Persistence of Cell Types in Monolayer Cultures of Dispersed Cells from the Pituitary Pars Distalis as Revealed by Immunohistochemistry B. L. BAKER, J. R. REEL, S. D. VAN DEWARK AND Y.-Y. YU Department of Anatomy, The University of Michigan Medical School, and Endocrine Section, Parke-Davis Research Laboratories, Ann Arbor, Michigan 48104 ABSTRACT The objective was to study the fate of specific secretory cell types of the rat hypophysis when grown in primary monolayer cultures for periods ranging up to 32 days. The cells were identified immunohistochemically using peroxidase-labeled antibody. Early in the culture period TSH-cells were scarce and by 12 days they could no longer be identified. In most cultures LH-cells were well stained and common for eight to 12 days, after which they underwent in- volution. Growth hormone cells were a prominent feature up to six days but by 12 days they were declining in number, size, and stainability; in contrast, pro- lactin cells proliferated and were large and intensely stained throughout the period of study, ultimately becoming the dominant secretory cell type. Cortico- tropic cells also continued throughout the period of study without regression. Thus drastic shifts occur with time in the relative proportions of cell types in monolayer cultures of rat pituitary cells. Cultures containing dispersed or isolated acid-Schiff technique and concluded that cells of the pars distaiis are being used almost all cells become chromophobes by with increasing frequency in the study of the end of the first week. Also, Rappay factors that regulate pituitary secretion. As et al. ('73) found that the ultrastructure observed by Vale et al. ('72), cultures of of rat pituitary cells grown in monolayer dispersed pituitary cells grown as mono- cultures for 11 to 27 days does not allow layers have two major advantages over the identification of specific cell types. those containing slices or fragments of the Since immunohistochemistry has helped gland. First, a homogeneous cell popula- to solve many problems of cell identifica- tion is obtainable which facilitates the con- tion in the intact hypophysis, in this study sideration of several variables in one exper- the method was applied to monolayer pitui- iment. Second, the dispersed ceSs are suf- tary cell cultures in order to demonstrate ficiently responsive to stimulating agents changes that occur in the cell population so that the amount of hormone released as the cultures age. Thereby, a better un- can be related directly to the amount of derstanding may be obtained of the capa- stimulating agent used. city of such preparations to respond to Both Vale et al. ('72) and Steinberger factors that stimulate or inhibit secretion. et al. ('73) have found that the capacity This represents the first attempt to analyze of monolayer cultures of pituitary cells to by means of immunohistochemistry the secrete certain hormones declines with cellular composition of normal pituitary time, this observation indicating that sig- tissue grown in vitro. nificant cytological alterations occur in the cell population. Thus far, histochemis- MATERIALS AND METHODS try and electron microscopy have not per- Holtzman female rats, in the diestrous mitted satisfactory analysis of the fate of stage of the cycle and weighing 200 to specific secretory cell types in monolayer cultures, For example, Kobayashi et al. Received July 11, '73. Accepted Oct. 12, '73. supported in part by NIH Research grant HD- ('71) stained with Giemsa or the periodic 03159. ANAT. REC., 179: 93-106. 93 94 B. L. BAKER, J. R. REEL, S. D. VAN DEWARK AND Y.-Y. YU 250 gm, served as the source of pituitary anti-rat prolactin by Dr. A. R. Midgley, Jr.; glands. After the rats were killed with COZ and anti-bTSH-p and anti-bLH-p by Dr. the hypophysis was excised and the pos- J. G. Pierce, Jr. For most preparations, terior hypophysis discarded. In some of 3,3’-diaminobenzidine was used as the sub- the earlier preparations portions of the strate for peroxidase. For double staining pars intermedia were retained with the a-naphthol was employed as substrate for pars distalis; however, in subsequent ex- staining of the second cell type. periments only the lateral portions of the Evidence for the specificity of these pro- pars distalis were used in order to elimi- cedures when applied to cells of the intact nate insofar as possible cells of the pars rat pituitary gland has been published intermedia. Monolayer cultures of cells from this laboratory as follows : for growth from the pars distalis were prepared by the hormone and prolactin cells (Baker et al., method of VaIe et al. (’72), which involves ’69; Baker, ’70), corticotropic and MSH- incubation of the tissue in a medium con- cells (Baker et al., ’70; Baker and Drum- taining hyaluronidase and collagenase mond, ’72); TSH-cells (Baker and Yu, followed by a 