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Endocrinology Vol. 141, No. 4 1515-1520
Copyright © 2000 by The Endocrine Society

ARTICLES

Localization of Leptin Receptor-Like Immunoreactivity in the Corticotropes, Somatotropes, and Gonadotropes in the Ovine Anterior Pituitary1

Javed Iqbal, Sueli Pompolo, Robert V. Considine and Iain J. Clarke

Prince Henry’s Institute of Medical Research (J.I., S.P., I.J.C.), Clayton, Victoria 3168, Australia; and Department of Medicine (R.V.C.), Indiana University, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Professor Iain J. Clarke, Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia. E-mail: iain.clarke{at}med monash.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin is a secreted product of the adipocytes that regulates a variety of functions. The presence of the leptin receptor (LR) has been demonstrated in the endocrine and neuroendocrine tissue, but only limited information is available regarding cell-specific expression in the anterior pituitary gland. We have used double-label immunofluorescence histochemistry to study the distribution of LR-like immunoreactivity (LR-ir) in the corticotropes, somatotropes, and gonadotropes of the ovine anterior pituitary. LR-ir was found in 34% of cells in the pars distalis and 94% of the cells in the pars tuberalis. In the pars distalis, LR-ir was present in 27% of corticotropes, 69% of somatotropes, and 29% of gonadotropes. In contrast, 90% of the gonadotropes in the pars tuberalis were immunopositive for LR. There was no alteration in the number of gonadotropes containing LR-ir during the various phases of the estrous cycle (n = 3/group) in the pars distalis (luteal phase, 36%; follicular phase, 32%; and estrous phase, 32%). In conclusion, we show that, in the pars distalis, LR-ir is expressed to a greater extent in the somatotropes than in the gonadotropes or corticotropes. This is in accordance with the documented effects of leptin on pituitary GH secretion. The differential expression of LR-ir between the gonadotropes of the pars distalis and pars tuberalis probably reflects the different phenotypes of the cells in these two regions. Lower levels of LR-ir expression in gonadotropes and corticotropes of the pars distalis may suggest that leptin does not substantially influence these particular cells, at least in this species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN is a hormone secreted from the adipocytes (1) that is involved in energy homeostasis (2, 3, 4) and in regulation of neuroendocrine (5, 6, 7, 8) and reproductive functions (9, 10, 11, 12). A deficiency in leptin or the leptin receptor (LR) causes disturbances in feeding behavior (13) and reproductive function (14) in various rodent models, and these effects can be corrected by exogenous leptin administration. Leptin can prevent the reduction in gonadotropin secretion that occurs with starvation (5, 15) and also appears to regulate GH secretion (7, 8, 16, 17). In rodents, leptin may also regulate the hypothalamo-pituitary-adrenal (HPA) axis (18) and the hypothalamo-pituitary-thyroid axis (19), but this is not the case in normally fed sheep (20).

In various species, imunohistochemical and in situ hybridization studies have demonstrated LR protein and its messenger RNA (mRNA) in several tissues including the hypothalamus and pituitary gland (21, 22, 23, 24, 25). A major site of leptin action is thought to be the hypothalamus where LR expression is seen in various neuropeptidergic neurons (25, 26, 27). Apart from a role in the regulation of appetite, mechanisms whereby leptin might influence neuroendocrine functions are not yet clear. Leptin could modulate the expression of neuropeptides that regulate the neuroendocrine system within the hypothalamus, but could also act directly on the anterior pituitary (6, 16, 18, 19, 21). In accordance with the latter possibility, LR mRNA has been localized to GH- secreting and PRL-secreting human pituitary adenomas (21). Although RT-PCR analysis showed presence of LR in the ovine pars distalis, there is no detailed information on localization to particular cell types (24). In fact, there have been no immunohistochemical studies in any species to indicate the presence of LR protein in the anterior pituitary gland.

