0163-769X/91/1204-0337$03.00/0 Endocrine Reviews Copyright© 1991 by The Endocrine Society

Vol. 12, No. 4 Printed in U.S.A.

Mammosomatotropes: Presence and Functions in Normal and Neoplastic Pituitary Tissue* L. STEPHEN FRAWLEY AND F. R. BOOCKFOR Division of Molecular and Cellular Endocrinology, Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, South Carolina 29425

I. Introduction II. Mammosomatotropes in Normal Pituitary Tissue A. Is the mammosomatotrope a significant constituent of the pituitary cell population in adults? 1. Rats 2. Cattle 3. Humans 4. Other species B. Is the mammosomatotrope a progenitor cell for the differentiation of classical mammotropes? 1. Rats 2. Humans 3. Mice C. Is the mammosomatotrope a transitional cell for the functional interconversion of GH and PRL secretors? 1. Physiological evidence 2. Experimental evidence III. Mammosomatotropes in Neoplastic Tissue A. Are mammosomatotropes a prominent cell type in neoplastic cell populations? 1. Human tumors 2. Animal tumors 3. Continuous cell lines B. What is the contribution of mammosomatotropes to hormone secretion? C. Are mammosomatotropes involved in a functional conversion of GH and PRL cells? IV. Overview A. What mechanism(s) might govern an interconversion of GH and PRL secretors? B. Is there an acidophil cycle?

is produced by a separate and distinct cell type (1). Consistent with this idea, the acidophilic staining cells (termed acidophils) of the adenohypophysis have been traditionally subdivided into two categories, those that secrete only GH and others that produce just PRL. This view was supported by considerable tinctorial evidence available at the time of Romeis' hypothesis and generated in the subsequent three decades (1-5). Moreover, data produced throughout the 1970s and early 1980s by the use of light microscopic immunocytochemistry (ICC) and immunofluorescence were generally consonant with the one cell-one hormone theory, except in the case of certain pituitary tumors (6-8). The presence of bihormonal acidophils in this latter situation was attributed to aberrant mechanisms attendant to the neoplastic condition. However, as will be discussed in detail later, the utilization of even newer techniques with greater powers of resolution and sensitivity has radically changed the unihormonal view of acidophil function. The purpose of this review is to consider and integrate more recent and earlier findings relevant to the existence and functions of bihormonal acidophils in normal and tumorous tissue. These dual cells have been termed mammosomatotropes because they possess the secretory characteristics of cells that release both PRL (mammotropes) and GH (somatotropes). Inasmuch as many of the issues surrounding mammosomatotropes have been controversial, it is imperative that the points of contention be expressed as objectively as possible. Accordingly, the organizational plan for this review is to pose and address specific questions that have been raised over the past few years by investigators working in this area. The article will begin with a review of mammosomatotropes in normal tissue and proceed to a consideration of these bihormonal cells in neoplastic pituitaries. Finally, an overview section will serve as a platform for addressing issues common to both situations.

I. Introduction

M

ORE than a half century has passed since Romeis first proposed the "one cell-one hormone" theory which holds that each of the major pituitary hormones Address correspondence and requests for reprints to: Dr. L. Stephen Frawley, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina, 29425. * Studies descriped herein that were conducted in the author's laboratories were supported by NIH Grants DK-38215 (to L.S.F.) and DK-41652 (to F.R.B.) as well as USDA Grant 9000720 (to L.S.F.). 337

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

338

FRAWLEY AND BOOCKFOR

II. Mammosomatotropes in Normal Pituitary Tissue The detection of mammosomatotropes in nontumorous adenohypophyseal tissue was made possible by the contemporaneous application of two innovative methodologies. The techniques successfully applied to the problem over the past 6-7 yr are the colloidal gold modification of dual-staining ICC (9) and reverse hemolytic plaque assays (10). The theoretical basis for ICC is widely known and will not be considered in detail here. Suffice it to say, use of the colloidal gold modification (in which gold particles of different sizes are directed to separate antibodies) made it feasible to perform duallabeling ICC at the electron microscopic (EM) level on the same pituitary section. Thus, it imparted a degree of sensitivity and resolution sufficient to unequivocally localize both GH and PRL to the same secretory granule, not just the same cell. In contrast to ICC, which detects hormone storage in fixed cells, the plaque assay enables microscopic visualization of hormone release from individual living cells. Because the principles underlying this methodology may not be as broadly understood, a brief description seems warranted and is provided in the legend to Fig. 1. A. Is the mammosomatotrope a significant constituent of the pituitary cell population in adults? The aim of this section is to review evidence favoring the existence of mammosomatotropes in normal pituitary tissue. For reasons described above, most of this evidence was generated during the latter half of the past decade, owing primarily to technological advances that enabled the detection of bihormonal acidophils. When appropriate, experimental details will be provided here and elsewhere to substantiate critical points. Otherwise, the reader is directed to the primary literature for this information. 1. Rats. The first direct evidence for the existence of pituitary cells that can concurrently release both GH and PRL was provided by Frawley et al. (10) who developed and utilized a sequential plaque assay (Fig. 1) for this purpose. They found that mammosomatotropes accounted for 8-15% of all cells in pituitary cultures derived from mature male and female rats. Moreover, the proportional abundance of these cells relative to the classical monohormonal somatotropes and mammotropes varied with the gender and physiological status of the pituitary donor (10,11). For example, the sequential plaque assay revealed that the relative number of GH-only, PRL-only, and mammosomatotropic cells was similar in mature male rats. This microscopic visualization of dual hormone secretors in the living state was corroborated both qualitatively and quantitatively by two additional strat-

Vol. 12, No. 4

egies. One of these was purely immunocytochemical and involved staining pituitary monolayers with GH and PRL antisera, alone or in combination, and then determining the mammosomatotrope population by subtraction. With the other approach, estimates of single and dual hormone secretors were made by a variation of the simultaneous plaque assay (see legend to Fig. 1). All three approaches yielded estimates for mammosomatotropes that were within a few percentage points of one another and thereby supported the same conclusion: that these dual secretors are indeed present in appreciable numbers within the male pituitary gland. Similar studies with cycling females revealed that although mammosomatotropes account for a similar fraction of all pituitary cells, they make up a smaller component of the total acidophil population due to the preponderance of PRLonly cells in this gender (11). Physiological fluctuations in the abundance of mammosomatotropes will be considered later in this article. By employing similar as well as new strategies for identifying mammosomatotropes, Lloyd and co-workers (12) confirmed the existence of dual-hormone secretors and observed proportions remarkably similar to those described above. In their first approach they used a simultaneous plaque assay to quantify the proportions of mammosomatotropes in pituitary dispersions from mature females and found that these cells accounted for approximately 15% of all pituitary cells present. Next, they performed double-staining ICC and estimated that about 10% of all pituitary cells stored both hormones. Finally, they ran ICC for one hormone on monolayers on which secretion of the other hormone had been identified by standard plaque assay. This approach, which quantifies and compares divergent aspects of the secretory process, showed at least 7% of pituitary cells to be mammosomatotropic. Leong and co-workers (13) also utilized a simultaneous plaque assay to provide evidence favoring the existence of mammosomatotropes, but found that they represented only 5% of all pituitary cells in males and females. This lower value may be attributable in part to their estimate of the total PRL cell population, which is considerably less than those made by others using plaque assays or ICC. Electron microscopic, immunocytochemical approaches have also been used with considerable success to investigate the existence of mammosomatotropes in rats (Fig. 2). According to Nikitovitch-Winer et al. (14), mammosomatotropes were consistently present in appreciable numbers in males, cycling females, and lactating rats. These dual cells were ultrastructurally distinct in that they possessed secretory granules that were considerably smaller (50-100 nm) than those found in traditional mammotropes (400-600 nm) or somatotropes (200-400 nm). This might explain why immunoreactive

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

339

F" FIG. 1. Direct evidence for the existence of cells that release both GH and PRL was provided by modifications of reverse hemolytic plaque assays, which enable microscopic visualization of hormone release from individual pituitary cells in culture. The technique is based on antibody-directed, complement-mediated lysis of protein A-coated erythrocytes (smaller cells above) in the vicinity of hormone secretors (larger cells). When pituitary cells and protein-A-coated erythrocytes are incubated as a monolayer with hormone-specific antibody, hormone released by the cells binds to antibody which in turn attaches to protein A. The presence of complement initiates a cascade that culminates in the lysis of erythrocytes around hormone secretors. The end result is the formation of a zone of hemolysis or plaque. In the sequential plaque assay shown here, the same pituitary cells are assayed first for GH (upper panel) and then PRL (lower panel). This is accomplished by adding fresh batches of antibody, complement, and erythrocytes in each sequence, and reidentification of the same cells is facilitated by the use of alpha-numeric coverslips. Note that cells that form plaques in the presence of both GH and PRL antisera are mammosomatotropes. In a simpler version of this system, termed the simultaneous plaque assay, different monolayers are incubated with each antiserum alone or both antisera together. The fraction of mammosomatotropes is then estimated indirectly by comparing the percentage of plaque formers detected when both antisera were applied simultaneously to the cumulative percentage obtained when each antiserum was used separately.

mammosomatotropes are more easily and reliably identified by EM than light microscopy (LM). Ishibashi and Shiino (15) observed a similar bihormonal, small granuled cell that was common in the pituitaries of pregnant but not in young virgin females (two animals at 8-weeks of age). Losinski et al. (16) also confirmed the observations of Nikitovitch-Winer and co-workers by using comparable techniques to detect numerous mammosomatotropes in pituitaries of adult males and females of two different rat strains. 2. Cattle. The existence of mammosomatotropes in cattle is also supported by both immunocytochemical and plaque assay data. Studies with colloidal gold ICC revealed that the lactating cow is quite unique among mammals examined thus far in that essentially all acidophils are mammosomatotropic to a certain extent. More specifically, Hashimoto et al. (17) reported that about 26% of all acidophils were conspicuously mammosomatotropic (i.e. contained secretory granules that labeled heavily for both hormones). However, even the acidophils that stored predominately GH (20%) or primarily PRL (54%) contained significant amounts of the

minority hormone, as determined by objective, quantitative criteria. Bovine mammosomatotropes are also distinct with respect to their morphology and location within the adenohypophyseal lobules (9). Morphologically, they tend to be multinucleated, which raises the possibility that they could have arisen as a postdifferentiation event by the fusion of preexisting classical somatotropes and traditional mammotropes. However, the observations that both hormones can be localized to the same secretory granules and trans-Golgi vesicles would argue against this view. As for "locale," mammosomatotropes are clustered in the central region of the lobules whereas their predominantly monohormonal counterparts occupy the peripheral area. Plaque assay data generated by Kineman et al. (18,19) show that an appreciable fraction of acidophils in both sexes of cattle are mammosomatotropic in terms of the capacity to release GH and PRL, and that their abundance is sexually dimorphic. In bulls (18), the PRL-only cell was predominant and accounted for 60% of all acidophils, followed by the traditional somatotrope (28%) and mammosomatotrope (9%). Interestingly, the ratio of PRL-only cells remained constant after castration,

