Molecular and Cellular Endocrinology, 76 (1991) 35-44 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
35
MOLCEL 02453
Function of prolactin cells in the individual rat pituitary gland is location dependent P. Mukherjee, T. Salada and W.C. Hymer Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A. (Received 8 October 1990; accepted 30 November 1990)
Key words: Prolactin; Prolactin cell heterogeneity; Pituitary gland; (Rat)
Summary Anterior pituitary glands from individual ovariectomized (ovx) or ovx-estrogen (E2) treated rats were sectioned into ~ cubes. Each section was incubated for four consecutive 15 min periods in order to measure the release of immunoreactive and bioactive prolactin (PRL); each individual section was then trypsinized into a single cell suspension for determination of PRL cell numbers in that section. Hormone release (rig PRL/1000 PRL cells) was not uniform throughout the gland; the consistency of the secretory patterns demonstrated that the amount of PRL release from the gland was location-dependent. Statistical analysis of the data showed that the most active cells were in the gland's left lobe, while the least active were in the right lobe. Within these lobes, the dorsal-caudal and ventral-rostral left lobe areas released the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas were least active. Injection of ovx rats with E2 for 2 days altered these secretory patterns. This sectioning procedure should prove useful in future studies addressing issues of cell-ceU interaction and geographic location as they relate to pituitary cell function.
Introduction
Prolactin (PRL) cells show considerable variation in the way they synthesize, store and secrete hormone. Assay techniques at the level of the single cell have provided one of the means to study this variability (Walker and Farquhar, 1980; Kendall and Hymer, 1987; Hymer and Motter, 1988; Chert et al., 1989). Considerable variability
Address for correspondence: W.C. Hymer, Department of Molecular and Cell Biology, 401 Althouse Lab., The Pennsylvania State University, University Park, PA 16802, U.S.A. Presented in part at the 72nd Annual Meeting of the Endocrine Society, Atlanta, GA, U.S.A., June 1990. Supported by NIH Grant CA-23248 (W.C.H.).
also exists in the activities of secreted PRL molecules themselves; potencies in biological and immunological assays are not always the same (Mitra, 1980; Lawson et al., 1982; Frawley et al., 1986; Subramaniam and Gala, 1986; Sylvester and Brisk, 1990). The probable importance of PRL cell location within the gland, in terms of its impact on differential secretory activity, was demonstrated in the pioneering studies of Papka and NikitovitchWirier (1986) and Boockfor and Frawley (1987). The novel approaches of Denef and his colleagues have also e~tablished the importance of ceU-cell communication in PRL secretion (Baes and Denef, 1987; Denef et al., 1989). A more complete understanding of the physiological significance of heterogeneity in the PRL
36
'system' will eventually require a method for the analysis of cell function in situ; i.e. in a situation where the PRL cell is in its native location and is in contact with its natural neighbors. Perez and Hymer (1990) have recently shown that these requirements can be met by using a method which involves sectioning of the individual male rat pituitary gland into eight pieces followed by their incubation. This approach has established that the function of growth hormone (GH) cells in vitro depends upon their location within the gland. In this report we extend the use of this sectioning procedure to study the issue of functional heterogeneity of lactotrophs in individual pituitary glands of the ovariectomized and estrogen treated rat. Materials and methods
Pituitary donors Fischer 344 rats, 38-42 days old, were used 10-14 days after ovariectomy (ovx). In some ex.periments ovx animals were injected with estrogen (E2) on the last 2 days prior to sacrifice according to Neill's protocol (Neill, 1972).
Sectioning procedure: ~th sections of individual glands The procedure of Perez and Hymer (1990) was used; this involved (a) removal of the neurointermediate lobe in situ, dissecting out the meningeal membrane from the pituitary gland and placement of the gland on the stage of a tissue slicer (SmithFarquhar, Dupont Instruments, Sorvall, CT, U.S.A.) in a drop of buffered spinners minimal essential medium (MEM) containing 0.1~ bovine serum albumin (BSA), pH 7.4 while maintaining tissue orientation; (b) addition of two drops of 4~ low melt (37°C) agarose (Bethesda Research Labs., Gaithersburg, MD, U.S.A.), cutting the gland into dorsal and ventral sections using the tissue slicer; (c) slicing each of these sections into rostral, caudal, left and right orientations using a scalpel (Fig. 1); (d) removal of the agar shell with a fine forceps from each of the eight sections and culturing for four successive 15 rain periods in multiwell plates (Falcon, Beckton Dickinson, N J, U.S.A.) containing 1 ml otMEM +0.1~ BSA, at 37°C in an atmosphere of humidified air + 5~ CO 2 with
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Fig. 1. Schematic representation of sectioning procedure: -~th sectioning of a single pituitary gland. See Perez and Hymer (1990) for more detail.
