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Endocrinology Vol. 143, No. 3 1066-1075
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

The Distribution of Betaglycan Protein and mRNA in Rat Brain, Pituitary, and Gonads: Implications for a Role for Betaglycan in Inhibin-Mediated Reproductive Functions

Leigh A. MacConell, Angela M. O. Leal and Wylie W. Vale1

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: . vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Betaglycan was reported by our laboratory to serve as an inhibin binding protein and to facilitate the antagonism of activin signaling. Although an accessory receptor for TGFß and inhibin, its distribution within reproductive tissues remains largely unexplored. Histochemical analyses reveal betaglycan protein and mRNA distributed throughout the rat reproductive axis. In the brain, betaglycan mRNA is localized in discrete regions of the forebrain and brain stem, including olfactory, septal, and hypothalamic nuclei. In the pituitary, moderate levels of betaglycan protein and mRNA were observed in the anterior and intermediate lobes. Betaglycan immunoreactivity was colocalized with all the pituitary cell subtypes, to the greatest extent with the gonadotrope population. In the gonads, betaglycan mRNA was localized in cellular compartments, coinciding with its protein for the most part. Moderate levels of mRNA were observed in ovarian granulosa cells, with lower expression in the thecal layer and the oocyte. In the testes, betaglycan mRNA was observed in the Leydig and tubule-specific germ cells. This is the first comprehensive report detailing the distribution of betaglycan in mammalian reproductive tissues. The present findings illustrate and support the hypothesis of a modulatory role for betaglycan in TGFß and/or inhibin effects in these tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INHIBIN, A GONADALLY derived hormone, is essential for the maintenance of normal mammalian reproductive function based on its ability to inhibit FSH synthesis and release in anterior pituitary gonadotropes (1, 2). Inhibin is also an autocrine/paracrine mediator within the gonads in the stimulation of both Leydig and thecal cell androgen production (3), the inhibition of oocyte maturation (4), and the inhibition of spermatogenesis (5). Inhibin acts as a tumor suppressor in the gonads (6) and adrenal cortex (7). Furthermore, inhibin-deficient mice present with severe liver pathologies (7). Based on sequence homology, inhibin belongs to the TGFß superfamily of growth and differentiation factors. Inhibin exists as a heterodimer of an 18-kDa {alpha}-subunit, disulfide-linked to one of two homologous 14-kDa ß-subunits (ßA and ßB), resulting in inhibin A or inhibin B (8). The structurally related activins are synthesized as hetero- or homodimers of the same ß-subunits, activin A, activin AB, and activin B (8). Inhibins are functional antagonists of the activins that stimulate FSH production and secretion in the pituitary gonadotropes (9).

TGFß family members bind to specific type I and type II serine-threonine kinase receptors (10, 11). The functional activin receptor complex consists of a constituitively phosphorylated type II component (ActRII, ActRIIB) (12, 13), which, upon ligand binding, recruits a type I receptor (ActRIB, ALK-4) (14) into a heteromeric complex formed at the cell surface (15, 16). Similarly, TGFß interacts with TßRI (ALK-5) and TßRII (17). In both systems, the ligand-bound, type II component transphosphorylates the type I receptor kinase, which initiates intracellular signaling via activation of downstream signaling proteins, termed Smads (18). Until recently, the existence of receptor components underlying inhibin action were unclear, and it has been proposed that inhibin blocks the actions of activin via binding ActRII and competing with activin, rather than through an inhibin-specific receptor (19, 20).

Our laboratory recently reported that the membrane-anchored proteoglycan, betaglycan, can serve as an accessory receptor for inhibin (21). Betaglycan is a membrane-anchored proteoglycan, originally characterized as the type III receptor for TGFß. The rat betaglycan gene encodes a 91.6-kDa protein core that is further modified by N-linked glycosyl residues and heparan and chondroitin sulfate side-chains. The betaglycan protein consists of a large extracellular domain, a single transmembrane domain, and a short intracellular domain (22, 23). Although the short 43-amino acid cytoplasmic domain lacks any obvious signaling motif, it is enriched with serines and threonines (22, 23). Betaglycan also exists in a soluble form that is released by cells and found in extracellular matrexes (24). We found that betaglycan binds inhibin A with high affinity, enhances inhibin A binding in cells coexpressing ActRII and betaglycan, and confers inhibin responsiveness to cells that otherwise lack sensitivity to inhibin (21). This betaglycan/inhibin/ActRII complex does not recruit the activin signaling receptor, ALK4, and thus cannot initiate downstream signaling by the Smads. We have proposed a model in which betaglycan enhances the stability of the inhibin/ActRII complex, and this ternary complex is better able to prevent access of activin to ActRII, thereby blocking activin’s effects.