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

340

FRAWLEY AND BOOCKFOR

FlG. 2. A high magnification electron micrograph of a mammosomatotrope from a cycling female rat. The colloidal gold method was used to localize GH (small gold spheres) and PRL (larger gold spheres) on the same section. Note that both hormones are frequently colocalized to the same secretory granule, not just the same cell. (22,900x). Photomicrograph kindly provided by Dr. M. B. Nikitovitch-Winer, Department of Anatomy and Neurobiology, University of Kentucky. [Reproduced with permission from M. B. Nikitovitch-Winer et al.: Endocrinology 121:625,1987(14). © The Endocrine Society]

whereas those of GH-only cells and mammosomatotropes underwent an exact reversal, suggesting that testosterone (or some other gonadal product) favors the traditional somatotrope over the mammosomatotrope. Mammosomatotrope values for cycling females (19) were intermediate to those found in bulls and steers. Thus, dual-hormone secretors are clearly present in the bovine pituitary, but not at the frequency that would be predicted by the immunocytochemical data. However, it is interesting to note that the proportion of acidophils determined by ICC (17) to be conspicuously mammosomatotropic in lactators (26%) is within the range of those found by plaque assays to actually release both hormones in males and cycling females. 3. Humans. As alluded to earlier, mammosomatotropes have been difficult to identify in normal pituitary tissue from humans as well as other species when LM-ICC was employed. Notable exceptions, however, were two very similar human studies separated by almost a decade. The strategy of both groups was to quantify on separate pituitary sections the percentage of all cells that contained GH or PRL by LM-ICC. One of these laboratories focused on normal pituitaries (20) while the other utilized paraadenomatous "normal" tissue (21). The results obtained by both groups were remarkably similar; the cumulative percentage of PRL cells and GH cells frequently exceeded 100% of all pituitary cells. In both studies, the

Vol. 12, No. 4

sum of the two values reach 150% for certain sections, supporting the conclusion that the two hormones could be synthesized and stored by the same cell. The latter group (21) substantiated this conclusion by staining adjacent tissue sections with antisera to each hormone and finding that many cells reacted with both antibodies, and subsequently by utilizing EM-ICC with colloidal gold to localize GH and PRL to many of the same cells and secretory granules (22). Lloyd et al. (23) expanded the repertoire of techniques applied to this problem by utilizing plaque assays and several variations of ICC, either alone or in combination, to identify unequivocally mammosomatotropes in humans. The results of this careful and comprehensive battery of studies demonstrated that mammosomatotropes are quite common in human pituitaries (accounting for 25-50% of all cells), and that their relative abundance in each sex was roughly equivalent to that of traditional mammotropes or somatotropes. 4. Other species. Mammosomatotropes were reported to be present but relatively rare in pituitaries of mature male and female mice by Sasaki and Iwama (24) who employed LM-ICC. Nevertheless, the size of this bihormonal population was of sufficient magnitude to inspire a subsequent report by the same laboratory (25) describing two subpopulations of murine mammosomatotropes, which were distinguishable by ultrastructural characteristics. Another group, using similar techniques (LMICC), readily detected mammosomatotropes in mice (sex not specified) and found roughly equal proportions of the three acidophilic cell types (26). Thus, there is agreement that mammosomatotropes are present in mice, but their abundance is presently controversial. EM-ICC also revealed cells in pituitaries of musk shrews that contained an intermixture of PRL granules and GH granules. Such bihormonal cells were found by Ishibashi and Shiino (15) to be present in pregnant and, to a lesser extent, lactating shrews and were not seen in virgin animals. Likewise, these same investigators detected mammosomatotropes in the adenohypophysis of the Japanese house bat (27). The abundance and secretory activity of these dual cells varied on a seasonal basis; they tended to be hypertrophied during pregnancy and accounted for the majority of PRL-containing cells during states of reproductive quiescence such as pre- and midhibernation, and intervening periods of arousal. Taken together, evidence obtained from several species demonstrates that the mammosomatotrope is a significant constituent cell-type in the adult pituitary. Moreover, these cells are sufficiently abundant to suggest that they probably play an important role in hormone secretion in adults.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

B. Is the mammosomatotrope a progenitor cell for the differentiation of classical mammotropes?

341

70

60

Historically, a developmental relationship between GH and PRL secretors has long been suggested by the ontogenic sequence in which these hormones first appear (see Refs. 28-31 for reviews of various species). However, experimental data to support this contention have come into being only recently. Nevertheless, a growing body of supportive evidence is now both extensive and convincing, and it derives primarily from the three species considered below. 1. Rats. The rat has been and continues to be an extremely popular model for studies on the differentiation of the pituitary gland in general and acidophils in particular. There is wide agreement that the synthesis and release of GH are initiated around day 19 of fetal development and that the proportion of GH secretors reaches a maximum (comparable to adult values) by the fourth or fifth day after birth (28, 32, 33). However, reports about the timing of PRL cell differentiation have been highly controversial with estimates ranging from approximately the midpoint of fetal development to several days after birth. Recent application of a plaque assay to the problem revealed that PRL secretors were extremely rare (accounting for considerably less than 1% of all cells) in pituitaries of fetal and 3-day-old rats, and ICC confirmed that synthesis and storage of the hormone were also absent up to that time (28). PRL cells first appeared in appreciable numbers by day 4 of neonatal life and by the next day accounted for 8-12% of all pituitary cells in each sex. Thus, GH cells differentiate approximately 1 week before the rather explosive appearance of PRL secretors in rats. This temporal relationship, coupled with observations that certain clonal lines of pituitary cells secrete both GH and PRL, raised the possibility that GH cells might give rise to PRL cells during development. In the event that such a lineage was operative, it would follow that the mammosomatotrope would function as an intermediate in this conversion and therefore should be present in the neonate. To test this logic, Hoeffler et al. (28) employed a sequential plaque assay to analyze the proportions of single and dualhormone secretors present in pituitaries derived from rats on the fifth day of neonatal life (1 day after PRL cells first appeared). Their results (illustrated in Fig. 3) show that of every 100 acidophils present in neonatal males, a proportion of 62.5 released GH only, 1.7 released PRL alone, and the remaining 35.8 released both of these hormones. Almost identical proportions were found for females, demonstrating that the sexual dimorphism of acidophils that exists in adults is not present at this time. These findings were quantitatively corroborated by use of a more simplistic version of plaque assay (which

50

I 40 30 20

.2 10 oa S a.

PRL

FIG. 3. Proportions of acidophils in 5-day-old (Sprague-Dawley-derived, Holtzman) rats that release PRL, GH, or both hormones. Values were determined in three separate experiments by sequential plaque assays. Note that virtually all of the PRL secretors present in pituitaries of both sexes were mammosomatotropes. [Reprinted with permission from J. P. Hoeffler et al: Endocrinology 117:187, 1985 (28). © The Endocrine Society]

is less prone to certain types of artifacts) and by doublestaining ICC. These results demonstrate conclusively that virtually all of the initial PRL cells in rats are mammosomatotropes. Moreover, the data are strongly supportive of the view that classical somatotropes give rise to traditional mammotropes by utilizing the mammosomatotrope as an intermediate. It should be noted, however, that the few PRL-only cells could contribute to the pool of traditional mammotropes by proliferation. Although the exact mechanism(s) governing the differentiation of the initial PRL secretors is unknown, recent experiments indicate that a subpopulation of GHonly secretors becomes presumptive mammosomatotropes (which secrete GH and contain PRL messenger RNA) at or before the time of birth. This conclusion is based not only on the aforementioned observation that the first PRL secretors also release GH, but also on the recent finding that a considerable amount of PRL message is present in the developing pituitary for several days before the initiation of PRL secretion. More specifically, Frawley and Miller (34) found that the quantity of PRL mRNA in pituitaries of newborn rats was at least as great as that measured in 10-day-old rats, in which 15-17% of all pituitary cells secreted the hormone. The virtual lack of PRL-containing or -releasing cells before day 4 of neonatal life indicates strongly that translation of the PRL message is posttranscriptionally blocked until this time. In an attempt to further explore this phenomenon, these same investigators fractionated cellular components from extracts of neonatal and adult pituitaries on sucrose gradients in order to separate mRNA associated with ribosomes from free cytoplasmic mRNA. They then quantified the relative amount of PRL message in each cellular fraction by cytodot hybridization with a rat

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

342

FRAWLEY AND BOOCKFOR

PRL cDNA probe. The results showed that most (88%) of the PRL mRNA from adults cosedimented with discrete ribosomal RNA species, whereas in the newborn only 24% of the PRL message was associated with these same fractions. The sedimentation profiles for GH mRNA were identical for newborns and adults. Thus, the apparent blockage of translation in the neonate is attributable, in a large part, to a lack of association of PRL message with ribosomes. On the basis of experiments with sea urchins and Xenopus (35-37), it is tempting to propose that this inhibition of translation is due to the absence of specific initiation factors, the presence of an inhibitory protein that "masks" the message, or a structural modification of the PRL message itself that renders it unable to bind to ribosomes. The precise intracellular mechanism notwithstanding, it is clear that a final step in the conversion of presumptive mammosomatotropes (that release GH alone) to cells that actually synthesize and release both GH and PRL is the dissolution of this translational blockage.1 Another unique twist to this developmental puzzle came to light recently when it was found that the timing for PRL cell differentiation on day 4 in rats might be dictated by an exogenous rather than an endogenous signal. Such a possibility was realized when Porter and co-workers (38) conducted fostering studies in which some pups were deprived of maternal influences specific to days 1-4 of lactation. This was accomplished by fostering litters of 1-day-old rat pups onto mothers that had been lactating for either 1 or 4 days. When the pups were 5 days of age, their pituitary cells were subjected to plaque assays in order to determine the percentages of PRL or GH secretors. The development of PRL cells proceeded normally in pups placed with day 1 mothers, whereas the process was severely retarded in companion litters placed on day 4 mothers; the percentage of PRL cells was 7-fold higher in the former group. The specificity of this response for PRL secretors was demonstrated by observations that the proportions of GH-secreting cells and body weights were essentially identical among untreated control and both experimental groups. In a subsequent study with a similar design, it was found that the magnitude of this maternal signal declined with the progression of lactation. Preliminary results indicate that the active principle is a small milk peptide that can exert its effects by a direct pituitary action (Porter, T. E., and L. S. Frawley, unpublished observations). Inas1 The authors recognize that the term "mammosomatotrope" is defined in large part by the methodology used to identify these bihormonal cells. Therefore, it becomes potentially problematic when a particular cell is shown to store (ICC) or release (plaque assay) one hormone, but only to produce the message for the other hormone. Accordingly, our working definition for a mammosomatotrope will be restricted to the capacity to store and/or release GH and PRL conconcurrently.

