The Intermediate Lactotroph: A Morphologically Distinct, Ghrelin-Responsive Pituitary Cell in the Dwarf (dw/dw) Rat
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《内分泌学杂志》
School of Biosciences (N.M.T., M.M.E.-K., T.W.), Cardiff University, Cardiff CF10 3US, United Kingdom
Laboratory for Cellular Endocrinology (I.H.-O., H.C.C.), Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
Abstract
Profound somatotroph hypoplasia in the dwarf (dw/dw) rat is accompanied by an estrogen-dependent induction of prolactin secretion by the GH secretagogue, GHRP-6. Using electron microscopy, we demonstrated that the reduction in the somatotroph population in the dw/dw pituitary is accompanied by the presence of a morphologically distinct lactotroph subpopulation. In these cells, which did not coexpress GH, the size, shape, and number of the secretory granules were between those of the type I and type II lactotrophs. We therefore called these cells intermediate lactotrophs. The intermediate lactotrophs accounted for up to 30% of the total prolactin-positive cell population in dw/dw males and up to 12% in females. Using tannic acid to quantify the fusion of secretory granules, we have shown that the intermediate lactotrophs are unresponsive to either GH-releasing factor (GRF) or TRH but exhibit a sexually dimorphic secretory response to acute ghrelin treatment, granular fusions being 4-fold higher in females. No cell matching the morphology of the novel lactotroph subpopulation was observed in the pituitary of the GRF-insensitive lit/lit mouse. However, ablation of GRF neurons with neonatal monosodium glutamate treatment had no effect on the population of intermediate lactotrophs in the dw/dw rat. Thus, the presence of the intermediate lactotrophs in the dw/dw pituitary appears to be independent of the function of the GRF neurons.
Introduction
THE DIFFERENTIATION OF secretory cell types during pituitary organogenesis is a multistep process regulated by the spatiotemporal expression of transcription factors, cytokines, and neuropeptides (reviewed in Refs.1, 2). Commitment to the somatotroph, lactotroph, and thyrotroph lineage is dependent on the sequential expression of the transcription factors, Prophet of Pit-1 (Prop-1) (3) and the pituitary transcription factor Pit-1 (4). Subsequently, somatotrophs and lactotrophs are thought to differentiate from a common bihormonal progenitor, the mammosomatotroph (5, 6), but knowledge of the factors regulating this process is incomplete. Proliferation of the monohormonal GH-containing cells is promoted by GH-releasing factor (GRF) (7), whereas expansion of the prolactin (PRL)-containing cell population is regulated by TGF (8), nerve growth factor (9), dopamine (10), and estrogen (11, 12). Mutations in either Prop-1 (3, 13) or Pit-1 (4, 13, 14) and reduction of hypothalamic GRF production (15, 16) usually lead to suppression of both somatotroph and lactotroph development and an associated dwarfism. In contrast, the dwarf (dw/dw) rat displays a unique adenohypophysial phenotype.
In the dw/dw rat, an unknown mutation leads to a profound reduction in somatotroph number (17, 18) and pituitary GH content (19), leading to low amplitude episodes of GH secretion (20, 21). Although pituitary PRL content in this model was initially thought to be reduced (19), subsequent evidence indicated that the suppression of somatotroph development is accompanied by an increase in both lactotroph number and PRL secretion (17, 18, 22, 23). Because it is unclear how these changes affect the distinct lactotroph subpopulations, we used immunogold labeling and electron microscopy (EM) to quantify the postnatal development of individual secretory cell types in the dw/dw rat.
Another unique feature of the dw/dw phenotype is that PRL secretion is elicited by the ghrelin mimetic, GHRP-6 (24). This effect is estrogen dependent, being female specific, abolished by ovariectomy, and induced in males by estrogen treatment (24). We therefore determined the sensitivity of the somatotrophic and lactotrophic axes in the dw/dw rat to a range of secretagogues, including ghrelin, the cognate endogenous ligand of the GH-secretagogue receptor (GHS-R1a) (25). Sensitivity has been determined in vivo by quantifying both circulating hormone levels and the responses of individual pituitary cell types, the latter being performed by tannic acid perfusion in conjunction with EM analysis. These experiments reveal a unique, morphologically distinct, ghrelin-responsive lactotroph in the pituitary of the dw/dw rat, which we have termed the intermediate lactotroph.
To establish whether the occurrence of these cells is regulated by the high levels of hypothalamic GRF, we analyzed the lactotroph populations in dw/dw rats after neonatal monosodium glutamate (MSG) treatment and in GRF-insensitive lit/lit mice (7). In addition, to determine whether the population of these cells is regulated in parallel with the somatotroph or lactotroph populations by the steroid hormones, we quantified the population of somatotrophs and lactotrophs in pregnant dw/dw rats. A preliminary account of sections of this work has previously been communicated (26).
Materials and Methods
Dwarf (dw/dw) and wild-type [Albino-Swiss (AS)] rats
The animal procedures described conformed to the institutional and national ethical guidelines for in vivo experiments in rats and were approved by local ethical review. Homozygous dw/dw rats, maintained on an AS background, and AS rats were derived from colonies at the National Institute for Medical Research (London, UK) and bred in the Transgenic Unit (School of Biosciences, Cardiff University). These rats were housed under conditions of 14-h light, 10-h dark (lights on at 0500 h), with rat chow and water available ad libitum.
Study 1: development of secretory cell populations in the dw/dw pituitary
Male and female dw/dw and AS rats were killed at 5, 30, and 60 d old. Pituitaries were excised and fixed immediately in 2.5% glutaraldehyde (EM grade) in PBS (pH 7.2) at room temperature. Pituitaries were subsequently transferred into 0.25% glutaraldehyde in PBS at 4 C until processing for EM and analysis of somatotroph, mammosomatotroph, and lactotroph populations (see below).
Study 2: neuroendocrine control of intermediate lactotrophs
Secretagogue-induced GH and PRL secretion in conscious dw/dw rats.
Male and female dw/dw rats [12 wk old; weighing 146–171 g (males) and 109–121 g (females)] were prepared with single-bore jugular vein cannulae under halothane anesthesia. After at least 48 h recovery, 120-μl blood samples were taken before and at 5, 15, and 30 min after a bolus iv injection of either vehicle [300 μl sterile saline containing BSA (1 mg/ml) and heparin (10 U/ml)], GRF [1 μg rat GRF(1–29)NH2 (Bachem, Bubendorf, Switzerland, generously supplied by Novo Nordisk A/S, Bagsvrd, Denmark)], ghrelin [10 μg rat ghrelin (generously supplied by Pharmacia & Upjohn, Uppsala, Sweden)], or TRH [1 μg Protirelin (Roche, Cambridge, UK)]. After 48 h this sampling procedure was repeated, each animal receiving an alternative treatment. Blood samples were taken into heparinized tubes and centrifuged, separated plasma subsamples being stored at –20 C for subsequent determination of plasma rat (r)GH and rPRL concentration (see below).
Secretagogue-induced responses in individual secretory cell types in dw/dw rats.
To quantify the cellular responses to each secretagogue treatment, dw/dw rats received a third (alternative) secretagogue treatment at least 48 h after the second period of sampling, in conjunction with tannic acid perfusion to capture exocytosed secretory granules. After secretagogue treatment, dw/dw rats were reanesthetized with sodium pentobarbitone (Euthatal, Vericore Ltd., Dundee, UK) and prepared for transcardial perfusion. Seven minutes after secretagogue treatment, the rats were perfused (5 ml/min) with heparinized saline (37 C for 3 min), followed by tannic acid (0.2% tannic acid in PBS at 37 C for 5 min). Pituitaries were excised, immersed in tannic acid for 2 min, fixed in 2.5% glutaraldehyde in PBS for 2 h, and stored in 0.25% glutaraldehyde in PBS at 4 C until processing for EM analysis.
Study 3: regulation of the intermediate lactotroph population by GRF
The effect of neonatal MSG treatment on somatotroph and lactotroph populations in dw/dw rats.
To determine whether the existence of the intermediate lactotrophs is brought about by the high levels of hypothalamic GRF expression in the dw/dw rat (16), we quantified the somatotroph and lactotroph populations in dw/dw rats after ablation of the arcuate GRF neurons with neonatal MSG treatment (27). Three litters of dw/dw rats (eight to 10 pups/litter) received ip injections of either vehicle (50 μl of 0.9% sterile saline) or MSG (4 mg/g body weight in 50 μl vehicle) on postnatal d 2, 4, 6, 8, and 10 and were carefully monitored for potential adverse effects. At 8 wk of age, these rats were weighed, stunned, and decapitated. Pituitaries were excised, weighed, and treated as in study 1 before processing for EM analysis. In addition, the liver was dissected and weighed, and the left tibiae were excised and the length measured using a handheld micrometer.