constant gyratory shaking (50 rpm). Elapsed th,_ from removal of the gland to the beginning of incubation was < 5 rain. PRL contents in each of the four incubation media were measured by both immunoassay and the Nb2 cell bioassay using NIH PRL B-6 (25 IU/mg) as standard (Tanaka et al., 1980; SignoreUa and Hymer, 1984). A trypsinization technique was used after the 1 h incubation period in order to determine the numbers of PRL cells in each section. Care was taken to fire polish the tips of the Pasteur pipette; gentle agitation (50 rpm) of the section in enzyme facilitated tissue dispersion into single cells (Perez and Hymer, 1990). Cells were counted by hemocytometry, their viability determined and the percentage of PRL cells in the suspensions determined on fixed cells using a flow cytometric immunofluorescence procedure (20,000 cells counted/sample (Hatfield and Hymer, 1985). Total cell yields from the combined eight sections from each gland ranged between 1.5 and 2.5 x 106/gland; cell viability was always > 95~. Morphological studies
Immunocytochernistry (ICC) An immunoperoxidase procedure (Hatfield and Hymer, 1985) was used to identify PRL cells either before or after incubation (5 x 104 cells/ coverslip). The rabbit anti-PRL serum, used at a final dilution of 1:106 has been validated (Hatfield and Hymer, 1985).
PRL cell counting A flow cytometric immunofluorescence procedure was used to count PRL cells (20,000 cells/trial) (Hatfield and Hymer, 1985). Measure° ments were made on an EPICS 753 flow cytome-
37
ter (Coulter Electronics, Hialeah, FL, U.S.A.) equipped with two argon ion lasers. Filters used were: a 488 long pass dichroic for perpendicular light scattering, a 457-504 laser blocking filter, a 590 long pass dichroic filter, with a 525 band filter for green fluorescence and a 610 long pass filter for red fluorescence. The laser was tuned to 488 nm at 100 mW of power. Typical machine high voltage settings for PRL cells were: 450 for perpendicular light scattering, 1000 for log peak green fluorescence (PRL specific) and 850 for integrated red fluorescence (propidium iodide).
Hormone assays Concentrations of immunoactive PRL in incubation media were estimated by enzyme immunoassay (Signorella and Hymer, 1984). The NIH rabbit anti-rat PRL antiserum (IC-3) was used at final dilution of 1:40,000. Concentrations of bioactive PRL in incubation media were determined by the Nb2 lymphoma cell bioassay using incorporation of [3H]thymidine into cellular DNA; 4 h pulse, 0.5 #Ci [3H]thymidine (specific activity 6.7 Ci/mmol; ICN, Irvin¢, CA, U.S.A.) as the end point (Russell et al., 1987). Lymphocytes were harvested onto pre-printed glass fiber filters using a Skatron semi-automatic cell harvester, and incorporation levels measured in a solid phase Beta counter (LKB, Rockville, MD, U.S.A.). Preliminary experiments indicated that estimates of PRL contents in incubation media, using either cell counting or thymidine incorporation as end points, varied less than 10~. Statistics Analyses of variance were done using the ANOVA program. Results Morphology Trypsinization of individual tissue sections after 1 h of incubation yielded cells which, by phase contrast microscopy, showed the appearance typical of freshly dispersed pituitary cells (Fig. 2A). The numbers of cells obtained from each of the eight sections (2.5 x 10 5 + 6.2 x 10 4, n = 16 experiments) were not significantly different, thus documenting reproducibility of section size. The
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A Fig. 2. (A) Appearanceof cells prepared from a 640 #m thick section after 1 h incubation followed by trypsinization. Refractile nature documents viability, x l000. (B) Immunocytochemicaistainingof PRL cellsprepared from a 640 #m thick section after 1 h incubation. Similar cell preparations were used to determinepercent PRL cellsby flowcytometry, x 1000,
PRL-specific cytoplasmic staining of these cells was not diminished after 1 h of incubation (Fig. 2B) relative to cells prepared from unincubated slices/sections (not shown). The percent and number of PRL cells as determined by immunofluorescence staining was uniform throughout the pituitary gland, both from ovx and E2 treated ovx rats (Fig. 3).
Incubation studies Glands from a total of eight ovx rats were sectioned and each of the s~ctions was incubated for four successive 15 min incubations prior to
38
dispersion of each slice into single cell suspensions. Glands from eight E 2 treated ovx rats were also sectioned and incubated as above.
Discmsion
Approaches tO the study of the relationship between structure and function in the in vitro rat pituitary gland have an interesting history. The hemipituitary incubation methodology which was used in the 1960s as the system to identify and characterize hypothalamic-hypophysiotropic factors probably fell out of favor in the 1970s for two reasons. First, Farquhar et al. (1975) identified cores of central tissue necrosis, caused by incomplete nutrient diffusion, as a severe limitation of the method. Second, Vale's demonstration that enzymaticaUy dispersed cells were exquisitely sensitive arid responsive to hypothalamic factors (Vale et al., 1970), while yielding data that were less variable than those obtained with hemipituitaries, resulted in virtual abandonment of the hemipituitary methodology. Not surprisingly, a very large database subsequently grew out of the use of dispersed pituitary cells (Hymer and Hatfield,
Ovariectomized rats Basal P R L release.
