0013-7227/91/1295-2757$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 5 Printed in U.S.A.
Contact-Dependent Intercellular Communication of Bovine Luteal Cells in Culture* DALE A. REDMERt, ANNA T. GRAZUL-BILSKA, AND LAWRENCE P. REYNOLDS Department of Animal and Range Sciences, North Dakota State University, Fargo, North Dakota 58105
ABSTRACT. Mammalian gap junctions permit exchange of nutrients, ions, and regulatory molecules of less than 1.5 kDa among contacting communication-competent cells and may be important for regulation of luteal function and maintenance of luteal homeostasis. The present studies were designed to evaluate gap junction-mediated intercellular communication between bovine luteal cells in culture. Using a dye-coupling technique along with interactive laser cytometry, selected luteal cells were studied for the rate of contact-dependent fluorescence redistribution after photobleaching. The rate of communication, reported as the rate of fluorescence recovery (percentage per min), was determined for steroidogenic cells as follows: 1) small luteal cells contacting only small luteal cells, 2) large luteal cells contacting only small luteal cells, and 3) large luteal cells contacting only large luteal cells. In addition, the effects of known regulators of luteal function [LH, prostaglandin F2a (PGF), and forskolin] on the rate of intercellular communication were determined. Small luteal cells communicated rapidly with each other, exhibiting an initial rate of fluorescence recovery of 4.1 ± 0.1%/min (n = 187). The rate of small cell-small cell communi-
cation was unaffected by LH and PGF. For large luteal cells contacting small luteal cells, however, LH and PGF stimulated (P < 0.02) the rate of communication compared with no hormone [1.6 ± 0.2 (n = 18) and 1.5 ± 0.6 (n = 20) vs. 0.8 ± 0.3%/min (n = 27), respectively]. LH and PGF in combination, however, did not enhance the rate (0.6 ± 0.2%/min; n = 19) of large cellsmall cell communication. In contrast, forskolin significantly stimulated both small cell-small cell and large cell-small cell communication rates compared with no forskolin [34% increase (n = 48) and 50% increase (n = 23), respectively]. Large luteal cells did not communicate with each other under any condition tested. Transmission electron microscopy revealed the presence of numerous gap junction-like structures in bovine luteal cells in culture. These data suggest that luteal cells are capable of intercellular communication and that the rate of communication may be influenced by hormones. Contact-dependent intercellular communication among luteal cells may, therefore, play a significant role in the regulation of luteal function. (Endocrinology 129: 2757-2766, 1991)
T
HE MAMMALIAN gap junction (junctio communicans, nexus) is a junction of communication or electrical coupling between adjacent cells, wherein the apposed cellular membranes are separated by an apparent gap of 3 nm, which is bridged by hexagonally organized connections (2, 3). Under transmission electron microscopy, gap junctions in most tissues, including corpora lutea, are typically pentalaminar in appearance (4, 5). Mammalian gap junctions permit the exchange of nutrients, ions, and regulatory molecules of less than approximately 1.5 kDa (e.g. Ca2+, Na+, and cAMP) among contacting communication-competent cells and are ubiquitous in multicellular organisims (6, 7). It has been suggested that gap junction-mediated intercellular communication plays an important role in the regulation of cell metabolism, proliferation, and signalling (6, 8, 9). The bovine corpus luteum (CL) is composed of several Received April 12,1991. * Journal article 1914 of the North Dakota Agricultural Experiment Station, Project 1780. This work was supported in part by USDA Competitive Grant 87-CRCR-1-2573 (to D.A.R. and L.P.R.). A preliminary communication of this work has been presented (1). t To whom all correspondence and requests for reprints should be addressed.
cell types, including small and large steroidogenic luteal cells, endothelial cells, connective tissue cells, and others (10). Much of the published work regarding luteal function has focused on regulation of. hormone synthesis and release by small and large steroidogenic luteal cells (for review, see Refs. 11-14). However, little information is available regarding interactions among cells of the corpus luteum, especially concerning metabolic connections {e.g. gap junctions), their control, and their role in maintaining luteal homeostasis. Based on ultrastructural studies, gap junctions have been reported in CL of mice, rats, rabbits, dogs, monkeys, and humans (5,15-22). Although not specifically characterized as gap junctions, McClellan et al. (23) and O'Shea et al. (24) have reported that numerous junctional structures exist among contacting ovine luteal cells. To the best of our knowledge, no reports have been published for any mammalian species regarding functional gap junction-mediated intercellular communication among luteal cells. The study of functional gap junctional communication between specific luteal cell types will probably lead to a better understanding of how regulatory signals are integrated among the various cell types contained within the CL. In this report
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS
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we present an important paradigm of cellular interaction, namely hormonal modulation of contact-dependent intercellular communication, applied to bovine luteal cells in culture.
