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Endocrinology, doi:10.1210/en.2003-0670
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Endocrinology Vol. 144, No. 12 5640-5649
Copyright © 2003 by The Endocrine Society

Betaglycan Localization in the Female Rat Pituitary: Implications for the Regulation of Follicle-Stimulating Hormone by Inhibin

Stacey C. Chapman and Teresa K. Woodruff

Department of Neurobiology and Physiology (S.C.C., T.K.W.) and Robert H. Lurie Comprehensive Cancer Center (T.K.W.), Northwestern University, Evanston, Illinois 60208; and Department of Medicine, Northwestern University Medical School (T.K.W.), Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Teresa K. Woodruff, Ph.D., Department of Neurobiology and Physiology, Northwestern University, O.T. Hogan 4-150, 2205 Tech Drive, Evanston, Illinois 60208. E-mail: tkw{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activin-stimulated FSH synthesis and secretion from the pituitary gonadotrope is negatively modulated by ovarian inhibin; however, the cellular mechanism of inhibin antagonism is unknown. Inhibin and activin share a common ß-subunit through which inhibin can compete with activin for binding to the activin type II receptor and prevent activin signal transduction. Although the affinity of inhibin for binding to the activin receptor is far lower than that of activin itself, inhibin is capable of inhibiting activin-stimulated FSH synthesis and secretion even at low or equimolar concentrations. It is now known that the TGFß type III receptor, betaglycan, acts as an inhibin coreceptor that binds the inhibins and increases their affinity for the activin type II receptor, thereby enhancing the antagonistic effect of inhibin on activin signal transduction. Yet, despite the characterization of betaglycan is an inhibin coreceptor in several cell models in vitro, the role of this protein in the regulation of FSH in vivo has not been demonstrated. In this study we sought to understand more fully the function of betaglycan in the control of FSH release by the gonadotrope by describing betaglycan immunolocalization in the pituitary and assessing its correlation to fluctuations in FSH and inhibin throughout the rat estrous cycle. In general, betaglycan immunoreactivity was present in the anterior pituitary at all estrous cycle time points, but was confined to the membrane of gonadotropes just before and after the primary and secondary FSH surges. Importantly, betaglycan localized to the gonadotrope membrane when inhibin must rapidly reduce FSH to basal levels after the secondary FSH surge. These data indirectly support a role for betaglycan in vivo as a coreceptor that is required for inhibin-modulated FSH release from the pituitary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH SYNTHESIS and secretion in the pituitary are tightly regulated throughout the female reproductive cycle by a balance of positive stimulation from the hypothalamus and the pituitary hormone activin and negative inhibition by locally produced follistatin (Fst) and inhibin from the ovary (1, 2, 3, 4, 5, 6). In female rats, FSH serum levels are maintained at basal levels during the early part of the 4-d estrous cycle, then surge on the afternoon of proestrus, concurrent with the LH surge, and again on the morning of estrus (7). The rat FSHß-subunit (FSHß) is a direct transcriptional target of autocrine or paracrine activin signals originating within the pituitary (8, 9, 10, 11), and several lines of evidence suggest that activin B is the pituitary form of activin involved in FSH regulation (8, 12, 13, 14).

It is possible that activin B synthesis and autocrine signaling in the pituitary fluctuate through the estrous cycle to direct increases and decreases in FSHß expression and serum FSH. Free activin A levels in the serum fluctuate throughout the rat estrous cycle, peaking on the evening of proestrus and the early morning of estrus, just before the primary and secondary FSH surges (15). A second hypothesis describes a tonic level of pituitary activin B production and signaling throughout the estrous cycle that is then negatively modulated by changing levels of inhibin and Fst. In female rats, both serum inhibin A and inhibin B concentrations negatively correlate with serum FSH, although inhibin B serum levels peak early in the estrous cycle, whereas circulating inhibin A increases gradually until just before the primary gonadotropin surge (16). Fst levels in the pituitary peak on the evening of proestrus and then fall by the morning of estrus, an expression pattern that would permit pituitary activin to stimulate the secondary FSH surge during estrus (15, 17).

Activin is a dimeric protein comprised of two ß-subunits, ßA or ßB, to produce three isoforms of mature activin: activin A (ßAßA), activin B (ßBßB), and activin AB (ßAßB). The activins signal through a heteromeric serine-threonine receptor kinase complex comprised of one of two ligand binding receptors, ActRIIA or ActRIIB, and the signaling activin type I receptor, activin-like kinase 4 (ALK4) (18). Although inhibin and activin are both members of the TGFß superfamily, an equivalent inhibin receptor complex or signaling pathway has not been described. Thus, inhibin has been largely characterized as an antagonist to activin-stimulated FSH synthesis and secretion in the pituitary.