As a coreceptor for the peptide hormone inhibin, betaglycan probably can serve an important modulatory role in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. However, the anatomical distribution, regulation, and overall physiological importance of betaglycan remain largely unexplored. In the present study we examined the expression pattern of betaglycan protein and mRNA in the adult rat brain, pituitary, and gonads. We report that betaglycan mRNA and protein show widespread cell-specific localization within reproductive tissues, in regions coinciding with inhibin- and TGFß-mediated effects. These data are consistent with a role of betaglycan in inhibin and/or TGFß action within their target tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, tissue preparation, and experimental protocols
Male and female Sprague Dawley rats (200–225 g) were purchased from Harlan Sprague Dawley, Inc. (San Diego, CA), and kept under standard housing, feeding, and lighting conditions (23 C; 12-h light, 12-h dark cycle, with lights on at 0600 h). For histological analyses, rats were deeply anesthetized and perfused transcardially with 4% paraformaldehyde in borate buffer (38.14 g sodium tetraborate/liter dH2O, pH 9.5). Tissues (brain, pituitary, ovary, and testes) were removed and postfixed overnight in the same fixative plus 10% sucrose. For brain tissue, frozen 30-µm coronal sections were cut on a sliding microtome, and free floating sections were mounted onto SuperFrost Plus slides (Fisher Scientific, Tustin, CA). For pituitary and gonadal tissues, tissues were frozen in Tissue-Tek OCT embedding medium (EM Science, Cherry Hill, NJ), and 10-µm sections were cut on a cryostat and mounted onto the same slides. For the isolation of pituitary tissue for ribonuclease (RNase) protection analysis (RPA), male Sprague Dawley rats (200–225 g) were obtained and housed as described above. Animals were killed by decapitation, and the anterior and intermediate/posterior pituitaries were removed for isolation of RNA. All animal procedures were performed in accordance with institution, local, state, federal, and NIH guidelines.

Generation of RNA probes
For generation of the riboprobe specific for rat betaglycan, a 665-bp fragment was excised from the full-length rat betaglycan cDNA, betaglycan7 (22), at the BglII (2227 bp) and AvrII (2892 bp) restriction sites, and the insert was subcloned into pBS SK+ (betaglycan.6). The probe corresponds to the cytoplasmic domain of the mature rat betaglycan protein. After linearization with BbsI, the antisense 33P-labeled RNA probe was generated by in vitro transcription from the T3 promoter of the linearized pBS SK+ subclone to a specific activity of 108 cpm/µg using an excess of [33P]UTP (2000 Ci/mmol) and used for in situ hybridization. Background levels were determined using a 33P-labeled sense RNA probe prepared by linearization with SacI and transcription from the T7 promoter. For RPA, the same linearized template was used to generate the rat betaglycan antisense 32P-labeled RNA probe by in vitro transcription in the presence of [32P]UTP (3000 Ci/mmol) and 20 µl cold UTP to yield a protected fragment of 331 nucleotides. A rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense riboprobe was synthesized using the T3 promoter, resulting in a protected fragment of 134 nucleotides, and was used as an internal control.

Primary antisera
For the immunohistochemical detection of betaglycan, a commercially available goat antihuman betaglycan antibody was used at a concentration of 5 µg/ml (R\|[amp ]\|D Systems, Inc., Minneapolis, MN).

For the detection of the pituitary hormones, affinity-purified polyclonal antibodies generated in rabbits against rat PRL (AFP-4251091; 1:5000), rat TSH (AFP-1274789; 1:500), rat FSH (AFP-HFSH6; 1:100), and rat GH (AFP-5672099; 1:2000) were obtained from the National Hormone and Pituitary Program of NIDDK, NIH (Bethesda, MD). For the detection of rat ACTH hormone, an affinity-purified polyclonal antibody produced in rabbits, specific for amino acids 1–24 of the mature peptide, was produced in our laboratory (code 238-131; 1:500).