Materials and Methods Materials For cell cultures, Dulbecco's Modified Eagle's medium (DMEM), Ham's F-10 medium, Ca2+- and Mg++-free Hank's Balanced Salt Solution (HBSS), fetal bovine serum, calf serum, crystalline bovine insulin, trypan blue stain (0.4%), and penicillin-streptomycin (10,000 U penicillin G sodium salt and 10,000 ng streptomycin sulfate/ml) were purchased from Gibco (Grand Island, NY). BSA (fraction V), collagenase (type I), forskolin, dimethylsulfoxide (DMSO), transferrin, hydrocortisone, etiocholan-3j9-ol-17-one, nitro blue tetrazolium, and NAD+ were purchased from Sigma (St. Louis, MO). 5-Carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) was purchased from Molecular Probes, Inc. (Eugene, OR). LH (USDA bovine LH B-5) was a gift from the USDA Animal Hormone Program and the National Hormone and Pituitary Program (Beltsville, MD). Prostaglandin F2a (PGF) was purchased from Upjohn Corp. (Kalamazoo, MI). For electron microscopy, cacodylate buffer, osmium tetroxide, uranyl acetate, Epon-Araldite plastic, and lead citrate were purchased from Tousimis Research Corp. (Rockville, MD). For progesterone RIA, progesterone standard was purchased from Sigma, and tritiated progesterone from DuPont/New England Nuclear Products (Boston, MA). The progesterone antibody (GDN-337) was provided by Dr. G. D. Niswender (Colorado State University, Fort Collins, CO). Dissociation of luteal cells
Ovaries were collected from midcycle nonpregnant cows at a local slaughterhouse, and luteal tissue was isolated and minced using methods similar to those described previously (25, 26). Minced luteal tissue was incubated in dissociation medium (1 g tissue/3 ml medium; 2 CL/dispersion; n = 6 dispersions; 12 cows) consisting of Ca2+- and Mg2+-free HBSS containing collagenase (150-300 U/ml), BSA (2%, wt/vol), and antibiotics (100 U penicillin and 100 fig streptomycin/ml) in a shaking (100 cycles/min) water bath at 35-37 C in capped 50-ml siliconized Erlenmeyer flasks. After an initial 40-min incubation, medium containing dispersed luteal cells was aspirated, and fresh dissociation medium was added to the remaining tissue. After this initial collection and replacement, medium was aspirated and replaced every 10-15 min. This process was continued for about 3 h. At the end of each incubation, medium containing dispersed luteal cells was centrifuged (300 x g) for 5 min at 22 C. The pellet was washed three times with Ca2+and Mg2+-free HBSS containing antibiotics and then resuspended in the same medium and dispersed additionally by trituration with a siliconized Pasteur pipette. The cell suspension was filtered through a sterile metal screen (no. 4320, Gelman, Ann Arbor, MI) to remove pieces of tissue and then centrifuged (300 X g; 5 min; 22 C). Cells were washed three times in Ca2+- and Mg2+-free HBSS with antibiotics, filtered
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through a nylon filter with 70-jim pores (Tetko, New York, NY), and resuspended in plating medium [DMEM containing 5% (vol/vol) fetal bovine serum, 5% (vol/vol) calf serum, and antibiotics (100 U penicillin and 100 fig streptomycin/ml)] for use in Exp 1 and 2 (see below). Luteal cells were counted using a hemocytometer, and viability was estimated by trypan blue exclusion. The viability of freshly dispersed luteal cells was 88.2 ± 3.3%. An additional 12 CL were dispersed (2 CL/dispersion; n = 6 dispersions) as described above, but were resuspended in Ca2+- and Mg2+-free HBSS with antibiotics for isolation of the large luteal cells used in Exp 3 and 4. Isolation of large steroidogenic luteal cells
Large steroidogenic luteal cells were separated from other luteal cells using a Beckman JE-6B elutriator rotor (Palo Alto, CA) equipped with a standard elutriation chamber, as described previously for ovine luteal cells (27). Suspensions of enzymatically dispersed cells containing 0.5-1.5 x 108 total cells in 20-ml Ca2+- and Mg2+-free HBSS were applied to the elutriation chamber and immediately subjected to elutriation with Ca2+- and Mg2+-free HBSS at a flow rate of 16 ml/min and a rotor speed of 1800 rpm. After 2.5 min, the flow rate was changed to 17 ml/min (rotor speed, 1800 rpm) for 11.8 min, followed by 27 ml/min at 1400 rpm for 9.3 min and 27 ml/min at 1200 rpm for 9.3 min. Large steroidogenic luteal cells were then recovered from the elutriation chamber in 250 ml Ca2+and Mg2+-free HBSS at a flow rate of 25-19 ml/min at 800 rpm. After collection, the large luteal cell fraction was centrifuged (300 x g; 5 min; 22 C), and the pellet was resuspended in plating medium. The concentration, cell size, and viability of cells were assessed using a hemocytometer, ocular micrometer, and trypan blue exclusion, respectively. The elutriated large cell fraction in suspension contained primarily large luteal cells (diameter, >20 /xm), 17.4 ± 2.2% (range, 10-24%) small luteal cells (diameter, 10 to 1 0 0 w
80
O LL
60
*
-e
-e
8
12
40
20
16
20
Time (min) FiG. 3. Fluorescence recovery curves for selected elutriated cells from Exp 3. All cells were present in one scanning site. In A, cell 1 (•) was a photobleached small cell in contact with a nonphotobleached small cell (—) and a nonphotobleached large cell (- - -)• Note that cell 1 recovered fluorescence after photobleaching, and each adjacent nonphotobleached cell lost a small amount offluorescence,which indicates that a portion of the fluorescent probe from the nonphotobleached cells had moved into the photobleached cell. In B, a photobleached large cell (O) was in contact only with a nonphotobleached large cell (•) and did not exhibit fluoresence recovery after photobleaching. The lack of change in fluorescence values over 20 min in both cells substantiates that large cells did not communicate and did not loose fluoresence due to repetitive laser scanning or leakage of fluorescent probe.
Areas where cell membranes were in close association with each other, as shown by transmission electron microscopy (Fig. 4), were common in a preparation of cultured luteal cells from Exp 1. At greater magnification (Fig. 4B), classical pentalaminar gap junction-like structures were observed in these areas of cell to cell contact. Also present were sites at which junctional structures existed, in which the intercellular space appeared to be trilaminar (not shown). Progesterone concentrations in conditioned medium after 16-24 h of incubation with hormone treatments are presented in Table 2. For incubations of nonelutriated luteal cells containing mixed cell types (Exp 1 and 2), LH, PGF, LH plus PGF, and forskolin increased (P < 0.05) progesterone secretion compared with that of un-
FiG. 4. Transmission electron micrograph of two adjacent luteal cells after 48 h in culture. In micrograph A (x24,600), note areas of close association of the two cell membranes (arrowheads). These areas lack a distinct intercellular space and are characteristic of junctional complexes, such as gap junctions. Also note the presence of large lipid droplets (L), a characteristic feature of luteal cells. Micrograph B shows a greater magnification (x82,000) of these junctional complexes (arrows). Note the pentalaminar structure which is characteristic of gap junctions. Scale bar: A, 0.5 jan; B, 0.25 fim.
treated cells. For elutriated large luteal cells (Exp 3 and 4), PGF and LH plus PGF (P < 0.05) and forskolin (P < 0.10) increased progesterone secretion.
Discussion In the present report it was shown that small luteal cells exhibit rapid contact-dependent communication with each other. Large luteal cells, however, exhibit contact-dependent communication with small luteal cells, but not with each other. In addition, it was shown that rates of communication between large and small cells can be hormonally regulated. It is generally accepted that metabolic coupling is most frequently associated with a specific junctional structure, the gap junction (8, 35). Previous reports have shown that gap junctional structures are common in CL of several species (5, 15-
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS TABLE 2. Effects of LH, PGF, and forskolin on progesterone secretion by bovine luteal cells in culture Progesterone (ng/104 cells • 24 h) Exp 1 (mixed cells) No hormone LH PGF LH + PGF
No forskolin Forskolin
38.5 ± 66.0 ± 70.5 ± 68.0 ±
7.1 7.6" 9.06 8.6*
Exp 3 (large cells) 66.2 ± 102.2 ± 120.4 ± 128.7 ±
10.3 17.1 21.96 21.4"
Exp 2 (mixed cells)
Exp 4 (large cells)
40.0 ± 5.6 68.5 ± 7.5*
73.4 ± 15.2 116.0 ± 18.4C
Progesterone concentrations were determined in conditioned medium collected after 16-24 h of incubation with treatments and immediately before the rate of intercellular communication was determined. " See footnote a of Table 1 for treatments. b Mean ± SE are different from no hormone or no forskolin (P < 0.05). c Mean ± SE is different from no forskolin (P < 0.10).
