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BOR - Papers in Press, published online ahead of print September 22, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030171
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BIOLOGY OF REPRODUCTION 72, 172–178 (2005)
DOI: 10.1095/biolreprod.104.030171
© 2005 by the Society for the Study of Reproduction, Inc.

Messenger RNA and Protein Expression Analysis of Betaglycan in the Pituitary and Ovary of the Domestic Hen1

Sheila A. Sweeney, and Patricia A. Johnson2

Department of Animal Science, Cornell University, Ithaca, New York 14853


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Betaglycan was originally characterized as the type III receptor for TGFß, yet recent research has indicated that betaglycan can serve as an accessory receptor for inhibin. To understand better the action of inhibin in avian follicular development, we have investigated the expression of betaglycan in the pituitary gland and ovary of the hen. In experiments 1 and 2, betaglycan mRNA was detected at 6 kilobases (kb) by Northern blot analysis (n = 5) in chicken pituitary, granulosa, and theca layers and whole ovary. Expression of betaglycan was greatest in the pituitary gland in experiment 1 and greater in the granulosa layer of small yellow follicles (SYF) compared with the granulosa layer of larger follicles. In experiment 2, betaglycan mRNA was more abundantly expressed in the theca layer compared with the granulosa layer for all follicle sizes, although there was no significant difference in betaglycan expression in the theca layer among follicle sizes. In experiment 3, immunohistochemical analysis revealed betaglycan protein in the anterior pituitary as well as in the ovary (n = 4) and SYF (n = 4). Colocalization studies revealed a high abundance of cells within the anterior pituitary expressing both betaglycan and FSH (n = 4). Betaglycan protein was found in the granulosa layer; however, markedly enhanced staining was observed in the theca layer of ovarian follicles. Our results provide evidence for expression of betaglycan mRNA and protein colocalization with FSH in the anterior pituitary, consistent with known inhibin effects. Ovarian localization of betaglycan, particularly in the theca layer, suggests a paracrine role for inhibin in the hen.

female reproductive tract, granulosa cells, inhibin, ovary, pituitary, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin and activin are related but distinct dimeric proteins that share a common beta subunit that classifies them as members of the transforming growth factor ß (TGFß) superfamily [1]. Inhibin is a heterodimer composed of an {alpha} subunit, unique to inhibin and one of the ß subunits forming inhibin A ({alpha}ßA) or inhibin B ({alpha}ßB). Activin is formed by the disulfide linkage of two ß subunits, resulting in either activin A (ßAßA), activin AB (ßAßB), or activin B (ßBßB). TGFß family members have a variety of actions on cell growth, differentiation, and function from early embryonic stages through adulthood [2–4]. It is well documented that inhibin regulates FSH release from the pituitary in an endocrine fashion while also having a paracrine role in regulating follicular selection and steroidogenesis [2–6].

Although the mechanism of activin signaling has been well studied in mammals, the mechanism of inhibin action is unclear. Activins bind a type II receptor (ActRII or ActRIIB), which then recruits and transphosphorylates a type I receptor (ALK4) leading to phosphorylation of downstream smads [7]. Inhibins are known antagonists to activins in many cases. Inhibins can bind the activin type II receptors via their shared ß subunit; however, this ligand-receptor interaction does not lead to the recruitment and phosphorylation of the type I activin receptor [7–9]. Recent data suggest that not all of the actions of inhibin are a result of functional antagonism. For example, Murata and others [10] have shown that, when all pituitary activin is neutralized by an activin-specific antibody, inhibin is still able to block FSH release. Moreover, inhibin is unable to antagonize all of activin's actions. Specifically, inhibin does not antagonize activin-stimulated liver cell apoptosis [11] or activin-induced granulosa cell growth [12]. Interestingly, both activin and inhibin promote oocyte maturation [13] and Leydig cell P45017{alpha} mRNA accumulation [14]. Taken together, these data support the possibility of an alternate pathway of action for inhibin via a unique receptor complex.