Quantification of somatotroph and lactotroph populations in male lit/lit mice.
To investigate whether intermediate lactotrophs are detectable in other models of GRF insensitivity, we quantified the somatotroph and lactotroph subpopulations in pituitaries from 8-wk-old male wild-type (C57BL/6J) and homozygous lit/lit (C57BL/6J-Ghrhr) mice (Jackson Laboratories, Bar Harbor, ME). lit/lit mice bear an inactivating point mutation in the GRF receptor (7). These mice were killed by cervical dislocation and the pituitaries excised and treated as in study 1 before shipment from Jackson Laboratories to Oxford UK, and subsequent processing for EM analysis.
Study 4: effect of pregnancy on somatotroph and lactotroph populations in the dw/dw pituitary
To determine whether the population of intermediate lactotrophs is regulated in parallel with the somatotrophs or lactotrophs by the steroid hormones, the populations of these secretory cells were determined in female dw/dw rats during pregnancy. Female dw/dw rats (12–15 wk old) were mated overnight with dw/dw males, mating being confirmed by the presence of a vaginal plug. After 2 wk these pregnant rats and a group of virgin female littermates were anesthetized with halothane and killed by decapitation. Pregnancy was confirmed post mortem, and pituitaries were excised and treated as in study 1 before processing for EM analysis.
Tissue processing and EM
Pituitaries for EM analysis were processed and analyzed as previously described (28, 29). Briefly, the tissue was postfixed in osmium tetroxide [1% (wt/vol) in 0.1 M PBS], contrasted with uranyl acetate [2%(wt/vol) in distilled water], dehydrated in ethanol, and embedded in Spurr’s resin. Ultrathin sections (50–80 nm) were taken using a Reichart-Jung ultracut microtome and mounted on nickel grids. Sections from dw/dw rat pituitaries were incubated for 2 h at room temperature with primary antibodies for either rGH [1:2000 dilution of monkey antirat GH; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)] or rPRL (1:2000 dilution of rabbit antirat PRL; NIDDK) followed by a 1-h incubation with either a 15-nm protein A gold-conjugated goat antimonkey or a 5-nm protein A gold-conjugated goat antirabbit secondary antibody (British Biocell, Cardiff, UK). For identification of bihormonal rGH- and rPRL-positive cells, sections were labeled for rPRL on d 1 and for rGH on the following day. All antibodies were diluted in 0.1 M PBS (containing 0.1% egg albumin), and in control sections the primary antibody was replaced with nonimmune rabbit serum. Sections from lit/lit mouse pituitaries were immunolabeled using the same antibodies used above because these cross-react well with mouse PRL and GH. Finally, sections were counterstained with lead citrate and uranyl acetate and examined on a JOEL 1010 transmission electron microscope (JOEL USA Inc., Peabody, MA).
For each pituitary, four randomly orientated sections, each containing six or nine grid squares, were counted for each hormone in rats and mice, respectively. The total secretory cell population was determined by counting the number of cells per grid, with individual somatotrophs and lactotrophs identified using established identification criteria (30, 31) in conjunction with the immunogold labeling. In brief, somatotrophs contain large numbers of large (>300 nm diameter) spherical electron-dense secretory granules; type I lactotrophs contain small numbers of large (>300 nm diameter) irregularly shaped granules; type II lactotrophs contain large numbers of small (<200 nm diameter) spherical granules. In addition, the bihormonal mammosomatotrophs were identified by the presence of granular immunogold labeling for both GH and PRL. The secretory response of individual cells from tannic acid perfused rats was determined by counting the number of exocytosed granules fused with the plasma membrane of each cell, with a minimum of six cells counted for each cell type per pituitary.
Plasma hormone analysis
Plasma GH and PRL concentrations were determined by RIA using delayed addition of label. The results are expressed in terms of the reference preparations RP-2 (rGH) and RP-3 (rPRL), using the reagents generously supplied by National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) [intraassay variation 6.04% (rGH) and 5.04% (rPRL); range 0.12–250 ng/ml (rGH) and 0.0480–100 ng/ml (rPRL)].
Statistical analysis
All data shown are mean ± SEM (n = 3–5 for EM analyses; n = 5–6 for hormone and tissue analyses), with multiple statistical comparisons performed by either unpaired Student’s t test, or one-way ANOVA followed by either Dunnett’s (vs. control) or Bonferroni’s (for selected pairs) post hoc tests, as indicated in figure legends.
Results
Study 1: development of secretory cell populations in the dw/dw pituitary
During postnatal pituitary development, a 50–60% reduction in number of secretory cells per grid was seen in AS rats (Fig. 1, A and G), which normally accompanies secretory cell enlargement. A similar, though less pronounced, decline (25–30%; P < 0.001) was also observed in dw/dw rats (Fig. 1, A and G), the total secretory cell number remaining higher than in their wild-type counterparts at 30 and 60 d of age (P < 0.001).
In AS rats the proportion of somatotrophs increased rapidly between 5 and 60 d (Fig. 1B; P < 0.001). In contrast, there was no significant increase in the proportion of somatotrophs in either male (Fig. 1B) or female (Fig. 1H) dw/dw rats, the proportion of somatotrophs remaining less than 15% of that in their AS counterparts (P < 0.001). A similar developmental pattern in the small proportion of mammosomatotrophs was seen in AS and dw/dw males (Fig. 1C), but in AS and dw/dw females, the proportion of mammosomatotrophs remained constant (Fig. 1I). However, none of the means for mammosomatotrophs were significantly different.
The combined proportion of all PRL-positive cells was higher in dw/dw males at 5 and 60 d of age (P < 0.001) and dw/dw females at 60 d of age (P < 0.05). However, the total proportion of PRL-positive cells in dw/dw rats was transiently lower at 30 d of age (P < 0.01).
In AS males the proportion of type II lactotrophs increased 4-fold between 5 and 30 d of age (Fig. 1E; P < 0.001), but was halved by 60 d (P < 0.01). A similar biphasic pattern was observed in AS females (Fig. 1K; P < 0.001), although the proportion of type II lactotrophs was 30–40% lower than in AS males (P < 0.05). At 5 d old, the proportion of type II lactotrophs in dw/dw males was double that in AS males (Fig. 1E; P < 0.001) and, after an initial decline (P < 0.01), was 70% higher than in AS males at 60 d (P < 0.001). In dw/dw females, the proportion of type II lactotrophs progressively declined and was 70% lower than in AS females at 30 and 60 d old (Fig. 1K; P < 0.001).
A similar developmental pattern in the proportion of type I lactotrophs was seen in AS males (Fig. 1D). In dw/dw males, the proportion of type I lactotrophs increased over the period studied (Fig. 1D; P < 0.001), this population being similar to that seen in AS males at 60 d of age. A progressive increase in type I lactotrophs was observed in both AS and dw/dw females (P < 0.001), these cells accounting for 35–40% of the total secretory cell population at 60 d (Fig. 1J).
Interestingly, we identified a population of lactotrophs in the dw/dw pituitary whose morphology did not conform to the established identification criteria (30, 31). These cells had a granular appearance between that of the two established lactotroph types, i.e. containing large numbers of large (>300 nm diameter), regularly shaped, electron-dense granules (Fig. 2, B, E, and G) and for this reason are subsequently referred to as intermediate lactotrophs. These granules were positively labeled for PRL (Fig. 2, B and E, insets) but did not coexpress GH. The intermediate lactotrophs were found to occur in direct juxtaposition with either type I (Fig. 2G) or type II (Fig. 2E) lactotrophs but seldom in association with somatotrophs or mammosomatotrophs. The proportion of intermediate lactotrophs was highest at 30 d, accounting for 7 and 3% of the total secretory cell population in males and females, respectively (Fig. 1, F and L). Although the number of these cells declined in the female dw/dw pituitary by 60 d, the population was maintained in dw/dw males. An occasional cell matching this description was also observed in the normal rat pituitary at 30 d (1%; Fig. 1, F and L).
Study 2: neuroendocrine control of intermediate lactotrophs
Secretagogue-induced GH and PRL secretion in conscious dw/dw rat.