In situ hybridization
In situ hybridization was employed to detect betaglycan mRNA in fixed-frozen sections of rat brain, pituitary, and gonads. Details concerning the in situ hybridization procedure have been reported previously (25). Briefly, processed slide-mounted sections were hybridized for 16 h at 60 C to the sense or antisense 33P-labeled RNA probe specific for rat betaglycan. After hybridization, nonspecific binding was reduced with RNase A treatment (20 µg/ml) and a low salt/high temperature wash (15 mmol/liter NaCl-1.5 mmol/liter sodium citrate at 65 C for 30 min). Dehydrated sections were exposed to Bio-Max film (Amersham Pharmacia Biotech, Arlington Heights, IL) for 5–7 d. The slides were then exposed to nuclear track emulsion (NTB-2, Kodak, Eastman Kodak Co., Rochester, NY) at 4 C for 2–4 wk, photographically processed, stained with hematoxylin (Richard Allen Scientific, Kalamazoo, MI), and coverslipped. The emulsion-coated and counterstained sections were photographed using Kodak Elite Chrome 35-mm film (Eastman Kodak Co.), and slides were analyzed for the cellular localization of silver grain deposits.

Immunofluorescence
Conventional immunofluorescence was employed to detect betaglycan protein in fixed-frozen sections of rat pituitary. Briefly, the sections were washed in potassium PBS (0.45 g KH2PO4; 3.8 g KaHPO4H2O; 9 g NaCl/liter dH2O, pH 7.4) and incubated in blocking buffer [0.5 M NaCl, 0.01 M phosphate buffer (pH 7.3), 3% BSA, 0.1% sodium azide, and 0.3% Triton X-100] containing 10% normal donkey serum overnight to minimize background fluorescence. The following day, sections were incubated overnight at room temperature with fresh blocking buffer containing 4% normal donkey serum and 5 µg/ml betaglycan antiserum. After incubation with primary antiserum, tissue sections were rinsed in KPBS (four times for 5 min each), followed by a 1-h incubation with indocarbocyanine (Cy3)-conjugated donkey antigoat IgG secondary antiserum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:800 dilution in 4% normal donkey serum/blocking buffer at room temperature. Sections were then coverslipped, and the slides were stored at 4 C in the dark until analysis.

For colocalization studies, betaglycan antiserum was applied to tissue in cocktails with primary antisera against the various pituitary hormones in 4% normal donkey serum/blocking buffer. Appropriate secondary antisera included both Cy3-conjugated donkey antigoat IgG (1:800; Jackson ImmunoResearch Laboratories, Inc.) for visualization of betaglycan protein and fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc.) to detect pituitary hormone staining. Three negative control procedures (exclusion of the primary antiserum, application of normal goat IgG, and preabsorption of the betaglycan antisera with a 100-fold excess of antigen) were run simultaneously to confirm the specificity of the immunostaining and the lack of bleed-through of the fluorescent markers.

Tissue analysis
To determine the extent of colocalization of betaglycan protein with each pituitary cell type, an Optiphot-2 microscope (Nikon, Tokyo, Japan) equipped with a mercury light source and Texas Red and FITC filter systems to visualize the red and the green immunofluorescence, respectively, was used. The expression of betaglycan immunoreactivity (betaglycan-ir) within each individual cell labeled for the various pituitary hormones was visualized by switching between the two filter systems. To estimate the percentage of cells expressing betaglycan-ir, 5 cells were visually counted in each of 10 randomly selected areas in a representative pituitary section for each animal (n = 5). Only cells with a visible nucleus were counted. The results provide a relative number (mean percentage ± SEM) of each cell type that is immunopositive for betaglycan and not the actual number of cells. The sections were also examined, and images were generated using an IX70 confocal microscope (Olympus Corp., Melville, NY).

