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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 10 5002-5008
Copyright © 2003 by The Endocrine Society

Expression of Betaglycan, an Inhibin Coreceptor, in Normal Human Ovaries and Ovarian Sex Cord-Stromal Tumors and Its Regulation in Cultured Human Granulosa-Luteal Cells

Jianqi Liu, Tiina Kuulasmaa, Veli-Matti Kosma, Ralf Bützow, Teemu Vänttinen, Christel Hydén-Granskog and Raimo Voutilainen

Department of Pathology (J.L., R.B., R.V.), Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland; Departments of Pediatrics (T.K., T.V., R.V.) and Pathology and Forensic Medicine (V.-M.K.), Kuopio University Hospital and University of Kuopio, FIN-70211 Kuopio, Finland; Department of Pathology, Center for Laboratory Medicine, Tampere University Hospital (V.-M.K.), FIN-33521 Tampere, Finland; and Department of Obstetrics and Gynecology, Helsinki University Central Hospital (R.B., C.H.-G.), FIN-00290 Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. Jianqi Liu, Department of Pathology, P.O. Box 21 Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: jiangi.liu{at}helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFß type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E2 and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E2, probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to activin signal transduction in human ovaries.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVINS AND INHIBINS are disulfide-linked dimers; activins ß-subunit dimers and inhibins heterodimers of one {alpha}- and one ß-subunit. The biological function of activins can be antagonized by inhibins in many tissues. The best known opposing functions of activins and inhibins relate to the reproductive axis: activins and inhibins exert opposite effects on FSH release from the pituitary, steroidogenesis in the gonads, proliferation of ovarian granulosa cells, and secretion of placental hormones (1, 2). The mechanisms of this antagonism can occur at different levels, such as ligand biosynthesis, and receptor binding, and activation (3). The net effect of activins and inhibins can be determined by preferential production of one subunit, leading to the biosynthesis of the corresponding mature protein. This phenomenon can be seen in developing ovarian follicles, where the {alpha}-subunit is produced in a 10- to 20-fold excess of the ß-subunits in FSH-recruited follicles, favoring the assembly of inhibin dimers over activin ones during the follicular maturation (1, 4). Another level of antagonism is determined by receptor interaction. Activins bind to specific type II receptors (ActRII or ActRIIB), promoting the recruitment and phosphorylation of the type I receptor serine kinase, which then regulates gene expression by activating intracellular mediators, Smad proteins (2). Inhibins also bind to type II activin receptors, but do not recruit type I receptor serine kinase, providing a competitive model for antagonizing the effect of activins. The TGFß type III receptor, betaglycan, can function as an inhibin coreceptor with ActRII. Inhibin binds betaglycan with high affinity, which enhances the binding of inhibin with ActRII. This inhibin/betaglycan/ActRII complex prevents the activins from binding to their own receptors (3, 5, 6, 7). In addition, betaglycan also enables inhibin to compete with bone morphogenetic proteins (BMPs; structurally related to activins and inhibins) for binding to the BMP-specific as well as to the activin-type II receptors and thus to prevent BMP signaling (8).

Activins and inhibins are implicated as autocrine and paracrine regulators of ovarian function. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation, up-regulation of FSH and LH receptor expression, stimulation of P450arom activity, induction of estrogen synthesis, and enhancement of oocyte maturation. Granulosa cell-derived inhibins can sensitize thecal cells to LH, thereby enhancing androgen production, an essential requirement for follicular estrogen synthesis. Activins can oppose this effect and suppress thecal cell androgen production (4, 9). Female mice homozygous for the null allele of inhibin {alpha}-subunit gene developed granulosa-thecal cell tumors, showing an important role of inhibin in the control of granulosa cell proliferation (10). Both type I and II activin receptors are expressed in human granulosa cells (11, 12, 13). Expression of betaglycan has also been found in human, porcine, and rat ovaries as well as in human granulosa cell tumors (5, 14, 15, 16). The function of activin/inhibin system is precisely regulated in the ovaries. The expression of each activin/inhibin subunit gene is modulated in a distinct manner by gonadotropins in granulosa cells (4, 11, 17). However, regulation at the receptor level is still poorly known. To shed more light on the antagonistic mechanisms of activins and inhibins in human ovaries, we studied the expression of betaglycan gene in normal and tumorous ovaries in vivo and its regulation pattern in cultured granulosa-luteal cells treated with different agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovarian tissues and cell cultures