Two potential inhibin receptors have been proposed: betaglycan, which is also known as TGFß type III receptor [15], and inhibin binding protein (InhBP), formerly referred to as p120 [16, 17]. Experiments have shown that InhBP can form a complex with ALK4, potentially functioning as an inhibin B-specific receptor by allowing inhibin B, but not inhibin A, to block activin-stimulated gene transcription [17, 18]. Recent data have shown that, although originally purified by affinity for inhibin, InhBP does not bind either inhibin A or B in standard receptor-binding assays [19]. In contrast with the results for InhBP, inhibin A has been shown to bind betaglycan with high affinity and, when in the presence of an activin type II receptor (ActRII), forms a highly stable complex [15]. The betaglycan-activin type II receptor complex is undisturbed by activin, which allows inhibin to antagonize activin action by competing for the type II receptor [15]. Lewis et al. [15] have shown that expression of betaglycan by activin-responsive cells previously insensitive to inhibin restores inhibin sensitivity and inhibits activin action. MacConell and colleagues [20] have shown the coexpression of immunoreactive betaglycan in the majority of FSH-immunostained gonadotropes. Moreover, the FSH-positive gonadotropes appear to be the predominant cell type immunopositive for betaglycan. Tissue-specific localization of betaglycan immunoreactivity has been shown in Leydig cells [20, 21], follicular granulosa and theca cells [20], and the oocyte [20], in accordance with documented inhibin responsivness in these cell types. These studies are consistent with a role for betaglycan as an important component of the signaling system for inhibin [20].

The site of inhibin production, both mRNA and protein, in the ovary of the hen is well characterized [22–24]. In the hen, as in mammals, the granulosa layer is the primary source of ovarian inhibin [25] and selective follicle-removal experiments have shown that the large preovulatory follicles are the major source of immunoreactive inhibin A in the hen ovary, while the small yellow follicles are the source of inhibin B [26–28]. To understand better the action of inhibin in avian reproductive physiology, we have investigated the expression of betaglycan as an indication of its role as a putative mediator of inhibin action. The objectives of the current study were to: 1) study the expression of betaglycan mRNA in chicken pituitary, granulosa, theca, and whole ovary tissue and 2) localize betaglycan protein in inhibin target tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Anterior pituitary and ovarian tissue was obtained from 1-yr-old single-comb White Leghorn hens of the Babcock B300 strain with regular laying cycles. The hens were individually caged, with egg records maintained at 2-h intervals during the daylight. All experimental animals had free access to water and food in rooms with controlled temperature and a lighting schedule of 15L:9D. Animal procedures were approved by the Institutional Animal Care and Use Committee of Cornell University.

Tissue Collection and RNA Extraction

Following euthanasia, the anterior pituitary gland and the complete ovary were removed and the follicles were placed in ice-cold Krebs-Ringer bicarbonate buffer (pH 7.4) for dissection. The pituitary and the remaining ovarian body were immediately placed in a guanidine isothiocyanate solution and frozen at –70°C until RNA extraction. In experiment 1, the granulosa layer was examined for betaglycan expression because it is the most abundant source of inhibin in the domestic hen. The granulosa layer was separated from the theca layer in all of the hierarchical follicles (F1– F5 follicles) as well as in the small yellow follicles (SYF; 6–12 mm) and pooled into three size categories; F1+F2, F3+F4+F5, and SYF. In experiment 2, we collected separate pools of theca tissue as well as granulosa tissue for the same follicle-size categories for comparison of expression of betaglycan mRNA. All granulosa and theca tissue pools were then frozen in ice-cold guanidine isothiocyanate solution at –70°C until RNA extraction. Tissues were homogenized and RNA was extracted using the guanidine isothiocyanate/phenol-chloroform method as previously described by Chomczynski and Sacchi [29]. For both experiments 1 and 2, tissue was collected from three birds for each replicate and this collection procedure was repeated to give five total replicates (n = 5). Separate hens were used (n = 4) as a source of pituitary and ovarian tissue for experiment 3.