Ribonuclease protection analysis
Total RNA was extracted from pituitary tissues (anterior or posterior and intermediate lobes) using the RNeasy kit (QIAGEN, Hilden, Germany) according to the manufacturer’s procedures, and assays were performed using 10 µg total RNA to assess the presence of betaglycan mRNA. Briefly, RNA samples were precipitated, vacuum-dried, and resuspended in hybridization buffer (4 M NaCl, 400 mM PIPES, 10 mM EDTA, and 80% formamide) containing 5 x 105 cpm of the betaglycan probe and 4 x 104 of the GAPDH probe. Samples were incubated at 85 C for 5 min and then at 42 C overnight. Samples were then incubated in 350 µl RNase A digestion buffer (5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl, 35 µg RNase A, and 10 µg RNase T1) for 1 h at room temperature. Next, 10 µl 20% SDS and 2 µl proteinase K (20 mg/ml) were added to each sample and incubated for 15 min at 37 C. Samples were extracted with 400 µl phenol/chloroform and precipitated with 100% ethanol. Vacuum-dried RNA pellets were resuspended in 5 µl loading buffer (80% formamide, 10x Trizma/borate/EDTA buffer (0.09 M Trizma base, 0.09 boric acid, 1.6 ml 0.5 M EDTA), 1 mg/ml bromophenol blue, and 1 mg/ml xylene cyanol), incubated at 85 C for 5 min, and resolved on a 5% polyacrylamide/8 M urea gel. The presence of RNA was assessed using the PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA) and ImageQuant 4.0 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue-specific distribution of betaglycan expression
Brain.
The localization pattern of mRNA encoding betaglycan was examined in the adult male rat brain using in situ hybridization. Structures were identified using the rat brain atlas of Paxinos and Watson (26). Betaglycan mRNA signal was widely distributed in discrete regions throughout the brain. A series of high power photomicrographs (Fig. 1Go) details the pattern of betaglycan mRNA signal throughout the brain. In olfactory regions, moderate levels of betaglycan mRNA expression were detected in the glomerular layer of the olfactory bulb, the accessory olfactory bulb, and the lateral olfactory tract. In addition, discrete hybridization patterns were found in the primary olfactory cortex, including the olfactory tubercle, tenia tecta, and piriform cortex, with abundant mRNA signal distributed throughout these three structures (Fig. 1AGo).



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  		Figure 1. High magnification localization of betaglycan mRNA signal in nuclei of the normal adult male rat brain. Photomicrographs of emulsion-dipped slides show betaglycan mRNA signal (white grains in a darkfield) in the olfactory cortex (A), septum (B), hypothalamus (C–E), and brainstem (F). Betaglycan mRNA signal is present in the piriform cortex (Pir), lateral septal nucleus, dorsal (LSD), choroid plexus (chp), supraoptic nucleus (SO), PVN, ventromedial hypothalamic nucleus (VMH), arcuate nucleus (ARC), ME, area postrema (AP), hypoglossal nucleus (12 ), and NTS of the normal male rat brain. Other structures labeled for reference are the anterior commissure (ac), corpus collosum (cc), optic chiasm (ox), and third ventricle (3V). Bars, 200 µm.

 
Betaglycan mRNA signal was localized in the septal region with abundant levels of expression observed in the dorsal and intermediate portions of the lateral septal nucleus (Fig. 1BGo). Positive signal also appeared to be localized to the choroid plexus (Fig. 1Go, B and D). As shown in Fig. 1Go, abundant betaglycan gene expression is detectable throughout the hypothalamus, with discrete hybridization patterns in the medial preoptic area, supraoptic nucleus, paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial hypothalamic nucleus, and median eminence (ME; Fig. 1Go, C–E).

In addition to the forebrain, moderate to abundant levels of betaglycan mRNA were expressed in the brainstem. Betaglycan mRNA expression was observed in the medial vestibular nucleus, solitary tract nucleus (NTS), reticular formation, and area postrema (Fig. 1FGo). Moderate levels of betaglycan mRNA were also observed in the hypoglossal nucleus (Fig. 1FGo) and the dorsal motor nucleus of vagus. Positive signal above background levels was not observed when the sense probe was applied to the tissue (data not shown).

Pituitary.
Using immunohistochemical and in situ hybridization analyses, pituitary areas expressing betaglycan protein and mRNA were examined in detail in the adult male rat. A summary of the localization patterns of betaglycan mRNA and protein in the pituitary is shown in Table 1Go, and typical examples of their distribution are presented in Fig. 2Go. As determined by in situ hybridization, abundant levels of betaglycan gene product were distributed throughout the anterior lobe of the pituitary (Fig. 2BGo). However, the intermediate and posterior lobes of the pituitary did not contain specific hybridization signal (Fig. 2BGo). Positive signal was not observed when the sense probe was applied to the tissue (data not shown), confirming the specificity of the hybridization signal generated by the antisense probe. RPA was used to confirm the expression of betaglycan mRNA within the individual lobes of the pituitary. In contrast to that observed by in situ hybridization, positive signal corresponding to betaglycan mRNA could be detected in both the anterior and intermediate lobes of the pituitary (Fig. 3Go).


