Heterogeneity of pituitary lactotrophs: immunocytochemical identification of functional subtypes

Acta histochem. 99, 277-289 (1997) Gustav Fischer Verlag

Heterogeneity of pituitary lactotrophs: immunocytochemical identification of functional subtypes Ana Lucia De Paul, Patricia Pons, Agustin Aoki and Alicia Ines Torres Laboratory of Cellular Neuroendocrinology and Electron Microscopy Center, Cordoba National University, RA 5000 Cordoba. Argentma Accepted 13 April 1997

Summary The existence of functional lactotroph subpopulations was confirmed in primary pituitary cell cultures of female rats submitted to estrogen treatment and stimulation with thyrotrophin releasing hormone (TRH) and angiotensin II (A-II). In cell cultures of pituitary tissue, prolactin (PRL) producing cells represent about 50% of the total cell count, most of which (90%) correspond to a typical lactotroph subpopulation characterized by large secretory granules, 500-900 nm in diameter, and well developed rough endoplasmic reticulum (RER) and Golgi complex. Few atypical lactotrophs were detected with a quiescent appearance and containing smaller secretory granules, often indistinguishable from granular content of other pituitary cells. Depletion of endogenous estrogen caused by ovariectomy (OYX) decreased the pituitary lactotroph population about 34%, with a relative increase of atypical forms (56%). Replacement therapy with benzoate estradiol (EB) to OVX rats did not reverse the proportion of typical and atypical lactotrophs gauged in control pituitary glands. The predominant lactotroph population of OVX rat was an atypical PRL producing cell which displayed a quiescent appearance compatible with a reduced secretory activity. By contrast. estrogen administration to OVX rats caused a striking development of the RER, a hypertrophy of the Golgi complex and an increased storage of mature and immature secretory granules in the majority of lactotrophs. These features are compatible with a reactivated protein synthesis. Estrogen also enhanced significantly (p < 0.05) the responsiveness of lactotrophs to A-II and the PRL secretion in both intact and OYX + EB treated rats increased by 40% and 30% respectively. By contrast, A-II did not produce any statistically significant response of lactotrophs from OVX female rats. At variance to this observation, in all models tested TRH increased significantly the PRL secretion (p < 0.05). The correlation of PRL secretion and morphology of different lactotroph SUbtypes authenticates the existence of a lactotroph subpopulation unresponsive to A-II in pituitary cell cultures from rats depleted of estrogen.

Key words: estrogen - prolactin - thyrotropin-releasing hormone - angiotensin II lactotroph subpopulations Correspondence to. A. 1. Torres

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Introduction The different cell populations constituting the adenohypophysis were identified by their ultrastructure and hormone production (Hymer and Motter, 1988). However, these cell populations are not homogeneous and several subtypes have been characterized morphologically by immunocytochemistry (Nogami and Yoshimura, 1980, 1982; St John et aI., 1986; Maldonado and Aoki, 1994) and functionally by their response to secretagogues (Frawley and Boockfor, 1991; Kazemzadeh et aI., 1992; Arita, 1993; Caldini et aI., 1996). In isolated lactotrophs, a striking heterogeneity was demonstrated correlating the fine structure of the cells with the levels of prolactin secretion (Velkeniers et aI., 1994). Also various lactotroph subtypes displaying significant differences in their transcriptional and secretory activities were isolated by gravity sedimentation (Velkeniers et aI., 1988). In addition, we have reported the occurrence of various sUbpopulations of lactotrophs in male rats (De Paul et aI., 1997). In keeping with observations of Takahashi (1995) it is highly probable that such heterogeneity observed in lactotroph subpopulations discloses functional differences occurring at various phases of lactotroph maturation or related to the production of the PRL molecular variants described in the literature (Wallis, 1988). Correlation of morphometric analysis, ultrastructural immunocytochemistry and quantification of PRL by RIA, has opened new horizons in the recognition and understanding of the functional significance of lactotroph cell heterogeneity. In the present study this approach has been applied to investigate the secretory behavior of lactotroph sUbpopulations in cell culture and allowed the identification of an atypical lactotroph subtype in pituitary gland of female rats, exhibiting differential responses to A-II, closely associated to estrogen levels.