GRF elevated circulating GH in both male and female dw/dw rats (P < 0.01), the response being twice as high in females (Fig. 3, A and C). In contrast, the robust ghrelin-induced GH secretion was similar in both sexes (Fig. 3, A and C). Injection of TRH had no significant effect on GH secretion.
As expected, TRH treatment elevated circulating PRL in dw/dw rats, eliciting a small increase in males (Fig. 3B; P < 0.05) and a more robust (P < 0.01) response in females (Fig. 3D; P < 0.01). In male dw/dw rats, ghrelin treatment had no effect on circulating PRL secretion (Fig. 3B), but in females ghrelin elicited a similar response to TRH (Fig. 3D; P < 0.05 vs. vehicle-treated controls). PRL secretion was unaffected by GRF treatment.
Secretagogue-induced responses in individual secretory cell types in dw/dw rat.
In conjunction with the circulating hormone data, we performed quantification of granular fusions in individual cell types after secretagogue treatment. Somatotrophs responded consistently to both GRF and ghrelin treatment, GRF-induced granular fusion being higher in females than males (Fig. 4, A and E; P < 0.05). TRH did not elicit granular fusion in somatotrophs. Type I lactotrophs were unresponsive to secretagogue treatment in males (Fig. 4D) and produced only a small, inconsistent (<1 granule/cell) response to ghrelin in females (Fig. 4H). TRH treatment elicited a robust granular fusion in type II lactotrophs in both sexes (Fig. 4, B and F). Type II lactotrophs were unresponsive to GRF (Fig. 4, B and F), but ghrelin induced a small granular response in females (Fig. 4F).
The morphologically distinct intermediate lactotrophs identified in the dw/dw pituitary displayed a unique response pattern to secretagogue treatment. Although unresponsive to either GRF or TRH, these cells showed significant granular fusion after ghrelin administration in both male and female dw/dw rats (Fig. 4, C and G). This response was 4-fold higher in females (P < 0.001). Examples of the granular fusion elicited by ghrelin treatment in male and female dw/dw rats are shown in Fig. 5, C and D.
Study 3: regulation of the intermediate lactotroph population by GRF
The effect of neonatal MSG treatment on somatotroph and lactotroph populations in dw/dw rats.
Although there was no significant effect on body weight, neonatal MSG treatment caused a small reduction in tibial length (Table 1; P < 0.05), accompanied by an elevation in visceral adiposity (P < 0.001; data not shown).
Neonatal MSG treatment had no effect on either pituitary weight (Table 1) or the proportion of somatotrophs (Fig. 6). However, MSG treatment resulted in a decline in the proportion of type I lactotrophs in male (25%; P < 0.01) and female (10%; P < 0.05) dw/dw rats (Fig. 6). The proportion of type II lactotrophs was halved by MSG treatment in dw/dw males (P < 0.001) but more than doubled in females (P < 0.05). Although the mean proportion of intermediate lactotrophs in MSG-treated males was only half of that in the vehicle-treated males, the means were not significantly different. In dw/dw females the proportion of intermediate lactotrophs was not significantly reduced by MSG treatment.
Quantification of somatotroph and lactotroph populations in male lit/lit mice.
To investigate whether this population of intermediate lactotrophs arises from an insensitivity to GRF, we examined the somatotroph and lactotroph populations in male lit/lit mice. As expected, the proportion of somatotrophs in lit/lit mice was only a third of that in wild-type mice (Fig. 7; P < 0.01), and the proportion of mammosomatotrophs was halved (P < 0.01). Conversely, the proportion of type II lactotrophs was almost doubled (Fig. 7; P < 0.01), and there was a small increase in the proportion of type I lactotrophs (P < 0.05). However, we were unable to detect any lactotrophs in the pituitaries of lit/lit mice corresponding to the morphology of the intermediate lactotroph.
Study 4: effect of pregnancy on somatotroph and lactotroph populations in the dw/dw pituitary
To determine whether the intermediate lactotrophs are regulated in parallel with the somatotroph or lactotroph populations by the steroid hormones, we quantified the secretory cell populations in 2-wk pregnant dw/dw rats. Although the proportion of intermediate lactotrophs in dw/dw females was almost doubled during pregnancy (Fig. 8; P < 0.01), the proportion of somatotrophs and types I and II lactotrophs were not significantly altered.
Discussion
Phenotypic analysis of mutant murine models has greatly facilitated our understanding of pituitary organogenesis and the transcription factors regulating cell lineage determination. However, our knowledge of the factors regulating the postnatal expansion of secretory cell populations is less advanced. Failure in somatotroph development is usually accompanied by lactotroph hypoplasia (4, 13, 14, 15), but in the dw/dw rat, the profound reduction in somatotrophs has been reported to be accompanied by lactotroph hyperplasia (17, 18, 22, 23). Our use of EM to quantify the development of secretory cell subpopulations has led to the identification of a novel lactotroph in the dw/dw pituitary, the intermediate lactotroph. The presence of this cell and its unique pattern of neuropeptide responses may contribute to some of the endocrine peculiarities of the dw/dw phenotype.
In the nonneoplastic anterior pituitary, the lactotrophs account for up to 50% of the total secretory cell population and are significantly higher in females (Fig. 1). Our examination of secretory cell development in dw/dw rats confirmed that profound somatotroph hypoplasia is accompanied by a significant expansion of the lactotroph population (study 1), which is seen most clearly in males. Although it is possible that the observed lactotroph hyperplasia is the result of expressing the secretory cell populations in relative terms, in the context of a profound reduction in the predominant secretory cell type (somatotrophs) (32), our data are consistent with the reported increase in pituitary PRL content (22, 23) in the dw/dw rat.
PRL-containing cells can be subdivided into distinct subpopulations on the basis of either morphology or function. Morphological subdivision is achieved under EM using established identification criteria (30, 31), which are based on the size, shape, and number of the electron-dense secretory granules. Briefly, type I lactotrophs contain sparse, large (>300 nm diameter), irregularly shaped granules, and type II lactotrophs display a large number of smaller (<200 nm diameter) spherical granules (Fig. 2). Using these criteria, the most remarkable feature we observed in the dw/dw pituitary was the presence of a subpopulation of lactotrophs with distinct morphological features that did not conform to the established type I and type II lactotrophs or to the less well-described type III lactotrophs (33). These novel cells, which did not coexpress GH, exhibited large numbers of large (>300 nm diameter), regularly shaped, electron dense granules (Fig. 2, B, E, and G) and accounted for up to 30% of the total PRL-positive cell population in dw/dw males and up to 12% in females, the highest proportion being observed at 30 d of age (Fig. 1, F and L). Due to their morphological appearance being between that of the types I and II lactotrophs, we called these cells intermediate lactotrophs.
Subdivision of lactotrophs on the basis of morphology does not correspond exactly with functional classification (34, 35). To characterize the neuroendocrine regulation of PRL secretion in the intermediate lactotrophs, we quantified secretory activity of individual cells under EM after neuropeptide treatment in conjunction with tannic acid to capture exocytosed granules. This revealed that the intermediate lactotrophs displayed a distinctive pattern of responses to secretagogue exposure. As we have previously reported in normal rats (36, 37), type I lactotrophs were almost completely unresponsive to neuropeptide treatment (Fig. 4, D and H), whereas type II lactotrophs showed a robust exocytotic response to TRH (Fig. 4, B and F). In contrast to these established lactotroph subtypes, the intermediate lactotrophs were unresponsive to TRH but displayed a prominent secretory response to ghrelin (Fig. 5), which was particularly marked in females (Fig. 4G). This ghrelin-induced granular fusion was 4-fold higher than that seen in either type I or type II lactotrophs (Fig. 4, H and F).
It should be noted that although ghrelin also induced granular fusion in male intermediate lactotrophs (Fig. 4C), a significant increase in circulating PRL was observed only in females (Fig. 3D). This may be due to a higher expression of PRL in individual lactotrophs of dw/dw females, as suggested by previous data (the differential in pituitary PRL content between male and female dw/dw rats being 4-fold higher than the differential in lactotroph number) (23). Should a higher PRL expression in individual lactotrophs of dw/dw females be confirmed, this might also explain the larger increase in circulating PRL seen in response to TRH (Fig. 3D). Alternatively, the difference between the granular responses and the observed changes in circulating PRL may result from a higher sensitivity to ghrelin in all lactotroph subtypes in female dwarves.