Reverse Transcription-PCR

To generate a cDNA probe for betaglycan, PCR primers were designed (Primer Design software) and synthesized by Invitrogen Life Technologies (Carlsbad, CA), based on the previously reported chicken sequence (GenBank L01121; [30]). The 505-basepair (bp) cDNA probe spans 304 bp of the extracellular region and the entire transmembrane and cytoplasmic region with the forward primer positioned at 2166 bp of the chicken sequence (5'CACATGCGCAGAAGGAC) and the reverse primer positioned at 2670 bp of the chicken sequence (ACAAGGTCGTCGTCACG3'). Complementary DNA was obtained by first treating 1 µg of chicken anterior pituitary RNA with DNase I (1 U/µl) and then completing reverse transcription with SuperScript II RNase H-Reverse Transcriptase (200 U/ µl) according to the manufacturer's instructions (Invitrogen). The PCR reaction for chicken betaglycan was completed using Qiagen reagents (Valencia, CA) and the PCR program consisted of a jump start at 95°C for 15 min followed by 40 cycles of 94°C for 60 sec, 55°C for 60 sec, and 72°C for 60 sec, and a final extension at 72°C for 10 min in an AmplitronII Thermolyne thermocycler (Barnstead, Dubuque, IA). Control PCR reactions were the same except they contained no cDNA or used primers for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR product for betaglycan was ligated into the pGEM-T Easy vector using a TA cloning kit (Promega, Madison, WI) and purified using a Qiagen Plasmid Midi kit (Valencia, CA) according to the manufacturer's instructions. The predicted sequence and orientation of the PCR product within the vector were confirmed by automated sequence analysis completed at the Biotechnology Resource Center at Cornell University.

Northern Blot Analysis

For each replicate in experiments 1 and 2, approximately 30–40 µg of total RNA were prepared, separated on a 1.5% denaturing formaldehyde gel, blotted by capillary transfer, and ultraviolet cross-linked to GeneScreen Plus nylon membrane (NEN, Boston, MA). Northern analysis was done with the chicken betaglycan cDNA probe labeled with 5 µCi 32P-dCTP (DuPont, Boston, MA) by a random primer kit (Prime-It II, Stratagene, La Jolla, CA) to a specific activity of approximately 0.8–1.2 x 109 dpm/µg. The free 32P-dCTP was removed by a Sephadex G-25 column (NAP-5 column; Pharmacia Biotech, Uppsala, Sweden). The membranes were hybridized with the 32P-dCTP-labeled chicken betaglycan cDNA probe at 42°C for 24 h, stringently washed, exposed to film (Kodak BioMax MS; Eastman Kodak, Rochester, NY), and the film developed as previously described [23]. To verify and correct for equality of RNA loading and transfer, the final hybridization of the blots was done with a chicken GAPDH cDNA probe [28]. Exposure times were experimentally determined to be approximately 3–10 days for betaglycan and 6 h for GAPDH films. Autoradiograph signals on the films were scanned (ScanJet Plus; Hewlett-Packard Co., Boise, ID) and the intensity of the main transcript for betaglycan and GAPDH was estimated by Scion Image densitometry software (Scion Corporation, Frederick, MD). For an individual signal on each blot, the intensity of the sample was expressed relative to the strongest signal on the blot, which was rated as one. Relative values were averaged across replicate blots.