Materials and Methods Animals, experimental design. Adult 3 month-old Wistar rats were divided in 3 groups: 1) a control group of intact female rats, 2) rats ovariectomized (OVX) 21 days earlier and 3) OVX rats implanted with estradiol benzoate (EB) for 10 days (OVX + EB). Gonadectomy was performed under deep ether anesthesia and the EB was implanted subcutaneously in slow releasing capsules made of medical grade silastic tubing (0.030ID . 0.065 OD, Dow Corning), 7 mm long filled with EB crystals and sealed with silastic cement. All rats were kept under controlled temperature and light conditions (14 h light/l0 h dark) with access to commercial lab chow and tap water ad libitum. The pituitaries from each group were pooled and the cells isolated for in vitro cultures. About 10 . 106 cells were distributed in equal parts in 5 wells which were tested as described below. The NIH Guide for the Care and Use of Laboratory Animals was followed for the maintenance and treatment of the animals. Cell dissociation, separation and culture. After decapitation and removal the posterior lobe, the anterior pituitaries were rapidly excised under sterile conditions and placed in freshly prepared minimal essential medium (MEM). All culture media were filtered through a 0.2 !lm membrane (Nalgene, Nalge, New York) before use. The glands were minced with a razor blade into small blocks, rinsed in MEM and then incubated with 0.4 % trypsin for 20 min at 37°C. Tissues were treated with trypsin inhibitor and deoxyribonuclease (1 mg/ml) for 3 min, followed by two washing in MEM at room temperature. The cells were mechanically dispersed with a siliconized Pasteur pipette, washed and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 3% fetal calf serum and 8% horse serum (Gibco). The cell yield was 1.5-2.106 per pituitary and the cell viability, tested with trypan blue exclusion technique, was better than 90%. The final suspension was adjusted to 1 . 106 cells/ml of medium. Five hundred microliters of the cell suspension were seeded in 35-mm sterile cell culture wells (Corning, NY) and maintained at 37°C, in a humidified atmosphere of 5% CO 2 - 95% air. Culture medium was changed on the 3rd and 4th day of culture. On the 5th day the medium was replaced by 1 ml fresh DMEM without sera and allowed to stabilize for 2 h (time 2) Thereafter, fresh medium without (control) or with 5 nM angiotensin II or 30 nM TRH were added to the test wells (n = 5 for each treatment) and then, the pituitary cells were incubated for 1 h (time 3).

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Samples of the medium (1 mJ) replaced every h up to 5 h were frozen and stored for radioimmunoassay (RIA) of PRL. All replacing media were made at 37 °C. Unless stated. Reagents for tissue culture used in this investigation were purchased from Sigma Chemical Company (St. Louis. MO, USA). Electron Microscopy, Immunocytochemistry. At the end of each experiment, the cells were scraped. washed and fixed in a mixture of 4% formaldehyde and 1.5% glutaraldehyde in 0.1 M cacodylate buffer plus 7% sucrose for 2 h (Karnovsky. 1965). Then, the cells were spun down by centrifugation, treated with 1 % OS04 for 1-2 h, dehydrated in an acetone series and embedded in Araldite. For immunocytochemistry. thin sections cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome were mounted on nickel grids and etched with 10% hydrogen peroxide for 7 min. The sections were then incubated overnight on a drop of anti-rat PRL rabbit serum (NIH National and Hormone and Pituitary Program) diluted 1: 2 500, at 4 °C for 24 h. Immunostaining was performed with protein A/colloidal gold complex (diluted 1: 18) for 30 min. To enhance the labelling. the immunoreaction was bridged with anti-protein A serum (at 1: 100 dilution) for 1 h followed by protein Ncolloidal gold complex (1 : 27) for 30 min. Colloidal gold particles, 16 nm in diameter. were prepared according to Frens (1973) with sodium citrate as reducing agent. Preparation of immunogold complexes and other details of the immunocytochemical procedure were described elsewhere (Maldonado and Aoki, 1986). The morphometric analysis of lactotroph subpopulations was performed by electron microscopy. To aVOid repetItive cell counting, only one thin section cut at various levels of the pellet thickness was mounted on the nickel grids. The same immunostaining procedure was applied to cells after 5 days of culture in 3 separate experiments. At least 300 cells were analyzed in triplicate for each experimental model. The lactotrophs counted were classified as typical (subtype I) and atypical (subtype II or III) sub populations. Data are expressed as percent positive cells. Radioimmunoassay, statistics. All experiments were performed at least three times unless otherwise stated. Prolactin in frozen media was quantified by RIA applying a double antibody technique performed at two dose levels and the results were expressed in terms of rat PRLlml of medium with reagents donated by NIDDK-NIH (Bethesda, MD, USA; NIDDK-r-PRL-I-6, anti-rPRL-S-9, and rPRL-RP-3). All the samples studied were processed simultaneously to avoid inter-assay variations. The intra-assay coefficient of variatIOns was less than 10%. To compare the prolactin release III paired wells. a paired t-test was applied to determine the average difference between individual observations. Level of significance was established at p < 0 05. Results were expressed as mean ± SEM of three expenments. Morphometry of lactotroph subpopulatlOns was analized by one-way analysis of variance and the differences between means were determined applying Tukey's test. Significance was reported at p < 0.Q1 and results expressed as mean ± SEM of three different preparations.