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  		Table 1. Localization of betaglycan-immunoreactive protein staining and mRNA signal in rat pituitary and gonadal cell groups and its comparative spatial distribution with inhibin/activin {alpha}-, ßA-, and ßB-subunit, activin receptors type II, and IIB (ActRII, IIB) mRNA and protein and with inhibin binding in the rat

 


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  		Figure 2. High magnification localization of betaglycan immunostaining and mRNA signals in the normal adult male rat pituitary gland. Bright- and darkfield photomicrographs demonstrate the distribution of betaglycan mRNAs (white grains) by in situ hybridization (B) and the morphology by hematoxylin-eosin staining (A) in a fixed-frozen male rat pituitary section. Cellular staining for betaglycan-ir (red fluorescence) is shown in the anterior and intermediate lobes of an adjacent fixed-frozen male rat pituitary section (C, E and F). F, Higher magnification of E. The positive immunostaining for betaglycan (C) is abolished when the antiserum is preabsorbed with excess antigen (D), confirming the specificity of the antiserum. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe.

 


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  		Figure 3. A representative image of an RPA. Total RNA (10 µg) from adult male rat anterior or intermediate and posterior pituitary lobes was hybridized with rat betaglycan and GAPDH (used as an internal control) riboprobes and resolved on a 5% polyacrylamide/8 M urea gel.

 
Betaglycan-ir was examined in adjacent pituitary sections. As shown in Fig. 2Go (C, E, and F), moderate levels of betaglycan-ir were observed throughout the anterior lobe, coinciding with its mRNA expression. In some cells of the anterior pituitary, betaglycan-ir was cytoplasmic, whereas immunolabeling was specific to the cell periphery of other cells, suggestive of membrane staining. In the intermediate lobe, intense cytoplasmic immunostaining was observed coincident with the expression of the corresponding mRNA in this region, as shown by RPA, but not in situ hybridization (Fig. 2Go, C, E, and F). Betaglycan-ir was not detected in cells of the posterior lobe of the pituitary (Fig. 2Go, C–E). Positive immunostaining for betaglycan in both anterior and intermediate lobes (Fig. 2CGo) was abolished when the primary antiserum was preabsorbed with soluble betaglycan (Fig. 2DGo), indicating the specificity of the antibody.

The extent to which the different cell types of the male rat pituitary express betaglycan-ir was determined. Double label immunofluorescence of anterior pituitary sections with representative examples of betaglycan-immunopositive gonadotropes, lactotropes, somatotropes, thyrotropes, and corticotropes is shown in Fig. 4Go. Betaglycan-ir colocalized with all pituitary cell types examined, albeit to greatly varying degrees. As shown in Fig. 5Go, betaglycan-ir was coexpressed in 97 ± 0.7% of gonadotropes, 23 ± 10.4% of lactotropes, 23 ± 16.3% of somatotropes, 5 ± 0.7% of thyrotropes, and 1 ± 0.0.7% of corticotropes. Interestingly, whereas very few corticotropes were found to be positive for betaglycan-ir in the anterior lobe, the ACTH-ir melanotropes of the intermediate lobe were strongly immunopositive for betaglycan, with 100% of these intermediate lobe cells coexpressing betaglycan-ir (data not shown). No double labeling was observed in tissue when the primary antiserum was either omitted or replaced with normal goat IgG serum.



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  		Figure 4. High magnification localization of betaglycan immunostaining to particular cell types in the normal adult male rat pituitary gland. Each column represents triple images of the same fixed-frozen section of the adult male rat anterior pituitary. In each case, the relevant pituitary hormone is visualized as a green fluorescent stain (top row), and betaglycan is visualized by red fluorescence (middle row). The merged image is shown in the bottom row, and double-labeled cells appear yellow. Typical examples (but not all) of dual labeled cells are indicated by arrows.

 


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  		Figure 5. Betaglycan immunostaining is localized in various pituitary cell types. The relative number (percentage ± SEM) of each rat anterior pituitary cell type expressing betaglycan-ir (sampling of five rats).