The inhibins are dimers comprised of a unique {alpha}-subunit and one of the two activin ß-subunits, to produce inhibin A ({alpha}ßA) or inhibin B ({alpha}ßB). As both inhibin and activin dimers are assembled from a common pool of ß-subunits, one proposed mechanism for inhibin antagonism of activin-stimulated FSH is based on competition for the ß-subunit during ligand assembly. Additionally, inhibin can bind to the activin type II receptors with low affinity, presumably through the single ß-subunit it shares with activin, but does not stimulate phosphorylation of ALK4 (19, 20, 21, 22). Thus, if the concentration of inhibin exceeds that of activin, inhibin could functionally antagonize activin signaling by competing with activin binding to its own receptor or by interfering with additional activin receptor complex assembly (23).

In some cases inhibin is incapable of antagonizing activin action (22, 24), whereas in other studies, inhibin prevents activin signaling at low or equimolar concentrations (4, 25, 26). These observations suggested that inhibin responsiveness may require the presence of inhibin binding accessory proteins or coreceptors that increase the affinity of inhibin binding to the activin type II receptors and facilitate functional antagonism of activin signaling (22, 24, 27, 28). Indeed, the TGFß type III receptor, betaglycan, is now recognized as an inhibin coreceptor that binds both inhibin A and inhibin B with very high affinity, increases the affinity of the inhibins for the activin type II receptors, and allows low or equimolar levels of inhibins to functionally antagonize activin signaling (29, 30, 31). The inhibin-binding properties of betaglycan differ with respect to the isoform of inhibin involved as well as the type of activin receptor present.

During the female reproductive cycle, changes in the availability of inhibin and the inhibin binding coreceptors in the pituitary gonadotrope would be expected to influence activin-stimulated FSH expression and secretion. Betaglycan mRNA is present in both anterior and intermediate lobes of the male rat pituitary; however, the majority of betaglycan protein is localized to the intermediate lobe (32, 33). Betaglycan colocalizes with the gonadotropin hormones as well as with GH and prolactin in the somatotrophs and lactotrophs of the male rat pituitary (33). In light of our recent data indicating that betaglycan function may depend on the available inhibin isoform (31), and the discordance of inhibin A and inhibin B secretion throughout the female reproductive cycle, we used immunofluorescence histochemistry to correlate pituitary betaglycan localization with circulating inhibin concentrations, FSHß expression in the gonadotrope, and serum FSH levels throughout the rat estrous cycle.

We predicted that betaglycan protein levels in the female rat pituitary gonadotrope would be highest during metestrus and diestrus before the primary FSH surge. During this time inhibin antagonism of activin-stimulated FSH is essential to maintain low levels of the gonadotropin. In addition, betaglycan activity in the pituitary would be necessary after the secondary FSH surge in late estrus, when inhibin levels are slowly rising but FSH must rapidly return to basal levels. The betaglycan immunoreactivity pattern throughout the estrous cycle, superimposed upon fluctuating serum levels and pituitary content of the inhibins and FSH, will give us a more complete picture of when, where, and how betaglycan function is likely to be important within the pituitary during the female reproductive cycle in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Adult male and female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were housed in a temperature-controlled room, with lights on from 0500–1900 h, at Northwestern University (Evanston, IL). Animals were housed four per cage and were provided with food and water ad libitum. Estrous cyclicity was monitored by daily examination of vaginal cytology. Animals exhibiting at least three consecutive 4-d estrous cycles were used in these experiments. One female rat from each time point was deeply anesthetized and perfused transcardially with 4% paraformaldehyde, and pituitaries were removed and postfixed overnight at 4 C. All other animals were asphyxiated in a CO2 chamber and decapitated. Trunk blood was collected and allowed to coagulate overnight at 4 C, and serum was collected after centrifugation, aliquoted, and stored at -80 C. Pituitaries were either snap-frozen on powdered dry ice or fixed in 4% paraformaldehyde overnight. All fixed pituitaries were cryoprotected in 30% sucrose and then frozen in Tissue-Tek OCT embedding medium (EM Science, Cherry Hill, NJ). Ten-micrometer pituitary sections were cut on a cryostat and thaw-mounted onto SuperFrost Plus slides (Fisher Scientific, Pittsburgh, PA). Pituitary samples for hormone assays were obtained by homogenizing one whole pituitary in 0.5 ml 0.85% NaCl (wt/vol) as described previously for the ovary (34). All animals were treated in full accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Hormone assays
Serum and pituitary LH and FSH and serum estradiol and progesterone were measured by RIA (Center for Research in Reproduction, University of Virginia, Charlottesville, VA). The FSH RIA had a detection range of 0.6–18 ng/ml and an intraassay variation of 2.5–1.7%. The LH RIA had a detection range of 0.07–37.4 ng/ml and an intraassay variation of 3.4–7.1%. Whole pituitaries were homogenized, then diluted 1:100 for the FSH assay and 1:1000 for the LH assay in 0.85% NaCl. Serum and pituitary inhibin A and inhibin B were measured by ELISA (Diagnostic Systems Laboratories, Webster, TX). The inhibin assays had a sensitivity of 10–1000 pg/ml. Intraassay variations for inhibin A and inhibin B were 5.12% and 4.67%, respectively. Interassay variations for inhibin A and inhibin B were 21.33% and 22.97%, respectively. Total activin A was also measured by ELISA (Serotec, Oxford, UK). The inhibin and activin assays were validated using recombinant human (rh-) inhibin A (WHO 91/624), rh-inhibin B (National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK), and rh-activin A standards (Northwestern University, Evanston, IL).