Results Morphometry and immunocytochemistry of lactotroph subpopulations. Electron microscopy of pituitary cell cultures revealed remarkable morphological variations in the lactotroph popUlation and in the percentage of the main lactotroph subtypes in the three models studied. Morphometric data estimated for each experimental condition are summarized in Table 1. Table 1. Morphometry of lactotroph sub populations Experimental model

Lactotroph population

Typical Lactotrophs

Atypical Lactotrophs

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51 ± 1.5% 34 ± 1.2% 38 ± 1.9%

90± 1.5% 44±0.6% 48 ± 1.8%

1O±0.5% 56±0.6% 52± 1.5%

Percentage of lactotroph subpopulations in culture of rat anterior pituitary cells under different experimental condition identified at the electron microscopy level applying Protein A-coloidal gold technique. The lactotroph populatIOn counted was classified as typical (subtype 1) and atypical (subtypes II and III) sUbpopulations. Mean ± SEM of 3 expenments. each one performed in triplicate on different experimental models. P < 0.01 was considered sigmficant.

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Fig. 1. Immuno-electron micrograph of a lactotroph subtype I from a control female rat. This cell exhibits a prominent nucleus with a hypertrophic nucleolus. In the cytoplasm, large secretory granules (500-900 nm) with irregular shape are stained specifically for PRL with the immunogold technique. A well-developed Golgi Complex contains numerous immature granules associated with its trans face. x 21.000.

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Fig. 2. Electron microscopy of a subtype I lactotroph from OVX female rat. The numerous mature secretory granules accumulated III the cytoplasm due to blockage of exocytosis. The secretory granules are intensely labelled for PRL with protein A/colloidal gold techlllque. The scarcely developed RER and GOlgl complex are additional features of a lower activity caused by estrogen depletion. x 14.600. Fig. 3. Electron micrograph of pituitary cells from OVX rats. An atypical lactotroph characterized by small sphencal secretory granules, immunostained for PRL The cell in the upper part is a typical somatotroph with unlabelled secretory granules as a negative control. x 14.600.

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Fig. 4. Atypicallactotroph from OVX + EB rat showing many spherical secretory granules in the 150250 nm range. These granules accumulate mainly near the plasmalemma and some of them are seen in the phase of exocytosis. The estrogen replacement therapy induced cellular hypertrophy of cytoplasmic organelles with a striking development of RER and Golgi complex. x 15.000. Fig. 5. Electron micrograph of typical lactotroph from OVX + EB female rat stimulated with A-II. The cytoplasm presents a well developed RER and Golgi complex. Note the presence of many immature and the scarce mature granules. x 15.000.

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.~ Fig. 6. Electron microscopic Immunocytochemistry of a typical lactotroph from a control female rat after TRH stimulus, The secretory granules are concentrated at the plasmalemma where they will exocytosed, x 15,600. Fig. 7, Typical lactotroph of pituitary gland from OVX + EB female rat after A-ll treatment. Several characteristic mature secretory granules are seen aligned along the plasmalemma. The inset shows a polymorphic mature secretory granule during exocytosis. x 15.600.

In intact female rat, PRL producing cells represented about 50% of the total number of the pituitary cells, the majority of which corresponded to subtype I (90%) , This sUbpopulation was characterized by cells containing large and polymorphic mature secretory granules (about 500-900 nm in diameter) and numerous immature granules associated with the Golgi trans face (Fig, 1). A highly developed rough endoplasmic reticulum (RER) and a prominent Golgi complex, characteristic of enhanced biosynthetic activity. were commonly observed in subtype I lactotrophs.

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Atypical lactotrophs, sUbtype II characterized by the presence of spherical granules ranging 150-250 nm diameter and subtype III with smaller (100 nm) secretory granules, comprised about 10% of the total cell count in control animals.

