Pituitary lactotroph sedimentation profiles and in vitro secretory activity after ablation of the medial basal hypothalamus.код для вставкиСкачать
THE ANATOMICAL RECORD 215365-373 (1986) Pituitary Lactotroph Sedimentation Profiles and In Vitro Secretory Activity After Ablation of the Medial Basal Hypothalamus CAROL J. PHELPS AND WESLEY C . HYMER Department of Molecular and Cell Bwlogy, The Pennsytuania State University, University Park, PA ABSTRACT Pituitary cells from adult male rats subjected to chronic (6 and 10 weeks) medial hypothalamic ablation (MHA)were analyzed by unit gravity sedimentation to assess distribution of size and density of lactotrophs, and for subsequent in vitro prolactin (PRL) release in primary culture. Tindorial staining (Herlant’s tetrachrome) showed that initial preparations of cells from MHA rats were small and relatively undifferentiated. MHA cells did not sediment as far into the gradient as did cells from intact control pituitaries. Intracellular PRL content was lower in all gradient fractions of MHA cells. At 6 weeks after surgery, peak recovery of PRL was also in the upper portions of the gradient. In the 10-weeks group, however, peak PRL recovery from MHA cells was in a population that sedimented further, but more restrictedly, in comparison with control cells. At both postsurgical intervals, the majority of tinctorially or immunocytochemically identified lactotrophs from lesioned rats were lower in the gradient, indicating enlarged and denser cells. Relative numbers of lactotrophs (per pituitary) were increased 10 weeks after MHA. In vitro PRL release, over a maximum of 21 days culture, was comparable for cells from MHA rats and intact controls, according to daily per cell secretion rates and “production index” (hormone releaseainitial hormone content). By comparison, luteinizing hormone (LH) release was suppressed in culture compared to intact controls, and LH was recovered from gradient fractions of smaller cells. The results indicate that chronic removal of hypothalamic influence results in gradual prolactin cell hypertrophy and decreased hormone retention and in relative increase in numbers. Since PRL release in vitro proceeded a t a normal rate, the primary effect of such a lesion appears to be increased hormone turnover. The data also emphasize the autonomous capacity of lactotrophs, relative to other pituitary cell types, to adjust cellular mechanisms in order to continue secretory function in the absence of hypothalamic influence. Large neurosurgical lesions of the hypophysiotropic hypothalamus have been employed for studying basal and stimulated secretion of several adenohypophysial hormones. Such medial hypothalamic ablation (MHA) has been shown to be compatible with persistent basal secretion of corticosterone (Dunn and Critchlow, 1973), growth hormone (Dunn and Arimura, 1974), and luteinizing hormone (LH) (Turpen et al., 1978), although typical stress-evoked changes in these hormone levels were compromised. The effects of such a lesion on secretion of most adenohypophysial hormones might well be expected to be detrimental, but to result in stimulation of prolactin (PRL) cell function, since PRL-inhibitory (MacLeod, 1976) dopaminergic neurons of the tuberoinfundibular system (Bjorklund and Nobin, 1973) are destroyed by such surgery. Indeed, several investigations have reported elevated circulating PRL levels shortly (1-14 days) following such lesions (Bishop et al., 1972; Cheung and Weiner, 1976; Turpen and Dunn, 1976). However, after long-term (4 and 6 weeks) MHA, PRL 0 1986 ALAN R. LISS, INC. levels measured in serum collected under strict nonstress conditions were not different from PRL levels of intact controls (Turpen and Dunn, 1976; Turpen et al., 1978). Subsequent in vitro PRL cell supersensitivity to the inhibitory effects of dopamine (DA) has been demonstrated in pituitaries from rats subjected to such lesions (Cheung and Weiner, 1978; Cheung et al., 1981). However, particular characteristics such a s relative numbers, size, and hormone content, of adenohypophysial cells, following hypothalamic lesions, have not been assessed. The present studies were therefore undertaken to evaluate the effect of chronic MHA on lactotroph numbers and size and subsequent in vitro secretory function. Received January 16,1986; accepted March 10, 1986. Address reprint requests to Dr. Carol J. Phelps, Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Carol J.Phelps is the former Carol Turpen. 