Immunofluorescence microscopy
Sections mounted on slides were washed in 1x PBS and incubated in 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 1x PBS and 0.3% Triton X-100 (PBST) overnight at 4 C. Sections were incubated overnight at room temperature with primary antibodies or antisera diluted in 4% normal donkey serum in PBST (see detailed protocols below). After four washes in 1x PBS, slides were incubated for 1 h at room temperature with the appropriate fluorescently labeled secondary antibody diluted in 4% normal donkey serum in PBST. Sections were washed twice in 1x PBS and coverslipped with Vectashield containing 4',6-diamido-2-phenylindole hydrochloride (Vector Laboratories, Inc., Burlingame, CA). Exclusion of the primary antibody and application of normal goat IgG were run as negative controls to confirm the specificity of the immunostain, as indicated in the figure legends. Slides were viewed using fluorescence microscopy, and digital images were collected using a SpotRT monochrome digital camera (Diagnostic Instruments, Sterling Heights, MI) and the Metamorph image analysis system (version 4.5, Universal Imaging Corp., West Chester, PA).

Localization of inhibin subunits was carried out by incubating tissues in mouse monoclonal antihuman {alpha}-subunit (Diagnostic Systems Laboratories), rabbit antihuman {alpha}-subunit IgG, rabbit antihuman ßA-subunit IgG, or rabbit antihuman ßB-subunit IgG (gifts from W. W. Vale, The Salk Institute, La Jolla, CA) diluted 1:200 in PBST. After four 1x PBS washes, tissues were incubated with fluorescein isothiocyanate (FITC)-conjugated donkey antimouse IgG (1:200 in PBST; Jackson ImmunoResearch Laboratories, Inc.), or Cy3-conjugated donkey antirabbit IgG (diluted 1:800 in PBST; Jackson ImmunoResearch Laboratories, Inc.).

Colocalization of FSHß and betaglycan was achieved by applying cocktails of affinity-purified goat antihuman TGFß type III receptor IgG (5 µg/ml in PBST; R&D Biosystems, Minneapolis, MN) and rabbit antisera directed against rat FSHß (diluted 1:50 in PBST; provided by Dr. A. Parlow, National Hormone and Pituitary Program) to tissues. After four washes in 1x PBS, tissues were subsequently incubated with Cy3-conjugated donkey antigoat IgG (diluted 1:800 in PBST; Jackson ImmunoResearch Laboratories, Inc.) and FITC-conjugated donkey antirabbit IgG (1:200 in PBST; Jackson ImmunoResearch Laboratories, Inc.).

Colocalization of betaglycan and cadherin was carried out by incubating tissue sections with cocktails of affinity-purified goat antihuman TGFß type III receptor IgG (5 µg/ml in PBST; R&D Biosystems) and mouse anti-(pan)cadherin (diluted 1:500 in PBST; AbCam Ltd., Cambridge, UK) to tissues. After four washes in 1x PBS, tissues were subsequently incubated with Cy3-conjugated donkey antigoat IgG (diluted 1:800 in PBST; Jackson ImmunoResearch Laboratories, Inc.) and FITC-conjugated donkey antimouse IgG (1:200 in PBST; Jackson ImmunoResearch Laboratories, Inc.).

