behrman, Endocrinology 115, in press (1984). 95 H. R. Behrman, A. K. Hall, S. L. Preston, and S. D. Gore, Endocrinology 110, 38 (1982). 96 j. L. Luborsky and H. R. Behrman, Endocrinology 115, in press (1984).
312
HORMONALLY RESPONSIVE CELLS
[25]
FIG. 3. A higher magnification view of the luteal cells shown in Fig. 2, to show larger luteal cells (LC), smaller luteal cells (lc), and probable endothelial cells (E) which are also present. Luteal cells typically have a more granular cytoplasm than endothelial cells. Nomarski optics. × 1200.
receptor aggregation decreased in the presence of PGF2~ and increased in the presence of (Bu)2cAMP96 suggesting that L H receptor aggregation is also related to cell function. With this preparation of luteal cells we showed a direct action of PGF2~ on L H stimulated cAMP and progesterone production. PGF2~ rapidly inhibits the acute action of L H by inhibition of cAMP production. 59,62 Luteal cells have specific receptors for PGF2a. 97'98 L H R H and D-Trp 6 L H R H also inhibit LH stimulated cAMP and progesterone production. 7° The rapid inhibition by PGF2~ and L H R H was not due to a decrease in LH binding. However, following the initial rapid inhibition, PGF2~ was shown to reduce the subsequent equilibrium binding level of [~25I]hCG (i.e., after 2 to 3 hr) by about 10%. This decrease was not due to a change in the initial association or dissociation rate but resulted in a decreased binding capacity for LH. 94 Upon initial binding, LH increases the number of 97 j. L. Luborsky, K. Wright, and H. R. Behrman, Adv. Exp. Biol. Med. 112, 633 (1979). 98 K. Wright, J. L. Luborsky, and H. R. Behrman, Mol. Cell. Endocrinol. 13, 25 (1979).
[25]
ISOLATED LUTEAL CELLS
313
~
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m e.~
6
~.~
314
HORMONALLY RESPONSIVE CELLS
[25]
~×
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oq
[25]
ISOLATED LUTEAL CELLS
3 15
available receptors by a similar small increment (about 10%) and this is blocked by PGF2~ .94 Thus, inhibition of LH action by PGF2~ occurs at the level of the coupling mechanism between the L H receptor and adenylate cyclase. In long-term cultures, LH-dependent increases in cAMP and progesterone production are inhibited by a prior pulse (24 hr earlier) of LH, PGF2~, L H R H , o r D - T r p 6 L H R H . An associated loss of L H receptors, similar to that seen in vivo, was not observed. 7° In this same study, untreated cells exhibited an increased (100x) sensitivity to L H at 24 hr. We also reported with luteal cells the first evidence that adenosine amplified the action of a polypeptide hormone. 2°,99 In rat luteal cells, adenosine amplifies L H action partly by an intracellular action (about 80%) and partly by interaction with a cell surface catalytic site. 99 Leydig cells do not respond to adenosine with an amplification of LH. 99 Adenosine also amplifies F S H action in human and rat granulosa cells and LH action in human luteal cells. 2° In addition, adenosine attenuates the inhibition of L H by PGF2,,, but this action is not associated with a change in L H receptor binding or prostaglandin synthesis. 95 Adenosine is less active in rat and human luteal cells from older corpora lutea. 2°,95 The basis for this purine amplification of LH action in luteal cells appears to be due to a rapid increase in ATP levels which follows a similar time course to the increase in cAMP production in response to L H . 93 Neither L H nor PGF2~, however, changes ATP levels. Thus, the ability of the cell to produce cAMP appears to be dependent on ATP levels and adenosine increases this capacity. The mechanism of action of antigonadotropins such as PGF2~ and L H R H was further investigated with respect to their possible action on transmembrane ion flux cations. Ouabain and monensin, inhibitors of Na + flux, inhibited LH-dependent increases in cAMP and progesterone production in intact cells but did not inhibit cAMP production in isolated membranes.l°° Prior treatment with L H or removal of extracellular Ca 2÷ prevented subsequent inhibition by ouabain and monensin. Thus, Ca 2+ appeared to regulate inhibition of L H action. This was confirmed in another study with PGF2~ and LHRH. 42 LH-stimulated cAMP production is enhanced in the absence of extracellular Ca 2+, the calcium ionophore A23187 inhibited L H stimulated cAMP, and LH-stimulated adenylate cyclase is directly inhibited by C a 2+ in isolated membranes. Verapamil, which blocks Ca 2+ channels, did not prevent inhibition of LH action by PGF2~ or L H R H . Thus acute increases in intracellular C a 2+, rather than an influx of extracellular Ca 2+, appear to inhibit activation of adenylate 99 A. K. Hall, S. L. Preston, and H. R. Behrman, J. Biol. Chem. 256, 10390 (1981). 100 S. D. Gore and H. R. Behrman, Endocrinology 114, 2020 (1984).
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• [26]
cyclase. This suggests that an increase in intracellular Ca 2÷, independent of an extracellular source, mediates the action of PGF2~ and LHRH. In conclusion, we have used isolated luteal cells to show a relationship between L H receptor topography and cell function, PGF2~ and L H R H inhibition of L H action, activation of adenylate cyclase by LH is regulated by the intracellular Ca 2÷ concentration, and that adenosine amplifies LH and FSH action by an increase in ATP levels. Acknowledgments Supported by NIH Grants HD 10718 (HRB) and HD 14098 (JLL).
[26] C u l t u r e a n d C h a r a c t e r i s t i c s o f H o r m o n e - R e s p o n s i v e N e u r o b l a s t o m a × G l i o m a H y b r i d Cells
By BERND HAMPRECHT, THOMAS GLASER, GEORG RElSER, ERNST BAYER, and FRIEDRICH PROPST
Due to the complexity of the mammalian nervous system, results from biochemical or pharmacological experiments with pieces or homogenates of nervous tissue are difficult to interpret. Especially it is hard to assign a certain effect observed to a certain cell type. The most desirable situation would be to have at one's disposal the numerous cell types as homogenous cell populations for studying their individual differentiated functions and their mechanisms of intercellular communication. Only recently a few cases have been reported of apparently relatively homogeneous populations of certain cell types from nervous tissue. Therefore, the hope to be able to study molecular mechanisms of nervous tissue functions has been resting completely on model systems derived from tumors of the nervous system. Initially, Sato's laboratory developed clonal rat glioma ~ and mouse neuroblastoma 2 cell lines that still expressed differentiated functions known to occur in glial cells and neurons, respectively. Other groups i p. Benda, J. Lightbody, G. Sato, L. Levine, and W. Sweet, Science 161, 370 (1968). 2 G. Augusti-Tocco and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 64, 311 (1969).
METHODS IN ENZYMOLOGY, VOL. 109
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2