366 C.J. PHELPS AND W. C. HYMER To that end, pituitary single-cell preparations from MHA rats were analyzed by the techniques of separation by unit gravity sedimentation (Hymer et al., 1973) and primary cell culture. Unit gravity sedimentation has been employed to demonstrate size/density differences among lactotrophs, which reflected the previous physiologic history of the donor animal (Hymer et al., 1973, 1974); separated lactotrophs retain hormone and morphologic integrity and may subsequently be cultured to assess secretory activity (Snyder et al., 1976). In a n initial study in which pituitaries from MHA rats were analyzed by these methods 6 weeks after surgery, it was found that although cell sedimentation was reduced, subsequent in vitro PRL release was unaffected. The experiments were then repeated, with pituitaries from greater numbers of MHA rats, 10 weeks following surgery; in the second experiment, pituitary cells were maintained in vitro for a longer period, to further test the secretory capacity of lactotrophs from lesioned rats. MATERIALS AND METHODS Experimental and control animals were adult (250280 gm) male Sprague-Dawley-derived rats (Charles River) maintained on a 12-hr light/l2-hr dark cycle with water and Purina rat chow available ad libitum. Following surgery, experimental (MHA) and control rats were housed in individual cages. Medial Hypothalamic Ablation Animals were subjected to ablation of the medial basal hypothalamus with a modified triangular Halasz-Pupp knife (Dunn and Critchlow, 1973) bearing a t its base a horizontal crossbar that disrupts the island circumscribed by the original deafferentation procedure. The dimensions of the instrument and hypothalamic area destroyed by it are illustrated in Figure 1.With the rat’s head held in a Kopf stereotaxic apparatus so that the base of the brain was in a horizontal position (nosebar at -3 mm), the MHA instrument was lowered, with its anterior tip 1mm posterior to bregma, to the base of the brain, rotated 360” several times, and removed in its entry position. MHA and intact control rats were killed 6 and 10 weeks after surgery by cervical dislocation and decapitation. Brains were removed from MHA rats and immersed in 10% formalin for subsequent histologic assessment of lesion placement. Whole brains were subsequently dehydrated, embedded in paraffin, and coronal 10-pm sections were cut on a rotary microtome. Sections were stained with thionin for microscopic examination. Preparation of Anterior Pituitary Cells Pituitaries from MHA and intact rats were aseptically removed, and respective MHA or control glands were placed together for enzymatic dissociation; the posterior pituitaries (neural and intermediate lobes) were discarded. The tissue was manually cut into small pieces (1 mm’), placed in calcium-free (“Spinner’s”) minimal essential medium (Grand Island Biological Corp.) containing 0.1% trypsin (Difco 1:250) and 0.3% bovine serum albumin (BSA) (Nutritional Biochemicals Co., fraction V), and subjected to continuous agitation in Spinner flasks at 37”C, as previously described (Hymer et al., 1973). The dispersed cells were centrifuged (200g for 10 min, room temperature) from the dissociation medium, and resuspended in sterile medium 199 with 0.1% BSA. Aliquots of initially dissociated cells (designated “starts”) were taken from each cell pool for 1)hormone extraction and assay, 2) cytocentrifuge preparation of microscope slides, 3)velocity sedimentation at unit gravity, and 4) cell culture. For each experimental or control cell pool, triplicate suspensions of 250,000-300,000 cells were extracted with cold (4°C) 0.01 N NaOH for 1hr, then centrifuged (1,OOOg for 40 min, at 4°C). The supernatant extract was diluted 1%in phosphosaline (0.1 M, buffered to pH 7.4) and stored frozen (-20°C) for subsequent radioimmunoassay (RIA), as previously described (Hymer et al., 1974). Differential Quantitation of Recovered Cells Samples of initial cell preparations and cells recovered from each gradient fraction (see below) were diluted to a concentration of approximately 400,000 cells per ml in phosphosaline with 0.1% BSA for preparation of microscope slides (approximately 80,000 cells per slide, in 0.2 ml) with a Shandon cytocentrifuge (1,200 rpm, for 7 min). The slides were fixed for 16-18 h r with BouinHollande sublimate, then strained