Vol. 12, No. 4

much as the final differentiation of PRL secretion is held in abeyance until day 4 by a translational blockage of the PRL message (34) subsequent to initiation of PRL gene transcription around the time of birth, it seems reasonable to speculate that this peptide, transmitted to the pups via the milk, acts directly or indirectly to override this blockage. 2. Humans. A developmental relationship between GH and PRL cells is also suggested from studies with human pituitary tissue. The ontogenic pattern of GH and PRL cells in humans is qualitatively similar to that observed in the rat; the major difference in the case of humans is that the differentiation of both cell types is completed in utero. It has been known for quite some time that the initial appearance of GH-containing cells in humans precedes that of PRL cells by at least 1 month; PRL cells first appear in appreciable numbers around week 16 of gestation. With this as a background, Mulchahey and Jaffe (29) performed sequential plaque assays on five fetal pituitaries (ages 18-22 weeks) in which the initial PRL cells had already differentiated. They found that of every 100 acidophils present, 69.9 were classical somatotropes, 21.7 were mammosomatotropes, and 8.7 were traditional mammotropes. Thus, even several weeks after their initial appearance, better than two-thirds of the PRL-secretors were mammosomatotropes. This compares favorably with the situation in rats (28). These same investigators corroborated the existence of mammosomatotropes in the human fetus by combining a plaque assay with ICC for PRL at the LM level, and also by dual-staining ICC at the EM level. However, due to the limited sample size for this morphological study (cells from a single pituitary gland were examined) the authors refrained from comparing these findings with the frequencies of single and dual-hormone secretors detected by sequential plaque assays. An ultrastructural and EM-ICC study by Asa et al. (30) confirmed the presence of mammosomatotropes in the human fetus, more clearly delineated their initial appearance, and also monitored their apparent transition to more traditional mammotropes. This group of investigators examined 63 pituitary glands from human fetuses, ranging in age from 5 weeks of gestation to term. They found that cells that contained only PRL could not be detected reliably before 23 weeks of gestational age. Nevertheless, mammosomatotropes were occasionally observed as early as 12 weeks, and these dual-hormone secretors became more abundant between 15 and 20 weeks of gestation. Mammosomatotropes remained numerous until weeks 26-28, after which their proportions began to decline. The diminution in abundance of dualhormone secretors was approximately coincident with the appearance of traditional mammotropes, which, in

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

turn, underwent a striking increase in frequency between 36 weeks of gestation and term. 3. Mice. Studies with mice have been extremely supportive of the idea that GH cells give rise to PRL secretors during pituitary differentiation. Indeed, such a relationship was first suggested by studies with Snell and Ames dwarf mice, which were found to be deficient in PRL as well as GH (39-42). Moreover, the developmental pattern of acidophils in mice is similar to that of rats in that the ontogeny of GH gene expression precedes that of PRL by about 2 weeks, and that only the former process is initiated in utero (31). However, the most important contribution of the mouse to this research area is that it provides a powerful model for elucidating developmental lineages through the use of transgene technologies. Two such studies have recently provided what may be the most direct, compelling evidence that PRL cells can arise from GH secretors during development. In the first of these studies, Behringer and co-workers (26) generated transgenic dwarf mice by using the GH promotor sequence to direct the cell-specific expression of a toxin gene in developing somatotropes. This was accomplished by fusing the enhancer/promoter region of the rat GH gene to the structural gene for diphtheria toxin and then using this construct for injection into the pronuclei of fertilized mouse eggs. The efficacy of this procedure was evidenced by observations that animals produced in this manner virtually lacked immunocytochemically identifiable GH cells within the pituitary and GH was undetectable in the serum. Moreover, circulating levels of insulin-like growth factor-1 were 8-fold lower in transgenics. Interestingly, the proportion of PRL-containing cells was also greatly reduced in these animals, suggesting a developmental pathway common with that of GH. However, the presence of some PRL cells in transgenics lacking GH gene expression led these investigators to propose that there might be two pathways for developing mammotropes: a major one, which derives from the traditional somatotrope and employs the mammosomatotrope as an intermediate, and a minor one in which the mammotrope arises directly from an acidophilic stem cell and thereby circumvents the GH-secreting cell as an intermediate. This latter possibility is consistent not only with the transgenic data for mice (26), but also with the situation in developing rats where a very small minority (1-2%) of initial PRL secretors in 5-day-old rats does not appear to release GH (28). Of course, an alternative interpretation of the rat study is that the few traditional mammotropes observed in neonates may simply be among the first to differentiate from mammosomatotropes. The results of a subsequent study with transgenically generated dwarfs suggest that the fraction of PRL secre-

343

tors that arises from GH cells may in fact approach unity. Borrelli et al. (43) utilized the rat GH or the rat PRL promoter to direct cell-specific expression of the herpes virus 1 thymidine kinase (HSV1-TK) gene. The intracellular presence of this gene product imparts pharmacological sensitivity to 1- (2-deoxy -2-fluoro-/?-6-aribinofuranosyl) -5-iodouracil (FIAU) which is converted to a metabolite lethal to dividing cells. Transgenic mice bearing the rat GH promotor-HSVl-TK fusion gene and treated with FIAU develop into dwarfs, as verified by criteria similar to those described above. The pituitaries of these animals were nearly devoid of both GH and PRL cells. When a similar strategy was used on transgenics carrying the HSV1-TK gene under control of the rat PRL promoter, the relative abundance of GH cells and PRL cells within the adenohypophysis was essentially normal. Inasmuch as cytotoxicity with the TK obliteration system requires cell division, these investigators concluded that PRL expression and mammotrope differentiation from GH cells are postmitotic events. Moreover, since deletion of GH cells led to a virtually complete elimination of PRL cells, these same authors also conclude that their results support a model of direct descent of mammotropes from GH secretors rather than the binary pathway proposed by Behringer et al. (26).2 In conclusion, there is very strong evidence that GH secretors give rise to PRL secretors during pituitary development and that the mammosomatotrope is by far the primary, if not the obligatory, intermediate in the process. This conclusion is based on observations that 1) development of GH cells invariably precedes that of PRL; 2) obliteration of GH cells severely attenuates or completely prevents differentiation of PRL secretors; and 3) the vast majority of the initial PRL secretors also release GH. C. Is the mammosomatotrope a transitional cell for the functional interconversion of GH and PRL secretors? This final question in this section focuses on the prospect that GH and PRL secretors do not become terminally differentiated shortly after birth but instead continue to undergo interconversions into adulthood, possibly without intervening cell divisions. Such a process, termed transdifferentiation (see Ref. 44 for review), might provide a mechanism whereby the long-term needs of an animal for GH or PRL could be met without a dramatic increase in the absolute number of pituitary cells. To date, most evidence supportive of this idea of "pituitary plasticity" is indirect and derives primarily from observations that the proportions of GH and/or 2 It is interesting to note that removal of FIAU in this paradigm eventuates in the restoration of somatotropes, indicating that the stem cell for the somatotrope/mammosomatotrope lineage survives ablation.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

344

FRAWLEY AND BOOCKFOR

PRL secretors vary with the physiological status of an organism. However, current studies have provided more direct evidence to suggest that this process can also be induced experimentally. Accordingly, the available literature will be reviewed here using these two categories of evidence as subheadings. 1. Physiological evidence. A prescient study conducted in 1969 by Goluboff and Ezrin (45) laid the groundwork for the notion that GH and PRL cells might be functionally interconvertible. These investigators utilized tinctorial stains to study acidophilic subtypes in pituitaries obtained from humans in a number of physiological states. They discovered that a reciprocal relationship existed between the absolute numbers of somatotropes and mammotropes, and that this pattern was most striking during the third trimester of pregnancy and the post partum period. They also noted that some cells exhibited the staining characteristics of both somatotropes and mammotropes and suggested that these putative multipotential cells might be involved in this process. This possibility was tested experimentally 5 yr later when Stratmann and co-workers (46) attempted to induce somatotrope "transformation" to mammotropes by estrogen treatment. More specifically, they injected normal male rats with [3H]thymidine, found that the label was taken up by morphologically identified somatotropes but not mammotropes, and that the label would remain in these cells for 3 weeks. However, if estrogen treatment was commenced shortly after thymidine labeling and continued for 3 weeks, the numbers of labeled somatotropes and mammotropes detected were approximately equal. On the basis of these results, they proposed the existence of an uncommitted mammosomatotrope (bearing the morphological characteristics of a somatotrope) which can convert to a mammotrope after exposure to estrogen. This exciting interpretation was based on the assumption that PRL cells were relatively amitotic in the control males. Unfortunately, a subsequent study by some of the same investigators (47) revealed that 3.3% of PRL cells were able to incorporate [3H]thymidine under control conditions similar to those used previously. Thus, the conclusion about possible somatotrope to mammotrope transformation had to be modified accordingly. The issue of acidophil interconversion was resurrected more recently when Porter and co-workers (48) used plaque assays to monitor changes in the ratios of GH or PRL secretors during the transition from nonpregnancy through lactation in rats. Their results, which confirmed and extended the immunocytochemical data of others, showed that proportions of GH or PRL secretors increased and decreased, respectively, by approximately the same extent, from the beginning of pregnancy until

Vol. 12, No. 4

the end of lactation. The same group conducted subsequent analysis of single and dual-hormone secretors (Fig. 4) and uncovered a rather interesting pattern. In the transition from virgin to midgestational female, there was an increase in the proportion of PRL-only cells that was exactly equivalent to the decrement in the mammosomatotrope population. Thus, PRL-only cells seemed to have arisen from mammosomatotropes without an overall change in the total number of PRL secre-

100 -i

j| ONLY GH • GH AND PRL YA ONLY PRL

80 -

Ui

O 60 -

5

O

40 -

LU O OC

Ui

QL

20 -

EL

LL

FIG. 4. Percentages of all pituitary cells that release GH only, PRL alone or both hormones during the physiological transition from the nonpregnant state through late lactation in the rat. Measurements were made in five separate experiments by simultaneous plaque assays. The physiological states on the abscissa are designated as follows: V (virgin, random cycling); G (gestating, >15 days pregnant); EL (early lactating, 5 or 6 days postpartum); LL (late lactating, 15 or 16 days postpartum). [Reprinted with permission from T. E. Porter et al: Endocrinology 127:2789,1990 (48). © The Endocrine Society]

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

tors (mammosomatotropes and PRL-only cells combined). As gestation progressed to early lactation, the mammosomatotrope population was replenished, apparently at the expense of GH-only cells, and the fraction of cells that released PRL alone remained constant. The net effect, then, was an overall increase in the proportion of PRL secretors. Next, as early lactation moved into late lactation, the fraction of PRL-only cells increased again concurrent with a commensurate decline in mammosomatotropes. Because these shifts occurred without any change in the absolute number of acidophils, the results of Porter et al. (48) invite speculation that GHreleasing cells can convert to PRL secretors by way of a transitional intermediate, the mammosomatotrope. The possibility that a similar process might be operative in other species is evidenced by reports of reciprocal shifts between mammosomatotropes and GH-only cells during gestation in bats (27) and musk shrews (15). Finally, it is conceivable that these acidophilic shifts are bidirectional since the divergent proportions of GH and/or PRL secretors observed at the end of lactation in rats return to prepregnancy values within 4 days of weaning (49). In a conceptually similar study, Kineman et al. (19) observed that the percentage of all pituitary cells that released PRL in plaque assays fluctuated during the course of the bovine estrous cycle. These changes were not attributable to variations in the relative abundance of PRL-only cells (which remained constant) but rather to reciprocal shifts in the proportions of GH-only and mammosomatotropic cells. This relationship between ratios of acidophilic subpopulations and stage of reproductive cycle indicates that ovarian steroids may regulate this phenomenon. 2. Experimental evidence. To date, few experimental studies have focused on the possibility that GH and PRL cells might be interconvertible and that the mammosomatotrope could function as a transitional intermediate. Available data bearing on this issue are limited and restricted to two categories of agents known to act directly at the pituitary level to influence acidophil secretion, namely hypothalamic regulatory peptides and steroids of gonadal and placental origins. A role for reproductive steroids as regulators of acidophilic subtypes is suggested by the aforementioned observations that 1) there is a sexual dimorphism in the ratios of single- and dual-hormone secretors; 2) castration of males significantly influences these proportions; 3) reciprocal shifts in GH and PRL secretors occur concurrently with rising gonadal and placental steroid levels during pregnancy; and 4) the ratios of GH and PRL cells fluctuate in a manner consistent with gonadal steroid patterns during the estrous cycle. In addition to this correlative evidence, there has been one study that addressed directly the