Results from eight separate studies, each involving a single pituitary gland, showed (a) that significantly more immunoreactive PRL was released from some sections than others (e.g., sections 4 and 8); and (b) that levels of both bPRL and iPRL declined with incubation time, the effect being most pronounced in release of bioactive hormone (Fig. 4). Estrogen treated rats Basal P R L release.
Results from eight separate studies, each involving a single pituitary gland, showed (a) that sigTdficantly more PRL was released from some secd.r~ns than others (e.g., sections 2, 4 and 7); and (b) that levels of both bPRL and iPRL declined with incubation time (Fig. 5).
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39
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Fig, 4. A m o u n t s of immunoactive and bioactive PRL released from ~th sections of single ovx rat pituitary glands expressed on the basis of lactotroph n u m b e r of the ~th sections. Orientation of ~th sections within gland is given in insert, n = 8 animals. Different letters in superscript indicate significant differences at p < 0.05.
1983; Hymer and Motter, 1988; Chen et al., 1989). While dispersed cells are obviously not nutrient diffusion limited and are well suited for experimental designs which test secretagognes, they suffer from (a) destruction of normal tissue architecture, (b) enzymatic degradation of cell surface components, and (c) production of cells which may 'leak' hormone. There are several reasons why we believe that our sectioning methodology gives results which are not artifactual and may more accurately reflect the state of the female rat pituitary gland in vivo. First, early studies by Farquhar et al. (1975) showed that cellular integrity was maintained in small tissue blocks (1.0 mm 3) up to 1 h of incubation; our experimental protocols 'fit' within these
limits. Second, our morphologic data show excellent tissue integrity; there is no evidence of central tissue necrosis. Third, cells prepared from the slices after 1 h of incubation are typical of those prepared from glands of the ovariectomized rat (Baes et al., 1987) in terms of total yield (range of 1.5-2.5 x 106/gland), viability (> 95%), percentage PRL cells (35-55%), and appearance by phase microscopy and immunocytochemistry (Fig. 2A and B). Fourth, the sectioning p~-ocedure used here was validated for the male rat pit,fitary gland (Perez and Hymer, !999). Fifth, nume~,ous studies document that single PRL cells are functionally heterogenous (Frawley et al., 1986; Boockfor and Fraw!~y, 1987; Kendall and Hymer, 1987; Hymer and Motter, 1988; Sato, 1980). Indeed, the inter-
40
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Fig. 5. Amountsof immunoactiveand bioactive PRL released from ~th sections prepared from single estrogenizedpituitaryglands expressed on the basisof lactotrophnumberof the eight sections, n = 8 animals.
esting study by Boockfor and Frawley (1987) established that PRL cell heterogeneity has a regional component within the gland. The pioneering study by Papka and Nikitovitch-Winer (1986) is particularly relevant because it established functional heterogeneity of PRL cells within intact tissue. The results of our current study offer a new way to quantitatively describe functional heterogeneity of PRL cells in intact tissue; in this regard they confirm and extend the earlier observations with cells (Boockfor and Frawley, 1987; Hymer and Motter, 1988; Denef et al., 1989) and tissue (Papka and Nikitovitch-Winer, 1986). The results of our short term incubation studies document that significant and repeatable dif-
ferences exist in PRL release from the ~ pituitary sections of individual ovx rats (Fig. 4). To further evaluate the data we adopted a modelling strategy which used the average of the eight PRL release values at any given incubation period and arbitrarily defined 1 standard deviation away from that mean as a 'hot' or a 'cold' area. The most interesting finding to emerge from that analysis is that virtually all of the immunoactive (8/8) and bioactive (6/7) 'hot' areas are on the left lobe of the gland while all of the 'cold' areas are in the right lobe (Fig. 6), In most cases, areas represented by sections 4 and 8 on the left and sections 1, 3 and 7 on the right seem most active. From these data we conclude that even though
41
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Fig. 6. Modelling of PRL release from -~th sections of ovx pituitary gland.
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n = 80VX + E-2 treated animals
Fig. 7. Modelling of PRL release from ~ th sections of estrogenized pituitary gland.
42
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Fig. 8. Bioactive/immunoactive (B/I) activity ratios of prolactin released from ~th sections of (A) ovx and (B) ovx-E2 treated individual rat pituitary gland, n = 8 animals for each group. Different letters in superscript indicate significant differences at p < 0.05.