through means either of Herlant’s tetrachrome method (Herlant, 1964) or of immunocytochemistry for PRL, by the unlabeled antibody peroxidase-antiperoxidase technique (Sternberger, 1979). The criteria used for simultaneous definition of cell types by the tetrachrome method included assessment of cytoplasmic granules, estimated ratios of nuclear to cell diameters, and relative cell diameters, which have been described in detail elsewhere (Hymer et al., 1973, 1974). “Definite” lactotrophs were distinguished from similar-sized somatotrophs, which contain yellow granules, by the presence of large (1-2 pm) red cytoplasmic granules; in addition, degranulated lactotrophs, included in final counts, were cells with scant blue cytoplasm and a yellow juxtanuclear Golgi area. Basophils were cells with cytoplasmic blue granules and smaller (1:4) nuclear-to-cytoplasmic ratios; no attempt was made to differentiate among the three basophilic cell types (gonadotrophs and thyrotrophs). In “start” (nonseparated) samples, and from gradient fractions where adequate cell numbers permitted, lactotrophs were also identified by prolactin immunocytochemistry. For this method, fixed cell preparations were incubated successively in phenylhydrazine (0.15%) for 60 min at room temperature, to inactivate endogenous peroxidase; in normal goat serum (0.5%)for 120 min at 4”C, to inhibit nonspecific binding of rabbit IgG, in primary antiserum (NIADDK rabbit anti-rat PRL IC-1, 1:4,000) overnight (16-18 hr, a t 4°C); in secondary antibody (goat anti-rabbit IgG, Cappel Labs, 1:200) for 60 min a t 37 “C; and in peroxidase-antiperoxidase (PAP, 12300, Miles-Yeda)for 60 min at 37°C. Color was developed with 3-3’diaminobenzidine (0.40 mg/ml containing 0.03% H202) for 5-20 min. Washes between incubations were conducted with Tris buffer containing 0.025% Triton X-100, to facilitate intracellular entry of antibodies. Controls for the immunocytochemical reaction included substitution of nonimmune rabbit serum for primary antiserum, and preabsorption of primary antiserum with 1.0 pg/ml of purified antigen (NIADDK rat PRL for iodination); either procedure abrogated specific staining. Lactotrophs were scored as cells with (brown) positive stain, by means of light and phase contrast microscopy PITUITARY CELL ANALYSIS AFTER HYPOTHALAMIC LESIONS of each field; no counterstain was used. On coded slides, 500-1,000 cells were counted, by two investigators, of two to three replicate preparations. Thus, a duplicate count of 21,500 cells per preparation was conducted. Insufficient cell numbers precluded immunocytochemistry of additional cell types. Velocity Sedimentation at Unit Gravity Cell suspensions were diluted to the following concentrations in 10 ml total volume of medium 199 0.1% BSA; a t 6 weeks after surgery, 4.7 x lo6 cells (MHA) and 9.2 x lo6 cells (intact controls); a t 10 weeks, 8.6 x lo6 cells (MHA) and 10.3 x lo6 cells (controls). These preparations were applied to a continuous density BSA (0.3-3.0%) gradient as described by Hymer and associates (1973). In this method, cells separate primarily on the basis of differences in cell size (Hymer et al., 1973). A total settling time of 2.25 h r was used to separate cells. Aliquots from each 30-ml fraction (14 fractions collected) were used to prepare microscope slides €or differential staining, and for radioimmunoassay of intracellular hormone content. Total cell recovery from the gradient was 65% among MHA cells a t 6 weeks (cell recovery is reduced when less than 5.0 x lo6 cells are applied) and 8 3 4 5 % in all other groups. + Cell Culture From each pooled experimental or control group, triplicate cultures, containing 25,000 pituitary cells each, were initiated in 35-mm dishes (Corning) containing 3 ml medium 199 plus 20% fetal calf serum, and were maintained at 37°C under 5% COz/95% air humidified atmosphere. Culture medium was replaced each 3 days for a 9-day period from the 6-week group, and for a 21day period from the 10-week group. Media samples were stored frozen for subsequent assay. At the termination of cell culture, intracellular hormone was extracted in 0.01 N NaOH as described above, and stored frozen until subsequent radioimmunoassay . Hormone Assay Quantity of prolactin and luteinizing hormone in gradient fractions and cell culture samples was determined by double antibody RIA methods using material and protocol provided by the NIADDK Rat Pituitary