Immunohistochemistry

In experiment 3, betaglycan protein was localized using standard immunhistochemical protocols. Anterior pituitary (n = 4), whole ovary (n = 4), and SYF tissue (n = 4) were collected in processing cassettes, preserved in 10% formalin, embedded in paraffin, and sectioned (4 µm) onto Probe-On Plus polylysine slides (Fisher Scientific, Pittsburgh, PA) by the Histology Laboratory at Cornell University's College of Veterinary Medicine. Some sections for each tissue type were also stained with hematoxylin-eosin to observe tissue morphology. Briefly, sections were dewaxed at 64°C for 10 min and then subjected to consecutive washes in xylene, ethanol, and PBS to rehydrate. For antigen retrieval, slides were placed in boiling 0.01 M citrate buffer (pH 6) and sections were then blocked with 5% donkey serum for 30 min at 37°C in a humidified chamber. Sections were incubated for 1 h at 37°C with the primary antibody, a polyclonal antibody made in sheep against chicken transforming growth factor ß III (TGFßIII) receptor (X1484P, 1:50 from stock; Exalpha Biologicals, Inc., Boston, MA). After washing, the slides were incubated with Alexa Fluor 488 donkey anti-sheep IgG (A-11015; Molecular Probes, Inc., Eugene, OR) at 1:2000 dilution for 1 h at 37°C. The sections were washed, a coverslip was applied, and the slides were examined under fluorescent light using a Nikon Eclipse E600W microscope (Nikon Corporation, Tokyo) and SPOT RT camera and software v3.5.1 (Spot Diagnostic Instruments, Stering Heights, MI). Control sections were incubated with normal sheep IgG (1:50, #12-515 from Upstate Cell Signaling Solutions) and without primary antibody. For further analysis, SYF and whole-ovary sections were incubated with propidium iodide (1 µg/ml in PBS, which stains nuclei red) for 15 min at room temperature following secondary antibody incubation.

For dual-label fluorescent immunohistochemistry of betaglycan and FSH, deparaffinization, rehydration, and antigen retrieval were performed as described above. Anterior pituitary tissue sections were first blocked with a Tris buffered saline (TBS) blocking buffer containing 5% donkey serum and 5% goat serum for 1 h at room temperature in a humidified chamber. Sections were incubated overnight (at room temperature and in a humidified chamber), with a primary antibody cocktail composed of the sheep anti-chicken TGFßIII receptor polyclonal antibody (as previously described) and mouse anti-chicken FSH monoclonal antibody (obtained as a gift from Dr. John A. Proudman; diluted 1:100 from ascites). Following a 5-min incubation at 4°C, paraffin sections were washed three times with TBS and further incubated with Alexa Fluor 488 donkey anti-sheep IgG for 2 h at room temperature in a humidified chamber. Sections were rinsed briefly in TBS and incubated with Alexa Fluor 555 goat anti-mouse IgG (1:2000; Molecular Probes, Inc.) for 2 h at room temperature in a humidified chamber. After incubation, sections were washed with TBS three times, a coverslip was applied, and slides were examined. Slides were examined for TGFßIII-receptor staining (green) and FSH staining (red) as described above.

Statistical Analysis

ANOVA was used to assess differences in RNA expression in Northern blots using the General Linear Model Procedure of Statistical Analysis Systems with replicate and tissue as factors. Protected least-significant difference test was used to compare differences among tissues.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of mRNA for Betaglycan

In experiment 1, the betaglycan cDNA probe hybridized to one major band at approximately 6 kilobase (kb) in the total RNA of anterior pituitary, whole ovary, and granulosa tissue of all follicle pools. In addition, smaller sized bands could be visualized when betaglycan was abundant. A representative Northern blot is presented in Figure 1. After quantification by densitometry and correction for RNA loading with GAPDH, the mean ± SD (n = 5) relative intensity of the 6-kb betaglycan band is shown in Figure 2. The relative density for the anterior pituitary was significantly different from all tissues (P < 0.05) except for the whole-ovary tissue. The whole ovary was also not significantly different from the granulosa of SYF. The relative density values for the granulosa from F1+F2 and F3+F4+F5 were not significantly different from each other.