In the hypothalamo-pituitary GH axis, ghrelin induces GH secretion predominantly through activation of arcuate GRF neurons (38, 39), with a smaller direct action on the somatotrophs (25, 40). The induction of PRL secretion by ghrelin in dw/dw rats is unlikely to be mediated by GRF because, in contrast to the somatotrophs (Fig. 4, A and B), none of the lactotroph subtypes were responsive to GRF (at a dose that produced a comparable granular response to ghrelin in somatotrophs). This suggests that in the dw/dw pituitary, the intermediate lactotrophs may express the cognate receptor for ghrelin (GHS-R1a) (25, 41), although this remains to be established. We propose that the presence of the novel intermediate lactotroph in dw/dw rats and its unique pattern of secretagogue regulation may contribute to the estrogen-dependent elevation in PRL secretion elicited by ghrelin and the synthetic GH-secretagogues in this model (24).
However, our observation of a population of morphologically and functionally distinct lactotrophs may have wider implications for the regulation of both cell lineage determination and plasticity between the pituitary cell types. A number of ontological mechanisms may give rise to the presence of this novel lactotroph in the dw/dw rat. These are summarized in Fig. 9.
First, the novel lactotroph may represent an intermediate form between that of the type I and type II lactotrophs (Fig. 9A), as suggested from the shape, size, and number of the secretory granules (Fig. 2). This possibility is also supported by the parallel regulation of these cells with type I and II lactotrophs during pregnancy (Fig. 8) when interconversion between the established lactotroph subtypes is known to occur (42). However, the lack of responsiveness of these cells to TRH contrasts sharply with the prominent response in type II lactotrophs (Fig. 4).
It has previously been reported that during pregnancy there is an inverse relationship between the population of somatotrophs and lactotrophs, lactotroph hyperplasia resulting from the transdifferentiation of somatotrophs (43, 44, 45). Given the profound somatotroph hypoplasia in the dw/dw pituitary, it is possible that the population of novel lactotrophs may arise from a similar process of transdifferentiation (Fig. 9C). Alternatively, because it has been suggested that the mammosomatotrophs are obligatory intermediates in this interconversion (46), the intermediate lactotrophs may arise from the differentiation of these bihormonal cells (Fig. 9B). Although we have no direct evidence to support either of these origins, there are notable structural and functional similarities between the intermediate lactotroph and the somatotroph, and the proportion of somatotrophs that coexpress GH and PRL is considerably increased in the dw/dw pituitary (23). A fourth option is that the intermediate lactotroph represents the product of transdifferentiation from another unspecified secretory cell type (Fig. 9D).
In this ontological context, the location of the intermediate lactotrophs relative to the neighboring secretory cells is worthy of consideration. Recent evidence from a transgenic mouse model with somatotroph-specific green fluorescent protein expression (47) indicates that all somatotrophs are directly associated with a neighboring somatotroph, forming a continuous somatotrophic network (48). It is probable that an equivalent lactotrophic network also exists. In the present study, the novel PRL-producing cells were always found in juxtaposition to other lactotrophs (both types I and II) but rarely in association with either somatotrophs or mammosomatotrophs (Fig. 2). In light of these considerations, it is our view that the intermediate lactotrophs are most likely to represent a transitional form in the lactotrophic lineage.
In addition to considering the ontological origin of the intermediate lactotroph, there are a number of potential trophic factors that may promote the presence of this novel cell type.
First, because there is some evidence linking GRF overexpression with lactotroph hyperplasia (49, 50), the intermediate lactotroph may result from the marked elevation in arcuate GRF expression in the dw/dw rat (16, 51). This is possible because, although cAMP responses to GRF in dw/dw somatotrophs are impaired (18), the structure of the GRF receptor is normal in this model (52) and some of the actions of GRF are mediated by cAMP-independent pathways (23, 53, 54). Our data, however, do not support this hypothesis. Although the lack of lactotrophs matching the intermediate morphology in the lit/lit mouse implies a role for GRF in the generation of these cells, the failure of neonatal MSG treatment to affect the proportion of intermediate lactotrophs (Fig. 6) strongly suggests the contrary.
Some caution is necessary in interpreting the data obtained with MSG treatment. Although neonatal MSG treatment destroys 70–90% of neuronal perikarya in the arcuate nuclei, including the majority of GRF-positive neurons (27, 55), it clearly affects other neuronal populations in these heterogeneous nuclei. Nevertheless, it is interesting to note in passing that, despite evidence of suppressed residual GH secretion (Table 1), MSG treatment had no effect on the number of somatotrophs in the dw/dw pituitary. This implies that dw/dw somatotrophs are insensitive to the trophic influence of GRF and may equate to the previously described somatotroph progenitors (7) (STp in Fig. 9).
It is now clear that there is a subtle difference in phenotype between those models in which the primary defect is an absence of (or insensitivity to) GRF and those in which the GRF neurons are either functionally suppressed or ablated. Suppression or ablation of the GRF neurons results in lactotroph hypoplasia or reduced pituitary PRL content (15, 16), whereas in the dw/dw rat, the lit/lit mouse, and the GRF-knockout mouse (56), lactotroph number is increased, with either an elevated or unchanged pituitary PRL content. Thus, an alternative neuroendocrine product of the GRF neurons, such as GRF-related peptide (57), may promote lactotroph proliferation. However, the absence of lactotrophs matching the intermediate morphology in the lit/lit mouse and the failure of MSG treatment to modify the proportion of the intermediate lactotrophs in the dw/dw rat (Fig. 6) indicates that this particular subpopulation arises through a mechanism independent of the GRF neurons.
Third, our data do not provide consistent support for the hypothesis that the presence of the intermediate lactotrophs is estrogen dependent. Whereas the proportion of these cells was elevated during pregnancy (Fig. 8), in younger animals at least, these cells are more abundant in males (Fig. 1). That said, the sensitivity of the intermediate lactotrophs to ghrelin (Fig. 4) is, like that for the synthetic mimetic, GHRP-6 (24), sexually dimorphic and probably estrogen dependent. Although hypothalamic expression of the cognate receptor, GHS-R, is up-regulated in dw/dw rats, it is not sexually dimorphic (58). Whether this is so in the pituitary, in which the level of GHS-R transcripts is much lower, has yet to be determined. Indeed, we cannot exclude the possibility that ghrelin-induced PRL secretion in the dw/dw rat may be mediated by a receptor other than GHS-R1a.
Although we have no direct evidence, it seems unlikely that elevated tuberoinfundibular dopamine in the dw/dw rat (22) gives rise to the intermediate lactotrophs because dopamine exerts a powerful antiproliferative influence on the pituitary, particularly on the lactrotroph lineage (10, 59). Thus, we are unable at present to provide conclusive evidence of the factors resulting in the presence of this unusual cell type. In this context, it is likely that the unknown autosomal dominant mutation that gives rise to the phenotypic peculiarities now described in the dw/dw rat interrupts the normal progression of cell fate determination in the pituitary. Because none of the rodent models with defined genotypic abnormalities have been shown to share this precise pituitary phenotype, the mechanism giving rise to the intermediate lactotroph and the significance of this cell in the process of normal pituitary development will be elucidated only by the identification of the dw/dw mutation and the subsequent cloning of the normal gene.
In summary, we have demonstrated the presence of a novel, morphologically distinct lactotroph, the intermediate lactotroph in the pituitary of the dw/dw rat. The responsiveness of these cells to ghrelin in female dwarves may contribute to the estrogen-dependent GHS-induced PRL secretion previously observed in this strain. The absence of lactotrophs bearing this intermediate morphology in normal rats or similar rodent models of dwarfism suggests that this cell may be uniquely observable in the dw/dw rat.
Acknowledgments
The authors thank Dr. Karin Fhlenhag (Pharmacia, Uppsala, Sweden, now at Biovitrum, Stockholm, Sweden) for the generous gift of rat ghrelin; Novo Nordisk A/S (Bagsvrd, Denmark) for the gift of rGRF; National Institute of Diabetes and Digestive and Kidney Diseases for the provision of rGH and rPRL assay reagents; Phill Blanning and Dr. Stanislav Glasewski (Cardiff University) for help with transcardial perfusions; and Miss Sarah Rogers and Mrs. Lynne Scott (Oxford University) for technical support.
Footnotes
This work was supported by the Biotechnology and Biosciences Research Council (United Kingdom; Grant 72/S11914; Research Committee Studentship Grant 99/B1/S/05486) and Wellcome Trust Grant 051887/C/97/Z/MW/NP/JF (to H.C.C.).