Hormone Program. Each sample was assayed in two dilutions, in duplicate. All samples (i.e., cell extracts and culture media) were assayed together, for each experiment (i.e., at 6 weeks and at 10 weeks after MHA). Values for hormone levels are expressed as Rat PRL-RP2, and Rat LH-RP-1. Grouped data are reported as mean standard error of the mean. Statistical differences were determined by Student’s t test. RESULTS A typical MHA lesion is illustrated in Figure 1, which shows the neurosurgical instrument (A) and a coronal section through the midpoint of the lesioned area (B). Hypothalamic destruction included a n area from the suprachiasmatic nuclei rostrally to the premammillary region caudally; laterally, the lesion extended to the plane of the fornix. Arcuate and ventromedial nuclei were consistently ablated. As shown, the knife dimen- 367 sions avoid destruction of the hypothalamic median eminence and its vasculature; these delicate structures are often destroyed in dissection of such lesioned brains. Rats subjected to MHA surgery commonly develop both diabetes insipidus and hyperphagia, and growth is stunted (Dunn and Arimura, 1974). In the group killed 10 weeks after MHA, body weights averaged 610.2 f 20.6 gm, compared with intact control weights of 527.2 f 11.4 g m (P < 0.05); nasoanal lengths of MHA rats after 10 weeks averaged 24.5 f 0.4 cm; controls measured 27.0 k 0.3 cm (P < 0.005). Data comparing characteristics of initial cell preparations from control and experimental groups are shown in Table 1. The total number of cells recovered from pituitaries after enzymatic dissociation was 1.7 x lo6 cells er pituitary for MHA rats at 6 weeks and 2.4-2.6 x 10 cells per gland for other groups. (Routine recovery for the dissociation procedure is 2.5 x lo6 cells. In 1974, Hymer and co-workers estimated that a recovery of 2.53.0 x lo6 cells per gland represented 60-70% of total cells, based on a n estimated 4.6 x lo6 cells per pituitary, derived from counts of nuclei in pituitary homogenates and from total gland DNA measurements.) The intracellular content of PRL in samples from MHA rats’ pituitary cells were consistently, but not significantly, lower than in cells from control animals. Actual prolactin content was lower in both 6-weeks groups (as it was in gradient fractions; see Fig. 4),but the relative decrease among MHA cells was comparable a t both postsurgical intervals. Numbers of lactotrophs, identified by the tetrachrome method, were greater in glands from lesioned animals; the difference was significant (P < 0.05) 10 weeks after MHA. The numbers of chronically identified basophils were not significantly affected by the surgery. The results of unit gravity separation of cells from MHA rats after 6 and 10 weeks are compared with profiles from intact controls in Figure 2. Peak cell recovery occurred in fraction rV (4 x 30 ml initial gradient fluid) in all instances. However, a greater percentage of cells from MHA pituitaries was recovered from upper gradient fractions; decreased overall size of cells from MHA rats was thus indicated. This differential was more notable in the group killed 6 weeks after surgery; at 10 weeks after MHA, a small number (0.6%) of pituitary cells sedimented as far as fraction X. Figure 3 presents comparative photomicrographs of Herlant’s-stained acutely dissociated (“start”) preparations from control and MHA a t the 10-weeks interval. Smaller, relatively undifferentiated cells are seen in the MHA preparation. In Figure 4 the unit sedimentation profiles of prolactin cells are shown. Prolactin cell sedimentation was estimated by three independent criteria: by relative recovery of total PRL from the gradient (top panel), by calculation of PRL content per 1,000 pituitary cells (middle panel), and finally by percentage PRL cells determined by differential counts of tetrachrome-stained gradient fractions. Six weeks after surgery, a greater percentage of recovered hormone was associated with smaller cells, in MHA preparations, than among cells from intact rats. At 10 weeks following MHA, PRL was recovered from cells sedimenting slightly further into the gradient. Although absolute values for intracellular PRL (center panel, Fig. 4) were much lower a t the six- B 368 C.J. PHELPS AND W. C. HYMER B A I 1 I - - - - - -3.5 mm Fig. 1. With the rat's head positioned in a stereotaxic apparatus so that the base of the brain was in a horizontal