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  		FIG. 1. Representative autoradiogram of the Northern analysis of betaglycan mRNA compared with GAPDH (n = 5). One major band of expression was seen for betaglycan at 6 kb in the tissues examined. PIT, Pituitary; F, follicle (the subscripted number represents the hierarchical follicle order); SYF, small yellow follicles (6–12 mm diameter); and OVY, whole ovary after removal of yolky follicles



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  		FIG. 2. Quantitative analysis of betaglycan expression in pituitary, ovary, and granulosa layer of follicles relative to GAPDH. Bars with no common letters are significantly different from one another (P < 0.05). For all tissues, n = 5, except for OVY, where n = 3. Abbreviations are the same as in the Figure 1

In experiment 2, the betaglycan cDNA probe also hybridized to one major transcript at approximately 6 kb in the total RNA of the granulosa and theca tissue of all follicle pools. Figure 3 displays a representative Northern blot comparing relative expression levels of betaglycan mRNA in granulosa and theca tissue according to follicle size. The mean (±SD) relative density of the 6-kb betaglycan transcript for the granulosa (G) and theca (T) layers, after correction with GAPDH, is shown in Figure 4. In all follicle categories, expression of betaglycan was significantly (P < 0.05) higher in the theca layer compared with granulosa. There was no significant difference in expression of betaglycan mRNA in theca or granulosa layer among follicle-size categories. Because betaglycan mRNA was expressed at a high level in the theca layer, films were developed earlier to prevent burn out, while in studies examining betaglycan mRNA expression in the granulosa layer (experiment 1), films were exposed longer to determine expression levels. For this reason, the granulosa betaglycan mRNA expression levels were not optimized in this experiment.



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  		FIG. 3. Representative autoradiogram of betaglycan mRNA expression in granulosa and theca layer of various sized ovarian follicles. One major band of expression was seen for betaglycan at 6 kb throughout the tissues examined. Abbreviations are the same as in Figure 1 and 2, except that G represents the granulosa layer and T represents the theca layer



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  		FIG. 4. Quantitative analysis of betaglycan expression in the granulosa and theca layer of follicles relative to GAPDH. Values with different letters are significantly different from one another (P < 0.05). For all tissues, n = 5, except for F1+F2 T, n = 2; SYF G, n = 4; and SYF T, n = 4. Abbreviations are the same as in previous figures

Immunohistochemical Localization of Betaglycan Protein

Immunohistochemical analysis in experiment 3 showed that betaglycan protein was expressed in the pituitary gland of the adult hen. Parts C, E, and G of Figure 5 depict a field in the central part of the pituitary gland; in comparison, parts D, F, and H of Figure 5 depict a field from a more peripheral area of the pituitary gland. Abundant levels of betaglycan (green fluorescence) were distributed throughout the anterior pituitary (Fig. 5, C–D). FSH expressing gonadotopes (red fluorescence; Fig. 5, E–F) were observed throughout the anterior pituitary, with increased abundance in the central area of the gland. Dual-label immunofluorescence of anterior pituitary sections colocalized betaglycan protein to FSH-expressing gonadotropes (yellow fluorescence) in the anterior pituitary as shown in Figure 5, G and H. Interestingly, there was an increased abundance of dual labeling toward the central portion of the anterior pituitary section (thick white arrows, Fig. 5, G–H) when compared with peripheral areas of the anterior pituitary, where colocalization was less abundant (thin white arrows, Fig. 5H). No staining was observed when the tissues were incubated with normal sheep IgG or when the primary antiserum was omitted (Fig. 5B).



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  		FIG. 5. Immunohistochemistry in the normal adult hen pituitary gland. A) Hematoxylin-eosin staining shows nests (arrow) of cuboidal cells organized near venous sinusoids. B) No specific staining is evident for betaglycan when pituitary sections were incubated with normal sheep IgG. C, E, G) A field in the central part of the pituitary gland, a site of abundant FSH-containing gonadotropes. D, F, H) A field from a more peripheral area of the pituitary gland. C, D) Betaglycan (green) is abundantly expressed throughout the anterior pituitary. E) FSH-containing cells (red) are more abundant in the central portion of the pituitary gland as compared with a more peripheral area (F). G, H) Colocalization of betaglycan and FSH is shown as yellow fluorescence, as indicated by thick white arrows. Note increased abundance of colocalized betaglycan and FSH on section taken from central portion of pituitary (G) as compared with (H), where cells negative for colocalization are indicated by thin white arrows. Scale bar = 50 µm