22). Based on ultrastructural evaluation, the presence of classical pentalaminar gap junctions (4, 5) was demonstrated in the present study for bovine luteal cells in culture. However, other than the present report, data demonstrating functional intercellular communication among luteal cells of any species is, surprisingly, nonexistent. The method used to study functional contact-dependent intercellular communication in the present studies has a distinct advantage over the more classical ultrastructural, dye-coupling, and electro-coupling methods (6, 28). With the aid of an interactive laser cytometer, selected luteal cells that are in contact only with specific types of cells can be analyzed not only for their ability to communicate, but also for their rate of communication. Since gap junctions can be in open or closed states, dye transfer among cells indicates that gap junctional channels are in the open state (36). The rate of communication, therefore, depends not only on the number of gap junctional structures, but also on the number of open gap junctional channels. Thus, methods to measure the rate of intercellular communication, such as those used in the present studies, can give a clear indication of the potential volume of metabolic transfer between live cells over a given period of time. The effects of biological modulators on channel status, rather than simply on numbers of gap junctions, can, therefore, be studied. Albeit that dye coupling between contacting cells is not a direct measure of the flow of biologically important modulators from one cell to another, it does unequivocally demonstrate contact-dependent transfer of hydrophilic molecules between cells (28). Thus, the strong possibility exists that biologically important molecules of low mol wt also are exchanged between cells that
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exhibit dye coupling (30). Unfortunately, determining the rate of transfer of fluorescently labeled, biologically active molecules between cells would be difficult, since those same molecules would probably affect the status of junctional channels. Use of nonbiological probes, therefore, has an advantage over biological markers for determining rates of contact-dependent communication. The techniques used to determine communication between cells in the present studies were not confounded by the presence of contaminating cell types, since only specific cells in contact with the desired cell types were analyzed for communication. Therefore, it is important to remind the reader that the observed effects of hormones on progesterone secretion were the result of a much less specific response (i.e. probably responses of both large and small cells) than the specific effects of hormones on contact-dependent intercellular communication. As stated by Trautmann (36), "gap junctions allow a direct cell-to-cell communication, and are sometimes thought to confer a syncitial behavior to a coupled ensemble of cells." This proposal is important in considering an endocrine organ such as the CL, which contains several cell types of different origin and with different types and numbers of hormone receptors (10, 11, 37). Fletcher et al. (9) demonstrated that, as long as they were in contact, all ovarian carcinoma cells responded to hCG with dissociation of cAMP-dependent protein kinase subunits, even though not all cells exhibited hCG binding. Thus, metabolic coupling of luteal cells via gap junctions would allow for coordination of hormonal responses among cells without the requirement that all cells have the same types or numbers of receptors. As shown by Lawrence et al. (38) granulosa cells and mouse myocardial cells in coculture exhibit contact-dependent communication, as demonstrated by the fact that exposure of the cocultures to hormone specific for one cell type caused a response in the other cell type. In agreement with the present data, endocrine regulation of the number and function of gap junctions has been observed in several other cell types, including ovarian granulosa cells (9, 38, 39). In the present studies, small luteal cells, which probably contain both LH and PGF receptors (37), communicated rapidly with each other, and the rate of communication was not influenced by LH and/or PGF, even though both hormones stimulated progesterone secretion. Thus, gap junctional channels between small luteal cells appear to be open normally and may, therefore, be involved in the maintenance of steady state conditions (36). In contrast, the rate of communication between large luteal cells and small luteal cells was stimulated by LH and PGF, which suggests that gap junctional channels are partially closed under resting conditions and
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS
open during hormonal stimulation. Many factors have been shown to regulate gap junctional conductance, including second messengers, such as Ca2+, cAMP, and diacylglycerol (4, 40, 41). Forskolin, which is known to stimulate the catalytic moiety of adenylate cyclase (42) resulting in increased cAMP and progesterone accumulation in luteal cells (43), increased rate of communication between contacting small cells and between large cells in contact with small cells in the present study. Elevated levels of cAMP have previously been shown to stimulate the rate of gap junctional communication in other cell types (44-46). Surprisingly, forskolin was the only compound tested in the present study that had an effect on small cell to small cell communication. Although levels of cAMP were not measured in the present studies, we have found that treatment of luteal cells with forskolin results in much greater cellular concentrations of cAMP than treatment with LH (Grazul-Bilska, A. T., L. P. Reynolds, and D. A. Redmer, unpublished), which may explain why forskolin, but not LH or PGF, stimulated communication between small luteal cells. It should also be noted that the design of the present studies permitted sufficient time, after the addition of hormones, for gap junctions to form between cells (47, 48). Therefore, hormonal effects on the rate of communication may also have been related to increased numbers of gap junctions, rather than to direct hormonal effects on the status (open vs. closed) of existing gap junctional channels. Large luteal cells failed to communicate with each other under any of the conditions tested. The lack of detectable communication between large cells is an important observation. The absence of gap junctional communication is frequently associated with abnormal tissue growth (49), because of the inability of cells to maintain homeostasis. However, the lack of communication between large luteal cells may be important functionally, for constitutive maintenance of progesterone production (11, 50). On the other hand, small cells readily communicate with large cells. Since small cells contain the majority of the LH receptors (11, 50), whereas large cells produce larger amounts of progesterone, the transfer of regulatory signals between small and large cells may be important during critical periods of luteal function. The scenario of small cell-large cell interactions may best be demonstrated by several studies that have clearly shown a synergistic effect on progesterone production when large and small luteal cells were cultured together (27, 51,52). In addition, the transfer of regulatory (luteolytic) signals from large cells to small cells has previously been hypothesized (12, 53). Of perhaps greater significance than its role in the function of the mature CL, is the role gap junctional communication may play in the growth and differentia-
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tion of luteal tissue. Regardless of cell type, elevations in the rate of gap junctional communication lead to an enhanced control of growth (6, 54) and differentiation (6). After ovulation, the central core of the follicle is rapidly infiltrated with blood vessels concomitant with phenomenal growth of parenchymal cells (12, 55). The granulosa and thecal cells must proliferate and differentiate to form a complete 4- to 5-g (bovine) endocrine organ in less than 14 days (26), making the CL one of the fastest growing normal tissues of the body. By only 3 days after ovulation, proliferating follicular cells begin functional differentiation, as detected by increasing systemic concentrations of progesterone (56). Other changes that may take place include further differentiation of small cells into large cells (57) as well as production of a variety of hormones by small and large cells. At the same time, a limit is imposed on the final size (degree of cellular growth) of the CL. The orchestration of events such as those just described for the CL is dependent on intercellular communication (58). The route of communication can be either humoral (e.g. endocrine or paracrine) or by direct transfer of small mol wt molecules through gap junctions (58). Since contact-dependent intercellular communication between homologous and heterologous cell types has been associated with the maintenance of a homeostatic environment and synchronization of cellular responses during cell and tissue growth, differentiation, and regeneration (7,59,60), the potential role of gap junctions in luteal function is apparent. This report, in which we demonstrate that bovine luteal cells in culture exhibit contact-dependent intercellular communication, suggests that communication with the surrounding cellular "society" should be an important aspect of regulation in vivo in this metabolically active endocrine organ.