In sheep, an alteration in adiposity generally affects the secretion of GH (28, 29, 30) and gonadotropins (29, 31, 32, 33, 34, 35, 36) with no effect on the plasma levels of PRL (29), TSH, or cortisol (36). It seems possible, therefore, that the gonadotropes and/or somatotropes of the pituitary gland are potential targets for leptin. The HPA axis is up-regulated by undernutrition in rodents (5), but this is not the case in sheep (36, 37). Accordingly, we sought to determine the extent to which somatotropes and gonadotropes express LR-ir in the female sheep pituitary. We also studied, for comparison, LR-ir expression in corticotropes, since the available data suggest that the HPA axis is not regulated by alteration in adiposity, or by leptin administration, in this species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection
All procedures were carried out with the prior approval of the Animal Experimentation Ethics Committee of Monash University and Victoria Institute of Animal Science.

Study 1. Four pituitaries (including the median eminence/pars tuberalis) were obtained from adult Corriedale ewes of similar age and weight, during the luteal phase of the estrous cycle. The tissue was collected as previously reported (38). Briefly, the sheep were administered 25,000 IU of heparin iv and were killed 5 min later by an overdose of sodium pentobarbital (2.5 ml/kg body weight, iv) (Lethabarb; May & Baker Pty Ltd, Melbourne, Australia). The heads were flushed with 2 liters of heparinized (12,500 IU/liter) 0.9% saline via carotid arteries followed by 2 liters of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) and 1.5 liters of 4% paraformaldehyde in PB containing 20% sucrose. The tissues were removed, transferred to a solution containing 4% paraformaldehyde plus 30% sucrose, and left at 4 C for 5 days. The tissues were then frozen on powdered dry ice and kept at -20 C until used. Sections of 20 µm were cut on a cryostat (CM 1850, Leica Corp., Deerfield, IL) at -20 C and thaw-mounted onto gelatin-chrom-coated slides, left overnight at room temperature, and stored at -20 C until processed for immunohistochemistry. Sections (20 µm) of median eminence/pars tuberalis tissue were similarly obtained and stored in cryoprotectant solution at -20 C until processed for free-floating immunohistochemistry.

Study 2. Analysis of the tissue sections in Study 1 revealed that 90% of the gonadotropes expressed LR-ir in the pars tuberalis, whereas only 30% of the gonadotropes were double labeled in the pars distalis. To investigate whether expression of LR-ir in gonadotropes varied across the estrous cycle, additional pituitaries and median eminence/pars tuberalis tissues were collected (as above) during the luteal, follicular, and surge phases of the estrous cycle (n = 3/group) and examined for LR-ir expression.

Antibodies
We used a rabbit polyclonal LR antibody raised against a peptide corresponding to amino acids 874–886 of LR (39), which is common to both long (OB-Rb) and short (OB-Ra) isoforms of LR. The characterization and use of the antibody for localization of LR have been reported previously (23, 39). The specificity of LR-ir in the sheep anterior pituitary was tested by preabsorption of LR antiserum with the peptide used for immunization at a final concentration of 5, 10, and 20 µg/ml. The use and specificity of GH and LHß and ACTH antibodies for the ovine anterior pituitary have been described previously (38).