Statistical analysis
Correlations were determined by linear regression analysis using GraphPad PRISM software (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum and pituitary hormone profiles for key reproductive hormones throughout the estrous cycle
The rats in this study showed the expected changes in serum FSH and LH (Fig. 1AGo) and estrogen and progesterone levels (Fig. 1BGo) across the estrous cycle (7). Pituitaries from at least four rats from each cycle time point were homogenized and assayed for pituitary gonadotropin content. Both LH and FSH pituitary levels rose through metestrus and diestrus and peaked on the morning of proestrus, just before the expected primary rise in serum gonadotropins by 1600 h on proestrus (Fig. 1Go, C and D). The comparison of FSH serum levels and pituitary content is consistent with that reported previously, although a second peak in pituitary FSH on the afternoon of proestrus was not observed (35). Serum inhibin A and inhibin B levels corresponded well to previously reported profiles (Fig. 1EGo) (16, 36), and both inhibin isoforms were negatively correlated to serum FSH levels (Fig. 1Go, F and G). Total activin A levels in serum were undetectable by ELISA (data not shown).



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  		FIG. 1. Serum and pituitary gonadotropin and serum estradiol, progesterone, and inhibin profiles during the 4-d rat estrous cycle. Serum or pituitary homogenates collected from cycling female rats at the indicated time points were assayed for hormones. A, Serum LH ({square}) or FSH ({bullet}), and B, estradiol (E2; {square}) or progesterone (P; {bullet}) were measured by competitive RIA. Serum ({square}) and pituitary ({blacksquare}) homogenates were assayed for FSH (C) or LH (D) content by competitive RIA. E, Serum inhibin A ({square}) and inhibin B ({bullet}) contents were determined by two-site ELISA. Serum FSH ({diamondsuit}) was compared with serum inhibin A (F; {square}) or inhibin B (G; {bullet}). Note that data in F and G are identical to those in A, D, and E. Arrows indicate the primary (1°) and secondary (2°) FSH surges. n = 4–7 animals/time point. M, Metestrus; D, diestrus; P, proestrus; E, estrus.

 
Inhibin subunit localization in the rat pituitary
Although the inhibin {alpha}-subunit has been localized to the rat anterior pituitary, others have since demonstrated that mature inhibin is not secreted from pituitary cells in culture (12, 13, 37). Thus, the prevailing assumption is that although the pituitary synthesizes {alpha}-subunit precursors and mature {alpha}-subunit, it does not secrete mature inhibin dimers. Both ßA- and ßB-subunits are expressed in the pituitary gonadotrope, although only the ßB-subunit localizes to the cytoplasm, and activin B is the major secretory product of cultured pituitary cells (12, 13). In this study pituitary homogenates from male rats and from cycling female rats were assayed for activin A, inhibin A, and inhibin B content by ELISA. Because activin B is the predicted ligand produced by the pituitary, none of these ligands was detected in any pituitary sample (data not shown).

The presence or absence of inhibin and activin subunits in rat pituitary was confirmed by immunofluorescence. For these experiments, two antibodies directed against the {alpha}-subunit, a mouse monoclonal antibody and a polyclonal rabbit IgG, were used to detect this protein in pituitary tissue from male (Fig. 2Go) and female (Fig. 3Go) rats. In male pituitary, neither antibody was able to detect {alpha}-subunit (Fig. 2Go, A and D) with either a 4 C or room temperature overnight incubation or at various primary antibody concentrations. Little or no ßA-subunit was detected in the male rat pituitary in this study (Fig. 2Go, B and C); however, the ßB-subunit was highly expressed in the intermediate lobe and in a distinct population of cells within the anterior pituitary (Fig. 2Go, E and F). In the female, no {alpha}-subunit protein was observed in either metestrous or proestrous pituitaries (Fig. 3Go, A–D). Given previous data suggesting that activin ß-subunit mRNA is not regulated across the cycle (17), we found it compelling that both ßA- and ßB-subunits were present in the proestrous pituitary, but were undetectable in metestrous animals (Fig. 3Go, E–L). As in males, the ßB-subunit was detected in proestrous female anterior pituitary in a diffuse pattern (Fig. 3LGo).