1 I.H.-O. and H.C.C. contributed equally to this work.
Abbreviations: AS, Albino-Swiss; EM, electron microscopy; GHS-R, GH-secretagogue receptor; GRF, GH-releasing factor; MSG, monosodium glutamate; PRL, prolactin; r, recombinant.
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Laboratory for Cellular Endocrinology (I.H.-O., H.C.C.), Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
Abstract
Profound somatotroph hypoplasia in the dwarf (dw/dw) rat is accompanied by an estrogen-dependent induction of prolactin secretion by the GH secretagogue, GHRP-6. Using electron microscopy, we demonstrated that the reduction in the somatotroph population in the dw/dw pituitary is accompanied by the presence of a morphologically distinct lactotroph subpopulation. In these cells, which did not coexpress GH, the size, shape, and number of the secretory granules were between those of the type I and type II lactotrophs. We therefore called these cells intermediate lactotrophs. The intermediate lactotrophs accounted for up to 30% of the total prolactin-positive cell population in dw/dw males and up to 12% in females. Using tannic acid to quantify the fusion of secretory granules, we have shown that the intermediate lactotrophs are unresponsive to either GH-releasing factor (GRF) or TRH but exhibit a sexually dimorphic secretory response to acute ghrelin treatment, granular fusions being 4-fold higher in females. No cell matching the morphology of the novel lactotroph subpopulation was observed in the pituitary of the GRF-insensitive lit/lit mouse. However, ablation of GRF neurons with neonatal monosodium glutamate treatment had no effect on the population of intermediate lactotrophs in the dw/dw rat. Thus, the presence of the intermediate lactotrophs in the dw/dw pituitary appears to be independent of the function of the GRF neurons.
Introduction
THE DIFFERENTIATION OF secretory cell types during pituitary organogenesis is a multistep process regulated by the spatiotemporal expression of transcription factors, cytokines, and neuropeptides (reviewed in Refs.1, 2). Commitment to the somatotroph, lactotroph, and thyrotroph lineage is dependent on the sequential expression of the transcription factors, Prophet of Pit-1 (Prop-1) (3) and the pituitary transcription factor Pit-1 (4). Subsequently, somatotrophs and lactotrophs are thought to differentiate from a common bihormonal progenitor, the mammosomatotroph (5, 6), but knowledge of the factors regulating this process is incomplete. Proliferation of the monohormonal GH-containing cells is promoted by GH-releasing factor (GRF) (7), whereas expansion of the prolactin (PRL)-containing cell population is regulated by TGF (8), nerve growth factor (9), dopamine (10), and estrogen (11, 12). Mutations in either Prop-1 (3, 13) or Pit-1 (4, 13, 14) and reduction of hypothalamic GRF production (15, 16) usually lead to suppression of both somatotroph and lactotroph development and an associated dwarfism. In contrast, the dwarf (dw/dw) rat displays a unique adenohypophysial phenotype.
In the dw/dw rat, an unknown mutation leads to a profound reduction in somatotroph number (17, 18) and pituitary GH content (19), leading to low amplitude episodes of GH secretion (20, 21). Although pituitary PRL content in this model was initially thought to be reduced (19), subsequent evidence indicated that the suppression of somatotroph development is accompanied by an increase in both lactotroph number and PRL secretion (17, 18, 22, 23). Because it is unclear how these changes affect the distinct lactotroph subpopulations, we used immunogold labeling and electron microscopy (EM) to quantify the postnatal development of individual secretory cell types in the dw/dw rat.
Another unique feature of the dw/dw phenotype is that PRL secretion is elicited by the ghrelin mimetic, GHRP-6 (24). This effect is estrogen dependent, being female specific, abolished by ovariectomy, and induced in males by estrogen treatment (24). We therefore determined the sensitivity of the somatotrophic and lactotrophic axes in the dw/dw rat to a range of secretagogues, including ghrelin, the cognate endogenous ligand of the GH-secretagogue receptor (GHS-R1a) (25). Sensitivity has been determined in vivo by quantifying both circulating hormone levels and the responses of individual pituitary cell types, the latter being performed by tannic acid perfusion in conjunction with EM analysis. These experiments reveal a unique, morphologically distinct, ghrelin-responsive lactotroph in the pituitary of the dw/dw rat, which we have termed the intermediate lactotroph.
To establish whether the occurrence of these cells is regulated by the high levels of hypothalamic GRF, we analyzed the lactotroph populations in dw/dw rats after neonatal monosodium glutamate (MSG) treatment and in GRF-insensitive lit/lit mice (7). In addition, to determine whether the population of these cells is regulated in parallel with the somatotroph or lactotroph populations by the steroid hormones, we quantified the population of somatotrophs and lactotrophs in pregnant dw/dw rats. A preliminary account of sections of this work has previously been communicated (26).
Materials and Methods
Dwarf (dw/dw) and wild-type [Albino-Swiss (AS)] rats
The animal procedures described conformed to the institutional and national ethical guidelines for in vivo experiments in rats and were approved by local ethical review. Homozygous dw/dw rats, maintained on an AS background, and AS rats were derived from colonies at the National Institute for Medical Research (London, UK) and bred in the Transgenic Unit (School of Biosciences, Cardiff University). These rats were housed under conditions of 14-h light, 10-h dark (lights on at 0500 h), with rat chow and water available ad libitum.
Study 1: development of secretory cell populations in the dw/dw pituitary
Male and female dw/dw and AS rats were killed at 5, 30, and 60 d old. Pituitaries were excised and fixed immediately in 2.5% glutaraldehyde (EM grade) in PBS (pH 7.2) at room temperature. Pituitaries were subsequently transferred into 0.25% glutaraldehyde in PBS at 4 C until processing for EM and analysis of somatotroph, mammosomatotroph, and lactotroph populations (see below).
Study 2: neuroendocrine control of intermediate lactotrophs
Secretagogue-induced GH and PRL secretion in conscious dw/dw rats.
Male and female dw/dw rats [12 wk old; weighing 146–171 g (males) and 109–121 g (females)] were prepared with single-bore jugular vein cannulae under halothane anesthesia. After at least 48 h recovery, 120-μl blood samples were taken before and at 5, 15, and 30 min after a bolus iv injection of either vehicle [300 μl sterile saline containing BSA (1 mg/ml) and heparin (10 U/ml)], GRF [1 μg rat GRF(1–29)NH2 (Bachem, Bubendorf, Switzerland, generously supplied by Novo Nordisk A/S, Bagsvrd, Denmark)], ghrelin [10 μg rat ghrelin (generously supplied by Pharmacia & Upjohn, Uppsala, Sweden)], or TRH [1 μg Protirelin (Roche, Cambridge, UK)]. After 48 h this sampling procedure was repeated, each animal receiving an alternative treatment. Blood samples were taken into heparinized tubes and centrifuged, separated plasma subsamples being stored at –20 C for subsequent determination of plasma rat (r)GH and rPRL concentration (see below).
Secretagogue-induced responses in individual secretory cell types in dw/dw rats.
To quantify the cellular responses to each secretagogue treatment, dw/dw rats received a third (alternative) secretagogue treatment at least 48 h after the second period of sampling, in conjunction with tannic acid perfusion to capture exocytosed secretory granules. After secretagogue treatment, dw/dw rats were reanesthetized with sodium pentobarbitone (Euthatal, Vericore Ltd., Dundee, UK) and prepared for transcardial perfusion. Seven minutes after secretagogue treatment, the rats were perfused (5 ml/min) with heparinized saline (37 C for 3 min), followed by tannic acid (0.2% tannic acid in PBS at 37 C for 5 min). Pituitaries were excised, immersed in tannic acid for 2 min, fixed in 2.5% glutaraldehyde in PBS for 2 h, and stored in 0.25% glutaraldehyde in PBS at 4 C until processing for EM analysis.
Study 3: regulation of the intermediate lactotroph population by GRF
The effect of neonatal MSG treatment on somatotroph and lactotroph populations in dw/dw rats.
To determine whether the existence of the intermediate lactotrophs is brought about by the high levels of hypothalamic GRF expression in the dw/dw rat (16), we quantified the somatotroph and lactotroph populations in dw/dw rats after ablation of the arcuate GRF neurons with neonatal MSG treatment (27). Three litters of dw/dw rats (eight to 10 pups/litter) received ip injections of either vehicle (50 μl of 0.9% sterile saline) or MSG (4 mg/g body weight in 50 μl vehicle) on postnatal d 2, 4, 6, 8, and 10 and were carefully monitored for potential adverse effects. At 8 wk of age, these rats were weighed, stunned, and decapitated. Pituitaries were excised, weighed, and treated as in study 1 before processing for EM analysis. In addition, the liver was dissected and weighed, and the left tibiae were excised and the length measured using a handheld micrometer.