Immunohistochemistry
Slide-mounted pituitary tissue sections and free-floating median eminence/pars tuberalis tissue sections were processed for immunofluorescence double labeling according to previously reported protocols (38) with some modification. Briefly, the sections were washed in 0.05 M PBS to remove the fixative and treated with 1% sodium borohydride (Sigma-Aldrich Corp., St. Louis, MO) in 0.01 M PB for 20 min. After washing, the sections were incubated in blocking solution (10% normal goat serum, 1% BSA, 0.1% Triton X-100 in PB) for 30 min and then with LR antiserum (1:500) overnight at room temperature. After washing in PBS, the sections were incubated with Texas Red-conjugated goat antirabbit IgG (1:250, Molecular Probes, Inc., Eugene, OR) in PB for 2 h, washed, and then incubated in blocking solution containing 10% normal donkey serum for 30 min. Sections were then incubated either with mouse monoclonal anti-ACTH (1:50, Novacastro, Newcastle on Tyne, UK), anti-LHß (1:2500, kindly donated by Dr. J. F. Roser, University of California, Davis, CA). or anti-GH (1:10, kindly donated by Dr. M. Brandon, University of Melbourne, Australia). After washing, the sections were then incubated in biotinylated horse antimouse IgG (1:800, DAKO Corp., Carpinteria, CA) for 1 h, washed, and then incubated with fluorescein thiocyanate (FITC)-conjugated avidin (1:500, Pierce Chemical Co., Rockford, IL) for 1 h and washed again. For the purpose of presenting representative photomicrographs, one section from both pars distalis and pars tuberalis of each animal was also stained with 1% Sudan Black B (Sigma-Aldrich Corp.) for 10 min to block autofluorescence (40). In addition, and to show that some cells did not express LR-ir, representative sections processed for LR-ir only were briefly counterstained with 4',6' diamidine-2-phenylindole dihydrochloride (DAPI) for 10 min (1:10, Molecular Probes, Inc., Eugene, OR). This allowed clear identification of cells that either did or did not contain LR-ir. The slide-mounted pituitary sections were then cover-slipped with anti-fade medium (DAKO Corp.). Free-floating median eminence/pars tuberalis tissue sections were mounted onto gel-chrome-subbed slides and cover slipped with antifade medium (DAKO Corp.). Slides were stored at 4 C in the dark until analyzed. In all cases, negative controls (omission of the primary antiserum) were run simultaneously to confirm the specificity of the immunostaining and lack of bleed through of the fluorescent markers.

Tissue analysis
Tissue analysis was carried out using a BMX 50 microscope (Olympus Corp., Lake Success, NY) equipped with a mercury light source and Texas Red and FITC band filter systems to visualize the red and green immunofluorescence, respectively. The nuclear staining with DAPI was assessed using the UV light. The expression of LR-ir within each individual cell labeled either for ACTH, GH, or LHß was visualized by switching between the Texas Red and FITC filter systems. To estimate the percentage of cells expressing LR-ir in the pars distalis, 50 cells were visually counted in each of the five randomly selected area of a representative section for each animal. Only cells that had discernable nuclei were counted. There was a clear difference between cells that stained with either fluorophor and background, with no cells showing equivocal staining.

To obtain an estimation of the level of expression of LR-ir in somatotropes and gonadotropes, 50 cells were visually counted in each of 5 randomly selected areas in a representative section for each animal. Since ACTH immunoreactive cells are not as abundant, 10 cells were counted in each of 10 randomly selected areas. All observations were made by a single investigator, and each counted cell had a clearly defined nucleus.

Statistical analysis
The data were not corrected for double-labeling cell count (41). After quantification of LR-immunoreactive cells, the data were subjected to statistical analysis using one-way ANOVA. Our results provide a relative number (mean % ± SEM) of each cell type that is immunopositive for LR and not the actual number of cells in the pars distalis or pars tuberalis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study 1
No specific LR-ir was seen after the preabsorption of the LR antiserum with antigen peptide or after the omission of primary antiserum (Fig. 1Go). All autofluorescence was completely blocked after counterstaining of the tissue with Sudan Black B (data not shown).



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  		Figure 1. Photomicrographs demonstrating cellular staining for LR-ir in the pars distalis. Panel A shows positive staining; panel B shows the lack of staining after preabsorption of the LR antiserum with the antigen peptide; and panel C shows lack of staining after the omission of the primary antiserum. Scale bar, 10 µm.

 
Double labeling of pituitary sections with DAPI and LR antiserum is shown in Fig. 2Go, A–D. In the pars distalis, LR-ir was found in 34.5 ± 1.71% of the cells whereas 94.4 ± 1.32% of the cells in the pars tuberalis expressed LR-ir (P < 0.01).