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  		FIG. 2. The inhibin and activin ßB-subunit localizes to the intermediate and anterior lobes of the male rat pituitary. Inhibin {alpha}-subunit was not detected in the male rat pituitary by either a mouse monoclonal (green; A) or a rabbit polyclonal (red; D) antibody directed against the {alpha}-subunit. B and C, ßA-Subunit was also not detected. E and F, ßB-Subunit immunoreactivity was observed in the intermediate lobe and in discrete cells within the anterior lobe, but was not detected in the posterior lobe. To verify the specificity of binding, tissues were incubated with secondary antibody alone (G, FITC-conjugated antirabbit IgG; H and I, Cy3-conjugated antirabbit IgG). Nuclei were stained blue with 4',6-diamido-2-phenylindole hydrochloride. Scale bar in E, 500 µm (also applies to A, B, D, G, and H); scale bar in F, 500 µm (also applies to C and I. C, F, and I, Anterior lobe tissue only. A, Anterior lobe; I, intermediate lobe; P, posterior lobe.

 


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  		FIG. 3. Inhibin and activin ß-subunits were detected in the female rat pituitary on the evening of proestrus. Pituitary sections from female rats in metestrus (1000 h; A, B, E, F, I, J, M, and N) or proestrus (1830 h; C, D, G, H, K, and L) were incubated with polyclonal antibodies directed against the {alpha}-subunit (A–D), the ßA-subunit (E–H), or the ßB-subunit (I–L) or with secondary antibody alone (M and N). A–D, The {alpha}-subunit was not detected in female rat pituitary. E–H, ßA-Subunit was detected in the intermediate (I; arrows) lobe on the evening of proestrus. I–L, The ßB-subunit localized to the intermediate and anterior (arrows) lobes of female rats in proestrus, but was not detected in metestrous animals. Nuclei were stained blue with 4',6-diamido-2-phenylindole hydrochloride. Scale bar in K, 500 µm (also applies to A, C, E, G, I, and M); scale bar in L, 500 µm (also applies to B, D, F, H, J, and N). All images represent anterior lobe tissue unless otherwise labeled. A, Anterior lobe; I, intermediate lobe.

 
Betaglycan immunoreactivity in male and female rat pituitary gonadotropes
The results of previous studies with regard to the localization of betaglycan in the rat pituitary gonadotrope are conflicting. In studies carried out in our laboratory, betaglycan immunoreactivity was present in the anterior pituitary of both male and female rats but did not colocalize with FSHß (32). A subsequent study demonstrated betaglycan colocalization with 98% of FSHß-positive gonadotropes in male rat pituitary (33). Both reports describe high levels of betaglycan protein in the intermediate lobe, a finding that was recapitulated in this study (data not shown). The immunofluorescence histochemistry protocols used in these studies differed in several potentially significant ways, including primary antibody concentration, incubation time and temperature, and the conjugated secondary antibody used. Based on the results of our reevaluation of each protocol to detect FSHß and betaglycan immunoreactivity in male mouse pituitary tissue (data not shown), all immunofluorescence histochemistry experiments described here used the protocol described by MacConell et al. (33).

FSHß and betaglycan colocalized in male anterior rat pituitary, verifying previous observations, although a few FSHß-positive cells did not express betaglycan (Fig. 4Go, arrow). In cycling females, the localization and colocalization of betaglycan and FSHß in pituitary were assessed (Fig. 5Go). Significantly, changes in the intensity of FSHß immunoreactivity in the pituitary across the estrous cycle (Table 1Go) reflected those previously reported for pituitary FSHß mRNA levels (35) and preceded the rise in FSH pituitary content during diestrus and proestrus (Fig. 6AGo). FSHß immunoreactivity correlated positively with serum FSH levels throughout the estrous cycle (Fig. 6BGo).



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  		FIG. 4. FSHß and betaglycan colocalize in the male rat anterior pituitary. FSHß (A; green) and betaglycan (B; red) were detected in the male anterior pituitary. C, Double-labeled cells (yellow) indicate betaglycan expression in the male pituitary gonadotrope. Note that some FSHß-positive cells were negative for betaglycan staining (arrow). As controls for specific antibody binding, tissues were incubated with secondary antibody alone (D and E) or with goat IgG in the place of the primary antibody (F). Scale bar in C, 400 µm (applies to all panels in this figure).