 
Gonads.
Using in situ hybridization, the localization pattern of betaglycan mRNA expression was examined in the adult rat testis and ovary. Figures 6Go and 7Go display the representative in situ hybridization data obtained in the rat testis and ovary sections, respectively, and Table 1Go shows a summary of the protein and mRNA localization patterns. As illustrated in Fig. 6Go, high levels of betaglycan mRNA signal were associated with germ cells of specific tubule sections of the rat testis. Expression of mRNA was specific to certain stages of spermiogenesis, with moderate levels of signal specific to the primary spermatocytes (leptotene and pachytene) and more intense hybridization signal specific to rounded spermatids, but neither spermatogonia nor elongated spermatids were observed to express betaglycan mRNA (Fig. 6EGo). The expression of betaglycan mRNA to the germ cells appeared specific to earlier stages of the seminiferous epithelial cycle (stages I–VI). Hybridization signal was not apparent over Sertoli cells. Leydig cell clusters exhibited high intensity betaglycan mRNA signal (Fig. 6Go, D and E). In the ovary, light hybridization signal was present in primordial follicles, with more moderate levels seen in follicular granulosa and thecal cells as well as in the oocytes of primary and secondary follicles (Fig. 7Go). Expression of betaglycan mRNA in the granulosa cells of secondary follicles was slightly greater than that observed in the primary follicles. Light to moderate levels of betaglycan mRNA were also detected in granulosa and thecal cells as well as the oocyte of Graafian follicles (Fig. 7Go). Lastly, high levels of signal for betaglycan were localized to the ovarian interstitial cells, and positive signal was not apparent over the corpus luteum. Again, positive signal above background levels was not observed in either testicular or ovarian tissue sections when the labeled sense strand of betaglycan was used as a probe (data not shown).



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  		Figure 6. High magnification localization of betaglycan mRNA signal in the normal adult rat testis. Pairs of bright- and darkfield photomicrographs demonstrate the distribution of betaglycan mRNAs (white grains) by in situ hybridization (B and D) and the morphology by hematoxylin-eosin staining (A and C) in fixed-frozen sections of the normal rat testis. B, View at lower magnification to illustrate hybridization to specific tubules. D, Positive signal in germ cells of a single tubule and the interstitial Leydig cells. E, Brightfield photomicrograph illustrating positive signal (black grains) over Leydig cells, spermatocytes, and round spermatids. GC, Germ cells; LC, Leydig cells; RS, round spermatids; SP, spermatocytes; ST, seminiferous tubule.

 


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  		Figure 7. High magnification localization of betaglycan mRNA signal in the normal adult rat ovary. A pair of bright- and darkfield photomicrographs demonstrates the distribution of betaglycan mRNAs (white grains) by in situ hybridization (B) and the morphology by hematoxylin-eosin staining (A) in a fixed-frozen section of the normal rat ovary. Higher magnification brightfield views are shown in the bottom two panels (C and D), illustrating positive hybridization signal (black grains) over granulosa cells of a secondary follicle (C) and the oocyte of a mature follicle (D). A, Antrum; CO, cumulus oophorus; GC, granulosa cells; I, interstitium; O, oocyte; T, thecal layers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study histological techniques have been applied to evaluate the tissue-specific expression of betaglycan protein and mRNA throughout the adult rat HPG axis. The results show that both betaglycan protein and mRNA are expressed in discrete cell types throughout reproductive tissues. Consistent with its potential role as an accessory receptor for inhibin, the distribution of betaglycan coincides with that reported for inhibin {alpha}- and ß-subunit proteins and mRNAs as well as with ActRII and -IIB gene expression in many areas (Table 1Go). In addition, the results show that the pattern of betaglycan expression is compatible with its role in modulating the reproductive functions of inhibin and TGFß in many tissues.

Whereas previous studies have described the presence of betaglycan protein and/or mRNA in cell lines (23, 27), those looking at its distribution and regulation in mammalian tissues remain limited, especially with respect to the reproductive organs. Northern analysis has revealed betaglycan mRNA in cultured porcine Leydig cells (28); rat lung, kidney, and ovary (29); and neonatal and adult rat forebrain and brainstem (30). In addition, various histological studies have shown betaglycan expression in rat peridontium (31, 32), chick heart (33), and human cornea (34), bone (35), breast (36), and intestine (37). A recent publication also described immunohistochemical analysis of betaglycan in adult rat pituitary (38).

In the present study betaglycan mRNA expression was observed throughout the rostral-caudal extent of the adult rat brain. This widespread distribution is consistent with RPA data indicating the presence of betaglycan mRNA in the rat forebrain and brainstem (30). Overall, a unifying generalization that can be made with regard to the pattern of betaglycan gene expression in the rodent forebrain is that regions associated with olfactory and neuroendocrine systems were found to be the dominant sites of betaglycan expression. In the primary olfactory cortex, the receptor is expressed in the olfactory bulb, tenia tecta, piriform cortex, and olfactory tubercle. This expression spatially correlates with that of inhibin {alpha}-subunit mRNA and inhibin ß-subunit-immunostained fibers in the olfactory cortex (39). While an involvement of the inhibin system in olfactory sensory transduction has not been reported, it is well established that olfactory sensory cues are important with regard to reproductive behavior as well as gonadotropin secretion (40). Given the neuroendocrine nature of the inhibin system, we may speculate that betaglycan, in addition to the other inhibin system factors, is in an anatomical position within olfactory-associated nuclei to potentially regulate olfactory-related reproductive behaviors in the brain.