Fig. 8. Electron micrograph of two immunolabeled lactotroph cells from OVX + EB rat. These cells display significant differences m their hormonal contents. The cell at the top con tams numerous typical mature secretory granules stored in the cytoplasm and some fusing immature granules. In contrast. the cell at the bottom is almost depleted of mature secretory granules but shows hypertrophic RER and Golgi complex. x 21.000.

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The depletion of estrogen consequent to ovariectomy, induced remarkable changes in the fine structural organization of lactotrophs. The quiescent appearance acquired by typical PRL cells was validated by the accumulation of secretory granules with uniform size and round profile, and a small and disorganized Golgi complex with a few immature granules (Fig. 2). The main subtype in OVX rats was an atypicallactotroph that contained smaller secretory granules (Fig. 3). All the features described in typical and atypical subtypes were compatible with a reduced secretory activity. Morphometry of cell cultures derived from OVX rats revealed that the total lactotroph popUlation decreased to 34%, a result that was concurrent to a significant increase in the percentage (56%) of atypical forms with respect to control values. Estrogen replacement therapy to OVX rats did not reverse the proportion of lactotroph subtypes to control values of cell cultures from intact female rats. However, the fine structure of both typical and atypical lactotrophs revealed that the estrogen treatment induced a striking development of RER cisternae, hypertrophy of the Golgi complex and generation of numerous immature granules, cytoplasmic features associated with a reactivated protein synthesis (Figs. 4, 5). Stimulation with TRH and A-II of lactotrophs from intact and OVX + EB female rats provoked several cytological changes related to an increased secretory activity, including the mobilization of mature PRL granules toward the plasmalemma and their release by exocytosis (Figs. 6, 7). In these experimental models the coexistence of lactotrophs at different functional stages is frequently observed in the same section. Lactotrophs with large stores of secretory granules were seen next to cells depleted of secretory granules by specific stimuli (Fig. 8). Radioimmunoassay. Prolactin released from pituitary cells into culture media of intact. OVX and OVX + EB rats after incubation with 30 nM TRH and 5 nM A-II, is shown in Figs. 9, 10 and 11. Stimulation with TRH induces a significant increase of PRL secretion (p < 0.05) of lactotroph cells from intact, OVX and OVX + EB rats when compared to previous secretory levels to stimulus (time 2 vs time 3) in paired wells. The effect of TRH was similar in intact and OVX + EB rats (between 4550%), but in OVX rats the secretory response was even higher (66%). The addition of A-II to cell cultures also increased significantly the release of PRL, reaching 40% and 30% (p < 0.05) in both control and OVX + EB female rats, respectively. By contrast, A-II did not cause any statistically significant response of lactotrophs from OVX rats.

Discussion Our study shows that lactotrophs of female rats in cell culture display remarkable heterogeneity when subjected to treatments with estrogen and regulatory neuropeptides. In control rats, typical lactotrophs (subtype I) are the predominant subpopulation comprising over the 90% of the total cell count. These cells exhibit the highest reactivity to both TRH and A-II as judged by the high levels of PRL synthesis and release. The increased secretory activity can be correlated with the frequent observation of exocytosed secretory granules. Ovariectomy decreased in the proportion of SUbtype I lactotrophs in pituitary cells in culture, while the number of atypical cells increased significantly. The accumulation of unreleased mature secretory granules, the sparse immature granules and the scarcely developed RER and Golgi complex in both subpopulations, are characteristic features of cells with low secretory activity, a secretory pattern similar to that of the male rats (De Paul et aI., 1997). In OVX rats as in male, TRH enhances significantly the PRL discharged into the culture medium, but A-II was ineffective to stimulate PRL secretion.