345

possibility that gonadal steroids might serve as a driving force for acidophil interconversions. In this study (50), pituitary cells derived from male rats were exposed to 17/3-estradiol or vehicle for 6 days and then subjected to a sequential plaque assay. Exposure to estrogen caused a striking increase in the proportion of mammosomatotropes and a commensurate decrease in the fraction that released GH alone, with the net effect being an overall increment in the number of PRL secretors. These results demonstrate that estradiol can act directly on anterior pituitary cells to evoke a shift in acidophilic subtypes and indicate that these changes can come about by a conversion of traditional somatotropes to mammosomatotropes. There are two lines of evidence to indicate that hypothalamic peptides might also function as physiological regulators of acidophil interconversions. The first of these comes from in vitro studies in which chronic (6day) treatment of rat pituitary cells with LHRH caused an increase in the percentage of PRL secretors and an equal decrease in the GH cell population, as determined by plaque assays (51). This effect persisted even when the experiments were repeated in the presence of cytosine arabinoside (an inhibitor of DNA synthesis and thus cell proliferation) suggesting that the shifts observed were the consequence of direct trans-differentiation (52). The second line of evidence involves a study with transgenic mice. In this experimental study, mice transgenic for human GRF were found by Stefaneanu et al. (53) to exhibit a massive hyperplasia of mammosomatotropes; GH-only cells were relatively rare in these animals. This finding led to the proposal that GH secretors proliferate and convert to mammosomatotropes after chronic exposure to GRF. To summarize, there is both physiological and experimental evidence to indicate that GH and PRL secretors can undergo functional interconversions in a regulated manner. Inasmuch as a significant change in the proportion of either GH-only or PRL-only cells is generally accompanied by an equal but opposite shift in mammosomatotropes, it seems logical to conclude that these dual-hormone secretors serve as transitional intermediates in this process. It may be prudent, however, to temper this conclusion in light of two final considerations. The first of these is that all acidophils may indeed be mammosomatotropes, as was previously suggested by the ICC studies of Hashimoto et al. (17). In this scenario, monohormonal acidophils might simply be mammosomatotropes that express GH or PRL at too low a level for detection. This in turn could be the result of local or cell-specific variation in transcriptional factor levels or activity. At the other extreme, the existence of the mammosomatotrope might merely be a reflection of the fact that a given cell may initiate the de novo secretion of one

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

346

FRAWLEY AND BOOCKFOR

gene product before cessation of the export (or depletion) of the other gene product. Thus, the mammosomatotrope might be more appropriately considered as a transitional state rather than as a distinct transitional cell type. These principally semantic considerations notwithstanding, the facts remain that bihormonal acidophils are present in appreciable numbers in several species and that their relative abundance fluctuates in a predictable manner with physiological status, indicating a transitional role for this phenotype. III. Mammosomatotropes in Neoplastic Tissue As with normal tissue, the reliable detection of mammosomatotropes in neoplastic cell populations has been made possible by development of several contemporary techniques. Recent use of such approaches as ICC, reverse hemolytic plaque assays, and in situ hybridization histochemistry has provided extensive information concerning this previously unrecognized cell type. In the following discussion, this information will be considered in relation to several questions that address certain functional aspects of these multipotential cells. A. Are mammosomatotropes a prominent cell type in neoplastic cell populations? 1. Human tumors. It is well established that certain pituitary tumors secrete both GH and PRL. In fact, the presence of one or both of these hormones in tumor tissue serves as one part of a system of classification that is used for pathological case descriptions. The presence of GH or PRL-only or a combination of these hormones, when considered with different ultrastructural features and other cellular characteristics, enables tissues to be classified into distinct groups. Using this approach, Kovacs and Horvath (54-56) and others (57) have defined at least nine different categories to enable ordered description of clinical findings with at least three of these categories dependent on the presence of GH and PRL in the tumor. These three categories (mixed somatotropiclactotropic adenomas, acidophil stem cell adenomas, and mammosomatotrope cell adenomas) have not been shown to comprise a large portion of all tumors but do represent tumor types that can be identified fairly regularly during clinical or pathological evaluations (54-57). For many years, the vast majority of evidence suggested that these pituitary adenomas were most frequently composed of distinct populations of GH or PRL cells and rarely contained cells that secreted both hormones (57-60). Many of these determinations were made using conventional LM techniques and supported the contention that each type of pituitary cell secreted a different hormone, a hypothesis generally accepted by most investigators. However, studies employing more

Vol. 12, No. 4

sophisticated technical approaches raised the possibility that dual-hormone cells were more frequent than previously recognized. Initial descriptions by Horvath and Kovacs (61), when combined with evidence obtained using double-staining ICC by Halmi (62) or immunocytochemical comparison of cells in adjacent sections by Kanie et al. (63), suggested that dual cells may be an important cell type in pituitary adenomas. Results consistent with these findings were provided by other LM or EM studies which enabled an indirect demonstration of the presence of GH and PRL in the same granule with the use of adjacent tissue sections (64-67). Direct demonstrations of mammosomatotropes in human pituitary adenomas, however, did not become possible until the use of colloidal gold immuno-electronmicroscopy which, as discussed earlier, enables the simultaneous identification of both cell types by employing gold beads of differing sizes that have been conjugated to separate antibodies. Using this approach, Felix and co-workers (68) reported that cells containing both GH and PRL were present in an adenoma of a patient with gigantism. Also, at about the same time, Bassetti et al. (69) found dual hormone-containing cells in five additional acromegalic patients. As these advanced methods of immunocytochemical analysis became more readily available, the presence of mammosomatotropes in various adenomatous tissues was confirmed by other investigators (22, 70-76). Although results from these initial studies clearly demonstrated the existence of dual cells, they did not provide an estimate of how frequently mammosomatotropes could be found in tumor tissue. In experiments by Lloyd and co-workers (23), reverse hemolytic plaque assays were used in conjunction with immunogold microscopic methods to demonstrate that mammosomatotropes were present in four out of five GH adenomas as well as in half of all mammotropic adenomas studied. These findings were supported by the studies of Bassetti et al. (70), which revealed that cells also positive for PRL were frequently present in GH-secreting adenomas, being detectable in 54% of the 22 tumors processed for immunogold EM-ICC. Quantitative analysis of five tumors in another study by this same laboratory (69) revealed that more than 50% of all cells present contained both hormones. Alternate approaches, such as use of in situ hybridization histochemistry have also enabled the extensive detection of mammosomatotropes by cellular localization of mRNA for both GH and PRL (77). Thus, the repeated demonstrations of these cells using a variety of approaches suggest that they are not a rare cell type, but comprise a significant portion of the cell population in human pituitary adenomas. 2. Animal tumors. Transplantable pituitary tumors were

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

347

developed in rats several years ago and have become valuable tools for the study of the phenotypic expression of GH and PRL in the pituitary. Tumors such as the MtT/F 4 , MtT/Wio, and MtT/W 15 were all induced by chronic administration of estrogenic compounds (78-80). Other tumors, such as the MtT/W 5 , were induced by xray exposure (81). Although many functional characteristics differ from tumor to tumor, it has become clear that each type is capable of secreting GH and PRL. In fact, the close association of the expression of these two hormones in various tumors led Furth and Clifton (82) to propose that cells were present in these tissues that secreted both hormones. Studies performed by these investigators and others in the next few years did not provide evidence to refute this hypothesis, and for a period of time it was generally accepted that dual cells were present in these tissues (80, 83-85). With the development of new methods of identification, other groups of investigators attempted to identify and quantitate the different cell types present in MtT/Wi 5 tumors but, surprisingly, were unable to K ilize cells that contained both GH and PRL (86). Other studies performed at that time using LM-ICC and EM provided results also suggesting that these dual cells were few in number or nonexistent in tumor tissue (87-90). The question of whether mammosomatotropes contributed markedly to the composition of rat pituitary tumor cell populations was not resolved until recently. Using plaque assays, Lloyd et al. (12) found that approximately 12% of all cells in MtT/Wi 5 tumors secreted both GH and PRL. Further analysis by these investigators revealed that MtT/F 4 tumors also contained dualhormone secretors and that these cells were present in fairly substantial numbers [19% of all cells present) (91)]. These findings were later confirmed by this group using in situ hybridization histochemistry (92, 93). The results of these and additional studies suggest that mammosomatotropes comprise a significant portion of cells present in rat pituitary adenomas, at least in the case of MtT/ W15 and MtT/F 4 and a few other tumor types (94-96). Further studies utilizing techniques that enable the reliable detection of dual cells must be performed extensively in different types of pituitary tumors to determine whether these cells are restricted to only certain tumor types or are present in all adenomatous acidophilic cell populations.

B. What is the contribution of mammosomatotropes to hormone secretion?

3. Continuous cell lines. Several different pituitary cell lines have been derived that are capable of secreting GH and PRL. The most widely used of these lines are cells that were obtained initially from the MtT/W 5 tumor (97). By alternate passages between cultures and animals, it was possible to isolate epithelial cell cultures that were found to synthesize and release GH and PRL

Many of the pathological changes that occur in animals or humans with neoplastic pituitary tissue result from hypersecretion of GH or PRL. Despite its potential clinical importance, very few studies have been conducted in which careful comparisons have been made between the ratios of dual and single secretors and concentrations of GH and PRL secreted. Recent reports by Bassetti et al.

without production and release of other hormones that are present in populations of normal pituitary cells. Continued propagation of these cells then enabled isolation of three clonal cell lines, GHi, GH4C1, and GH3 cells, each able to secrete GH and PRL (98, 99). From these cultures several other lines were also developed with similar properties (100). Although many attempts have been made to characterize these cell cultures, success has been limited because of technical problems. These clonal cells store only very small quantities of GH or PRL (101-103). Thus, traditional tinctorial and immunocytochemical techniques, which can only localize hormones that are contained intracellularly, are not completely effective in defining the types or quantity of cells present. In the absence of information about individual cells, investigators had proposed that all functionally active cells in these cultures produce both GH and PRL (101, 102). Several studies employing ICC were unable to confirm or disprove this hypothesis (103,104). It was shown, however, that GH3 cultures propagated from single cells gave rise to cultures that released GH and PRL suggesting that all cells were multipotential (100). Little additional information was forthcoming until Boockfor and Schwarz (105), using reverse hemolytic plaque assays, demonstrated that mammosomatotropes are present in GH3 cell cultures. Interestingly though, these investigators found that only a portion of all cells in culture released both hormones, as shown in Fig. 5. The presence of these dual secretors in GH3 cultures was later confirmed by others (93). Although the existence of dual cells in other GH clonal lines has not been established, it was reported recently that mammosomatotropes are present in the rPCO cell line (106). These cultures were obtained by continuous propagation of normal dispersed pituitary cells that were not exposed to chronic hormone or radiation treatment as was tissue from which GH cell lines were derived. Despite their different origins, both cell populations contain mammosomatotropes. In fact, Chomczynski and co-workers (106) reported that the majority of cells present in rPCO cultures are mammosomatotropic. Thus, as with other types of pituitary neoplastic tissue, mammosomatotropes appear to be a prominent cell type in clonal populations.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