Consistent with the potential role of betaglycan in inhibin-modulation of central neuroendocrine functions, there is a prevalence of betaglycan mRNA in discrete nuclei of the rat hypothalamus. This predominant hypothalamic expression of betaglycan mRNA is in accord with the expression of inhibin {alpha}- and ß-subunit protein and mRNAs, the localization of which generally mirrors that of betaglycan in the hypothalamus (39, 41). The results also show that betaglycan mRNA is expressed in the CRF-rich region of the PVN, and inhibin has been reported to block the facilitation of CRF release by activin in male rats (42). Betaglycan mRNA is present in the NTS of the brainstem, and data suggest that inhibin ß-subunit-containing fibers, originating from the NTS, project to the PVN and mediate suckling-induced oxytocin release (41, 43). Inhibin antagonizes activin A-stimulated GnRH secretion in vitro (44, 45), and substantial levels of betaglycan mRNA are expressed in the ARC nucleus and ME, areas implicated in the release of GnRH. In addition, inhibin is reported to decrease GHRH mRNA levels in the ARC nucleus of male rats (46). Although functional testing is needed before any precise central role can be established for betaglycan, the high level expression of betaglycan in multiple hypothalamic cell populations supports a role for the receptor in inhibin- and TGFß-mediated central reproductive functions.

As one would predict for an inhibin receptor, the present study shows substantial betaglycan immunostaining and levels of mRNA throughout the anterior pituitary and demonstrates coexpression of betaglycan-ir in the inhibin-responsive pituitary gonadotropes. Although all of the anterior pituitary cell types were observed to coexpress betaglycan to some degree, the predominant cell type found to be immunopositive for betaglycan was the FSH-positive gonadotropes (98%). It is well established that inhibin blocks activin-stimulated FSH synthesis and release in gonadotropes (9). Thus, the high level of expression of betaglycan in this cell type is consistent with a role for betaglycan as an accessory receptor for inhibin and suggests that pituitary betaglycan may be involved in the inhibin influence on gonadotropin secretion. Although TGFß1 inhibits PRL secretion and gene expression in lactotropes (47, 48, 49), only 23% of lactotropes were found to coexpress betaglycan-ir. Although betaglycan has been shown to increase TGFß1 responsiveness, it is not thought to be required for TGFß1 action (50). Interestingly, the anterior pituitary corticotrope cell population, which has not been reported to be responsive to either inhibin or TGFß, had the fewest number of cells that expressed betaglycan-ir (1%). These observations are in contrast to a recent publication that also documented betaglycan immunostaining throughout the anterior and intermediate lobes of the adult male rat pituitary, but failed to show a preponderance of FSHß-immunopositive cells that coexpressed betaglycan-ir (coexpression with the other pituitary cell types was not examined) (38). However, we have confirmed the presence of betaglycan-ir in the pituitary gonadotropes using a polyclonal antibody, generated in our laboratory, which is specific for a portion of the extracellular domain of rat betaglycan. Using this antisera, betaglycan staining is strongest in the anterior lobe, and the majority of FSH-immunostained gonadotropes are positive for betaglycan (less intense staining is evident in the intermediate lobe and the posterior lobe is devoid of betaglycan-ir; our unpublished observations). Hence, this discrepancy is indeed puzzling, but could be attributed to differences in assay sensitivity due to variances in tissue thickness as well as conditions pertaining to primary and secondary antibody incubations. Studies in our laboratory are currently underway to examine the expression of betaglycan in the female rat pituitary to determine whether pituitary betaglycan is expressed in a sexually dimorphic manner. In addition, studies suggest that the physiological action of inhibin on FSH secretion is age dependent (51, 52), so it will be interesting to determine whether pituitary betaglycan levels change with age, possibly mediating inhibin action in sexual maturation.

Interestingly, betaglycan protein staining was present in the intermediate lobe of the rat pituitary. Whereas RPA confirmed the presence of betaglycan transcript in the intermediate lobe, in situ hybridization did not reveal concordant betaglycan mRNA expression in these cells. It is conceivable that in situ hybridization was not sufficiently sensitive for the detection of potentially low levels or rapid turnover of betaglycan mRNA in the intermediate lobe cells, whereas immunohistochemical and RPA analyses provided adequate sensitivity to detect protein and mRNA, respectively. Furthermore, as betaglycan can exist in a soluble form (24), it is possible that some of the betaglycan protein observed by immunostaining in the intermediate lobe is transported there from an adjacent cell population. Studies are ongoing to delineate a potential action of inhibin and/or TGFß on the intermediate lobe melanotropes in vitro. Neither betaglycan protein nor gene expression was observed in the posterior lobe of the pituitary.