43
PRL cells from the ovx rat are uniformly distributed throughout the anterior lobes, those cells located in the dorsal-caudal and ventral-rostral left lobe areas release the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas are least active. Our studies offer little insight into the mechanism(s) responsible for regional differences in in vitro basal PRL release from the gland of the ovx rat. An asymmetric delivery system for dopamine (Adams et al., 1964) and higher dopamine concentrations in the central portal vessels (Portea' and Cramer, 1974; Reymond et al., 1983) might account for some of the differences in PRL release from different regions of the gland. Also, Denef (1989) showed that one cell type can alter functional properties of another cell type; obviously the cell types in contact with the lactotrophs in one section might vary from one to another and could therefore explain the differential release of prolactin which we observed. It is also possible that mammosomatotrophs could account for at least part of the heterogeneity of the PRL cells observed here. The results of the estrogen study are particularly interesting because they demonstrate plasticity in regional activity of PRL cells. Relative to glands from ovx rats the most important differences are the tendencies for loss of 'hot spots' in section 4 and their appearance or gain in frequency in sections 7 and 8 respectively (Fig. 7). While a total of 14/15 'hot' areas were found in the left lobe of the ovx gland, in vivo E2 treatment shifted this response so that 7/12 'hot' areas were not found in the left lobe of the gland. The results of studies by Frawley et al. (1986) and Subramaniam and Gala (1986) e:nphasized the importance of discrepancies in the biological/ immunological (B/I) activity ratio of secreted PRL in certain physiological situations. In our study, E 2 treatment dramatically affected that B/I activity ratio of PRL released fcom sections 2 and 4 up to 5-fold (Fig. 8A and B). Even in the studies with ovx glands PRL released from some sections had significantly different B / I ratios. It would be interesting to find out if this preferential activity has significance in regard to the generation of E2-induced rat pituitary tumors. In summary, our study supports the idea that
cell-cell interaction as well as location within the individual pituitary gland play important roles in PRL cell function in vitro. Almost 30 years ago, Adams et al. (1964) shuv:ed that groups of portal vessels running down the pituitary stalk supplied circumscribed areas in the pars distalis which they suggested might account for regional holmone secretion. New investigations using the methodology described in this report should be useful in furthering our understanding of the complexities involved in the control of pituitary gland function.
Acknowledgements Some of the immunochemical reagents used in this study were kindly furnished by the NIADDK. We thank Patricia Nye for technical help and Elaine Kunze for help with the flow cytometry.
References Adams, J.H., Daniel, P.M. and Prichard, M.L.M. (1964) Endocrinology 75, 120-126. Baes, M., Allaerts, W. and Denef, C. (1987) Endocrinology 120, 685-691. Boockfor, F.R. and Frawley, S.L. (1987) Endocrinology 120, 874-879. Chen, T.T., Kineman, R.D., Betts, J.G., Hill, J.B. and Frawley, L.S. (1989) Endocrinology 125, 1904-1909. Denef, C., Philippe, M., Allearts, W., Mignon, A., Robberecht, W., Swennen, L. and Carmeliet, P. (1989) Methods Enzymol. 168, 47-74. Farquhar, M.G., Skulelsky, E.H. and Hopkins, C.R. (1975) in The Anterior Pituitary (Tixien-Vidal, A. and Farquhar, M.G., eds.), pp. 84-135, Academic Press, New York. Frawley, L.S., Clark, C.L., Schoderbek, W.E., Hoeffler, J.P. and Boockfor, F.R. (1986) Endocrinology 119, 2867-2869. Hatfield, J.M. and Hymer, W.C. (1985) Cytometry 6, 137-142. Hymer, W.C. and Hatfield M.J. (1983) Methods Enzymol. 103, 257-287. Hymer, W.C. and Motter, K.A. (1988) Endocrinology 122, 2324-2338. Kendall, M.E. and Hymer, W.C. (1987) Endocrinology 121, 2260-2262. Lawson, D.M., Sensui, N., Haisenleder, D.H. and Gala, R.R. (1982) Life Sci. 31, 3063-3070. Mitra, I. (1980) Biochem. Biophys. Res. Commun. 95, 17501759. Neill, J.D. (1972) Endocrinology 90, 1154-1159. Papka, R.E., Yu, S.M. and Nikitovitch-Winer, M.B. (1986) Am. J. Anat. 175, 289-306. Perez, F. and Hymer, W.C. (1990) Endocrinology 127, 18771886. Porter, J.C., Ondo, J.G. and Cramer, O.M. (1974) in ttandbook of Physiology-Endocrinology IV, Part 1 (Greep, and Ast-
44 wood., eds.), pp. 33-43, American Physiological Society, Washington, DC. Reymond, M.J., Speeiak, ¢' G. and Porter, J.C. (1983) Endocrinology 122, 1958-1963. Russell, D.H., Buekley, A.R., Montgomery, D.W., Larson, N.A., Gout, P.W., Beer, C.T., Putnam, C.W., Zukoski, C.F. and Kibler, R. (1987) J. lmmunol. 138, 276-284. Sato, S. (1980) Endocrinol. Jpn. 27, 573-583. Signorella, A.P. and Hymer, W.C. (1984) Anal. Biochem. 136, 372-381. Subramaniam, M. and Gala, R. (1986) J. Clin. lmmunoassay 9, 42-52.
Sylvester, P.W. and Brisk, K.P. (1990) Endocrinology 126, 746-753. Tanaka, T., Shiu, R.P.C., Gout, P.W., Beer, C.T., Noble, R.L. and Friesen, H.G. (1980) J. Clin. Endocrinol. Metab. 51, 1058-1063. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guilleman, R. (1970) Endocrinology 91, 562-572. Walker, A.M. and Farquhar, M.G. (1980) Endocrinology 107, 1095-1104.