FRAWLEY AND BOOCKFOR

348 j

40i

111 U

o itr

30 -

s

uj 20 -

o 10 111

o cc

111 Q.

GH ONLY

DUAL

PRL ONLY

CELL TYPE FIG. 5. Proportions of cells present in GH3 cultures that release either GH or PRL individually or both hormones together. In plaque assays, antisera to GH and PRL were applied alone or together to monolayers of GH3 cells and indicator erythrocytes. A comparison was then made between the cumulative percentages of two individual assays and those obtained when both antisera were employed. Because a cell can be identified as a plaque former only once, regardless of whether one or two hormones are secreted, the differences in the proportions of secretors detected using these two approaches enabled the quantification of cells that secreted one or both hormones. These results (mean ± SE) were obtained in four separate experiments. [Reprinted with permission from F. R. Boockfor and L. K. Schwarz: Endocrinology 122:762, 1987 (105). © The Endocrine Society]

(70) demonstrated that a positive correlation exists between serum PRL levels and the percentages of mammosomatotropes present in human pituitary adenomas. These investigators used double immunocytochemical labeling with protein-A gold EM to identify the types of cells present in various adenoma cell populations. Their findings not only suggested a relationship between cell type and hormone levels but also revealed that the majority of cells identified as PRL cells also contained GH. Other investigators found positive correlations between dual secretors and PRL release in experiments utilizing animal tumor tissue. Although careful studies have not been conducted on all tumor cell types, it was demonstrated recently that MtT/F 4 cell populations contain approximately twice as many dual secretors as found in MtT/W 15 tumors (91, 93). Interestingly, these MtT/F 4 tumors have been shown to elevate serum levels of PRL in the host animals while MtT/Wi 5 tumors have been more closely associated with elevated levels of GH as evidenced by increased body weight and visceromegaly (12, 40). The association of increased serum levels of PRL with dual cells in these animal tissues, when viewed in light of similar findings in human adenomas, suggests that mammosomatotropes may contribute more to PRL than GH levels. A relationship between the proportion of mammoso-

Vol. 12, No. 4

matotropes and the amount of hormone secreted is also suggested by experiments in which tumor cell populations are altered by treatment with different agents. Chronic exposure of animals containing MtT/Wi 5 tumors to the potent estrogenic compound diethylstilbestrol results in highly elevated levels of GH and marked decreases in serum PRL (12, 107, 108). These changes occur with a concomitant reduction in the proportion of dual-secreting tumor cells (12, 93). Interestingly, treatment of MtT/Wis tumor-bearing animals with progesterone, which causes an increase in serum PRL levels and a decrease in serum GH concentrations, also induces an increase rather than a decrease in the number of mammosomatotropes in the tumor tissue (12). Recent studies from our laboratory using GH3 cells provide further evidence to support a positive relationship between PRL secretion and dual cells. We found that treatment with estradiol, which increases the amount of PRL secreted by these cultures, also increases the percentages of dual secretors (105). Although the influence of estradiol on GH3 cultures is opposite to that of MtT/ W15 tumors with regard to its effect on PRL secretion, it appears that a positive correlation exists between mammosomatotropes and PRL secretion whether PRL is increasing or decreasing in magnitude. Of course, additional studies must be performed to determine whether such a relationship also exists in other types of neoplastic tissue. However, the establishment of a direct association between a particular cell type and hormone levels may provide insight into the cause and future treatment of abnormalities in GH or PRL secretion. C. Are mammosomatotropes involved in a functional conversion of GH and PRL cells? It was recognized for many years that a reciprocal relationship exists between GH and PRL secretion. Using GH3 cell populations, Clausen et al. (109) demonstrated that chronic treatments that increased PRL secretion decreased GH release. In contrast, those treatments that stimulated GH secretion caused decreases in PRL release. These types of reciprocal changes were also observed with other types of neoplastic tissue (12, 107, 108). The cellular basis for these changes was not uncovered until the application of plaque assays to these cultures (Fig. 6). Using this approach, Boockfor et al. (110) found that treatment with either estradiol or TRH increased the proportions of PRL secretors and decreased the proportions of GH-releasing cells. In contrast, cortisol treatment caused an increase in GH secretors and a decrease in PRL cells. Interestingly, each of these shifts between GH and PRL cells, regardless of treatment, was found to be reciprocal. Other studies revealed that factors such as TRH could induce changes in hormone secretion

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991 60-

|

50-^

IING CEL

S

MAMMOSOMATOTROPES

I 40^ x

30 ^

f

40-

20"

PERCENT

60 50"

30-

10"

0

^

CONTROL

40 "

DC

.AQUE FG

i

349

GH ONLY

DUAL

PRL ONLY

CELL TYPE

T

"I 30" d

20"

40

ESTRADIOL-TREATED

H

UJ

u o

10-

5 Control

TRH

Estradiol Cortlsol

FIG. 6. The percentages of GH or PRL secretors present in GH3 cultures after treatment with maximal doses of estradiol, TRH, or cortisol for 6 days. Each bar represents the values obtained in four separate experiments. Note that a change in the proportions of one type of secretor was accompanied by a reciprocal change in the percentages of the other regardless of the treatment imposed. [Reprinted with permission from F. R. Boockfor et ai: Endocrinology 117:418,1985 (110). © The Endocrine Society]

without altering the number of cells in culture (111). Thus, when taken together, these findings raised the intriguing possibility that GH and PRL cells may be functionally interconvertible. Recent evidence also suggests that mammosomatotropes may play an important role in these functional shifts between GH and PRL cells. Boockfor and Schwarz (105), using sequential plaque assays to quantify single and dual secretors, found that the relative abundance of mammosomatotropes (Fig. 7) changed markedly after incubation of cultures with TRH or estradiol. Presumably, alterations in dual cell populations would contribute a great deal to the overall shifts in GH to PRL secretors that have been found previously to occur upon treatment with these agents. Moreover, the type and extent of changes that occurred in these studies would then be entirely consistent with a cell type that serves as an intermediate between these single secretors. This possibility is further supported by findings obtained in another type of neoplastic tissue. Using MtT/Wi 5 tumors Lloyd et al. (12) reported that DES administration for 3 weeks resulted in overall reductions in PRL releasing cells and increases in GH secreting cells. As with GH3

30 1 20 -

10 -

*

'ITITIT8.I Wlinillllll

GHONLY

DUAL CELL TYPE

PRL ONLY

TRH -TREATED

40 "

30 "

20 -

iHi

ill

10 "

ml



GH ONLY

DUAL CELL TYPE

PRL ONLY

FIG. 7. The effect of chronic treatment (6 days) with 17/S-estradiol or TRH on the proportions of single- and dual-hormone secretors present in GH3 cultures. The assays were performed as described previously in the legend to Fig. 5. As shown, shifts in GH3 cell populations after treatment involved mammosomatotropes as well as single secretors. Shown are the mean ± SE obtained in three separate experiments. [Reprinted with permission from F. R. Boockfor and L. K. Schwarz: Endocrinology 122:762,1987 (105). © The Endocrine Society]

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

FRAWLEY AND BOOCKFOR

350

cells, these reciprocal changes could be attributed in part to changes in mammosomatotropes. Although evidence has been obtained in a limited number of cell populations, it appears that alterations in the relative abundance of GH or PRL secretors, which may result from an interconversion of one type of cell to another, may be mediated by mammosomatotropic cells. Indeed, mammosomatotropes may serve as a transitional cell type during a shift between these two apparent functionally distinct secretors. IV. Overview It is clear from work performed to date that the mammosomatotrope is a prominent cell type in both normal and neoplastic tissue. Interestingly, initial identification of these multipotential cells in tumor cell populations led several investigators to propose that they were abnormal cells which give rise to pituitary neoplastic tissue. However, their presence in almost every type of pituitary tissue studied, regardless of species, demonstrates the normality of mammosomatotropes. Additionally, identification of neoplastic tissue that is devoid of extensive numbers of these cells (54-57, 112) would suggest that mammosomatotropes are probably not involved in tumorigenesis any more often than other acidophilic cell types. Although dual-hormone secretors in both types of tissue probably do not function exactly the same way, cells from each source appear to have at least one aspect in common, a major role in shifts between GH and PRL secretors. The potential importance of cellular interconversion in either physiological or in pathological situations has prompted several ancillary questions to be raised that begin to address the cellular process(es) which may underlie this phenomenon. Two of these deserve special consideration at this time and are addressed below. A. What mechanism(s) might govern an interconversion of GH and PRL secretors? Intuitively, one would expect that a mechanism enabling an interconversion of GH and PRL cells to occur would be composed of at least two parts: 1) a process containing functional components common to both cells that would facilitate a transition from one cell type to another, and 2) a process containing separate components that would allow each cell type to function independently before and after a shift occurs. Recent reports have provided evidence for each of these types of modulatory elements in pituitary cells. First, it has been established that GH and PRL genes have defined sequences in the 5'-flanking regions that confer cell-specific expression (113-120). Although in question until recently, a common transcription factor Pit-1/GHFl has

Vol. 12, No. 4

been shown to bind these regulatory regions and mediate tissue-specific expression of gene transcription (121125). The requirement for this factor is evidenced by demonstrations that its addition stimulates transcription from the GH promoter in extracts of HeLa cells which do not normally express GH (126). Also, it has been shown that fusion of GH3 cells with a non-GH expressing cell line which causes extinction of GHF1 expression also results in a loss of GH expression (127, 128). These findings, in addition to others for PRL, reveal that Pit1/GHFl is not only common to GH and PRL cells but is also necessary for gene expression. Such a factor may provide a mechanistic link between GH and PRL secretors that would be critical in an interconversion of cell types. The second type of component that may facilitate an interconversion of one cell type to another is one that would dictate the specific expression of only one gene at a time. Recent experiments reveal a transactivating substance, denoted LSF1, that appears to stimulate PRL gene transcription (117, 129). Although the structure of this substance has not been reported, it was shown to bind in a specific manner to several elements within the promoter region of the PRL gene. Many of these elements overlap with those found to bind Pit-1/GHFl, but functional studies have revealed that only the most proximal LSF1 binding site is required for accurate transcription of the PRL gene (130). Further study is needed to clearly establish whether this substance can induce the expression of PRL without affecting GH gene expression. In addition to a substance such as this one that has positive influences on gene transcription, other proteins may exist that act negatively on the expression of a particular gene. A regulatory protein functioning in this manner has been proposed to explain the loss of GH gene expression that results when pituitary cell/fibroblast hybrids are prepared (127). The possibility of a distinct genomic region that inhibits transcription of certain genes has been suggested to explain the restriction of PRL and GH gene expression in thyrotropes (131). Additional support for the involvement of negative regulators in gene expression is provided by experiments performed using cells from the pancreas, an endocrine tissue with many regulatory processes analogous to the pituitary. Preliminary results reveal that a single protein complex, when bound to a specific regulatory element, inhibits the expression of somatostatin gene sequences in non-somatostatin expressing islet cells of the pancreas (132). These examples, although few, suggest that certain factors may be present that can either activate or extinguish the expression of an individual gene. Recent studies suggest that other factors which work in concert with those above are involved in modulating gene expression. For example, substances such as TRH,