Acknowledgments We thank the USDA Animal Hormone Program and the National Hormone and Pituitary Program for providing bovine LH, and Dr. G. D. Niswender for providing progesterone antisera. The authors acknowledge J. D. Kirsch, K. C. Kraft, and D. S. Millaway for technical assistance; the North Dakota State University Cell Biology Center for use of facilities; the North Dakota State University Electron Microscopy Laboratory for assistance with electron microscopy; and J. Berg for typing the manuscript.
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS 3. Williams PL, Warwick R, Dyson M, Bannister LH (eds) 1989 Gray's Anatomy, ed 37. Churchill Livingstone, New York, p 24 4. Bennett MVL, Spray DC (eds) 1985 Gap Junctions. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 1-404 5. Fukushima M 1977 Intercellular junctions in the human developing preovulatory follicle and corpus luteum. Int J Fertil 22:206-216 6. Loewenstein WR1979 Junctional intercellular communication and the control of growth. Biochim Biophys Acta 560:1-65 7. Loewenstein WR 1981 Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev 61:829-913 8. Hertzberg EL, Lawrence TS, Gilula NB 1981 Gap junctional communication. Annu Rev Physiol 43:479-491 9. Fletcher WH, Byus CV, Walsh DA 1987 Receptor-mediated action without receptor occupancy: a function for cell-cell communication in ovarian follicles. In: Mahesh VB, Dhindsa DS, Anderson E, Kalra SP (eds) Regulation of Ovarian and Testicular Function. Plenum Press, New York, pp 299-323 10. O'Shea JD, Rodgers RJ, D'Occhio MJ 1989 Cellular composition of the cyclic corpus luteum of the cow. J Reprod Fertil 85:483-487 11. Niswender GD, Schwall RH, Fitz TA, Farin CE, Sawyer HR 1985 Regulation of luteal function in domestic ruminants: new concepts. Recent Prog Horm Res 41:101-151 12. Niswender GD, Nett TM 1988 The corpus luteum and its control. In: Knobil E, Neill J (eds) The Physiology of Reproduction. Raven Press, New York, pp 489-525 13. Hansel W, Convey EM 1983 Physiology of the estrous cycle. J Anim Sci [Suppl 2] 57:404-424 14. Hansel W, Alila HW, Dowd JP, Yang X 1987 Control of steroidogenesis in small and large bovine luteal cells. Aust J Biol Sci 40:331-351 15. Albertini DF, Anderson E 1975 Structural modifications of lutein cell gap junctions during pregnancy in the rat and mouse. Anat Rec 181:171-194 16. Van Blerkom J, Motta P 1978 A scanning electron microscope study of the luteo-follicular complex. Cell Tissue Res 189:131-153 17. Enders AC 1973 Cytology of the corpus luteum. Biol Reprod 8:158182 18. Abel JH, McClellan MC, Verhage HG, Niswender GN 1975 Subcellular compartmentalization of the luteal cell in the ovary of the dog. Cell Tissue Res 158:461-480 19. Abel JH, Verhage HG, McClellan MC, Niswender GN 1975 Ultrastructural analysis of the granulosa-luteal cell transition in the ovary of the dog. Cell Tissue Res 160:155-176 20. Crisp TM, Dessouky DA 1980 Fine structure of primate corpus luteum. In: Motta PM, Hafez ESE (eds) Biology of the Ovary. Martinus Nijhoff, Boston, pp 150-161 21. Gulyas BJ, Yuan L, Tullner WW, Hodgen GD 1976 The fine structure of corpus luteum from intact, hypophysectomized and fetectomized pregnant monkeys (Macaca mulatto) at term. Biol Reprod 14:613-626 22. Adams EC, Hertig AT 1969 Studies on the human corpus luteum. J Cell Biol 41:696-715 23. McClellan MC, Diekman MA, Abel JH, Niswender GD 1975 Luteinizing hormone, progesterone and the morphological development of normal and superovulated corpora lutea in sheep. Cell Tissue Res 164:291-307 24. O'Shea JD, Cran DG, Hay MF 1979 The small luteal cell of the sheep. J Anat 128:239-251 25. Redmer DA, Grazul AT, Kirsch JD, Reynolds LP 1988 Angiogenic activity of bovine corpora lutea at several stages of luteal development. J Reprod Fertil 82:627-634 26. Grazul AT, Kirsch JD, Slanger WD, Marchello MJ, Redmer DA 1989 Prostaglandin F2a, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effect of oxytocin, luteinizing hormone, prostaglandin F2a and estradiol17/3. Prostaglandins 38:307-318 27. Grazul-Bilska AT, Redmer DA, Reynolds LP 1991 Secretion of angiogenic activity and progesterone by ovine luteal cell types in vitro. J Anim Sci 69:2099-2107 28. Wade MH, Trosko JE, Schindler M 1986 A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232:525-528
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types from the corpus luteum of the sow in progesterone synthesis in vitro. J Reprod Fertil 64:315-323 Silvia WJ, Fitz TA, Mayan MH, Niswender GD 1984 Cellular and molecular mechanisms involved in luteolysis and maternal recognition of pregnancy in the ewe. Anim Reprod Sci 7:57-74 Bertram JS 1990 Role of gap junctional cell/cell communication in the control of proliferation and neoplastic transformation. Radiat Res 123:252-256 Bassett DL 1943 The changes in the vascular pattern of the ovary of the albino rat during the estrous cycle. Am J Anat 73:251-291 Donaldson LE 1970 Peripheral plasma progesterone concentration of cows during puberty, oestrous cycles, pregnancy and lactation,
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59. 60.