Immunohistochemical analysis also revealed betaglycan staining in the SYF and whole-ovary tissue, as shown in Figure 6. As illustrated in Figure 6C, very faint expression of betaglycan was apparent in the granulosa layer of a SYF, as indicated by thin white arrow, while very intense levels of betaglycan were expressed in the theca layer of the SYF, as indicated by the thick white arrow (Fig. 6, C and D). Within the ovary, very faint staining for betaglycan was seen in the granulosa of very small follicles (<1 mm diameter) in the ovarian stroma, as indicated by the thin white arrow (Fig. 6E). Staining for betaglycan in the theca layer of these follicles, however, was very abundant, as indicated by the thick white arrow (Fig. 6E). Again, positive signal above background levels was not observed in ovarian sections when incubated with normal sheep IgG or without primary antiserum (Fig. 6, B and F; propidium iodide indicates individual nuclei).



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  		FIG. 6. Immunohistochemical localization of betaglycan in the ovary of an adult hen. A) Hematoxylin-eosin staining of SYF shows follicle morphology (with yolk platelets within the oocyte). B) No specific betaglycan staining is evident in the negative control (nuclei are stained red with propidium iodide). C) Intense expression of betaglycan (green) in the theca layer (thick white arrow) with much lower expression in the granulosa layer of SYF (thin white arrow). D) Higher magnification of panel (C) shows betaglycan (green) expression in the theca layer with nuclei stained red with propidium iodide. E) Immunohistochemical localization of betaglycan in follicles (<1 mm diameter) within the stroma of the whole ovary from the normal adult hen. Betaglycan (green) was strongly localized in the theca layer (thick white arrow; E), with much lower expression in scattered positive granulosa cells (thin white arrow; E). F) The specific immunostaining for betaglycan (E) is abolished in the negative control. Propidium iodide staining was used to identify the nuclei (red). Scale bar = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, Northern blot analysis and immunohistochemistry have been used to evaluate the tissue-specific expression of betaglycan mRNA and protein in the hen. Previous studies have described the presence of betaglycan protein and/or mRNA in mammalian species [31–34], but there have been no studies examining the distribution of betaglycan within reproductive tissues of the hen. Northern blot analysis has previously shown betaglycan mRNA expression in the chick heart, lung, and eye [30]. The results of the present study show that both betaglycan mRNA and protein are expressed in the ovary and pituitary gland of the hen.

As one would expect for a potential inhibin receptor, Northern blot and immunohistochemical analysis revealed abundant expression of betaglycan mRNA and protein within the anterior pituitary. Moreover, betaglycan protein colocalized to FSH-expressing pituitary gonadotropes. Although betaglycan protein localization was widespread within the pituitary, we observed a higher abundance of colocalization with FSH-expressing gonadotropes toward the central portion of the anterior pituitary. This is likely the result of FSH-expressing cells being less abundant at the perimeter of sagittal sections, as was also observed by Proudman and colleagues [35]. Studies in the rat have shown that, although all anterior pituitary cell types coexpress betaglycan to some degree, the predominant cell type found to be immunopositive for betaglycan is the FSH-positive gonadotrope [20]. It is well established that inhibin acts in an antagonistic fashion to block activin-stimulated FSH synthesis and consequently release of FSH from gonadotropes [36]. Therefore, the high level of betaglycan expression in the FSH-expressing gonadotropes is consistent with a role for betaglycan as an accessory receptor in mediating the inhibin effect on FSH secretion.