Quantification of somatotroph and lactotroph populations in male lit/lit mice.
To investigate whether intermediate lactotrophs are detectable in other models of GRF insensitivity, we quantified the somatotroph and lactotroph subpopulations in pituitaries from 8-wk-old male wild-type (C57BL/6J) and homozygous lit/lit (C57BL/6J-Ghrhr) mice (Jackson Laboratories, Bar Harbor, ME). lit/lit mice bear an inactivating point mutation in the GRF receptor (7). These mice were killed by cervical dislocation and the pituitaries excised and treated as in study 1 before shipment from Jackson Laboratories to Oxford UK, and subsequent processing for EM analysis.
Study 4: effect of pregnancy on somatotroph and lactotroph populations in the dw/dw pituitary
To determine whether the population of intermediate lactotrophs is regulated in parallel with the somatotrophs or lactotrophs by the steroid hormones, the populations of these secretory cells were determined in female dw/dw rats during pregnancy. Female dw/dw rats (12–15 wk old) were mated overnight with dw/dw males, mating being confirmed by the presence of a vaginal plug. After 2 wk these pregnant rats and a group of virgin female littermates were anesthetized with halothane and killed by decapitation. Pregnancy was confirmed post mortem, and pituitaries were excised and treated as in study 1 before processing for EM analysis.
Tissue processing and EM
Pituitaries for EM analysis were processed and analyzed as previously described (28, 29). Briefly, the tissue was postfixed in osmium tetroxide [1% (wt/vol) in 0.1 M PBS], contrasted with uranyl acetate [2%(wt/vol) in distilled water], dehydrated in ethanol, and embedded in Spurr’s resin. Ultrathin sections (50–80 nm) were taken using a Reichart-Jung ultracut microtome and mounted on nickel grids. Sections from dw/dw rat pituitaries were incubated for 2 h at room temperature with primary antibodies for either rGH [1:2000 dilution of monkey antirat GH; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)] or rPRL (1:2000 dilution of rabbit antirat PRL; NIDDK) followed by a 1-h incubation with either a 15-nm protein A gold-conjugated goat antimonkey or a 5-nm protein A gold-conjugated goat antirabbit secondary antibody (British Biocell, Cardiff, UK). For identification of bihormonal rGH- and rPRL-positive cells, sections were labeled for rPRL on d 1 and for rGH on the following day. All antibodies were diluted in 0.1 M PBS (containing 0.1% egg albumin), and in control sections the primary antibody was replaced with nonimmune rabbit serum. Sections from lit/lit mouse pituitaries were immunolabeled using the same antibodies used above because these cross-react well with mouse PRL and GH. Finally, sections were counterstained with lead citrate and uranyl acetate and examined on a JOEL 1010 transmission electron microscope (JOEL USA Inc., Peabody, MA).
For each pituitary, four randomly orientated sections, each containing six or nine grid squares, were counted for each hormone in rats and mice, respectively. The total secretory cell population was determined by counting the number of cells per grid, with individual somatotrophs and lactotrophs identified using established identification criteria (30, 31) in conjunction with the immunogold labeling. In brief, somatotrophs contain large numbers of large (>300 nm diameter) spherical electron-dense secretory granules; type I lactotrophs contain small numbers of large (>300 nm diameter) irregularly shaped granules; type II lactotrophs contain large numbers of small (<200 nm diameter) spherical granules. In addition, the bihormonal mammosomatotrophs were identified by the presence of granular immunogold labeling for both GH and PRL. The secretory response of individual cells from tannic acid perfused rats was determined by counting the number of exocytosed granules fused with the plasma membrane of each cell, with a minimum of six cells counted for each cell type per pituitary.
Plasma hormone analysis
Plasma GH and PRL concentrations were determined by RIA using delayed addition of label. The results are expressed in terms of the reference preparations RP-2 (rGH) and RP-3 (rPRL), using the reagents generously supplied by National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) [intraassay variation 6.04% (rGH) and 5.04% (rPRL); range 0.12–250 ng/ml (rGH) and 0.0480–100 ng/ml (rPRL)].
Statistical analysis
All data shown are mean ± SEM (n = 3–5 for EM analyses; n = 5–6 for hormone and tissue analyses), with multiple statistical comparisons performed by either unpaired Student’s t test, or one-way ANOVA followed by either Dunnett’s (vs. control) or Bonferroni’s (for selected pairs) post hoc tests, as indicated in figure legends.
Results
Study 1: development of secretory cell populations in the dw/dw pituitary
During postnatal pituitary development, a 50–60% reduction in number of secretory cells per grid was seen in AS rats (Fig. 1, A and G), which normally accompanies secretory cell enlargement. A similar, though less pronounced, decline (25–30%; P < 0.001) was also observed in dw/dw rats (Fig. 1, A and G), the total secretory cell number remaining higher than in their wild-type counterparts at 30 and 60 d of age (P < 0.001).
In AS rats the proportion of somatotrophs increased rapidly between 5 and 60 d (Fig. 1B; P < 0.001). In contrast, there was no significant increase in the proportion of somatotrophs in either male (Fig. 1B) or female (Fig. 1H) dw/dw rats, the proportion of somatotrophs remaining less than 15% of that in their AS counterparts (P < 0.001). A similar developmental pattern in the small proportion of mammosomatotrophs was seen in AS and dw/dw males (Fig. 1C), but in AS and dw/dw females, the proportion of mammosomatotrophs remained constant (Fig. 1I). However, none of the means for mammosomatotrophs were significantly different.
The combined proportion of all PRL-positive cells was higher in dw/dw males at 5 and 60 d of age (P < 0.001) and dw/dw females at 60 d of age (P < 0.05). However, the total proportion of PRL-positive cells in dw/dw rats was transiently lower at 30 d of age (P < 0.01).
In AS males the proportion of type II lactotrophs increased 4-fold between 5 and 30 d of age (Fig. 1E; P < 0.001), but was halved by 60 d (P < 0.01). A similar biphasic pattern was observed in AS females (Fig. 1K; P < 0.001), although the proportion of type II lactotrophs was 30–40% lower than in AS males (P < 0.05). At 5 d old, the proportion of type II lactotrophs in dw/dw males was double that in AS males (Fig. 1E; P < 0.001) and, after an initial decline (P < 0.01), was 70% higher than in AS males at 60 d (P < 0.001). In dw/dw females, the proportion of type II lactotrophs progressively declined and was 70% lower than in AS females at 30 and 60 d old (Fig. 1K; P < 0.001).
A similar developmental pattern in the proportion of type I lactotrophs was seen in AS males (Fig. 1D). In dw/dw males, the proportion of type I lactotrophs increased over the period studied (Fig. 1D; P < 0.001), this population being similar to that seen in AS males at 60 d of age. A progressive increase in type I lactotrophs was observed in both AS and dw/dw females (P < 0.001), these cells accounting for 35–40% of the total secretory cell population at 60 d (Fig. 1J).
Interestingly, we identified a population of lactotrophs in the dw/dw pituitary whose morphology did not conform to the established identification criteria (30, 31). These cells had a granular appearance between that of the two established lactotroph types, i.e. containing large numbers of large (>300 nm diameter), regularly shaped, electron-dense granules (Fig. 2, B, E, and G) and for this reason are subsequently referred to as intermediate lactotrophs. These granules were positively labeled for PRL (Fig. 2, B and E, insets) but did not coexpress GH. The intermediate lactotrophs were found to occur in direct juxtaposition with either type I (Fig. 2G) or type II (Fig. 2E) lactotrophs but seldom in association with somatotrophs or mammosomatotrophs. The proportion of intermediate lactotrophs was highest at 30 d, accounting for 7 and 3% of the total secretory cell population in males and females, respectively (Fig. 1, F and L). Although the number of these cells declined in the female dw/dw pituitary by 60 d, the population was maintained in dw/dw males. An occasional cell matching this description was also observed in the normal rat pituitary at 30 d (1%; Fig. 1, F and L).
Study 2: neuroendocrine control of intermediate lactotrophs
Secretagogue-induced GH and PRL secretion in conscious dw/dw rat.
GRF elevated circulating GH in both male and female dw/dw rats (P < 0.01), the response being twice as high in females (Fig. 3, A and C). In contrast, the robust ghrelin-induced GH secretion was similar in both sexes (Fig. 3, A and C). Injection of TRH had no significant effect on GH secretion.