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and the effects of under-nutrition or exogenous oxytocin on progesterone concentration. J Endocrinol 48:599-614 Alila HW, Hansel W 1984 Origin of different cell types in the bovine corpus luteum as characterized by specific monoclonal antibodies. Biol Reprod 31:1015-1025 Trosko JE, Chang CC, Madhukar BV, Klaunig JE 1990 Chemical, oncogene and growth factor inhibition of gap junctional intercellular communication: an integrative hypothesis of carcinogenesis. Pathobiology 58:265-278 Schultz RM 1985 Roles of cell-to-cell communication. Biol Reprod 32:27-42 Pitts JD, Finbow ME 1986 The gap junction. J Cell Sci 4:239-266
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Vol. 129, No. 5 Printed in U.S.A.
Contact-Dependent Intercellular Communication of Bovine Luteal Cells in Culture* DALE A. REDMERt, ANNA T. GRAZUL-BILSKA, AND LAWRENCE P. REYNOLDS Department of Animal and Range Sciences, North Dakota State University, Fargo, North Dakota 58105
ABSTRACT. Mammalian gap junctions permit exchange of nutrients, ions, and regulatory molecules of less than 1.5 kDa among contacting communication-competent cells and may be important for regulation of luteal function and maintenance of luteal homeostasis. The present studies were designed to evaluate gap junction-mediated intercellular communication between bovine luteal cells in culture. Using a dye-coupling technique along with interactive laser cytometry, selected luteal cells were studied for the rate of contact-dependent fluorescence redistribution after photobleaching. The rate of communication, reported as the rate of fluorescence recovery (percentage per min), was determined for steroidogenic cells as follows: 1) small luteal cells contacting only small luteal cells, 2) large luteal cells contacting only small luteal cells, and 3) large luteal cells contacting only large luteal cells. In addition, the effects of known regulators of luteal function [LH, prostaglandin F2a (PGF), and forskolin] on the rate of intercellular communication were determined. Small luteal cells communicated rapidly with each other, exhibiting an initial rate of fluorescence recovery of 4.1 ± 0.1%/min (n = 187). The rate of small cell-small cell communi-
cation was unaffected by LH and PGF. For large luteal cells contacting small luteal cells, however, LH and PGF stimulated (P < 0.02) the rate of communication compared with no hormone [1.6 ± 0.2 (n = 18) and 1.5 ± 0.6 (n = 20) vs. 0.8 ± 0.3%/min (n = 27), respectively]. LH and PGF in combination, however, did not enhance the rate (0.6 ± 0.2%/min; n = 19) of large cellsmall cell communication. In contrast, forskolin significantly stimulated both small cell-small cell and large cell-small cell communication rates compared with no forskolin [34% increase (n = 48) and 50% increase (n = 23), respectively]. Large luteal cells did not communicate with each other under any condition tested. Transmission electron microscopy revealed the presence of numerous gap junction-like structures in bovine luteal cells in culture. These data suggest that luteal cells are capable of intercellular communication and that the rate of communication may be influenced by hormones. Contact-dependent intercellular communication among luteal cells may, therefore, play a significant role in the regulation of luteal function. (Endocrinology 129: 2757-2766, 1991)
T
HE MAMMALIAN gap junction (junctio communicans, nexus) is a junction of communication or electrical coupling between adjacent cells, wherein the apposed cellular membranes are separated by an apparent gap of 3 nm, which is bridged by hexagonally organized connections (2, 3). Under transmission electron microscopy, gap junctions in most tissues, including corpora lutea, are typically pentalaminar in appearance (4, 5). Mammalian gap junctions permit the exchange of nutrients, ions, and regulatory molecules of less than approximately 1.5 kDa (e.g. Ca2+, Na+, and cAMP) among contacting communication-competent cells and are ubiquitous in multicellular organisims (6, 7). It has been suggested that gap junction-mediated intercellular communication plays an important role in the regulation of cell metabolism, proliferation, and signalling (6, 8, 9). The bovine corpus luteum (CL) is composed of several Received April 12,1991. * Journal article 1914 of the North Dakota Agricultural Experiment Station, Project 1780. This work was supported in part by USDA Competitive Grant 87-CRCR-1-2573 (to D.A.R. and L.P.R.). A preliminary communication of this work has been presented (1). t To whom all correspondence and requests for reprints should be addressed.
cell types, including small and large steroidogenic luteal cells, endothelial cells, connective tissue cells, and others (10). Much of the published work regarding luteal function has focused on regulation of. hormone synthesis and release by small and large steroidogenic luteal cells (for review, see Refs. 11-14). However, little information is available regarding interactions among cells of the corpus luteum, especially concerning metabolic connections {e.g. gap junctions), their control, and their role in maintaining luteal homeostasis. Based on ultrastructural studies, gap junctions have been reported in CL of mice, rats, rabbits, dogs, monkeys, and humans (5,15-22). Although not specifically characterized as gap junctions, McClellan et al. (23) and O'Shea et al. (24) have reported that numerous junctional structures exist among contacting ovine luteal cells. To the best of our knowledge, no reports have been published for any mammalian species regarding functional gap junction-mediated intercellular communication among luteal cells. The study of functional gap junctional communication between specific luteal cell types will probably lead to a better understanding of how regulatory signals are integrated among the various cell types contained within the CL. In this report
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS
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we present an important paradigm of cellular interaction, namely hormonal modulation of contact-dependent intercellular communication, applied to bovine luteal cells in culture.