The present study is the first analysis of betaglycan expression in any of the ovarian follicles in the hen. As expected, betaglycan mRNA and its protein are expressed in gonadal cell types documented to be inhibin responsive, in particular granulosa and theca cells [37–39]. Betaglycan mRNA expression was highest in the theca layer when compared with the granulosa layer in all follicle-size categories, although betaglycan mRNA expression was not significantly different in the theca layer among follicle size categories. These data were further substantiated by immunohistochemical studies in which we saw intense signal for betaglycan protein within the theca layer of SYF (6–12 mm) and stromal-embedded follicles (<1 mm), with only a faint signal within the granulosa layer. In experiment 1, only when conditions were optimized for quantitation of granulosa RNA expression, the granulosa of SYF had significantly higher betaglycan mRNA levels than the granulosa layer of other follicle sizes. A recent study in the rat showed higher levels of betaglycan expression in the follicular granulosa cells and less expression in the thecal layer, while high levels of betaglycan localized to nonfollicular ovarian interstitial cells [20]. The dramatic difference in expression of betaglycan in the follicle layers of the hen ovary may be due to the different cell types involved in ovarian hormone production between mammals and birds. In contrast with mammalian species, the theca layer in the hen is not only the primary source of activin [24], it is also the most abundant source of estradiol and androgen [40]. In addition, while granulosa cells from prehierarchical follicles (3–8 mm) are steroidogenically incompetent [41], theca cells at all stages of follicular development are fully capable of steroidogenesis [42].

While the best-known example of the antagonistic relationship between activin and inhibin is FSH production by the pituitary [43], there is increasing evidence that inhibin also antagonizes the autocrine/paracrine actions of activin within the gonads, in particular steroidogenesis [44]. In the rat, inhibin stimulates while activin inhibits testosterone synthesis in testicular and theca cell cultures [37]. In addition, inhibin counteracts activin-stimulated 3ß-hydroxysteroid dehydrogenase (HSD) expression in porcine Leydig cells [14, 45]. Ying et al. [46] also observed that porcine inhibin reduced FSH-stimulated aromatase activity and progesterone production in rat granulosa primary culture, while activin has been shown to enhance progesterone and inhibin production as well as FSH-induced aromatase activity [47]. Jimenez-Krassel et al. [48] have also recently shown evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by altering aromatase activity in bovine granulosa cells.

The abundance of betaglycan expression within the theca layer of hierarchical and nonhierarchical follicles in the hen may be an indication of the modulatory role of inhibin in steroidogenesis. As previously outlined, inhibin is thought to bind with high affinity to betaglycan via its {alpha} subunit, which enhances binding of inhibin via its ß subunit to the type II activin receptor, forming a stable complex that interferes with activin heterodimerization of activin type I and II receptors [15]. It is possible that inhibin of granulosa origin modulates the production of steroids by the theca layer in vivo by antagonizing activin via association with betaglycan and activin type II receptors. In fact, there was a noticeable suppression of both basal and agonist- (LH, 8-bromo-cAMP) induced androstenedione production (and consequently, agonist-stimulated estradiol output) by theca cells incubated with granulosa cells of 6- to 8-mm hen follicles as compared with levels obtained from theca cells alone [41]. Clearly, examining inhibin binding interactions with betaglycan in these cell systems and subsequent steroid production will be important areas of future research.

In conclusion, betaglycan mRNA and protein are present in FSH-expressing pituitary cells and ovarian cell types likely responsive to inhibin, consistent with a role as an accessory receptor for inhibin in the hen. The abundance of betaglycan expression in the theca tissue suggests that it may be modulating inhibin-specific actions at the ovarian level, although functional assays need to be completed. Characterization of the pattern of betaglycan expression in the pituitary and ovary of the hen adds to our understanding of the mechanism of inhibin action and the role of inhibin in avian follicular development.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. John Proudman for providing the chicken FSH antibody for immunohistochemical studies and Dr. James Giles for immunohistochemistry assistance.


   FOOTNOTES
 
1 Supported by USDA NRI agreement 00-35203-9116, 03-35203-13397. Back

2 Correspondence: Patricia A Johnson, Department of Animal Science, Cornell University, 202 Morrison Hall, Ithaca, NY 14853. FAX: 607 255 9829; paj1{at}cornell.edu Back

Received: 26 March 2004.

First decision: 22 April 2004.

Accepted: 30 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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