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  		Figure 2. Localization of LR-ir to particular cell types in the ovine pitutary gland. The two left-hand columns and the two right-hand columns, respectively, represent dual images of the same section. Each row represents two sets of dual images. In each case, LR-ir is stained in red. DAPI staining is blue, and the staining for the relevant pituitary hormone is shown in green. Asterisks show some (but not all) examples of cells that are double labeled. Arrows indicate some (but not all) examples of cells that are single labeled. Photomicrographs A–D provide some examples of dual labeling for DAPI and LR-ir in the pars distalis (A and B) and the pars tuberalis (C and D). Photomicrographs E–H demonstrate examples of corticotropes in the pars distalis that contain LR-ir. A single corticotrope that does not contain LR-ir is shown in panel G, and cells containing only LR-ir are shown in panels F and H. Photomicrographs I and J provide examples of colocalization of LR-ir in somatotropes; cells that were singly labeled for either GH or LR-ir are shown in panels K or L, respectively. Panels M–P provide examples for pars distalis gonadotropes; double labeling is seen in cells in panels M and N, whereas single and double labeling is seen in panels O and P. Panels Q–T are examples of labeling of gonadotropes in the pars tuberalis. Double labeling of cells is seen in panels Q and R, and single labeling is seen in panels S and T.

 
The pattern of distribution of ACTH-, GH-, and LH-secreting cells was essentially similar to that reported previously (38). LR-ir was observed in some corticotropes, but not others (Fig. 2Go, E–H). Similarly, some representative examples of LR-ir-positive and LR-ir-negative somatotropes (Fig. 2Go, I–L) and gonadotropes (Fig. 2Go, M–T) are shown.

Expression of LR-ir was colocalized in 27.5 ± 2.5% of corticotropes, 69.0 ± 3.3% of somatotropes, and 29.3 ± 2.5% of gonadotropes in the pars distalis (Fig. 3Go). In the pars tuberalis, 90.0 ± 1.5% of gonadotropes contained LR-ir (Fig. 4Go)(P < 0.01 compared with pars distalis). No somatotropes were seen in the pars tuberalis, but a few corticotropes were seen as reported by Clarke et al. (42); these were not immunopositive for LR.



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  		Figure 3. The relative number (% ± SEM) of each type of cell expressing LR-ir in the pars distalis of the ovine anterior pituitary.

 


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  		Figure 4. The relative number (% ± SEM) of gonadotropes expressing LR-ir during the luteal, follicular, and surge phases of the estrous cycle (n = 3 per group) in the pars distalis and pars tuberalis of the ovine anterior pituitary gland (**, P < 0.01).

 
Study 2
There was no difference across the estrous cycle in the proportion of gonadotropes that expressed LR-ir in either the pars distalis or pars tuberalis (Fig. 4Go). As in Study 1, the number of gonadotropes that were immunopositive for LR were significantly (P < 0.01) greater in the pars tuberalis than in the pars distalis (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates expression of LR-ir in the corticotropes, somatotropes, and gonadotropes in the ovine anterior pituitary gland. Since no specific immunostaining was observed after control procedures and counterstaining with Sudan Black B blocked all autofluorescence, the LR-ir appears to be specific. Earlier work has indicated expression of LR mRNA in the pituitary gland of the sheep (24) and other species (21, 22). Shimon et al. (21) reported that mRNA for LR may be found in GH-secreting and PRL-secreting human pituitary adenomas using RT-PCR, but there are no other studies on localization to particular cells in normal pituitary cells.

There is a high level of expression of LR-ir in somatotropes (69%) of the ovine pars distalis and a lower level of expression of LR-ir in corticotropes (27%) and gonadotropes (29%). Since only one third of the gonadotropes were found to express LR in the pars distalis of sheep sampled during the luteal phase (Study 1), it remained possible that expression of LR-ir was regulated by steroids and varied across the estrous cycle. Accordingly, we examined expression of LR-ir in gonadotropes across the luteal, follicular, and estrous phases of the estrous cycle but found no cyclic variation. This strongly suggests that the ovarian steroids do not regulate expression of LR-ir in the gonadotropes.