 


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  		FIG. 5. FSHß and betaglycan colocalize in the cycling female rat pituitary. FSHß (green) was localized within discrete cells of the anterior lobe of the female rat pituitary. Betaglycan (red) immunoreactivity varied during the rat estrous cycle in a discrete population of cells within the anterior lobe. The highest level of betaglycan immunoreactivity was detected on at 1000 h metestrus on (M1000). Some degree of betaglycan expression was consistently observed in FSHß-positive gonadotropes at all time points (overlay). Note that at 1000 h on metestrus (M1000) and 1600 h on proestrus (P1600), betaglycan and FSHß colocalized to the cytoplasm (A, asterisks), whereas from 1000 h on diestrus (D1000) to P1230, betaglycan immunoreactivity was limited to the periphery of FSHß-positive gonadotropes (A, arrowheads). Cytoplasmic betaglycan immunoreactivity was high in the gonadotrope after the primary FSH surge at 0400 h on estrus (E0400) and again at E1000 (B, asterisks). Betaglycan and FSHß immunoreactivity localized to discrete compartments within the same cell on P1830 and on the morning of estrus (B, arrowheads). Scale bar in the lower right panel of the third column, 500 µm (applies to panels in the first three columns). M, Metestrus; D, diestrus; P, proestrus; E, estrus.

 

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  		TABLE 1. Betaglycan and FSHß localization in rat pituitary varied throughout the estrous cycle

 


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  		FIG. 6. FSHß immunoreactivity correlates with pituitary and serum FSH levels throughout the rat estrous cycle. FSH protein levels in pituitary homogenates (A, {blacksquare}) and serum (B, {diamondsuit}) were plotted against the immunofluorescent intensity of FSHß within the anterior lobe of the female rat pituitary ({circ}; data from Table 1Go). Serum and pituitary data are identical to those presented in Fig. 1Go. Note that some error bars are smaller than symbols. The correlation coefficients were -0.698 for pituitary FSH and FSHß immunofluorescence (A) and 0.078 for serum FSH and FSHß immunofluorescence (B). M, Metestrus; D, diestrus; P, proestrus; E, estrus.

 
The observed fluctuations in betaglycan immunofluorescence intensity throughout the cycle agreed with previous data describing slight changes in betaglycan mRNA levels, which decrease through metestrus and diestrus, then rise again after 0400 h on estrous (Fig. 5Go and Table 1Go) (32). However, overall betaglycan immunofluorescence intensity was not regarded as a reliable measure of betaglycan function, because betaglycan is also expressed in somatotropes and lactotropes, and low intensity signals correlated with betaglycan immunoreactivity limited to the periphery of the cells, where betaglycan would be expected to be the most active.

For this reason two distinct localization patterns of FSHß and betaglycan immunoreactivity were assessed in this study (Fig. 5Go and Table 1Go). It was immediately apparent that betaglycan and FSHß colocalized to some extent at each time point during the rat estrous cycle. Cells in which FSHß and betaglycan colocalized to the same cellular compartment, and thus produced a yellow overlay (Fig. 5Go, asterisks), were scored separately from those cells that showed both FSHß and betaglycan immunoreactivity but displayed no yellow overlay. In these cells FSHß localized to the cytoplasm, whereas betaglycan was detected exclusively on the cell boundary, resulting in green cells with distinct red margins (Fig. 5Go, arrowheads). The percentage of cells in which betaglycan immunoreactivity was limited to the membrane of FSHß-positive gonadotropes was highest during the early part of the estrous cycle, from 1000 h on diestrus to 1230 h on proestrus, fell during proestrus, then rose again at 0600 h on estrus (Table 1Go). This localization pattern inversely correlated with serum FSH and positively correlated with serum inhibin A and inhibin B (Fig. 7Go).



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  		FIG. 7. Relationship among serum and pituitary FSH, serum inhibins, and betaglycan immunoreactivity limited to the gonadotrope membrane. The percentage of FSHß-positive gonadotropes displaying betaglycan immunoreactivity limited to the cell membrane (% membrane BG; {blacksquare}; data from Table 1Go) was plotted against pituitary FSH (A, {blacksquare}), serum FSH (B, {diamondsuit}), serum inhibin A (C, {square}), and serum inhibin B (D, {bullet}). Pituitary and serum data are identical to those presented in Fig. 1Go. The correlation coefficients were 0.692 for pituitary FSH and % membrane BG (A), -0.185 for serum FSH and % membrane BG (B), 0.123 for inhibin A and % membrane BG (C), and 0.031 for inhibin B and % membrane BG (D). M, Metestrus; D, diestrus; P, proestrus; E, estrus.

 
The subcellular localization of betaglycan on the cell surface was verified by coimmunofluorescent localization of betaglycan and cadherin (Fig. 8Go). Cadherin is a transmembrane protein involved in cell adhesion and maintenance of cell-cell contacts and is expressed on the surface of various cell types throughout the body. Of note, cadherin immunoreactivity was observed in all cells of the anterior pituitary, with high levels of protein localized to the cell membrane. Because cadherin and betaglycan colocalized to some degree at all cycle time points (Fig. 8Go, arrowheads), betaglycan is most likely expressed on the cell surface of anterior pituitary cells, including somatotropes, lactotropes, and gonadotropes, throughout the rat estrous cycle.