Materials and Methods Materials For cell cultures, Dulbecco's Modified Eagle's medium (DMEM), Ham's F-10 medium, Ca2+- and Mg++-free Hank's Balanced Salt Solution (HBSS), fetal bovine serum, calf serum, crystalline bovine insulin, trypan blue stain (0.4%), and penicillin-streptomycin (10,000 U penicillin G sodium salt and 10,000 ng streptomycin sulfate/ml) were purchased from Gibco (Grand Island, NY). BSA (fraction V), collagenase (type I), forskolin, dimethylsulfoxide (DMSO), transferrin, hydrocortisone, etiocholan-3j9-ol-17-one, nitro blue tetrazolium, and NAD+ were purchased from Sigma (St. Louis, MO). 5-Carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) was purchased from Molecular Probes, Inc. (Eugene, OR). LH (USDA bovine LH B-5) was a gift from the USDA Animal Hormone Program and the National Hormone and Pituitary Program (Beltsville, MD). Prostaglandin F2a (PGF) was purchased from Upjohn Corp. (Kalamazoo, MI). For electron microscopy, cacodylate buffer, osmium tetroxide, uranyl acetate, Epon-Araldite plastic, and lead citrate were purchased from Tousimis Research Corp. (Rockville, MD). For progesterone RIA, progesterone standard was purchased from Sigma, and tritiated progesterone from DuPont/New England Nuclear Products (Boston, MA). The progesterone antibody (GDN-337) was provided by Dr. G. D. Niswender (Colorado State University, Fort Collins, CO). Dissociation of luteal cells
Ovaries were collected from midcycle nonpregnant cows at a local slaughterhouse, and luteal tissue was isolated and minced using methods similar to those described previously (25, 26). Minced luteal tissue was incubated in dissociation medium (1 g tissue/3 ml medium; 2 CL/dispersion; n = 6 dispersions; 12 cows) consisting of Ca2+- and Mg2+-free HBSS containing collagenase (150-300 U/ml), BSA (2%, wt/vol), and antibiotics (100 U penicillin and 100 fig streptomycin/ml) in a shaking (100 cycles/min) water bath at 35-37 C in capped 50-ml siliconized Erlenmeyer flasks. After an initial 40-min incubation, medium containing dispersed luteal cells was aspirated, and fresh dissociation medium was added to the remaining tissue. After this initial collection and replacement, medium was aspirated and replaced every 10-15 min. This process was continued for about 3 h. At the end of each incubation, medium containing dispersed luteal cells was centrifuged (300 x g) for 5 min at 22 C. The pellet was washed three times with Ca2+and Mg2+-free HBSS containing antibiotics and then resuspended in the same medium and dispersed additionally by trituration with a siliconized Pasteur pipette. The cell suspension was filtered through a sterile metal screen (no. 4320, Gelman, Ann Arbor, MI) to remove pieces of tissue and then centrifuged (300 X g; 5 min; 22 C). Cells were washed three times in Ca2+- and Mg2+-free HBSS with antibiotics, filtered
Endo • 1991 Vol 129-No 5
through a nylon filter with 70-jim pores (Tetko, New York, NY), and resuspended in plating medium [DMEM containing 5% (vol/vol) fetal bovine serum, 5% (vol/vol) calf serum, and antibiotics (100 U penicillin and 100 fig streptomycin/ml)] for use in Exp 1 and 2 (see below). Luteal cells were counted using a hemocytometer, and viability was estimated by trypan blue exclusion. The viability of freshly dispersed luteal cells was 88.2 ± 3.3%. An additional 12 CL were dispersed (2 CL/dispersion; n = 6 dispersions) as described above, but were resuspended in Ca2+- and Mg2+-free HBSS with antibiotics for isolation of the large luteal cells used in Exp 3 and 4. Isolation of large steroidogenic luteal cells
Large steroidogenic luteal cells were separated from other luteal cells using a Beckman JE-6B elutriator rotor (Palo Alto, CA) equipped with a standard elutriation chamber, as described previously for ovine luteal cells (27). Suspensions of enzymatically dispersed cells containing 0.5-1.5 x 108 total cells in 20-ml Ca2+- and Mg2+-free HBSS were applied to the elutriation chamber and immediately subjected to elutriation with Ca2+- and Mg2+-free HBSS at a flow rate of 16 ml/min and a rotor speed of 1800 rpm. After 2.5 min, the flow rate was changed to 17 ml/min (rotor speed, 1800 rpm) for 11.8 min, followed by 27 ml/min at 1400 rpm for 9.3 min and 27 ml/min at 1200 rpm for 9.3 min. Large steroidogenic luteal cells were then recovered from the elutriation chamber in 250 ml Ca2+and Mg2+-free HBSS at a flow rate of 25-19 ml/min at 800 rpm. After collection, the large luteal cell fraction was centrifuged (300 x g; 5 min; 22 C), and the pellet was resuspended in plating medium. The concentration, cell size, and viability of cells were assessed using a hemocytometer, ocular micrometer, and trypan blue exclusion, respectively. The elutriated large cell fraction in suspension contained primarily large luteal cells (diameter, >20 /xm), 17.4 ± 2.2% (range, 10-24%) small luteal cells (diameter, 10 to 1 0 0 w
80
O LL
60
*
-e
-e
8
12
40
20
16
20
Time (min) FiG. 3. Fluorescence recovery curves for selected elutriated cells from Exp 3. All cells were present in one scanning site. In A, cell 1 (•) was a photobleached small cell in contact with a nonphotobleached small cell (—) and a nonphotobleached large cell (- - -)• Note that cell 1 recovered fluorescence after photobleaching, and each adjacent nonphotobleached cell lost a small amount offluorescence,which indicates that a portion of the fluorescent probe from the nonphotobleached cells had moved into the photobleached cell. In B, a photobleached large cell (O) was in contact only with a nonphotobleached large cell (•) and did not exhibit fluoresence recovery after photobleaching. The lack of change in fluorescence values over 20 min in both cells substantiates that large cells did not communicate and did not loose fluoresence due to repetitive laser scanning or leakage of fluorescent probe.
Areas where cell membranes were in close association with each other, as shown by transmission electron microscopy (Fig. 4), were common in a preparation of cultured luteal cells from Exp 1. At greater magnification (Fig. 4B), classical pentalaminar gap junction-like structures were observed in these areas of cell to cell contact. Also present were sites at which junctional structures existed, in which the intercellular space appeared to be trilaminar (not shown). Progesterone concentrations in conditioned medium after 16-24 h of incubation with hormone treatments are presented in Table 2. For incubations of nonelutriated luteal cells containing mixed cell types (Exp 1 and 2), LH, PGF, LH plus PGF, and forskolin increased (P < 0.05) progesterone secretion compared with that of un-
FiG. 4. Transmission electron micrograph of two adjacent luteal cells after 48 h in culture. In micrograph A (x24,600), note areas of close association of the two cell membranes (arrowheads). These areas lack a distinct intercellular space and are characteristic of junctional complexes, such as gap junctions. Also note the presence of large lipid droplets (L), a characteristic feature of luteal cells. Micrograph B shows a greater magnification (x82,000) of these junctional complexes (arrows). Note the pentalaminar structure which is characteristic of gap junctions. Scale bar: A, 0.5 jan; B, 0.25 fim.
treated cells. For elutriated large luteal cells (Exp 3 and 4), PGF and LH plus PGF (P < 0.05) and forskolin (P < 0.10) increased progesterone secretion.