plane, the MHA instrument (A) was lowered midsagittally to the basisphenoid bone, 1 mm posterior to bregma, and rotated 360" several times. The resulting lesion is illustrated in B. Coronal section; X 4 . TABLE 1. Cell and hormone recovery in initial suspensions of pituitary cells from intact control and medial hypothalamicablated rats Parameter Number of rats Celldanterior pituitary ng Prolactid1,OOO cells % Prolactin cells % Basophils Interval (weeks) 6 10 6 10 6 10 6 10 6 10 Intact MHA 4 4 2.5 x lo6 2.4 X lo6 3.1 & 0.5 9.6 k 2.7 29.3 3.3 32.2 & 3.6* 6.0 f 0.8 7.0 + 0.9 3 8 1.7 X lo6 2.6 X lo6 2.3 & 0.2 7.3 0.2 35.5 f 1.4 42.0 1.7* 7.7 1.1 8.4 _+ 1.2 * + Rats were age-matched males of the Sprague-Dawley strain. Cell recovery was determined by counts of pooled, acutely dissociated glands from each experimental group. PRL content was determined by RIA, and expressed in terms of NIADDK RP-2. PRL cells and basophils were identified by Herlant's tetrachrome staining; quantities shown represent mean k SEM of counts by two investigators of triplicate preparations ( - 500 cells per slide). *These values are significantly different (P < 0.05). weeks postsurgery interval, relative content for MHA vs. intact control groups was comparable at the two intervals: less PRL was recovered from MHA cells, especially in upper gradient fractions. Likewise, identification of lactotrophs showed a similar pattern at 6 and at 10 weeks after MHA: peak recovery of lactotrophs among MHA cells shifted downward (indicating slightly larger cells) compared with cells from control rats. Sedimentation patterns of identified lactotrophs were similar, however, for control and experimental groups, the PITUITARY CELL ANALYSIS AFTER HYPOTHALAMIC LESIONS 6 WEEKS AFTER MHA t c 0 .c 40- I\ I\ I I --- intact rats MHA rats fraction Fig. 2. Total cell recovery following unit gravity sedimentation. Fractions indicated on the horizontal axis represent (in this and the following figures) 30 ml of gradient fluid; therefore, cells recovered from higher-numbered fractions sedimented further into the gradient. Cell recovery on the vertical axis is shown as percentage per fraction of total recovery. Total cell recoveries from the sedimentation chamber: at 6 weeks, 83%(of 9.2 x 10.6 cells applied) for control and 65% (of 4.7 x lo6 cells) for MHA; at 10 weeks, 83% (of 8.6 x lo6 cells) for control and 85% (of 10.3 X lo6 cells) for MHA tissue. majority PRL cells occupying a middle-sized category among pituitary cells and having a heterogeneous distribution. In Table 2, quantitation of Herlant's tetrachrome and immunocytochemically (ICC) identified lactotrophs is presented. In "start" samples, numbers of lactotrophs were similar according to the two techniques, if broad criteria €or chromic identification are used. An increase in PRL cell numbers was indicated 10 weeks after MHA; that increase was also visualized (although not significantly) by ICC quantitation, but was not reflected in heavily granulated lactotrophs. In gradient samples, peak numbers of lactotrophs occurred in fraction V €or control rats, and in fraction VI after MHA. In those peak fractions, lactotrophs identified by ICC are roughly half the quantity identified by "broad criteria" (see Methods) in chromic staining. In those fractions only, ICC percentages were comparable to numbers obtained by "strict" chromic identification (by the presence of red cytoplasmic granules). Secretory activity of acutely dispersed adenohypophysial cells is shown in Figure 5. Each point represents the mean (+ SEMI of four replicate cultures of 2.5 x lo4 cells. In the group sacrificed 6 weeks after MHA sur- 2 +I 369 C.J. PHELPS AND W. C . HYMER 370 Fig, 3. Photomicrographs of dissociated anterior pituitary cells. Aliquots of initial dispersions were deposited on microscope slides with a cytocentrifuge and were subsequently stained by Herlant's tetrachrome method. The photograph on the left shows a preparation from an intact control rat. The cells on the right were prepared from a rat subjected 10 weeks previously to MHA; these cells appear smaller and relatively undifferentiated compared to those from the normal animal. X 600. 