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  		FIG. 8. Betaglycan colocalizes with cadherin on the membrane of anterior pituitary cells from cycling female rats. Betaglycan (red) is expressed in discrete cells within the anterior lobe of the pituitary and colocalizes with membrane cadherin (green) at every stage of the rat estrous cycle (yellow, arrowheads). Scale bar in lower right panel, 400 µm (applies to all panels in this figure). M, Metestrus; D, diestrus; P,proestrus; E, estrus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although betaglycan has been shown to increase the affinity of the inhibins for the activin type II receptors (29, 30) and facilitate inhibin A antagonism of activin signaling (29), the role of betaglycan in vivo has not yet been demonstrated. To date, no betaglycan overexpression or null animal models have been created to directly address this question. Until these models are available, evidence indirectly linking betaglycan to inhibin action in the pituitary continues to be gathered and assessed. In this study we attempted to expand this body of evidence by characterizing the localization of betaglycan in the female rat pituitary and its correlation to inhibin and gonadotropin levels throughout the estrous cycle.

The rats in this study displayed the appropriate patterns of serum gonadotropin and steroid levels as measured by RIA. Inhibin A, inhibin B, and activin A were not detected by ELISA in the pituitary of male or female rats. Accordingly, inhibin {alpha}-subunit was not detected in either male or female rat pituitary by immunofluorescence histochemistry. Male rats only expressed the ßB-subunit, whereas female rats expressed both ßA- and ßB-subunits on proestrus, but not metestrus. These findings indirectly suggest that activin B or activin AB production by the pituitary is regulated across the cycle and support the hypothesis that an increase in these activin isoforms may be involved in autocrine or paracrine up-regulation of FSHß expression on proestrus. Confirmation of this hypothesis through the direct measurement of pituitary and serum activin B throughout the female reproductive cycle awaits further assay development.

The detection of betaglycan in the male rat pituitary gonadotrope was expected based on previous work (33), and it is clear from this study that betaglycan protein and its subcellular localization are regulated across the rat estrous cycle. A high percentage of cells with betaglycan and FSHß immunoreactive overlay was observed from 1000 h on estrus to 1000 h on diestrus of the next cycle and also from 1600 h on proestrus to 0400 h on estrus, whereas betaglycan localized exclusively to the gonadotrope cell periphery from 1000 h on diestrus to 1230 h on proestrus and 0600 h on estrus. This pattern of betaglycan localization is suggestive of fluctuations in betaglycan production (on the morning of estrus through metestrus of the next cycle and during late proestrus) and cell surface expression (during diestrus and early proestrus, and again just after the secondary FSH surge) in the gonadotrope. A high percentage of cells displaying betaglycan immunoreactivity limited to the cell surface of gonadotropes corresponded to low pituitary and serum FSH and high inhibin levels through metestrus and diestrus. Significantly, the highest number of gonadotropes with membrane-localized betaglycan was detected at 0600 h on estrus, when slowly rising inhibin levels may require the coreceptor to mediate the rapid drop in serum and pituitary FSH levels after the secondary FSH surge.

It is important to note that betaglycan immunoreactivity was present at the cell surface of anterior pituitary cells to some degree across the estrous cycle. At this time, the degree of betaglycan immunoreactivity on the surface of the gonadotrope vs. other anterior pituitary cells, or whether any change in this immunoreactivity reflects variations in betaglycan function in the gonadotrope during the estrous cycle cannot be ascertained. However, if betaglycan immunoreactivity limited to the gonadotrope membrane is indicative of betaglycan function, then these data support a model in which betaglycan mediates inhibin antagonism of activin-stimulated FSH synthesis and release during the early part of the rat estrous cycle and just after the secondary FSH surge in estrus. Further studies are necessary to confirm this model.