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

MAMMOSOMATOTROPES

calcium, cAMP, and estrogen receptors are clearly involved in enhancement of PRL gene expression and appear to act by binding to regions of the gene that overlap in many instances with Pit-1/GHFl binding sites (129, 133-140). In fact, the action of one of these substances, the estrogen receptor, has been associated with such large increases in PRL gene expression in the neonate that it has been proposed as a major controlling factor in the ontogeny of PRL gene expression (131). Thus, although only limited information is available at this time, it is clear that the process(es) enabling the expression of only one gene at a time, whether involving one or a combination of factors, is quite complex. The diversity of substances involved in transcriptional regulation in other mammalian cell types would suggest that a number of yet unidentified components are probably central to the modulation of GH and PRL gene expression (141, 142). Nevertheless, evidence for factors that regulate individual genes as well as those that are common to the expression of both would be quite consistent with a mechanism that would facilitate an interconversion of GH and PRL cells to occur. The synthesis and secretion of a particular hormone during this process may also be dependent on genomic structural considerations, such as the methylation state of DNA. Several investigators have reported that a relationship exists between the degree of methylation of cytosine residues in specific gene sequences and the activity of the gene; active genes have certain hypomethylated residues, and inactive genes have various hypermethylated residues (reviewed in Refs. 143 and 144). The association of GH and PRL expression and the state of methylation was demonstrated in several GH cell lines. In these experiments, treatment with 5-azacytidine or Neplanocin A, which cause demethylation of various cytosine residues, resulted in increases in the production of both GH and PRL (145-148). Site-directed DNA methylation attempts were also found to result in suppression of PRL gene expression (149). Different investigators were also able to show a positive correlation between hypomethylation and active transcription of GH or PRL genes in estrogen-treated tumor tissue (150) or other untreated clonal cell populations (151). Finally, evidence that the state of methylation may play a role in physiological changes was provided recently in studies by Kumar and Biswas (152). These investigators demonstrated that hypomethylation of several cytosine residues in the PRL gene was associated with pregnancy and lactation, states in which high levels of PRL were produced. In contrast, hypermethylation of the GH gene was observed in these same physiological periods when GH production was reduced. The cellular basis for these changes was later suggested by the work of Porter et al. (48), which revealed that increases in PRL and decreases

351

in GH normally occurring during lactation were accompanied by shifts from GH cells to mammosomatotropes and PRL cells. This relationship prompted these investigators to propose that changes in the methylation of cytosine residues associated with the GH and PRL gene may be involved in shifts between GH and PRL cells. Thus, the state of methylation of a particular gene may not only dictate the extent to which that gene is transcribed but may also be important in its cell-specific expression. This process, when coupled with other cellular mechanisms, may contribute markedly to an interconversion of GH and PRL cells. B. Is there an acidophil cycle? The seminal observation to indicate that acidophils might undergo a secretory cycle was made in 1937 by Severinghaus (153) who studied the evolution and eventual disappearance of the so called "pregnancy cell." This investigator cautioned colleagues that this was not a new type of cell. Instead, because of cyclic changes in secretory activity, this acidophil was "sometimes this, sometimes that." By extension, it is tempting to propose that all acidophils release GH and PRL at one time or another. To put it another way, it is conceivable that the three acidophilic subtypes identified to date might simply reflect different stages of a secretory cycle for the same cell! As discussed in detail earlier, there is considerable evidence to support the conversion of GH-only cells to mammosomatotropes in normal and neoplastic pituitary cells. Likewise, numerous experimental findings are consonant with the functional differentiation of mammosomatotropes to PRL-only cells. So the progression (developmental or otherwise) of traditional somatotrope —• mammosomatotrope —*• classical mammotrope is reasonably well characterized and established. There is also evidence that classical PRL secretors probably revert back to traditional GH cells; this is seen after weaning in rats and as a consequence of cortisol treatment in GH3 cells. Thus, the issue of whether an acidophil cycle exists boils down to how this latter step is accomplished. Unfortunately, the possible role of mammosomatotropes in the conversion of PRL-only to GH-only cells has not been examined in sufficient detail to support an unambiguous answer. Until such time as this evidence is forthcoming, it will be impossible to determine whether the shifts are the consequence of an acidophil cycle per se or of a bidirectional interconversion of GH and PRL secretors in which the mammosomatotrope would subserve as an obligatory transitional cell. In summary, one might envision the mammalian acidophil as being suspended as a pendulum (Fig. 8). As the pendulum swings to one extreme it becomes primarily a somatotrope that secretes GH, whereas displacement to

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

FRAWLEY AND BOOCKFOR

352

Vol. 12, No. 4

HYPOTHALAMIC FACTORS, STEROIDS AND GROWTH FACTORS

FIG. 8. A pituitary cell is shown suspended as a pendulum to illustrate the proposed functional relationship among cells that secrete GH, PRL, or both hormones. The surrounding environment created by local and systemic factors appears to dictate in a very precise manner which intracellular regulatory mechanisms become operational and which hormone or hormones are released. However, as illustrated by the pendulum, the secretory status of a cell is not fixed; it may acquire or lose the ability to secrete GH or PRL as the regulatory input changes.

TRANSCRIPTIONAL REGULATORY FACTORS

^MMOSOMATOTROPE

the other extreme results in the cell taking on the exclusive phenotype of a PRL-secreting mammotrope. When suspended at an intermediate point, the cell functions as a mammosomatotrope and releases both hormones concurrently. Unlike a true pendulum whose motion is driven by gravitational forces, the movement of our hypothetical pendulum is regulated by a series of gears, the most proximate of which represent transcriptional regulatory factors. These gears in turn are propelled by another series representing input by hypothalamic hypophysiotropic agents, growth factors, and gonadal as well as adrenal steroid hormones. Although this pendulum analogy is a bit frivolous, it appears to convey accurately what is suggested by the available physiological and experimental data: that GH and PRL cells share a common developmental lineage and are functionally interconvertible in the adult.

Acknowledgments The authors gratefully acknowledge the excellent secretarial assistance of Mary Ackerman. We also appreciate the comments of Dr. Tom Porter, Dr. Rhonda Kineman, and Bryan Hill who read the manuscript and offered a number of helpful suggestions for improvement. Finally, we thank Carmella Wiles who contributed the artwork to our final figure.

References 1. Romeis B 1940 Hypophyse. In: v Mollendorff W (ed) Handbuch der mikroskopischen anatomie des menschen. Springer, Berlin Heidelberg, vol 6, part 3 2. Purves HD 1966 Cytology of the adenohypophysis. In: Harris GW, Donovan BT (eds) The Pituitary Gland. Butterworths, London, vol 1:147 3. Racadot J 1963 Contribution a l'etude des types cellulaires du lobe anterieur de l'hypophyse chez quelques mammiferes. In: Benoit J, Da Lage C (eds) Cytologie de l'adenohypophyse. Centre National de la Recherche Scientifique, Paris, p 33

4. Landolt AM 1975 Ultrastructure of human sella tumors. Acta Neurochir [Suppl] (Wien) 22:1 5. Pelletier G 1971 Classification et physiopathologie des tumeurs hypophysaires. Union Med Can 100:1779 6. Tixier-Vidal A, Tougard C, Dufy D, Vincent JD 1982 Morphological, functional and electrical correlates in anterior pituitary cells. In: Muller EE, MacLeod RM (eds) Neuroendocrine Perspectives. Elsevier, Amsterdam, p 211 7. Sternberger LA, Joseph SA1979 The unlabelled antibody method: contrasting color staining of paired pituitary hormones without antibody removal. J Histochem Cytochem 27:1424 8. Nakane PK 1970 Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J Histochem Cytochem 18:9 9. Fumagalli G, Zanini A 1985 In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell. J Cell Biol 100:2019 10. Frawley LS, Boockfor FR, Hoeffler JP 1985 Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 116:734 11. Frawley LS 1989 Mammosomatotropes: current status and possible functions. Trends Endocrinol Metab 1:31 12. Lloyd RV, Coleman K, Fields K, Nath V 1987 Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay. Cancer Res 47:1087 13. Leong DA, Lau SK, Sinha YN, Kaiser DL, Thorner MO 1985 Enumeration of lactotropes and somatotropes among male and female pituitary cells in culture: evidence in favor of a mammosomatotrope subpopulation in the rat. Endocrinology 116:1371 14. Nikitovitch-Winer MB, Atkin J, Maley BE 1987 Colocalization of prolactin and growth hormone within specific adenohypophyseal cells in male, female, and lactating female rats. Endocrinology 121:625 15. Ishibashi T, Shiino M 1989 Co-localization pattern of growth hormone (GH) and prolactin (PRL) within the anterior pituitary cells in the female rat and female musk shrew. Anat Rec 223:185 16. Losinski NE, Horvath E, Kovacs K 1989 Double-labeling immunogold electronmicroscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries. Am J Anat 185:236 17. Hashimoto S, Fumagalli G, Zanini A, Meldolesi J 1987 Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary. J Cell Biol 105:1579 18. Kineman RD, Faught WJ, Frawley LS 1991 Mammosomatotropes are abundant in bovine pituitaries: influence of gonadal status. Endocrinology 128:2229 19. Kineman RD, Henricks DM, Faught WJ 1991 Fluctuations in the

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

20.

21. 22. 23.

24.

25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

36.

37. 38.

39. 40. 41. 42.

MAMMOSOMATOTROPES

proportions of growth hormone- and prolactin-secreting cells during the bovine estrous cycle. Endocrinology 129:1221 Zimmerman EA, Defendini R, Frantz AG 1974 Prolactin and growth hormone in patients with pituitary adenomas: a correlative study of hormone in tumor and plasma by immunoperoxidase technique and radioimmunoassay. J Clin Endocrinol Metab 38:577 Landolt AM, Minder H 1984 Immunohistochemical examination of the paraadenomatous "normal" pituitary. Pathol Anat 403:181 Zurschmiede C, Landolt AM 1987 Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis. Virchows Arch [Cell Pathol] 53:308 Lloyd RV, Anagnostou D, Cano M, Barkan AL, Chandler WF 1988 Analysis of mammosomatotropic cells in normal and neoplastic human pituitary tissues by the reverse hemolytic plaque assay and immunocytochemistry. J Clin Endocrinol Metab 66:1103 Sasaki F, Iwama Y 1988 Sex difference in prolactin and growth hormone cells in mouse adenohypophysis: stereological, morphometric, and immunohistochemical studies by light and electron microscopy. Endocrinology 123:905 Sasaki F, Iwama Y 1989 Two types of mammosomatotropes in mouse adenohypophysis. Cell Tissue Res 256:645 Behringer RR, Mathews LS, Palmiter RD, Brinster RL 1988 Dwarf mice produced by genetic ablation of growth hormoneexpressing cells. Genes Dev 2:453 Ishibashi T, Shiino M 1989 Subcellular localization of prolactin in the anterior pituitary cells of the female Japanese house bat, Pipistrellus abramus. Endocrinology 124:1056 Hoeffler JP, Boockfor FR, Frawley LS 1985 Ontogeny of prolactin cells in neonatal rats: initial prolactin secretors also release growth hormone. Endocrinology 117:187 Mulchahey JJ, Jaffe RB 1987 Detection of a potential progenitor cell in the human fetal pituitary that secretes both growth hormone and prolactin. J Clin Endocrinol Metab 66:24 Asa SL, Kovacs K, Horvath E, Losinski NE, Laszlo FA, Domokos I, Halliday WC 1988 Human fetal adenohypophysis. Neuroendocrinology 48:423 Slabaugh MB, Lieberman ME, Rutledge JJ, Gorski J 1982 Ontogeny of growth hormone and prolactin gene expression in mice. Endocrinology 110:1489 Frawley LS, Hoeffler JP, Boockfor FR 1985 Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay. Endocrinology 116:2355 Setalo G, Nakane P 1976 Functional differentiation of the fetal anterior pituitary cells in the rat. Endocrinol Exp (Bratisl) 10:155 Frawley LS, Miller III HA 1989 Ontogeny of prolactin secretion in the neonatal rat is regulated posttranscriptionally. Endocrinology 124:3 Smith LD, Richter JD 1985 Synthesis, accumulation, and utilization of maternal macromolecules during oogenesis and oocyte maturation. In: Metz C, Monroy A (eds) Biology of Fertilization. Academic Press, New York, vol 1:141 Colin AM, Brown BD, Dholakia JN, Woodley CL, Wabsa AJ, Hille MB 1987 Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs. Dev Biol 123:354 Auget RG, Goodchild J, Richter JD 1987 Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes. Dev Biol 121:58 Porter TE, Chapman LE, Van Dolah FM, Frawley LS 1991 Normal differentiation of prolactin cells in neonatal rats requires a maternal signal specific to early lactation. Endocrinology 128:792 Beamer WG, Eicher EM 1976 Stimulation of growth in the little mouse. J Endocrinol 71:37 Slabaugh MB, Lieberman ME, Rutledge JJ, Gorski J1981 Growth hormone and prolactin synthesis in normal and homozygous Snell and Ames dwarf mice. Endocrinology 109:1040 Roux M, Bartke A, Dumont F, Dubois MP 1982 Immunohistological study of the anterior pituitary gland—pars distalis and pars intermedia—in dwarf mice. Cell Tissue Res 223:415 Yashiro T, Arai M, Miyashita E, Yamashita K, Suzuki T 1988 Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice. Cell Tissue Res 251:249