As expected, TRH treatment elevated circulating PRL in dw/dw rats, eliciting a small increase in males (Fig. 3B; P < 0.05) and a more robust (P < 0.01) response in females (Fig. 3D; P < 0.01). In male dw/dw rats, ghrelin treatment had no effect on circulating PRL secretion (Fig. 3B), but in females ghrelin elicited a similar response to TRH (Fig. 3D; P < 0.05 vs. vehicle-treated controls). PRL secretion was unaffected by GRF treatment.
Secretagogue-induced responses in individual secretory cell types in dw/dw rat.
In conjunction with the circulating hormone data, we performed quantification of granular fusions in individual cell types after secretagogue treatment. Somatotrophs responded consistently to both GRF and ghrelin treatment, GRF-induced granular fusion being higher in females than males (Fig. 4, A and E; P < 0.05). TRH did not elicit granular fusion in somatotrophs. Type I lactotrophs were unresponsive to secretagogue treatment in males (Fig. 4D) and produced only a small, inconsistent (<1 granule/cell) response to ghrelin in females (Fig. 4H). TRH treatment elicited a robust granular fusion in type II lactotrophs in both sexes (Fig. 4, B and F). Type II lactotrophs were unresponsive to GRF (Fig. 4, B and F), but ghrelin induced a small granular response in females (Fig. 4F).
The morphologically distinct intermediate lactotrophs identified in the dw/dw pituitary displayed a unique response pattern to secretagogue treatment. Although unresponsive to either GRF or TRH, these cells showed significant granular fusion after ghrelin administration in both male and female dw/dw rats (Fig. 4, C and G). This response was 4-fold higher in females (P < 0.001). Examples of the granular fusion elicited by ghrelin treatment in male and female dw/dw rats are shown in Fig. 5, C and D.
Study 3: regulation of the intermediate lactotroph population by GRF
The effect of neonatal MSG treatment on somatotroph and lactotroph populations in dw/dw rats.
Although there was no significant effect on body weight, neonatal MSG treatment caused a small reduction in tibial length (Table 1; P < 0.05), accompanied by an elevation in visceral adiposity (P < 0.001; data not shown).
Neonatal MSG treatment had no effect on either pituitary weight (Table 1) or the proportion of somatotrophs (Fig. 6). However, MSG treatment resulted in a decline in the proportion of type I lactotrophs in male (25%; P < 0.01) and female (10%; P < 0.05) dw/dw rats (Fig. 6). The proportion of type II lactotrophs was halved by MSG treatment in dw/dw males (P < 0.001) but more than doubled in females (P < 0.05). Although the mean proportion of intermediate lactotrophs in MSG-treated males was only half of that in the vehicle-treated males, the means were not significantly different. In dw/dw females the proportion of intermediate lactotrophs was not significantly reduced by MSG treatment.
Quantification of somatotroph and lactotroph populations in male lit/lit mice.
To investigate whether this population of intermediate lactotrophs arises from an insensitivity to GRF, we examined the somatotroph and lactotroph populations in male lit/lit mice. As expected, the proportion of somatotrophs in lit/lit mice was only a third of that in wild-type mice (Fig. 7; P < 0.01), and the proportion of mammosomatotrophs was halved (P < 0.01). Conversely, the proportion of type II lactotrophs was almost doubled (Fig. 7; P < 0.01), and there was a small increase in the proportion of type I lactotrophs (P < 0.05). However, we were unable to detect any lactotrophs in the pituitaries of lit/lit mice corresponding to the morphology of the intermediate lactotroph.
Study 4: effect of pregnancy on somatotroph and lactotroph populations in the dw/dw pituitary
To determine whether the intermediate lactotrophs are regulated in parallel with the somatotroph or lactotroph populations by the steroid hormones, we quantified the secretory cell populations in 2-wk pregnant dw/dw rats. Although the proportion of intermediate lactotrophs in dw/dw females was almost doubled during pregnancy (Fig. 8; P < 0.01), the proportion of somatotrophs and types I and II lactotrophs were not significantly altered.
Discussion
Phenotypic analysis of mutant murine models has greatly facilitated our understanding of pituitary organogenesis and the transcription factors regulating cell lineage determination. However, our knowledge of the factors regulating the postnatal expansion of secretory cell populations is less advanced. Failure in somatotroph development is usually accompanied by lactotroph hypoplasia (4, 13, 14, 15), but in the dw/dw rat, the profound reduction in somatotrophs has been reported to be accompanied by lactotroph hyperplasia (17, 18, 22, 23). Our use of EM to quantify the development of secretory cell subpopulations has led to the identification of a novel lactotroph in the dw/dw pituitary, the intermediate lactotroph. The presence of this cell and its unique pattern of neuropeptide responses may contribute to some of the endocrine peculiarities of the dw/dw phenotype.
In the nonneoplastic anterior pituitary, the lactotrophs account for up to 50% of the total secretory cell population and are significantly higher in females (Fig. 1). Our examination of secretory cell development in dw/dw rats confirmed that profound somatotroph hypoplasia is accompanied by a significant expansion of the lactotroph population (study 1), which is seen most clearly in males. Although it is possible that the observed lactotroph hyperplasia is the result of expressing the secretory cell populations in relative terms, in the context of a profound reduction in the predominant secretory cell type (somatotrophs) (32), our data are consistent with the reported increase in pituitary PRL content (22, 23) in the dw/dw rat.
PRL-containing cells can be subdivided into distinct subpopulations on the basis of either morphology or function. Morphological subdivision is achieved under EM using established identification criteria (30, 31), which are based on the size, shape, and number of the electron-dense secretory granules. Briefly, type I lactotrophs contain sparse, large (>300 nm diameter), irregularly shaped granules, and type II lactotrophs display a large number of smaller (<200 nm diameter) spherical granules (Fig. 2). Using these criteria, the most remarkable feature we observed in the dw/dw pituitary was the presence of a subpopulation of lactotrophs with distinct morphological features that did not conform to the established type I and type II lactotrophs or to the less well-described type III lactotrophs (33). These novel cells, which did not coexpress GH, exhibited large numbers of large (>300 nm diameter), regularly shaped, electron dense granules (Fig. 2, B, E, and G) and accounted for up to 30% of the total PRL-positive cell population in dw/dw males and up to 12% in females, the highest proportion being observed at 30 d of age (Fig. 1, F and L). Due to their morphological appearance being between that of the types I and II lactotrophs, we called these cells intermediate lactotrophs.
Subdivision of lactotrophs on the basis of morphology does not correspond exactly with functional classification (34, 35). To characterize the neuroendocrine regulation of PRL secretion in the intermediate lactotrophs, we quantified secretory activity of individual cells under EM after neuropeptide treatment in conjunction with tannic acid to capture exocytosed granules. This revealed that the intermediate lactotrophs displayed a distinctive pattern of responses to secretagogue exposure. As we have previously reported in normal rats (36, 37), type I lactotrophs were almost completely unresponsive to neuropeptide treatment (Fig. 4, D and H), whereas type II lactotrophs showed a robust exocytotic response to TRH (Fig. 4, B and F). In contrast to these established lactotroph subtypes, the intermediate lactotrophs were unresponsive to TRH but displayed a prominent secretory response to ghrelin (Fig. 5), which was particularly marked in females (Fig. 4G). This ghrelin-induced granular fusion was 4-fold higher than that seen in either type I or type II lactotrophs (Fig. 4, H and F).
It should be noted that although ghrelin also induced granular fusion in male intermediate lactotrophs (Fig. 4C), a significant increase in circulating PRL was observed only in females (Fig. 3D). This may be due to a higher expression of PRL in individual lactotrophs of dw/dw females, as suggested by previous data (the differential in pituitary PRL content between male and female dw/dw rats being 4-fold higher than the differential in lactotroph number) (23). Should a higher PRL expression in individual lactotrophs of dw/dw females be confirmed, this might also explain the larger increase in circulating PRL seen in response to TRH (Fig. 3D). Alternatively, the difference between the granular responses and the observed changes in circulating PRL may result from a higher sensitivity to ghrelin in all lactotroph subtypes in female dwarves.