35
MOLCEL 02453
Function of prolactin cells in the individual rat pituitary gland is location dependent P. Mukherjee, T. Salada and W.C. Hymer Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A. (Received 8 October 1990; accepted 30 November 1990)
Key words: Prolactin; Prolactin cell heterogeneity; Pituitary gland; (Rat)
Summary Anterior pituitary glands from individual ovariectomized (ovx) or ovx-estrogen (E2) treated rats were sectioned into ~ cubes. Each section was incubated for four consecutive 15 min periods in order to measure the release of immunoreactive and bioactive prolactin (PRL); each individual section was then trypsinized into a single cell suspension for determination of PRL cell numbers in that section. Hormone release (rig PRL/1000 PRL cells) was not uniform throughout the gland; the consistency of the secretory patterns demonstrated that the amount of PRL release from the gland was location-dependent. Statistical analysis of the data showed that the most active cells were in the gland's left lobe, while the least active were in the right lobe. Within these lobes, the dorsal-caudal and ventral-rostral left lobe areas released the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas were least active. Injection of ovx rats with E2 for 2 days altered these secretory patterns. This sectioning procedure should prove useful in future studies addressing issues of cell-ceU interaction and geographic location as they relate to pituitary cell function.
Introduction
Prolactin (PRL) cells show considerable variation in the way they synthesize, store and secrete hormone. Assay techniques at the level of the single cell have provided one of the means to study this variability (Walker and Farquhar, 1980; Kendall and Hymer, 1987; Hymer and Motter, 1988; Chert et al., 1989). Considerable variability
Address for correspondence: W.C. Hymer, Department of Molecular and Cell Biology, 401 Althouse Lab., The Pennsylvania State University, University Park, PA 16802, U.S.A. Presented in part at the 72nd Annual Meeting of the Endocrine Society, Atlanta, GA, U.S.A., June 1990. Supported by NIH Grant CA-23248 (W.C.H.).
also exists in the activities of secreted PRL molecules themselves; potencies in biological and immunological assays are not always the same (Mitra, 1980; Lawson et al., 1982; Frawley et al., 1986; Subramaniam and Gala, 1986; Sylvester and Brisk, 1990). The probable importance of PRL cell location within the gland, in terms of its impact on differential secretory activity, was demonstrated in the pioneering studies of Papka and NikitovitchWirier (1986) and Boockfor and Frawley (1987). The novel approaches of Denef and his colleagues have also e~tablished the importance of ceU-cell communication in PRL secretion (Baes and Denef, 1987; Denef et al., 1989). A more complete understanding of the physiological significance of heterogeneity in the PRL
36
'system' will eventually require a method for the analysis of cell function in situ; i.e. in a situation where the PRL cell is in its native location and is in contact with its natural neighbors. Perez and Hymer (1990) have recently shown that these requirements can be met by using a method which involves sectioning of the individual male rat pituitary gland into eight pieces followed by their incubation. This approach has established that the function of growth hormone (GH) cells in vitro depends upon their location within the gland. In this report we extend the use of this sectioning procedure to study the issue of functional heterogeneity of lactotrophs in individual pituitary glands of the ovariectomized and estrogen treated rat. Materials and methods
Pituitary donors Fischer 344 rats, 38-42 days old, were used 10-14 days after ovariectomy (ovx). In some ex.periments ovx animals were injected with estrogen (E2) on the last 2 days prior to sacrifice according to Neill's protocol (Neill, 1972).
Sectioning procedure: ~th sections of individual glands The procedure of Perez and Hymer (1990) was used; this involved (a) removal of the neurointermediate lobe in situ, dissecting out the meningeal membrane from the pituitary gland and placement of the gland on the stage of a tissue slicer (SmithFarquhar, Dupont Instruments, Sorvall, CT, U.S.A.) in a drop of buffered spinners minimal essential medium (MEM) containing 0.1~ bovine serum albumin (BSA), pH 7.4 while maintaining tissue orientation; (b) addition of two drops of 4~ low melt (37°C) agarose (Bethesda Research Labs., Gaithersburg, MD, U.S.A.), cutting the gland into dorsal and ventral sections using the tissue slicer; (c) slicing each of these sections into rostral, caudal, left and right orientations using a scalpel (Fig. 1); (d) removal of the agar shell with a fine forceps from each of the eight sections and culturing for four successive 15 rain periods in multiwell plates (Falcon, Beckton Dickinson, N J, U.S.A.) containing 1 ml otMEM +0.1~ BSA, at 37°C in an atmosphere of humidified air + 5~ CO 2 with
N ° 8 ANIMALS ~o,.~~,yENTRAL
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Fig. 1. Schematic representation of sectioning procedure: -~th sectioning of a single pituitary gland. See Perez and Hymer (1990) for more detail.