353

43. Borrelli E, Heyman RA, Arias C, Sawchenko PE, Evans RM 1989 Transgenic mice with inducible dwarfism. Nature 339:538 44. Beresford WA 1990 Direct transdifferentiation: can cells change their phenotype without dividing? Cell Dif & Dev 29:81 45. Goluboff LG, Ezrin C 1969 Effect of pregnancy on the somatotroph and the prolactin cell of the human adenohypophysis. J Clin Endocrinol Metab 29:1533 46. Stratmann IE, Ezrin C, Sellers EA 1974 Estrogen-induced transformation of somatotrophs into mammotrophs in the rat. Cell Tissue Res 152:229 47. Corenblum B, Kovacs K, Penz G, Ezrin C 1980 The effects of estrogen on prolactin cells of the male rat pituitary. An immunocytologic and autoradiographic study. Endocr Res Commun 7:137 48. Porter TE, Hill JB, Wiles CD, Frawley LS 1990 Is the mammosomatotrope a transitional cell for the functional interconversion of growth hormone- and prolactin-secreting cells? Suggestive evidence from virgin, gestating, and lactating rats. Endocrinology 127:2789 49. Porter TE, Wiles CD 1991 Evidence for bidirectional interconversion of mammotropes and somatotropes: rapid reversion of acidophilic cell types to pregestational proportions after weaning. Endocrinology 129:1215 50. Boockfor FR, Hoeffler JP, Frawley LS 1986 Estradiol induces a shift in cultured cells that release prolactin or growth hormone. Am J Physiol 25O:E1O3 51. Hoeffler JP, Frawley LS 1987 Hypothalamic factors differentially affect the proportions of cells that secrete growth hormone or prolactin. Endocrinology 120:791 52. Frawley LS, Hoeffler JP 1988 Hypothalamic peptides affect the ratios of GH and PRL cells: role of cell division. Peptides 9:825 53. Stefaneanu L, Kovacs K, Horvath E, Asa SL, Losinski NE, Billestrup N, Price J, Vale W 1989 Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a histological, immunocytochemical, and electron microscopic investigation. Endocrinology 125:2710 54. Kovacs K, Horvath E 1987 Pathology of pituitary tumors. Endocrinol Metab Clin North Am 16:529 55. Horvath E, Kovacs K 1986 Pathology of prolactin cell adenomas of the human pituitary. Semin Diagn Pathol 3:4 56. Kovacs K, Horvath E 1988 Pathology of pituitary adenomas: In: Collo R, Brown GM, VanLoon GR (eds) Clinical Neuroendocrinology. Blackwell, Boston, Chapter 13, p 333 57. Melmed S, Braunstein GD, Horvath E, Ezrin C, Kovacs K 1983 Pathophysiology of acromegaly. Endocr Rev 4:271 58. Kovacs K, Horvath E, Ezrin C 1977 Pituitary adenomas. Pathol Annu 2:341 59. Guyda H, Robert F, Colle E, Hardy J 1973 Histologic, ultrastructural, and hormonal characterization of a pituitary tumor secreting both hGH and prolactin. J Clin Endocrinol Metab 36:531 60. Zimmerman EA, Defendini R, Frantz AG 1974 Prolactin and growth hormone in patients with pituitary adenomas. A correlative study of hormones in tumor and plasma by immunoperoxidase technique and radioimmunoassay. J Clin Endocrinol Metab 38:577 61. Horvath E, Kovacs K 1980 Pathology of the pituitary gland. In: Ezrin C, Horvath E, Kaufman E, Kovacs K, Weiss MH (eds) Pituitary Diseases. CRC Press, Boca Raton, FL, p 1 62. Halmi NS 1982 Occurrence of both growth hormone and prolactin immunoreactive material in the cells of human somatotropic pituitary adenomas containing mammotropic elements. Virchows Arch [Pathol Anat] 398:19 63. Kanie N, Kageyama N, Kuwayama A, Nakane T, Watanabe M, Kawaoi A 1983 Pituitary adenomas in acromegalic patients: an immunohistochemical and endocrinological study with special reference to prolactin secreting adenoma. J Clin Endocrinol Metab 57:1093 64. Ishikawa H, Nogami H, Kamio M, Suzuki T 1983 Single secretory granules contain both GH and prolactin in pituitary mixed type of adenoma. Virchows Arch [Pathol Anat] 339:221 65. Kameya T, Tsumura M, Adachi I, Abe K, Ichikizaki K, Toya S, Demura R 1980 infrastructure, immunohistochemistry and hormone release of pituitary adenomrs in relation to prolactin production. Virchows Arch [Pathol Ai ut] 387:31 66. Horvath E, Kovacs K, Killinger DW, Smyth HS, Weiss MH, Ezrin C 1983 Mammosomatotroph cell adenoma at the human

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

354

67. 68.

69.

70. 71.

72.

73. 74.

75. 76.

77. 78. 79. 80. 81.

82. 83. 84. 85. 86.

87. 88.

FRAWLEY AND BOOCKFOR pituitary: a morphologic entity. Virchows Arch [Pathol Anat] 398:277 Teramoto A 1980 Immunohistochemical studies on the functioning pituitary adenomas. Brain Nerve 32:1163 Felix A, Horvath E, Kovacs K, Smyth HS, Killinger DW, Vale J 1986 Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. A morphological study including immunoelectron microscopy. Acta Neuropathol (Berl) 71:76 Bassetti M, Spada A, Arosio M, Vailar L, Brina M, Giannattasio G 1986 Morphological studies on mixed growth hormone (GH) and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Clin Endocrinol Metab 62:1093 Bassetti M, Spada A, Arosio M, Brina M, Giannattasio G 1988 GH and PRL: hormone studies and immunocytochemical correlates. Adv Biosci 69:23 Robert F, Pelletier G, Serri 0, Hardy J 1988 Mixed growth hormone and prolactin-secreting human pituitary adenomas. A pathological, immunocytochemical, ultrastructural and immunoelectron microscopic study. Hum Pathol 19:1327 Holm R, Nesland JM, Attramadal A, Reinli S, Johannessen JV 1989 Mixed growth hormone and prolactin cell adenomas of the pituitary gland. An immunoelectrol microscopic study. J Submicrosc Cytol Pathol 21:339 Kovacs K, Horvath E, Asa SL, Stefaneau L, Sano T1989 Pituitary cells producing more than one hormone: human pituitary adenomas. Trends Endocrinol Metab 1:104 Beckers A, Courtoy R, Stevanaert A, Boniver J, Closset J, Frankenne F, Reznik M, Hennen G 1988 Mammosomatotropes in human pituitary adenomas as revealed by electron microscopic double gold immunostaining method. Acta Endocrinol (Copenh) 118:503 Lloyd RV 1988 Analysis of mammosomatotropic cells in normal and neoplastic human pituitaries. Pathol Res Pract 183:577 Cameron S, Allen I, Ozo C, Kennedy L, Atkinson B, Hadden D 1990 Clinical, biochemical, and immunoelectron microscopical evidence of dual hormone production in a mammosomatotroph cell adenoma. J Pathol 161:239 Lloyd RV, Cano M, Chandler WF, Barkan AL, Horvath E, Kovacs K 1989 Human growth hormone and prolactin secreting pituitary adenomas analysed by in situ hybridization. Am J Pathol 134:605 Clifton K, Meyer RD 1956 Mechanism of anterior pituitary tumor induction by estrogen. Anat Rec 125:65 Furth J 1969 Pituitary cybernetics and neoplasia. Harvey Lect 63:47 Furth J, Ueda G, Clifton KH 1973 The pathophysiology of pituitaries and their tumors: methological advances. Methods Cancer Res 10:210 Yokoro K, Furth J, Haran-Ghera H 1961 Induction of mammotropic pituitary tumors by x-rays in rats and mice. The role of mammotropes in development of mammary tumors. Cancer Res 21:178 Furth J, Clifton KH 1966 Experimental pituitary tumors. In: Harris GW, Donovan BT (eds) The Pituitary Gland. Butterworth & Co, Ltd, London, vol 2:460 Ito A, Furth J, May P 1972 Growth hormone-secreting variants of a mammotropic tumor. Cancer Res 32:48 Ueda G, Takizawa S, May P, Marolla F, Furth J 1968 Characterization of four transplantable mammotropic pituitary tumor variants in the rat. Cancer Res 28:1963 Ueda G, May P, Furth J 1973 Multihormonal activities of normal cell neoplastic pituitary cells as indicated by immunohistochemical staining. Int J Cancer 12:100 Parsons JA, Erlandsen SL, Carpenter A, Debault LE 1978 Heterogeneity of the MtTWis mammosomatotropic tumor. I. Light microscopic evaluation of cell types by means of immunocytochemistry, morphometric quantisation, fluorescence cytophotometry and radioimmunoassay. Anat Rec 190:719 Parsons JA 1974 Morphological and immunocytochemical evaluation of the MtTW^ mammosomatotropic pituitary tumor. Anat Rec 178:435 Parsons JA, Baskin DG, Erlandsen SL 1979 Heterogeneity of the MtTW15 mammosomatotropic tumor. II. Characterization of parenchymal cells by superimposition immunocytochemistry and electron microscopy. Anat Rec 196:301