In the hypothalamo-pituitary GH axis, ghrelin induces GH secretion predominantly through activation of arcuate GRF neurons (38, 39), with a smaller direct action on the somatotrophs (25, 40). The induction of PRL secretion by ghrelin in dw/dw rats is unlikely to be mediated by GRF because, in contrast to the somatotrophs (Fig. 4, A and B), none of the lactotroph subtypes were responsive to GRF (at a dose that produced a comparable granular response to ghrelin in somatotrophs). This suggests that in the dw/dw pituitary, the intermediate lactotrophs may express the cognate receptor for ghrelin (GHS-R1a) (25, 41), although this remains to be established. We propose that the presence of the novel intermediate lactotroph in dw/dw rats and its unique pattern of secretagogue regulation may contribute to the estrogen-dependent elevation in PRL secretion elicited by ghrelin and the synthetic GH-secretagogues in this model (24).
However, our observation of a population of morphologically and functionally distinct lactotrophs may have wider implications for the regulation of both cell lineage determination and plasticity between the pituitary cell types. A number of ontological mechanisms may give rise to the presence of this novel lactotroph in the dw/dw rat. These are summarized in Fig. 9.
First, the novel lactotroph may represent an intermediate form between that of the type I and type II lactotrophs (Fig. 9A), as suggested from the shape, size, and number of the secretory granules (Fig. 2). This possibility is also supported by the parallel regulation of these cells with type I and II lactotrophs during pregnancy (Fig. 8) when interconversion between the established lactotroph subtypes is known to occur (42). However, the lack of responsiveness of these cells to TRH contrasts sharply with the prominent response in type II lactotrophs (Fig. 4).
It has previously been reported that during pregnancy there is an inverse relationship between the population of somatotrophs and lactotrophs, lactotroph hyperplasia resulting from the transdifferentiation of somatotrophs (43, 44, 45). Given the profound somatotroph hypoplasia in the dw/dw pituitary, it is possible that the population of novel lactotrophs may arise from a similar process of transdifferentiation (Fig. 9C). Alternatively, because it has been suggested that the mammosomatotrophs are obligatory intermediates in this interconversion (46), the intermediate lactotrophs may arise from the differentiation of these bihormonal cells (Fig. 9B). Although we have no direct evidence to support either of these origins, there are notable structural and functional similarities between the intermediate lactotroph and the somatotroph, and the proportion of somatotrophs that coexpress GH and PRL is considerably increased in the dw/dw pituitary (23). A fourth option is that the intermediate lactotroph represents the product of transdifferentiation from another unspecified secretory cell type (Fig. 9D).
In this ontological context, the location of the intermediate lactotrophs relative to the neighboring secretory cells is worthy of consideration. Recent evidence from a transgenic mouse model with somatotroph-specific green fluorescent protein expression (47) indicates that all somatotrophs are directly associated with a neighboring somatotroph, forming a continuous somatotrophic network (48). It is probable that an equivalent lactotrophic network also exists. In the present study, the novel PRL-producing cells were always found in juxtaposition to other lactotrophs (both types I and II) but rarely in association with either somatotrophs or mammosomatotrophs (Fig. 2). In light of these considerations, it is our view that the intermediate lactotrophs are most likely to represent a transitional form in the lactotrophic lineage.
In addition to considering the ontological origin of the intermediate lactotroph, there are a number of potential trophic factors that may promote the presence of this novel cell type.
First, because there is some evidence linking GRF overexpression with lactotroph hyperplasia (49, 50), the intermediate lactotroph may result from the marked elevation in arcuate GRF expression in the dw/dw rat (16, 51). This is possible because, although cAMP responses to GRF in dw/dw somatotrophs are impaired (18), the structure of the GRF receptor is normal in this model (52) and some of the actions of GRF are mediated by cAMP-independent pathways (23, 53, 54). Our data, however, do not support this hypothesis. Although the lack of lactotrophs matching the intermediate morphology in the lit/lit mouse implies a role for GRF in the generation of these cells, the failure of neonatal MSG treatment to affect the proportion of intermediate lactotrophs (Fig. 6) strongly suggests the contrary.
Some caution is necessary in interpreting the data obtained with MSG treatment. Although neonatal MSG treatment destroys 70–90% of neuronal perikarya in the arcuate nuclei, including the majority of GRF-positive neurons (27, 55), it clearly affects other neuronal populations in these heterogeneous nuclei. Nevertheless, it is interesting to note in passing that, despite evidence of suppressed residual GH secretion (Table 1), MSG treatment had no effect on the number of somatotrophs in the dw/dw pituitary. This implies that dw/dw somatotrophs are insensitive to the trophic influence of GRF and may equate to the previously described somatotroph progenitors (7) (STp in Fig. 9).
It is now clear that there is a subtle difference in phenotype between those models in which the primary defect is an absence of (or insensitivity to) GRF and those in which the GRF neurons are either functionally suppressed or ablated. Suppression or ablation of the GRF neurons results in lactotroph hypoplasia or reduced pituitary PRL content (15, 16), whereas in the dw/dw rat, the lit/lit mouse, and the GRF-knockout mouse (56), lactotroph number is increased, with either an elevated or unchanged pituitary PRL content. Thus, an alternative neuroendocrine product of the GRF neurons, such as GRF-related peptide (57), may promote lactotroph proliferation. However, the absence of lactotrophs matching the intermediate morphology in the lit/lit mouse and the failure of MSG treatment to modify the proportion of the intermediate lactotrophs in the dw/dw rat (Fig. 6) indicates that this particular subpopulation arises through a mechanism independent of the GRF neurons.
Third, our data do not provide consistent support for the hypothesis that the presence of the intermediate lactotrophs is estrogen dependent. Whereas the proportion of these cells was elevated during pregnancy (Fig. 8), in younger animals at least, these cells are more abundant in males (Fig. 1). That said, the sensitivity of the intermediate lactotrophs to ghrelin (Fig. 4) is, like that for the synthetic mimetic, GHRP-6 (24), sexually dimorphic and probably estrogen dependent. Although hypothalamic expression of the cognate receptor, GHS-R, is up-regulated in dw/dw rats, it is not sexually dimorphic (58). Whether this is so in the pituitary, in which the level of GHS-R transcripts is much lower, has yet to be determined. Indeed, we cannot exclude the possibility that ghrelin-induced PRL secretion in the dw/dw rat may be mediated by a receptor other than GHS-R1a.
Although we have no direct evidence, it seems unlikely that elevated tuberoinfundibular dopamine in the dw/dw rat (22) gives rise to the intermediate lactotrophs because dopamine exerts a powerful antiproliferative influence on the pituitary, particularly on the lactrotroph lineage (10, 59). Thus, we are unable at present to provide conclusive evidence of the factors resulting in the presence of this unusual cell type. In this context, it is likely that the unknown autosomal dominant mutation that gives rise to the phenotypic peculiarities now described in the dw/dw rat interrupts the normal progression of cell fate determination in the pituitary. Because none of the rodent models with defined genotypic abnormalities have been shown to share this precise pituitary phenotype, the mechanism giving rise to the intermediate lactotroph and the significance of this cell in the process of normal pituitary development will be elucidated only by the identification of the dw/dw mutation and the subsequent cloning of the normal gene.
In summary, we have demonstrated the presence of a novel, morphologically distinct lactotroph, the intermediate lactotroph in the pituitary of the dw/dw rat. The responsiveness of these cells to ghrelin in female dwarves may contribute to the estrogen-dependent GHS-induced PRL secretion previously observed in this strain. The absence of lactotrophs bearing this intermediate morphology in normal rats or similar rodent models of dwarfism suggests that this cell may be uniquely observable in the dw/dw rat.
Acknowledgments
The authors thank Dr. Karin Fhlenhag (Pharmacia, Uppsala, Sweden, now at Biovitrum, Stockholm, Sweden) for the generous gift of rat ghrelin; Novo Nordisk A/S (Bagsvrd, Denmark) for the gift of rGRF; National Institute of Diabetes and Digestive and Kidney Diseases for the provision of rGH and rPRL assay reagents; Phill Blanning and Dr. Stanislav Glasewski (Cardiff University) for help with transcardial perfusions; and Miss Sarah Rogers and Mrs. Lynne Scott (Oxford University) for technical support.
Footnotes
This work was supported by the Biotechnology and Biosciences Research Council (United Kingdom; Grant 72/S11914; Research Committee Studentship Grant 99/B1/S/05486) and Wellcome Trust Grant 051887/C/97/Z/MW/NP/JF (to H.C.C.).
1 I.H.-O. and H.C.C. contributed equally to this work.
Abbreviations: AS, Albino-Swiss; EM, electron microscopy; GHS-R, GH-secretagogue receptor; GRF, GH-releasing factor; MSG, monosodium glutamate; PRL, prolactin; r, recombinant.
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