constant gyratory shaking (50 rpm). Elapsed th,_ from removal of the gland to the beginning of incubation was < 5 rain. PRL contents in each of the four incubation media were measured by both immunoassay and the Nb2 cell bioassay using NIH PRL B-6 (25 IU/mg) as standard (Tanaka et al., 1980; SignoreUa and Hymer, 1984). A trypsinization technique was used after the 1 h incubation period in order to determine the numbers of PRL cells in each section. Care was taken to fire polish the tips of the Pasteur pipette; gentle agitation (50 rpm) of the section in enzyme facilitated tissue dispersion into single cells (Perez and Hymer, 1990). Cells were counted by hemocytometry, their viability determined and the percentage of PRL cells in the suspensions determined on fixed cells using a flow cytometric immunofluorescence procedure (20,000 cells counted/sample (Hatfield and Hymer, 1985). Total cell yields from the combined eight sections from each gland ranged between 1.5 and 2.5 x 106/gland; cell viability was always > 95~. Morphological studies
Immunocytochernistry (ICC) An immunoperoxidase procedure (Hatfield and Hymer, 1985) was used to identify PRL cells either before or after incubation (5 x 104 cells/ coverslip). The rabbit anti-PRL serum, used at a final dilution of 1:106 has been validated (Hatfield and Hymer, 1985).
PRL cell counting A flow cytometric immunofluorescence procedure was used to count PRL cells (20,000 cells/trial) (Hatfield and Hymer, 1985). Measure° ments were made on an EPICS 753 flow cytome-
37
ter (Coulter Electronics, Hialeah, FL, U.S.A.) equipped with two argon ion lasers. Filters used were: a 488 long pass dichroic for perpendicular light scattering, a 457-504 laser blocking filter, a 590 long pass dichroic filter, with a 525 band filter for green fluorescence and a 610 long pass filter for red fluorescence. The laser was tuned to 488 nm at 100 mW of power. Typical machine high voltage settings for PRL cells were: 450 for perpendicular light scattering, 1000 for log peak green fluorescence (PRL specific) and 850 for integrated red fluorescence (propidium iodide).
Hormone assays Concentrations of immunoactive PRL in incubation media were estimated by enzyme immunoassay (Signorella and Hymer, 1984). The NIH rabbit anti-rat PRL antiserum (IC-3) was used at final dilution of 1:40,000. Concentrations of bioactive PRL in incubation media were determined by the Nb2 lymphoma cell bioassay using incorporation of [3H]thymidine into cellular DNA; 4 h pulse, 0.5 #Ci [3H]thymidine (specific activity 6.7 Ci/mmol; ICN, Irvin¢, CA, U.S.A.) as the end point (Russell et al., 1987). Lymphocytes were harvested onto pre-printed glass fiber filters using a Skatron semi-automatic cell harvester, and incorporation levels measured in a solid phase Beta counter (LKB, Rockville, MD, U.S.A.). Preliminary experiments indicated that estimates of PRL contents in incubation media, using either cell counting or thymidine incorporation as end points, varied less than 10~. Statistics Analyses of variance were done using the ANOVA program. Results Morphology Trypsinization of individual tissue sections after 1 h of incubation yielded cells which, by phase contrast microscopy, showed the appearance typical of freshly dispersed pituitary cells (Fig. 2A). The numbers of cells obtained from each of the eight sections (2.5 x 10 5 + 6.2 x 10 4, n = 16 experiments) were not significantly different, thus documenting reproducibility of section size. The
B C
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A Fig. 2. (A) Appearanceof cells prepared from a 640 #m thick section after 1 h incubation followed by trypsinization. Refractile nature documents viability, x l000. (B) Immunocytochemicaistainingof PRL cellsprepared from a 640 #m thick section after 1 h incubation. Similar cell preparations were used to determinepercent PRL cellsby flowcytometry, x 1000,
PRL-specific cytoplasmic staining of these cells was not diminished after 1 h of incubation (Fig. 2B) relative to cells prepared from unincubated slices/sections (not shown). The percent and number of PRL cells as determined by immunofluorescence staining was uniform throughout the pituitary gland, both from ovx and E2 treated ovx rats (Fig. 3).
Incubation studies Glands from a total of eight ovx rats were sectioned and each of the s~ctions was incubated for four successive 15 min incubations prior to
38
dispersion of each slice into single cell suspensions. Glands from eight E 2 treated ovx rats were also sectioned and incubated as above.
Discmsion
Approaches tO the study of the relationship between structure and function in the in vitro rat pituitary gland have an interesting history. The hemipituitary incubation methodology which was used in the 1960s as the system to identify and characterize hypothalamic-hypophysiotropic factors probably fell out of favor in the 1970s for two reasons. First, Farquhar et al. (1975) identified cores of central tissue necrosis, caused by incomplete nutrient diffusion, as a severe limitation of the method. Second, Vale's demonstration that enzymaticaUy dispersed cells were exquisitely sensitive arid responsive to hypothalamic factors (Vale et al., 1970), while yielding data that were less variable than those obtained with hemipituitaries, resulted in virtual abandonment of the hemipituitary methodology. Not surprisingly, a very large database subsequently grew out of the use of dispersed pituitary cells (Hymer and Hatfield,
Ovariectomized rats Basal P R L release.
Results from eight separate studies, each involving a single pituitary gland, showed (a) that significantly more immunoreactive PRL was released from some sections than others (e.g., sections 4 and 8); and (b) that levels of both bPRL and iPRL declined with incubation time, the effect being most pronounced in release of bioactive hormone (Fig. 4). Estrogen treated rats Basal P R L release.