Discussion In the present report it was shown that small luteal cells exhibit rapid contact-dependent communication with each other. Large luteal cells, however, exhibit contact-dependent communication with small luteal cells, but not with each other. In addition, it was shown that rates of communication between large and small cells can be hormonally regulated. It is generally accepted that metabolic coupling is most frequently associated with a specific junctional structure, the gap junction (8, 35). Previous reports have shown that gap junctional structures are common in CL of several species (5, 15-
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS TABLE 2. Effects of LH, PGF, and forskolin on progesterone secretion by bovine luteal cells in culture Progesterone (ng/104 cells • 24 h) Exp 1 (mixed cells) No hormone LH PGF LH + PGF
No forskolin Forskolin
38.5 ± 66.0 ± 70.5 ± 68.0 ±
7.1 7.6" 9.06 8.6*
Exp 3 (large cells) 66.2 ± 102.2 ± 120.4 ± 128.7 ±
10.3 17.1 21.96 21.4"
Exp 2 (mixed cells)
Exp 4 (large cells)
40.0 ± 5.6 68.5 ± 7.5*
73.4 ± 15.2 116.0 ± 18.4C
Progesterone concentrations were determined in conditioned medium collected after 16-24 h of incubation with treatments and immediately before the rate of intercellular communication was determined. " See footnote a of Table 1 for treatments. b Mean ± SE are different from no hormone or no forskolin (P < 0.05). c Mean ± SE is different from no forskolin (P < 0.10).
22). Based on ultrastructural evaluation, the presence of classical pentalaminar gap junctions (4, 5) was demonstrated in the present study for bovine luteal cells in culture. However, other than the present report, data demonstrating functional intercellular communication among luteal cells of any species is, surprisingly, nonexistent. The method used to study functional contact-dependent intercellular communication in the present studies has a distinct advantage over the more classical ultrastructural, dye-coupling, and electro-coupling methods (6, 28). With the aid of an interactive laser cytometer, selected luteal cells that are in contact only with specific types of cells can be analyzed not only for their ability to communicate, but also for their rate of communication. Since gap junctions can be in open or closed states, dye transfer among cells indicates that gap junctional channels are in the open state (36). The rate of communication, therefore, depends not only on the number of gap junctional structures, but also on the number of open gap junctional channels. Thus, methods to measure the rate of intercellular communication, such as those used in the present studies, can give a clear indication of the potential volume of metabolic transfer between live cells over a given period of time. The effects of biological modulators on channel status, rather than simply on numbers of gap junctions, can, therefore, be studied. Albeit that dye coupling between contacting cells is not a direct measure of the flow of biologically important modulators from one cell to another, it does unequivocally demonstrate contact-dependent transfer of hydrophilic molecules between cells (28). Thus, the strong possibility exists that biologically important molecules of low mol wt also are exchanged between cells that
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exhibit dye coupling (30). Unfortunately, determining the rate of transfer of fluorescently labeled, biologically active molecules between cells would be difficult, since those same molecules would probably affect the status of junctional channels. Use of nonbiological probes, therefore, has an advantage over biological markers for determining rates of contact-dependent communication. The techniques used to determine communication between cells in the present studies were not confounded by the presence of contaminating cell types, since only specific cells in contact with the desired cell types were analyzed for communication. Therefore, it is important to remind the reader that the observed effects of hormones on progesterone secretion were the result of a much less specific response (i.e. probably responses of both large and small cells) than the specific effects of hormones on contact-dependent intercellular communication. As stated by Trautmann (36), "gap junctions allow a direct cell-to-cell communication, and are sometimes thought to confer a syncitial behavior to a coupled ensemble of cells." This proposal is important in considering an endocrine organ such as the CL, which contains several cell types of different origin and with different types and numbers of hormone receptors (10, 11, 37). Fletcher et al. (9) demonstrated that, as long as they were in contact, all ovarian carcinoma cells responded to hCG with dissociation of cAMP-dependent protein kinase subunits, even though not all cells exhibited hCG binding. Thus, metabolic coupling of luteal cells via gap junctions would allow for coordination of hormonal responses among cells without the requirement that all cells have the same types or numbers of receptors. As shown by Lawrence et al. (38) granulosa cells and mouse myocardial cells in coculture exhibit contact-dependent communication, as demonstrated by the fact that exposure of the cocultures to hormone specific for one cell type caused a response in the other cell type. In agreement with the present data, endocrine regulation of the number and function of gap junctions has been observed in several other cell types, including ovarian granulosa cells (9, 38, 39). In the present studies, small luteal cells, which probably contain both LH and PGF receptors (37), communicated rapidly with each other, and the rate of communication was not influenced by LH and/or PGF, even though both hormones stimulated progesterone secretion. Thus, gap junctional channels between small luteal cells appear to be open normally and may, therefore, be involved in the maintenance of steady state conditions (36). In contrast, the rate of communication between large luteal cells and small luteal cells was stimulated by LH and PGF, which suggests that gap junctional channels are partially closed under resting conditions and
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS
open during hormonal stimulation. Many factors have been shown to regulate gap junctional conductance, including second messengers, such as Ca2+, cAMP, and diacylglycerol (4, 40, 41). Forskolin, which is known to stimulate the catalytic moiety of adenylate cyclase (42) resulting in increased cAMP and progesterone accumulation in luteal cells (43), increased rate of communication between contacting small cells and between large cells in contact with small cells in the present study. Elevated levels of cAMP have previously been shown to stimulate the rate of gap junctional communication in other cell types (44-46). Surprisingly, forskolin was the only compound tested in the present study that had an effect on small cell to small cell communication. Although levels of cAMP were not measured in the present studies, we have found that treatment of luteal cells with forskolin results in much greater cellular concentrations of cAMP than treatment with LH (Grazul-Bilska, A. T., L. P. Reynolds, and D. A. Redmer, unpublished), which may explain why forskolin, but not LH or PGF, stimulated communication between small luteal cells. It should also be noted that the design of the present studies permitted sufficient time, after the addition of hormones, for gap junctions to form between cells (47, 48). Therefore, hormonal effects on the rate of communication may also have been related to increased numbers of gap junctions, rather than to direct hormonal effects on the status (open vs. closed) of existing gap junctional channels. Large luteal cells failed to communicate with each other under any of the conditions tested. The lack of detectable communication between large cells is an important observation. The absence of gap junctional communication is frequently associated with abnormal tissue growth (49), because of the inability of cells to maintain homeostasis. However, the lack of communication between large luteal cells may be important functionally, for constitutive maintenance of progesterone production (11, 50). On the other hand, small cells readily communicate with large cells. Since small cells contain the majority of the LH receptors (11, 50), whereas large cells produce larger amounts of progesterone, the transfer of regulatory signals between small and large cells may be important during critical periods of luteal function. The scenario of small cell-large cell interactions may best be demonstrated by several studies that have clearly shown a synergistic effect on progesterone production when large and small luteal cells were cultured together (27, 51,52). In addition, the transfer of regulatory (luteolytic) signals from large cells to small cells has previously been hypothesized (12, 53). Of perhaps greater significance than its role in the function of the mature CL, is the role gap junctional communication may play in the growth and differentia-
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tion of luteal tissue. Regardless of cell type, elevations in the rate of gap junctional communication lead to an enhanced control of growth (6, 54) and differentiation (6). After ovulation, the central core of the follicle is rapidly infiltrated with blood vessels concomitant with phenomenal growth of parenchymal cells (12, 55). The granulosa and thecal cells must proliferate and differentiate to form a complete 4- to 5-g (bovine) endocrine organ in less than 14 days (26), making the CL one of the fastest growing normal tissues of the body. By only 3 days after ovulation, proliferating follicular cells begin functional differentiation, as detected by increasing systemic concentrations of progesterone (56). Other changes that may take place include further differentiation of small cells into large cells (57) as well as production of a variety of hormones by small and large cells. At the same time, a limit is imposed on the final size (degree of cellular growth) of the CL. The orchestration of events such as those just described for the CL is dependent on intercellular communication (58). The route of communication can be either humoral (e.g. endocrine or paracrine) or by direct transfer of small mol wt molecules through gap junctions (58). Since contact-dependent intercellular communication between homologous and heterologous cell types has been associated with the maintenance of a homeostatic environment and synchronization of cellular responses during cell and tissue growth, differentiation, and regeneration (7,59,60), the potential role of gap junctions in luteal function is apparent. This report, in which we demonstrate that bovine luteal cells in culture exhibit contact-dependent intercellular communication, suggests that communication with the surrounding cellular "society" should be an important aspect of regulation in vivo in this metabolically active endocrine organ.