Vol. 12, No. 4

89. Baskin DG, Erlandsen SL, Parsons JA 1980 Functional classification of cell types in the growth hormone and prolactin secreting rat MtTW^ mammosomatotropic tumor with ultrastructural immunocytochemistry. Am J Anat 158:455 90. McComb DJ, Ryan N, Horvath E, Kovacs K, Nagy E, Berczi I, Domokos I, Laszio FA 1981 Five different adenomas derived from the rat adenohypophysis: immunocytochemical and ultrastructural study. J Natl Cancer Inst 66:1103 91. Lloyd RV 1987 Analysis of prolactin and growth hormone production in the MtT/F 4 transplantable pituitary tumor by the reverse hemolytic plaque assay. Am J Pathol 129:441 92. Jin L, Song J, Lloyd RV 1989 Estrogen stimulates both prolactin and growth hormone in RNAs expression in the MtT/F4 transplantable pituitary tumor. Proc Soc Exp Biol Med 192:225 93. Song J, Jin L, Lloyd RV 1989 Effects of estradiol on prolactin and growth hormone messenger RNAs in cultured hormonal and neoplastic (MtT/W]5) and (GH3) rat pituitary cells. Cancer Res 49:1247 94. Lloyd RV, Jin L, Fields K, Kulig E 1990 Regulation of prolactin gene expression in a DMBA estrogen-induced transplantable rat pituitary tumor. Am J Pathol 137:1525 95. Losinski NE, Horvath E, Kovacs K 1989 Double-labeling immunogold electron-microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries. Am J Anat 185:236 96. Asa SL, Kovacs K, Stefaneanu L, Horvath E, Billestrup N, Gonsalez-Manchon C, Vale W 1990 Pituitary mammosomatotroph adenomas develop in old mice transgenic for growth hormone-releasing hormone. Proc Soc Exp Biol Med 193:232 97. Tashjian Jr AH, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342 98. Bancroft FC, Levine L, Tashjian Jr AH 1969 Control of growth hormone production by a clonal strain of rat pituitary cells: stimulation by hydrocortisone. J Cell Biol 43:432 99. Tashjian Jr AH, Bancroft FC, Levine L 1970 Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. J Cell Biol 47:61 100. Tashjian Jr AH 1979 Clonal strains of hormone-producing pituitary cells. Methods Enzymol 58:527 101. Hoyt RF, Tashjian Jr AH 1980 Immunocytochemical analysis of prolactin production by monolayer cultures of GH3 rat anterior pituitary tumor cells. I. Long-term effects of stimulation with thyrotropin releasing hormone (TRH). Anat Rec 197:153 102. Hoyt RF, Tashjian Jr AH 1980 Immunocytochemical analysis of prolactin production by monolayer cultures of GH3 rat anterior pituitary tumor cells. II. Variation in prolactin content of individual cell colonies, and dynamics of stimulation with thyrotropinreleasing hormone. Anat Rec 197:163 103. Gourdji D, Tougard C, Tixier-Vidal A 1982 Clonal prolactin strains as a tool in neuroendocrinology. In: Ganong WF, Martine L (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 7:317 104. Gautvik KM, Kriz M, Fossum S 1976 A microanalytical method for measurement of prolactin synthesis in individual cultured rat pituitary cells. Anal Biochem 74:52 105. Boockfor FR, Schwarz LK 1987 Cultures of GH3 cells contain both single and dual hormone secretors. Endocrinology 122:762 106. Chomczynski P, Brar A, Frohman LA 1988 Growth hormone synthesis and secretion by a somatomammotroph cell line derived from normal adult pituitary of the rat. Endocrinology 123:2276 107. Lloyd RV, Landefeld TD, Maslar I, Frohman LA 1985 Diethylstilbestrol inhibits tumor growth and prolactin production in rat pituitary tumors. Am J Pathol 118:379 108. Lloyd RV, Schmidt K, Coleman K, Wilson BS 1986 Prolactin and growth synthesis and thymidine incorporation in dissociated rat pituitary tumor cells. Proc Soc Exp Biol Med 181:18 109. Clausen OPF, Gautvik KM, Haug E 1978 Effects of cortisol, 170estradiol and thyroliberin on prolactin and growth hormone production, cell growth, and cell cycle distribution in cultured rat pituitary tumor cells. J Cell Physiol 94:205 110. Boockfor FR, Hoeffler JP, Frawley LS 1985 Cultures of GH3 cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors. Endocrinology 117:418 111. Gautvik KM, Fossum S 1976 Basal and thyroliberin stimulated

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

November, 1991

112. 113.

114.

115.

116. 117. 118. 119.

120. 121.

122.

123. 124.

125.

126. 127. 128.

129.

130.

131.

MAMMOSOMATOTROPES

prolactin synthesis in single cell cultures and in populations of rat pituitary cells. Biochem J 158:119 Kovacs K, Horvath E 1986 Pathology of growth hormone producing tumors of the human pituitary. Semin Diagn Pathol 3:18 Nelson C, Crenshaw III EB, Franco R, Lira SA, Albert VR, Evans RM, Rosenfeld MG 1986 Discrete cis-active genomic sequences dictate the pituitary cell type-specific expression of rat prolactin and growth hormone genes. Nature 332:557 Lira SA, Crenshaw III EB, Glass CK, Swanson LW, Rosenfeld MG 1988 Identification of rat growth hormone genomic sequences targeting pituitary expression in transgenic mice. Proc Natl Acad Sci USA 85:4755 Sharp ZD, Helsel S, Cao Z, Barron EA, Sanchez Y 1989 DNA recognition element required for PUF-1 mediated cell-type-specific transcription of the rat prolactin gene. Nucleic Acid Res 17:2705 Cao Z, Barron EA, Carillo AJ, Shapr ZD 1987 Reconstitution of cell type-specific transcription of the rat prolactin gene in vitro. Mol Cell Biol 7:3402 Gutierrez-Hartmann A, Siddigui S, Loukin S 1987 Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell free extracts. Proc Natl Acad Sci 84:5211 Lufkin T, Bancroft C 1987 Identification by cell fusion of gene sequences that interact with positive trans-acting factors. Science 237:283 West BL, Catanzaro DF, Mellon SH, Cattini PA, Baxter JD, Reudelhuber TL 1987 Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element. Mol Cell Biol 7:1193 Ye ZS, Samuels HH 1987 Cell and sequence specific binding of nuclear proteins to 5' flanking DNA of the rat growth hormone gene. J Biol Chem 262:6313 Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissuespecific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519 Nelson C, Albert VR, Elsholtz HP, Lu LI-W, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239:1400 Castrillo JL, Bodner M, Karin M 1989 Purification of growth hormone-specific transcription factor GHF-1 containing homeobox. Science 243:814 Mangalam HJ, Albert VR, Ingraham HA, Kapiloft M, Wilson L, Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein Pit-1, activates both growth hormone and prolactin promoters transcriptionally. Genes & Dev 3:946 Fox SR, Jong MTC, Cassanova J, Fe Z-S, Stanley F, Samuels HH 1990 The homeodomain protein Pit-1/GHFl is capable of binding to and activating cell specific elements of both the growth hormone and prolactin gene promoters. Mol Endocrinol 4:4069 Bodner M, Karin M 1987 A pituitary-specific transacting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells. Cell 50:267 Tripputi P, Guerin SL, Moore DD 1988 Two mechanisms for the extinction of gene expression in hybrid cells. Science 241:1205 McCormick A, Wu D, Castrillo J-L, Dana S, Strobl J, Thompson EB, Karin M 1988 Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific transactivator GHF-1. Cell 55:379 Keech CA, Gutierrez-Hartmann A 1989 Analysis of rat prolactin promoter sequences that mediate pituitary specific 3'5' cyclic adenosine monophosphate-regulated gene expression in vivo. Mol Endocrinol 3:832 Harvey C, Jackson SM, Siddiqui SK, Gutierrez-Hartmann A 1991 Structure function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements. Mol Endocrinol 5:836 Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS, Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes involve cell-specific Pit-1 in RNA translation and synergistic

355

interactions with other classes of transcription factors. Genes & Dev 4:695 132. Vallejo M, Miller CP, Penchuk L, Habener JF, Positive and negative transcriptional control elements regulate the cell-specific expression of the somatostatin gene in pancreatic islet cells. Program of the 73rd Annual Meeting of The Endocrine Society, Washington, DC, 1991, p 113 (Abstract) 133. Maurer RA, Notides AC 1987 Identification of an estrogen responsive element from the 5' flanking region of the rat prolactin gene. Mol Cell Biol 7:4247 134. Waterman ML, Adler S, Nelson C, Greene GL, Evans RM, Rosenfeld MG 1988 A single domain of the estrogen receptor confers deoxyribonucleic acid binding and transcriptional activation of the prolactin gene. Mol Endocrinol 2:14 135. Crenshaw III EB, Kalla K, Ingraham HA, Simmons DM, Swanson LW, Rosenfeld MG 1989 Cell specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between Pit-1 recognition elements. Genes & Dev 3:959 136. Camper SA, Yao YAS, Rottman FM 1985 Hormonal regulation of the bovine prolactin promoter in rat pituitary tumor cells. J Biol Chem 260:12246 137. Jackson AE, Bancroft C 1988 Proximal upstream flanking sequences direct regulation of the rat prolactin gene. Mol Endocrinol 2:1139 138. Day RN, Maurer RA 1990 Pituitary calcium channel modulation and regulation of prolactin-gene expression. Mol Endocrinol 4:736 139. Yan G-Z, Pan WT, Bancroft C 1991 Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein Pit-1. Mol Endocrinol 5:535 140. Iverson RA, Day KH, d'Emden M, Day R, Maurer RA 1990 Clustered point mutation analysis of the rat prolactin promoter. Mol Endocrinol 4:1564 141. Schaufele F, West BL, Reudelhuber TL 1990 Overlapping Pit-1 and Spl binding are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Spl binding. J Biol Chem 265:17189 142. Mitchell PJ, Tjian R 1989 Transcriptional regulation in mammalian cells by sequence specific DNA binding proteins. Science 245:371 143. Weintraub H 1985 Assembly and propagation of repressed and derepressed chromosonal states. Cell 42:705 144. Weissbach A, Word C, Bolden A 1989 Eukaryotic DNA methylation and gene expression. Current Top Cell Regul 30:1 145. Wolfson G, Chisholm J, Tashjian Jr AH, Fish S, Abies RH 1986 Neplanocin Action on S-adenosylhomocysteine hydrolase and on hormone synthesis by GH4Ci cells. J Biol Chem 261:4492 146. Ivarie RD, Morris JA 1982 Induction of prolactin-deficient variants of GH3 rat pituitary tumor cells by ethyl methanesulfonate: reversion by 5 azacytidine, a DNA methylation inhibitor. Proc Natl Acad Sci USA 79:2967 147. Lan NC 1984 The effects of 5 azacytidine on the expression of the rat growth hormone gene. J Biol Chem 259:11601 148. Laverrire JN, Muller N, Buisson C, Tougard A, Tixier-Vidal A, Martial J, Gourdji D 1986 Differential implication of deoxyribonucleic acid methylation in rat prolactin and growth hormone gene expression: a comparison between rat pituitary cell strains. Endocrinology 118:198 149. Zhang Z, Kumar V, Rivera RT, Pasion SG, Chisholm J, Biswas DK 1989 Suppression of prolactin gene expression in GH cells correlates with site specific DNA methylation. DNA 8:605 150. Durrin LK, Weber JL, Gorski J 1984 Chromatin structure, tran1 scription and methylation of the prolactin gene domain in pituitary tumors of Fischer 344 rats. J Biol Chem 259:7086 151. Strobl JS, Dannies PS, Thompson EB 1986 Rat growth hormone gene expression is correlated with an unmethylated CGCG sequence near the transcription initiation site. Biochemistry 25:3640 152. Kumar V, Biswas DK 1988 Dynamic state of site specific DNA methylation concurrent to altered prolactin and growth hormone gene expression in the pituitary gland of pregnant and lactating rats. J Biol Chem 263:12645 153. Severinghaus AE 1937 Cellular changes in the anterior hypophysis with special reference to its secretory activities. Physiol Rev 17:556

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 17:07 For personal use only. No other uses without permission. . All rights reserved.

Mammosomatotropes: presence and functions in normal and neoplastic pituitary tissue.

0163-769X/91/1204-0337$03.00/0 Endocrine Reviews Copyright© 1991 by The Endocrine Society Vol. 12, No. 4 Printed in U.S.A. Mammosomatotropes: Presen...
3MB Sizes 0 Downloads 0 Views