Results from eight separate studies, each involving a single pituitary gland, showed (a) that sigTdficantly more PRL was released from some secd.r~ns than others (e.g., sections 2, 4 and 7); and (b) that levels of both bPRL and iPRL declined with incubation time (Fig. 5).
1DRRT. D: DORSALI I 2DRL. V :. VENTRALI I 3 DCRT. ROSTRAq I 4 DCL, C i CAUDALI I § VCRT. RT: RIGHT I | 6 VCL. L : LEFT |
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39
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Fig, 4. A m o u n t s of immunoactive and bioactive PRL released from ~th sections of single ovx rat pituitary glands expressed on the basis of lactotroph n u m b e r of the ~th sections. Orientation of ~th sections within gland is given in insert, n = 8 animals. Different letters in superscript indicate significant differences at p < 0.05.
1983; Hymer and Motter, 1988; Chen et al., 1989). While dispersed cells are obviously not nutrient diffusion limited and are well suited for experimental designs which test secretagognes, they suffer from (a) destruction of normal tissue architecture, (b) enzymatic degradation of cell surface components, and (c) production of cells which may 'leak' hormone. There are several reasons why we believe that our sectioning methodology gives results which are not artifactual and may more accurately reflect the state of the female rat pituitary gland in vivo. First, early studies by Farquhar et al. (1975) showed that cellular integrity was maintained in small tissue blocks (1.0 mm 3) up to 1 h of incubation; our experimental protocols 'fit' within these
limits. Second, our morphologic data show excellent tissue integrity; there is no evidence of central tissue necrosis. Third, cells prepared from the slices after 1 h of incubation are typical of those prepared from glands of the ovariectomized rat (Baes et al., 1987) in terms of total yield (range of 1.5-2.5 x 106/gland), viability (> 95%), percentage PRL cells (35-55%), and appearance by phase microscopy and immunocytochemistry (Fig. 2A and B). Fourth, the sectioning p~-ocedure used here was validated for the male rat pit,fitary gland (Perez and Hymer, !999). Fifth, nume~,ous studies document that single PRL cells are functionally heterogenous (Frawley et al., 1986; Boockfor and Fraw!~y, 1987; Kendall and Hymer, 1987; Hymer and Motter, 1988; Sato, 1980). Indeed, the inter-
40
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Fig. 5. Amountsof immunoactiveand bioactive PRL released from ~th sections prepared from single estrogenizedpituitaryglands expressed on the basisof lactotrophnumberof the eight sections, n = 8 animals.
esting study by Boockfor and Frawley (1987) established that PRL cell heterogeneity has a regional component within the gland. The pioneering study by Papka and Nikitovitch-Winer (1986) is particularly relevant because it established functional heterogeneity of PRL cells within intact tissue. The results of our current study offer a new way to quantitatively describe functional heterogeneity of PRL cells in intact tissue; in this regard they confirm and extend the earlier observations with cells (Boockfor and Frawley, 1987; Hymer and Motter, 1988; Denef et al., 1989) and tissue (Papka and Nikitovitch-Winer, 1986). The results of our short term incubation studies document that significant and repeatable dif-
ferences exist in PRL release from the ~ pituitary sections of individual ovx rats (Fig. 4). To further evaluate the data we adopted a modelling strategy which used the average of the eight PRL release values at any given incubation period and arbitrarily defined 1 standard deviation away from that mean as a 'hot' or a 'cold' area. The most interesting finding to emerge from that analysis is that virtually all of the immunoactive (8/8) and bioactive (6/7) 'hot' areas are on the left lobe of the gland while all of the 'cold' areas are in the right lobe (Fig. 6), In most cases, areas represented by sections 4 and 8 on the left and sections 1, 3 and 7 on the right seem most active. From these data we conclude that even though
41
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Fig. 6. Modelling of PRL release from -~th sections of ovx pituitary gland.
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n = 80VX + E-2 treated animals
Fig. 7. Modelling of PRL release from ~ th sections of estrogenized pituitary gland.
42
01!1
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Fig. 8. Bioactive/immunoactive (B/I) activity ratios of prolactin released from ~th sections of (A) ovx and (B) ovx-E2 treated individual rat pituitary gland, n = 8 animals for each group. Different letters in superscript indicate significant differences at p < 0.05.