15- to 30-minute treatment ’71a,b) and TSH- and LH-cells (Baker with Viokase. The rate of dispersion is in- et al., ’72). In addition, numerous con- creased by gently drawing the fragments trols were employed in connection with in and out of a siliconized Pasteur pipette immunohistochemical staining of the cul- every ten minutes throughout the disper- tured cells, No staining was obtained sion procedure, Exceptions to the Vale under the following conditions : omission et al. procedure were the use of Hanks’ of the hormone-specific antiserum, prior calcium- and magnesium-free balanced absorption of the hormone-specific anti- salt solution (Grand Island Biological Co. ) serum with the hormone used as the anti- instead of HEPES buffer as the medium gen, and application to the slide of uncon- for cell dispersion. Since Hanks’ balanced j ugated sheep anti-rabbit-y-globulin prior salt solution was employed as the buffer, to application of the peroxidase-conjugated cell dispersion was carried out in a humidi- y-globulin. Prolactin represented an excep- fied COz incubator. In addition, penicillin tion to these generalizations because ab- G (100 units/ml) and streptomycin (100 sorption of anti-rat prolactin with ovine pg/ml) were added to the growth medium. prolactin greatly reduced staining intensity The cells were cultured in Flaskettes-Bio- but did not totally eliminate it. logical Growth Chambers (Lab-Tek Prod- ucts Division, Miles Laboratories). In gen- RESULTS eral the cells of 15 hypophyses were As noted by Vale et al. (’72), the dis- dispersed at a time, thus providing suffi- persed pituitary cells were spherical when cient material to prepare approximately 45 first placed in culture and this was their Flaske t tes. shape at the end of one day in our prepara- Seventy-four cultures were maintained tions. By the second day some cells had for 1, 2, 4, 6, 8, 12, 16, 20, 24, 30, or 32 become flattened and stellate. With pro- days. After removal of the incubation longation of the culture time a greater pro- medium each culture was stained immuno- portion of the cells were flattened. The histochemically for one to three cell types pituitary epithelioid cells tended to form by the method of Nakane and Pierce (’67). colonies which enlarged up to about six The cell types studied were: growth hor- days. Such cell groups were illustrated by mone cells, prolactin cells, corticotropic Steinberger et al. (’73) in ten-day cultures. cells, melanotropic (MSH-) cells, thyro- With advancing time fibroblasts became tropic (TSH-) cells, and luteinizing hor- mone (LH-) cells. Antiserums were pre- 2 We thank the following sources for the hofmones indicated: NIAMD Pituitarv Hormone Distnbution pared in our laboratory by the method of PFohr~mfor human erowth-hormone and bovine lu- Midgley et al. (’71) to human growth hor- T&%Gzing hoImoie; Organon, Inc., W. Orange, N.J., for ,31-z4-corticotropin (Cosyntropinm); and Professor H. J. mone (NIH-GH-HS 1395) ,2 p1-Z4-cortico- -Bein .~~~ and Doctor W. Rittel of Ciba-Geiay Ltd., Basle, tropin, and human p-melanotropin. Anti- Switzerland for ~~~-39-corticotropinand Pmelanotropin. 3 In abbreviations for hormones “b” indicates bovine human thyrotropin (anti-hTSH) was pro- origin ‘‘0” ovine, and “h” human. Antmerums are in- dicated by the prefix “anti-” added to the name of the vided by Dr. w. D. Odell; anti-oLH and hormone. PITUITARY MONOLAYER CULTURES 95 predominant in areas intervening between Cells stained with anti-p-MSH showed the colonies. However, immunohistochemi- the same characteristics as described above cal staining showed that secretory cells for corticotropic cells (fig. 13). Attempts occurred in the fibroblastic and other areas to demonstrate two separate cell types by also (fig. 10). double-staining with anti-p'-24-corticotropin Growth hormone cells. Between two and anti-p-hMSH were unsuccessful; thus (fig. 2) and four days (fig. 3) growth hor- it appears that in these cultures one cell mone cells did not show much change but type contained both corticotropin and at six days they were a prominent feature melanotropin. of epithelioid cell colonies (fig. 5). How- TSH-cells. TSH-cells were regularly ever, by 12 days in most cultures growth demonstrable with both anti-hTSH and hormone cells were clearly declining in anti-bTSH-p at one and two days and in relative number and staining intensity. By one of three cultures at each of the fol- 30 days the remaining growth hormone lowing times: four (fig. 14), six and eight cells were few, small, weakly stained and days. TSH-cells were undetectable in all generally stellate (fig. 6). Most growth other speciments at all other times. When hormone cells maintained their typical observed they were rare, small, stellate, ovoid shape for about 12 days in culture; and their size was never equivalent to that later a greater proportion were stellate.