Our previous data indicate that betaglycan facilitates inhibin A binding to both ActRIIA and ActRIIB, but augments inhibin B binding to ActRIIA only (23). In this study betaglycan immunoreactivity was highest on metestrus, when inhibin A is low relative to inhibin B. Based on the coreceptor model, we hypothesize that antagonism of activin-stimulated FSH by inhibin A requires the presence of betaglycan to augment inhibin A affinity for the activin type II receptors. Furthermore, antagonism of activin action in the pituitary by inhibin B may also require the presence of betaglycan if ActRIIA is the predominant activin type II receptor type in the pituitary. Using semiquantitative RT-PCR, we observed that the gonadotrope cell line, LßT2, expresses much higher levels of ActRIIA than ActRIIB (data not shown). Whole rat pituitary cultures also show far greater levels of ActRIIA expression than ActRIIB (38), and in the adult female rat, ActRIIA expression is confined to the intermediate and anterior lobes of the pituitary, whereas ActRIIB appears to be more diffusely expressed across all three lobes (39). The antibodies currently available for the detection of the activin type II receptor protein are not satisfactory for immunofluorescence histochemistry applications, and so the localization patterns of these receptors through the estrous cycle could not be assessed in the present study. However, we hypothesize that both receptors will be expressed in the pituitary gonadotrope, although perhaps, like betaglycan, these receptors will be available at the cell surface at different degrees throughout the estrous cycle.

An intriguing aspect of betaglycan function is that it is involved in both up-regulation of TGFß2 and down-regulation (via inhibin) of activin signal transduction. TGFß isoforms have been shown to compete with inhibin A for binding to betaglycan, leading to the hypothesis that TGFß may be able to modulate activin signaling by interfering with inhibin binding to the coreceptor (29, 30). Indeed, a recent study demonstrated that inhibin A antagonism of two activin-responsive reporter constructs could be rescued in a dose-dependent manner by increasing TGFß1 or TGFß2 in LßT2 cells (40). In the same study betaglycan-containing complexes were affinity labeled with [125I]inhibin A and could be competed by incubation with unlabeled inhibin A, TGFß1, or TGFß2, leading the researchers to conclude that TGFß competes with inhibin for binding to betaglycan and thereby rescues activin from antagonism. In addition, betaglycan has recently been implicated in mediation of inhibin antagonism of bone morphogenic protein (BMP) signaling through both activin type II receptors and BMP type II receptors (41). Thus, betaglycan can be considered a general regulator of TGFß superfamily signaling. As such, it is not surprising that betaglycan localizes to a diverse array of tissues, including lactotrophs and somatotrophs in the anterior pituitary, which may be targets of TGFß, BMPs, or the inhibins and activins.

The finding that betaglycan is involved in the modulation of several TGFß superfamily members particularly complicates the prediction of betaglycan knockout and transgenic overexpression model phenotypes. As both a TGFß and BMP coreceptor, the loss or overexpression of betaglycan would be expected to have a severe impact on animal development as well as on multiple aspects of adult physiology. To be specifically instructive of the role of betaglycan in inhibin antagonism in the reproductive axis, knockout or overexpression models would require the use of inducible, gonadotrope-targeted gene constructs.

In conclusion, this study demonstrated that betaglycan immunoreactivity on the surface of pituitary gonadotropes is highly correlated with high serum and pituitary inhibin levels and low FSHß and FSH during the early part of the estrous cycle and just after the secondary FSH surge. These findings are significant in that they provide strong evidence that betaglycan localizes within the pituitary gonadotrope at the appropriate time points within the female reproductive cycle to mediate inhibin antagonism of activin-stimulated FSH. We anticipate that the final proof that betaglycan acts as an inhibin coreceptor in vivo to modulate activin-stimulated FSH will be available with the creation of betaglycan overexpression and null mouse models.


   Acknowledgments
 
We thank Dr. W. W. Vale for the inhibin and activin subunit polyclonal antibodies. Thanks also to Dr. A. F. Parlow and the NIDDK National Hormone and Peptide Program for the FSHß antiserum, and to Diagnostic Systems Laboratories for the activin A ELISA kits and the monoclonal inhibin {alpha}-subunit antibody. Finally, I would like to express my sincere appreciation for the participation of Dr. Signe Kilen and Hilary Kenny in this study.


   Footnotes
 
This work was supported by NIH Grant HD-37096 (to T.K.W.) and by NIDDK/NIH through Cooperative Agreement U54-HD-28934 as part of the Specialized Cooperative Centers Program in Reproductive Research. During part of this study, S.C.C. was a fellow of the Northwestern University Robert H. Lurie Comprehensive Cancer Center Carcinogenesis (Training Grant T32-CA-009560).

Abbreviations: ALK4, Activin-like kinase 4; BMP, bone morphogenic protein; FITC, fluorescein isothiocyanate; Fst, follistatin; rh-, recombinant human.

Received May 29, 2003.

Accepted for publication September 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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