The present report extends our previous work describing localization of betaglycan protein in the rat ovary and testis (21). Betaglycan mRNA, like its protein, is expressed in gonadal cell types documented to be inhibin responsive (9). The gonadal distribution of the betaglycan gene is comparable to that of the protein and closely parallels reported [125I]inhibin A-binding sites, as shown by in vitro ligand binding studies (53, 54). In the testis the predominant site of betaglycan expression is the testicular Leydig cell, with strong signal also associated with the spermatocytes and rounded spermatids of some seminiferous tubules. The expression of betaglycan by germ cells at specific maturational stages closely parallels work by Woodruff et al. (55), who reported that FITC-conjugated inhibin binds primarily to spermatocytes (leptotene/zygotene, pachytene/diplotene) and early spermatids. The expression pattern of betaglycan mRNA in the rat testis also coincides with the recent identification of a high affinity inhibin A-binding complex, consisting of betaglycan, on a mouse Leydig cell line (56) and corresponds with Northern blot analyses showing betaglycan mRNA expression by rat Leydig and germ cells (57). In addition, this cellular distribution is consistent with reports that inhibin, produced by Sertoli cells (58), acts locally to stimulate steroidogenesis in Leydig cells (3) and to suppress spermatogonial mitosis (59). The expression of betaglycan by stage-specific spermatocytes and spermatids suggests that inhibin may also have an important paracrine effect on the regulation of germ cell meiosis/maturation in the adult rat testis. Although betaglycan mRNA is present in the testicular germ cells, betaglycan-ir was not detected in germ cells in our earlier study (21). This mismatch of expression may be attributed to potentially low translation or rapid degradation of protein in these cells. In the ovary, positive signal for betaglycan mRNA is demonstrable in follicular granulosa cells, consistent with the inhibin and TGFß responsiveness of this cell type (3, 60, 61, 62, 63) as well as the presence of betaglycan mRNA in cultured granulosa cells, as shown by Northern blotting (64). High levels of betaglycan mRNA signal are localized to the nonfollicular ovarian interstitial cells that are reported to respond to TGFß (65, 66). Light hybridization signal is also present over the thecal layer, and a diffuse betaglycan mRNA signal could be observed over the oocyte of developing follicles in accord with reported direct effects of inhibin on oocyte maturation (67, 68).

In conclusion, betaglycan is expressed in specific cell compartments throughout the HPG axis, consistent with a role for the accessory receptor in inhibin- and TGFß-regulated functions in these cell types. Although the intrapituitary role of betaglycan remains to be elucidated, its strong presence within the pituitary gonadotropes suggests that betaglycan may mediate inhibin action on FSH production and release, and changes in pituitary betaglycan expression could have important physiological consequences in regulation of the HPG axis. Although functional assays are needed before any explicit reproductive role can be assigned to betaglycan, elucidating the tissue-specific distribution of betaglycan establishes a morphological basis for better understanding the potential functional roles of the inhibin system in mammalian reproduction.


   Acknowledgments
 
We thank Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY) for providing the rat betaglycan cDNA (betaglycan7). The pituitary hormone antibodies were all generously donated by the National Hormone and Pituitary Program of the NIDDK and A. F. Parlow (Torrance, CA). We also thank Dr. Louise Bilezikjian for critical reading of the manuscript and Keith Anderson for his assistance with cell counting.


   Footnotes
 
This work was supported by NIH Grants DK-09980-02 (to L.A.M.) and HD-13527 (to W.V.), the Americas Fellowship Program of NIH (to A.L.), the Kleberg Foundation (to W.V.), and the Adler Foundation (to A.L.) and in part by the Foundation for Medical Research, Inc. (to W.V.).

1 Foundation for Medical Research, Inc., senior investigator. Back

Abbreviations: ActR, Activin receptor; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPG, hypothalamic-pituitary-gonadal; -ir, immunoreactivity; ME, median eminence; NTS, solitary tract nucleus; PVN, paraventricular nucleus; RNase, ribonuclease A; RPA, ribonuclease protection analysis.

Received July 2, 2001.

Accepted for publication November 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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