Only 30% of the gonadotropes express LR-ir in the pars distalis compared with a high (90%) level of expression in the pars tuberalis gonadotropes. The cells of the pars tuberalis are different in embryonic origin and gene expression, and regulation of the cells is different in the two tissues (43). This is exemplified by ontogenetic studies of the distribution of the transcription factor PIT-1 in the rat (44, 45) and studies on the expression of arylalkylamine N-acetyltransferase in the ovine anterior pituitary gland (46). It has also been known for some time that the pars tuberalis contains a very high percentage of gonadotropes (47), but other pituitary cell types are also present (42, 48). In the ovine, the pars tuberalis cells express a high level of melatonin receptors, whereas the pars distalis cells do not (49, 50). Further, it has been reported that variable endocrine conditions do not affect the number and distribution of gonadotropes in the pars tuberalis of the sheep (48), and this also appears to be true for the pars distalis. The high level of LR-ir expression in the pars tuberalis compared with that of the pars distalis could be due to the close proximity of the former to the neural components of the median eminence (hypothalamic neuronal endings) and the primary plexus of the hypophyseal portal system. Thus, it seems most likely that cells of pars tuberalis are differentially regulated to those of pars distalis. The results of the present study provide further evidence of the different phenotypes of gonadotropes in the pars tuberalis and pars distalis of the ovine pituitary.

Studies in vitro and in vivo have suggested that leptin may act directly on the pituitary gland to affect the secretion of GH (6, 7, 8, 16, 17, 21) and gonadotropins (6, 15), but PRL secretion is not altered (21). It is well established that leptin regulates the HPA axis in rodents (5, 6, 18) and may affect TSH levels in humans (19), but this is not the case in the sheep, at least with central administration of leptin (20). Collectively, these studies are in accordance with observations that an alteration in adiposity influences the GH (20, 28, 29, 30) and gonadotropin (LH and FSH) secretion (31, 32, 33, 34, 35, 36) in sheep, with no effect on PRL (29). Whereas the effect of leptin to regulate GH could reflect, in part, altered secretion of somatostatin (30), there could also be direct pituitary regulation as indicated by the in vitro studies (see above). Appropriate in vivo studies to demonstrate direct pituitary action of leptin in relation to GH and gonadotropin secretion remain to be done. The present study strongly suggests that a GH is most likely to be affected by leptin action at this level. The lower level of LR-ir expression in gonadotropes and corticotropes does not provide such a clear-cut indication. Neither is it clear at this stage as to whether these cell types express the short or the long form of the receptor.

The LR exists as multiple splice variants that differ in the length of their intracellular domain (51). The short isoform of the LR is reported to be present in the several peripheral tissues, and the long isoform exists mainly in the hypothalamus where it is thought to mediate signal transduction in the specific neurons (27). Using RT-PCR, presence of the both long and short isoforms of the LR have been demonstrated in the rat, human, and sheep anterior pituitary (21, 22, 23, 24). The LR antiserum we used in the present study recognizes both short and long isoforms of the LR, and further studies are required to determine which form predominates in the ovine anterior pituitary.

In summary, the present study demonstrates localization of LR-ir in the corticotropes (27%), somatotropes (69%), and gonadotropes (29%) of the pars distalis of the ovine anterior pituitary. A high level of expression of LR-ir was found in the gonadotropes (90%) of the pars tuberalis, further demonstrating the special nature of these cells. The proportion of gonadotropes that expressed LR-ir did not change across the luteal, follicular, and surge phases of the estrous cycle, suggesting a lack of regulation by ovarian steroids in the ovine pituitary.


   Acknowledgments
 
We thank Mr. Bruce Doughton and Ms. Karen Perkins for animal care and Dr. J. F. Roser and Professor M. Brandon for antibodies. We also appreciate the technical assistance of Sue Pankridge in preparing the figures.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council, Australia. Back

Received September 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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