10 WEEKS AFTER MHA -intact rots MHA r o t s 6 WEEKS AFTER MHA ,+, I I -intact rots ---MHA rots \ * 50 --- \ 0 c -" 0 ea 3 f roction B f roction 1 L fraction 20 10 I D fraction Fig. 4. Analysis of unit gravity gradient fractions for PRL recovery and PRL cell identification in two experiments. A. Six weeks after MHA surgery. B. Ten weeks after surgery. PRL in fractions is expressed as percentage of total hormone recovery (top panel), and hormoneicell number (middle panel); numbers of PRL cells (lower panel) were determined in Herlant's tetrachrome-stained sections. gery, cells were maintained in vitro for 9 days. Both release and cell content of PRL were comparable between cells from intact and experimental rats for the culture period. Since initial PRL content of these cells was very low, total hormone secretion over the 9-day period was quite high. Cells from controls secreted a total of 63.3 ng/1,000 cells, and cells from MHA rats released 58.5 ng/1,000 cells, in the 9-day period. Calculation of "production index" (hormone releasedhnitial intracellular content) gave a value of 20.4 + 2.3 for control cells, and 25.4 t- 2.4 for MHA cells; the values are not significantly different. For the group killed 10 weeks after surgery, cells were monitored in vitro for 21 days. In that group, MHA-derived cells released signifi- cantly less PRL ( P < O . O l ) beginning at 9 days in vitro; cell content of PRL was slightly less (NS), after the 21day period. The values represented (as total hormone released by 2.5 x lo4 cells) were 56 nglday, initially, for cells from intact rats, and 45 ng/day for cells from MHA rats; those secretory levels increased to 108 nglday for intact and 85 ng/day for MHA cells. Over the 21-day culture period, cells from intact rats released 196.8 ngl 1,000 cells; cells from lesioned rats released 125.3 ng/ 1,000 cells. Production indices were 20.5 f 1.3 for controls and 17.2 k 1.6 for MHA (NS) in this 10-weekslesioned group. For comparison, the same fractions (at 6 weeks postsurgical survival) were assessed for LH content and 371 PITUITARY CELL ANALYSIS AFTER HYPOTHALAMIC LESIONS -intact 10 WEEKS AFTER MHA x 0 6 WEEKS AFTER MHA rots --- MHA rots I I I 1 3 6 9 k- 3 m 6 12 9 days in culture 15 21 18 days in culture 0cells medium medium 8w ?.0 . 4 L intact rats c intact 0 MHA c rats rats MHA rots Fig. 5. PRL release from acutely dispersed (not separated) cells maintained in vitro, expressed as secretory rate (upper panel) and as total hormone recovery for the culture period (lower panel). A B 6 WEEKS AFTER MHA -intact 6 WEEKS AFTER MHA rots - f roction I 3 intoct rots I I 6 9 days in culture 0 cells a medium ” fraction 8 0.4 9 0.3 \ 3 0.2 #’ I II m ‘4 P PLpIImIxx-XIp f ractian c 0 & intoct rats MHA rots Ip Fig. 6. Assessment of LH cell characteristics and function after gradient separation (A) and in vitro maintenance (B). The lower left graph includes quantitation of all basophils. Samples and parameters analyzed identical to those in Figures 4 and 5, respectively. “basophil” (TSH, LH, and FSH) staining (Fig. 6). LH was recovered from MHA-rat cells that had slowed sedimentation rates (peak at fraction V) compared with intact rats (peak at fraction IX). LH secretion, in nonseparated cells from the 6-weeks-lesioned group, was initially much lower (P < 0.01) among cells from lesioned rates. Decline in LH release among pituitary cells from intact rats was precipitous over the 9-day sampling period release from MHA pituitaries was ini- tially low and remained low. Total LH secretion was lower among MHA glands (P < 0.01);final cell content was comparably low in intact and lesioned rats. DISCUSSION The marked obesity and reduced nasoanal length that occurred among MHA rats have been noted previously (Dunn and Critchlow, 1973). The lesion would likely 372 C.J. PHELPS AND W. C. HYMER destroy many hypothalmic cells containing GH-releasing factor (GHRF), since these have recently been localized, in the rat, to the arcuate and ventromedial nuclear areas (Merchenthaler et al., 1984) included in MHA. Thus, it is likely that the lesion effect included deficits in both GHRF and somatostatin regulation of pituitary GH secretion, as proposed by Dunn and Arimura (1974); reduced growth could therefore result from deficits in pulsatile GH secretion or somatomedin availability (Martin, 1983). Blood samples were not taken because it was not possible to optimize laboratory conditions for eliminating variable and nonspecific stress. PRL levels are elevated by stress in MHA rats, although the response is blunted (Turpen et al., 1978);intact control rats show a marked elevation of PRL in response to stress; comparisons are thus