43
PRL cells from the ovx rat are uniformly distributed throughout the anterior lobes, those cells located in the dorsal-caudal and ventral-rostral left lobe areas release the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas are least active. Our studies offer little insight into the mechanism(s) responsible for regional differences in in vitro basal PRL release from the gland of the ovx rat. An asymmetric delivery system for dopamine (Adams et al., 1964) and higher dopamine concentrations in the central portal vessels (Portea' and Cramer, 1974; Reymond et al., 1983) might account for some of the differences in PRL release from different regions of the gland. Also, Denef (1989) showed that one cell type can alter functional properties of another cell type; obviously the cell types in contact with the lactotrophs in one section might vary from one to another and could therefore explain the differential release of prolactin which we observed. It is also possible that mammosomatotrophs could account for at least part of the heterogeneity of the PRL cells observed here. The results of the estrogen study are particularly interesting because they demonstrate plasticity in regional activity of PRL cells. Relative to glands from ovx rats the most important differences are the tendencies for loss of 'hot spots' in section 4 and their appearance or gain in frequency in sections 7 and 8 respectively (Fig. 7). While a total of 14/15 'hot' areas were found in the left lobe of the ovx gland, in vivo E2 treatment shifted this response so that 7/12 'hot' areas were not found in the left lobe of the gland. The results of studies by Frawley et al. (1986) and Subramaniam and Gala (1986) e:nphasized the importance of discrepancies in the biological/ immunological (B/I) activity ratio of secreted PRL in certain physiological situations. In our study, E 2 treatment dramatically affected that B/I activity ratio of PRL released fcom sections 2 and 4 up to 5-fold (Fig. 8A and B). Even in the studies with ovx glands PRL released from some sections had significantly different B / I ratios. It would be interesting to find out if this preferential activity has significance in regard to the generation of E2-induced rat pituitary tumors. In summary, our study supports the idea that
cell-cell interaction as well as location within the individual pituitary gland play important roles in PRL cell function in vitro. Almost 30 years ago, Adams et al. (1964) shuv:ed that groups of portal vessels running down the pituitary stalk supplied circumscribed areas in the pars distalis which they suggested might account for regional holmone secretion. New investigations using the methodology described in this report should be useful in furthering our understanding of the complexities involved in the control of pituitary gland function.
Acknowledgements Some of the immunochemical reagents used in this study were kindly furnished by the NIADDK. We thank Patricia Nye for technical help and Elaine Kunze for help with the flow cytometry.
References Adams, J.H., Daniel, P.M. and Prichard, M.L.M. (1964) Endocrinology 75, 120-126. Baes, M., Allaerts, W. and Denef, C. (1987) Endocrinology 120, 685-691. Boockfor, F.R. and Frawley, S.L. (1987) Endocrinology 120, 874-879. Chen, T.T., Kineman, R.D., Betts, J.G., Hill, J.B. and Frawley, L.S. (1989) Endocrinology 125, 1904-1909. Denef, C., Philippe, M., Allearts, W., Mignon, A., Robberecht, W., Swennen, L. and Carmeliet, P. (1989) Methods Enzymol. 168, 47-74. Farquhar, M.G., Skulelsky, E.H. and Hopkins, C.R. (1975) in The Anterior Pituitary (Tixien-Vidal, A. and Farquhar, M.G., eds.), pp. 84-135, Academic Press, New York. Frawley, L.S., Clark, C.L., Schoderbek, W.E., Hoeffler, J.P. and Boockfor, F.R. (1986) Endocrinology 119, 2867-2869. Hatfield, J.M. and Hymer, W.C. (1985) Cytometry 6, 137-142. Hymer, W.C. and Hatfield M.J. (1983) Methods Enzymol. 103, 257-287. Hymer, W.C. and Motter, K.A. (1988) Endocrinology 122, 2324-2338. Kendall, M.E. and Hymer, W.C. (1987) Endocrinology 121, 2260-2262. Lawson, D.M., Sensui, N., Haisenleder, D.H. and Gala, R.R. (1982) Life Sci. 31, 3063-3070. Mitra, I. (1980) Biochem. Biophys. Res. Commun. 95, 17501759. Neill, J.D. (1972) Endocrinology 90, 1154-1159. Papka, R.E., Yu, S.M. and Nikitovitch-Winer, M.B. (1986) Am. J. Anat. 175, 289-306. Perez, F. and Hymer, W.C. (1990) Endocrinology 127, 18771886. Porter, J.C., Ondo, J.G. and Cramer, O.M. (1974) in ttandbook of Physiology-Endocrinology IV, Part 1 (Greep, and Ast-
44 wood., eds.), pp. 33-43, American Physiological Society, Washington, DC. Reymond, M.J., Speeiak, ¢' G. and Porter, J.C. (1983) Endocrinology 122, 1958-1963. Russell, D.H., Buekley, A.R., Montgomery, D.W., Larson, N.A., Gout, P.W., Beer, C.T., Putnam, C.W., Zukoski, C.F. and Kibler, R. (1987) J. lmmunol. 138, 276-284. Sato, S. (1980) Endocrinol. Jpn. 27, 573-583. Signorella, A.P. and Hymer, W.C. (1984) Anal. Biochem. 136, 372-381. Subramaniam, M. and Gala, R. (1986) J. Clin. lmmunoassay 9, 42-52.
Sylvester, P.W. and Brisk, K.P. (1990) Endocrinology 126, 746-753. Tanaka, T., Shiu, R.P.C., Gout, P.W., Beer, C.T., Noble, R.L. and Friesen, H.G. (1980) J. Clin. Endocrinol. Metab. 51, 1058-1063. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guilleman, R. (1970) Endocrinology 91, 562-572. Walker, A.M. and Farquhar, M.G. (1980) Endocrinology 107, 1095-1104.