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The replacement therapy with estradiol for 10 days was insufficient to revert the proportion of lactotroph sUbpopulations to control values. However, in spite of fewer subtype I lactotrophs (48%), estrogen enhanced the response to A-II stimulation. The functional heterogeneity of pituitary PRL cells appears to be an integrated balance between the effects of hypothalamic and steroid hormones on PRL synthesis and secretion. In this process other secretagogues might also be involved. The behavior of a heterogeneous population of PRL cells is still not fully understood. Angiotensin II did not modify the PRL secretion from lactotrophs of OVX rats, but stimulated the secretion of PRL when the OVX rats were submitted to an estrogen pretreatment. As expected, marked ultrastructural changes in lactotroph subtypes were effected by estrogen, mainly in organelles involved in the processing of PRL and mobilization of secretory granules. In the present study, the correlation of PRL secretion and morphometry of the different lactotroph subtypes let us to confirm the occurrence of a lactotroph subpopulation unresponsive to A-II in pituitary cell cultures derived from OVX rats. In these rats depleted of estrogen, the replacement therapy reversed the behavior of this lactotroph subtype to stimulation with A-II. Luque et al. (1986) applying reverse hemolytic plaque assay (RHPA) of individual pituitary cells reported a bimodal distribution of plaque sizes suggesting that the amount of hormone released from each cell may differ among PRL cells from the same pituitary gland. Until recently, PRL producing cells have been accepted to be a single cell type containing the largest secretory granules among pituitary cells. At present, there is a general agreement on the existence of several morphological lactotroph SUbtypes. Velkeniers et a1. (1988) separated PRL cell subpopulations into high-density and low-density popUlation, using a discontinuous Percoll gradient, and found that low-density PRL cells have a high basal secretory activity and a higher PRL mRNA content, and that high-density PRL cells have a low basal secretory activity and a lower PRL mRNA content but a higher responsiveness to vasoactive intestinal polypeptide. Furthermore, PRL in the pituitary gland occurs in two main

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molecular forms, designated as big and small prolactin, which have been associated with well-differentiated cytoplasmic pools and their release can be closely correlated with dynamic states of the lactotroph (Torres and Aoki, 1985; 1987). The introduction of specific and improved techniques of cell biology and application to cellular endocrinology, such as immunocytochemistry for identification of pituitary cell types (St John et al., 1986); the RHPA for the detection of hormone secretion by individual cells (Boockfor et al., 1986; Luque et al., 1986; Boockfor and Frawley, 1987) and in situ hybridization correlated with RIA (Velkeniers et al., 1988), have allowed the identification of new subtypes of PRL producing cells. The analysis of lactotroph cell heterogeneity supports the interpretation that this particular characteristic, expressed at various levels, from the molecular to the subcellular, appears to be designed to provide the necessary flexibility to meet effectively the prolactin demands for each particular requirement.

Acknowledgments The authors particularly thank Mercedes Guevara and Lucia Artino for their excellent technical assistance and Prof. Fernanda Dutari for the preparation of the manuscript. This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), the Consejo de Investigaciones de la Provincia de C6rdoba (CONICOR), Argentina. The antisera used for immunocytochemistry were kindly supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIHDDK) and the National Hormone Program at the University of Maryland, School of Medicine.

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Maldonado C, and Aoki A (1994) Occurence of atypicallactotrophs associated with levels of prolactin secretory activity. Biocell 18: 83-95 Maldonado C, and Aoki A (1986) Improvement of prolactin immuno-Iabelling m osmium-fixed acrylic-embedded pituitary gland. Bas Appl Histochem 30: 301-305 Nogami H, and Yoshimura F (1980) Prolactin immunoreactivity of acidophils of the small granule type. Cell Tissue Res 211: 1-4 Nogami H, and Yoshimura F (1982) Fine structural criteria of prolactin cells identified immunocytochemically in the male rat. Anat Rec 202: 261-274 St 10hn PA. Dufy-Barbe L, and Barker lL (1986) Anti-prolactm cell surface immunoreactivity Identifies a subpopulation of lactotrophs from the rat anterior pitUitary. Endocrinology 113: 2783-2795. Takahashi S (1995) Development and heterogeneity of prolactin cells. Internat Rev Cyto1157: 33-97 Torres A, and Aoki A (1985) Subcellular compartmentation of prolactin in rat lactotrophs. 1 Endocnnol105: 219-225 Torres A. and Aoki A (1987) Release of big and small molecular forms of prolactin: dependence upon dynamic state of the lactotroph. J Endocrinol114: 213-220 Velkeniers B, Hooge-Peters EL, Hooghe R. Belayew A, Smets G, Claeys A, Robberecht P, and Vanhaelst L (1988) Prolactin subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical and physiological characterization. Endocrinology 123: 1619-1630. Velkeniers B, Kazemzadeh M, Vanhaelst L, and Hooge-Peters EL (1994) Functional heterogeneity with respect to oestrogen treatment in prolactin cell subpopulatlOn separated by Percoll gradient centnfugation. 1 Endocrinol141. 251-258 Walhs M (1988) Mechanisn of actIOn of prolactin. In: Cooke BA, Kmg RlB. van der Molen, Hl (Eds) Hormones and their actions. Elsevier, Amsterdam, Vol 2, pp 295-319