obviated. Although the focal point of the present study was PRL cell function, some general effects of MHA were observed. Although MHA pituitaries are smaller (Cronin et al., 1982; Turpen and Dunn, 1976), the total number of cells recovered after trypsinization was affected in the present study only a t 6 weeks after MHA, a result that might be accounted for by the small number of pituitaries used. The result implies general reduction in cell volumes. Total cell recovery after MHA was shifted to upper portions of the gradient, indicating generally smaller pituitary cells. This difference was less noticeable a t 10 weeks than at 6 weeks after MHA. In pituitaries from intact rats, 90% of cells sedimented in fractions IV-IX; 90% of MHA cells sedimented to fractions IIIVI. Tinctorial identification of cell types and gradient hormone recovery indicated a slight downward shift in lactotroph, and a n upward shift in LH, cell populations after MHA. Somatotrophs decreased from 32.6%to 18.6% at 10 weeks after MHA (data not shown); total basophils maintained comparable populations among cells identified after long-term lesioning. The relative size of the basophil population reported here is comparable to that determined recently by immunochemical means; Dada et al. (1984) reported a range of 9.3-11.3% of total LH, FSH, and TSH cells in the male rat pituitary. Recent immunocytochemical counts estimate the PRL cell population to be much larger than once was assumed in Sprague-Dawley adult male rats: 26.9 f 3.1% when quantitated in dispersed cultures (Phelps, 1986), and as high as 49.8% when assessed in histologic sections (Dada et al., 1984). Tinctorial identification of lactotrophs in this study was based on less stringent criteria than originally described (Herlant, 1964): scored as PRL cells were those cells described by a blue cytoplasm and prominent yellow Golgi apparatus. These criteria result in a comparable correlation with immunofluorescence and immunoperoxidase staining for PRL cells, as evaluated in cycle-staged and in lactating female rats (Hymer et al., 1974); this study corroborates the use of such criteria in the male rat pituitary. In acutely dissociated pituitary cells, ICC scores corroborated numbers based on broad, but not strict, tinctorial criteria. In peak fraction (V or VI) samples from all groups, ICC numbers were closer to those based on strict Herlant criteria (Herlant, 1964). The explanation for this discrepancy is not apparent; the broad chemical nature and polymeric storage forms of intracellular PRL may explain differences in chromic and immunologic identification that occur in this medium-sized lactotroph population; perhaps antigenic sites are obscured in cells in this physio- logic state. After gradient separation, such a difference would be revealed, whereas it is obscured by variance in nonseparated populations. Sedimentation of basophils (which includes gonadotrophs, thyrotrophs, and possibly corticotrophs) 6 weeks after MHA indicated smaller and/or less dense cells; the number of basophils per fraction was reduced. LH from MHA cells was recovered from upper portions of the gradient, and LH release andlor synthesis was lower among MHA pituitary cells. Lower testicular weight in MHA rats has been previously noted (Turpen and Dunn, 1976); reduced gonadotroph activity would contribute to such a result. With regard to the specific effect of MHA on pituitary lactotrophs, differences occurred between 6 and 10 weeks after MHA, in both cell separation and culture. Six weeks after MHA, nearly 90% of PRL was recovered from fractions IV and V, although PRL cells were heterogeneously distributed through the gradient. The cellular content of PRL was generally reduced. When cells from this group were maintained in vitro, however, MHA and control cells were comparable as to secretory activity. Thus, the smaller size and initial PRL content of MHA cells may have reflected a n increased hormone release rate, as would be expected after removal of dopaminergic inhibition. Ten weeks after MHA peak PRL recovery had shifted to lower gradient fractions compared with cells from intact controls or with MHA cells at 6 weeks after surgery. Intracellular PRL was still reduced compared with control levels, and identified cell distribution remained heterogeneous and comparable to that of controls. Although PRL release into culture medium was reduced, production indices