Acknowledgments We thank the USDA Animal Hormone Program and the National Hormone and Pituitary Program for providing bovine LH, and Dr. G. D. Niswender for providing progesterone antisera. The authors acknowledge J. D. Kirsch, K. C. Kraft, and D. S. Millaway for technical assistance; the North Dakota State University Cell Biology Center for use of facilities; the North Dakota State University Electron Microscopy Laboratory for assistance with electron microscopy; and J. Berg for typing the manuscript.
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INTERCELLULAR COMMUNICATION OF LUTEAL CELLS 3. Williams PL, Warwick R, Dyson M, Bannister LH (eds) 1989 Gray's Anatomy, ed 37. Churchill Livingstone, New York, p 24 4. Bennett MVL, Spray DC (eds) 1985 Gap Junctions. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 1-404 5. Fukushima M 1977 Intercellular junctions in the human developing preovulatory follicle and corpus luteum. Int J Fertil 22:206-216 6. Loewenstein WR1979 Junctional intercellular communication and the control of growth. Biochim Biophys Acta 560:1-65 7. Loewenstein WR 1981 Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev 61:829-913 8. Hertzberg EL, Lawrence TS, Gilula NB 1981 Gap junctional communication. Annu Rev Physiol 43:479-491 9. Fletcher WH, Byus CV, Walsh DA 1987 Receptor-mediated action without receptor occupancy: a function for cell-cell communication in ovarian follicles. In: Mahesh VB, Dhindsa DS, Anderson E, Kalra SP (eds) Regulation of Ovarian and Testicular Function. Plenum Press, New York, pp 299-323 10. O'Shea JD, Rodgers RJ, D'Occhio MJ 1989 Cellular composition of the cyclic corpus luteum of the cow. J Reprod Fertil 85:483-487 11. Niswender GD, Schwall RH, Fitz TA, Farin CE, Sawyer HR 1985 Regulation of luteal function in domestic ruminants: new concepts. Recent Prog Horm Res 41:101-151 12. Niswender GD, Nett TM 1988 The corpus luteum and its control. In: Knobil E, Neill J (eds) The Physiology of Reproduction. Raven Press, New York, pp 489-525 13. Hansel W, Convey EM 1983 Physiology of the estrous cycle. J Anim Sci [Suppl 2] 57:404-424 14. Hansel W, Alila HW, Dowd JP, Yang X 1987 Control of steroidogenesis in small and large bovine luteal cells. Aust J Biol Sci 40:331-351 15. Albertini DF, Anderson E 1975 Structural modifications of lutein cell gap junctions during pregnancy in the rat and mouse. Anat Rec 181:171-194 16. Van Blerkom J, Motta P 1978 A scanning electron microscope study of the luteo-follicular complex. Cell Tissue Res 189:131-153 17. Enders AC 1973 Cytology of the corpus luteum. Biol Reprod 8:158182 18. Abel JH, McClellan MC, Verhage HG, Niswender GN 1975 Subcellular compartmentalization of the luteal cell in the ovary of the dog. Cell Tissue Res 158:461-480 19. Abel JH, Verhage HG, McClellan MC, Niswender GN 1975 Ultrastructural analysis of the granulosa-luteal cell transition in the ovary of the dog. Cell Tissue Res 160:155-176 20. Crisp TM, Dessouky DA 1980 Fine structure of primate corpus luteum. In: Motta PM, Hafez ESE (eds) Biology of the Ovary. Martinus Nijhoff, Boston, pp 150-161 21. Gulyas BJ, Yuan L, Tullner WW, Hodgen GD 1976 The fine structure of corpus luteum from intact, hypophysectomized and fetectomized pregnant monkeys (Macaca mulatto) at term. Biol Reprod 14:613-626 22. Adams EC, Hertig AT 1969 Studies on the human corpus luteum. J Cell Biol 41:696-715 23. McClellan MC, Diekman MA, Abel JH, Niswender GD 1975 Luteinizing hormone, progesterone and the morphological development of normal and superovulated corpora lutea in sheep. Cell Tissue Res 164:291-307 24. O'Shea JD, Cran DG, Hay MF 1979 The small luteal cell of the sheep. J Anat 128:239-251 25. Redmer DA, Grazul AT, Kirsch JD, Reynolds LP 1988 Angiogenic activity of bovine corpora lutea at several stages of luteal development. J Reprod Fertil 82:627-634 26. Grazul AT, Kirsch JD, Slanger WD, Marchello MJ, Redmer DA 1989 Prostaglandin F2a, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effect of oxytocin, luteinizing hormone, prostaglandin F2a and estradiol17/3. Prostaglandins 38:307-318 27. Grazul-Bilska AT, Redmer DA, Reynolds LP 1991 Secretion of angiogenic activity and progesterone by ovine luteal cell types in vitro. J Anim Sci 69:2099-2107 28. Wade MH, Trosko JE, Schindler M 1986 A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232:525-528
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