for controls and MHA rats were similar. It thus appears that PRL secretory function was unaffected after long periods in MHA-lesioned pituitaries, although lactotroph morphology, reflecting hormone storage, generally declined. Cells were maintained in vitro for a long period in this group; studies of PRL cell survival in vitro (Snyder et al., 1976) indicate that PRL cells are maintained at constant relative proportion in vitro, rather than showing relative proliferation or degeneration. There is no evidence that such a phenomenon was affected by MHA. In acutely dispersed preparations, however, PRL cell numbers increased a t the 10-weeks postsurgery interval. It should be noted that this increase reflects a relative population among pituitary cells; it is possible that a decline in numbers of other cell types (such as somatotrophs) may account for the increase in PRL cells. Cronin and associates (1982) reported a volume percentage increase among PRL cells 2 or 3 weeks after hypothalamic lesions in ovariectomized female rats, which could have been the result of either hyperplasia or hypertrophy of lactotrophs. The present report indicates that both phenomena may occur 10 weeks after MHA, since both sedimentation and numbers of morphologically identified lactotrophs increased slightly at that interval; the latter indices were only slight at 6 weeks after surgery. Comparison of the present results with those of Cronin et al. (1982) may be inappropriate, not only because postsurgical intervals differed, but because the lesions evidently were not comparable. Although those authors cite a n identical author (Dunn and Critchlow, 1973) for the neurosurgical procedure, neither dimensions of the instrument nor histologic evaluations of the lesion were given. Since their animals showed significant decreases in body weight, PITUITARY CELL ANALYSIS AFTER HYPOTHALAMIC LESIONS rather than the marked hyperphagia and obesity noted here and in previous reports (Dunn and Critchlow, 1973; Dunn and Arimura, 1974; Turpen and Dunn, 1976), it is likely that their lesions extended further laterally into the basal hypothalamus than those accomplished in the present experiments. Lesions in lateral hypothalamus, alone or in combination with medial hypothalamic lesions are known to damage feeding centers, resulting in hypophagia (Anand and Brobeck, 1951). As in the present report, those investigators found no significant change in tinctorially or immunochemically identified gonadotrophs. Again, comparison of the results obtained in ovariectomized females vs. males should be viewed with caution. In view of possible lactotroph hyperplasia in either study, Burdman and colleagues (1984) have reported that prolactin cell proliferation is dependent on estrogenic stimulation. Lieberman et al. (1982) reported a similar phenomenon in vitro. That the PRL cell population is tonically held in abeyance (presumably by hypothalamic dopamine) regarding proliferation, as well as secretory function, is a widely held but largely undocumented view. The few studies that have addressed this question used in vitro pituitary isolation. In 1976, Snyder et al. noted that mammotrophs accounted for 45% of pituitary cells after 3 days in culture, but 70% of cells after 30 days maintenance; they commented that it was unknown whether that relative increase was due to degeneration of other epithelial types or mitosis of mammotrophs. In 1984, Wilfinger et al. noted increased DNA levels (30-50%) in pituitary cell cultures maintained 9 days; the DNA increase was attributed to fibroblast proliferation. Antakly et al. (1980) quantitated stable numbers of PRL cells over 12-day periods in culture. Thus, the effect of long-term removal of medial basal hypothalamic influence on PRL cell function appears to be surprisingly slight. Some lactotroph hypertrophy and increased hormone retention, coupled with reduced PRL release, occurred 10 weeks after MHA. However, hormone production indices for MHA cells were comparable to those of controls. The increase in lactotroph numbers seen at 10 weeks following surgery was only relative to other cell types, which were adversely affected by the lesion. It appears that lactotroph disinhibition after lesions of tuberoinfundibular neurons pertains primarily to hormone release rates, with only incidental effect on cell morphology and proliferation. 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