Normal human ovarian tissues (n = 6) were from women with regular cycles obtained during cervical cancer operations without preoperative irradiation or medication, as described previously (18, 19). Tumor samples were obtained from women who underwent surgery for either granulosa cell tumors (n = 29) or thecomas and fibromas (n = 42) at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, as described previously (19). A tissue microarray was prepared from the tumors as previously described (20). In brief, a representative tumor area in the original donor block was selected based on a hematoxylin-eosin-stained section from the same block. Core tissue specimens (diameter, 0.8 mm) were taken from these areas of the individual donor blocks and precisely arrayed into a new recipient paraffin block with a custom-built precision instrument (Beecher Instruments, Silver Spring, MD). Four tissue biopsies were obtained from each tumor specimen. After the recipient block construction was completed, 5-µm sections were cut, and the presence of core tumor tissue on the arrayed samples was verified by hematoxylin-eosin staining. Human granulosa-luteal cells were harvested during follicular aspiration from women undergoing oocyte retrieval for in vitro fertilization (IVF), as described previously (17). The cells were pooled, enzymatically dispersed, separated from red blood cells, and cultured for 5–10 d before initiation of hormonal stimulation. At this culture stage progesterone production and activin/inhibin components are optimally responsive to gonadotropin and prostaglandin (PG) treatments (21, 22, 23). The research ethics committees of Helsinki and Kuopio University Hospitals approved the study protocols, and the women gave informed written consent.

Recombinant human FSH (rhFSH) and LH (rhLH) were gifts from Serono-Nordic (Vantaa, Finland), and recombinant human activin A peptide was obtained from R&D Systems (Minneapolis, MN). Dibutyryl cAMP [(Bu)2cAMP], prostaglandin E2(PGE2), PGF2{alpha}, and 12-O-tetradecanoyl phorbol 13-acetate (TPA) were purchased from Sigma-Aldrich (St. Louis, MO); Butaprost was obtained from Cayman Chemical (Ann Arbor, MI); and staurosporine was purchased from Roche (Mannheim, Germany).

Northern blot analysis

Extraction of cytoplasmic RNA, Northern blotting, and hybridization conditions were previously described (24). A 30-mer oligonucleotide probe was used to detect the betaglycan mRNA in Northern hybridization. The oligonucleotide sequence for betaglycan was 5'-CTGTTTCTGCTGTCAAGGAGAAGTTTGCTG-3', complementary to the human betaglycan mRNA (GenBank accession no. L07594) (25). Ribosomal 28S RNA cDNA was used for controlling RNA loading (26). The autoradiographs were scanned, and the relative intensities of the signals were quantified using Quant Mode in the MacBAS software (Fuji Photo Film Co., Tokyo, Japan). All mRNA data shown were normalized with the respective 28S RNA values.

RT-PCR

Due to the poor availability of normal ovarian tissues, RT-PCR analysis of previously isolated RNA from normal granulosa, thecal, and stroma cells (27) was used to investigate betaglycan expression in different normal ovarian compartments as well as in fresh granulosa-luteal cells from IVF patients. RNA was reverse transcribed with the First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). Betaglycan PCR was carried out in a final volume of 20 µl containing 50 ng cDNA, 1x reaction buffer, 0.2 mM deoxy-NTP mix, 0.4 µM of each primer, 2.5 mM MgCl2, and 1.5 U Taq DNA polymerase (MBI Fermentas). The primer set with a 7-base restriction enzyme recognition sequence at each 5'-end was 5'-TGGATCCCAAGGGAATCTGGTGAAGTG-3' (forward; GenBank accession no. L07594) and 5'-CGAATTCCACCTCTTCTGGCTCTCTGA-3' (reverse). Due to the influence of the 7-base restriction enzyme recognition sequence on the annealing temperature, we used a two-step PCR program. The amplification was performed with denaturation at 95 C for 5 min; 5 cycles of 95, 57, and 72 C for 30 sec each; an additional 35 cycles of 95 C for 30 sec, 62 C for 30 sec, and 72 C for 45 sec; and extension at 72 C for 10 min. PCR products were resolved in 1.5% agarose gels. The expected PCR product length from RNA was 374 bp. DNA contamination was ruled out by different PCR product sizes of the cDNA and genomic DNA due to a 1.7-kb intron in the PCR-amplified genomic DNA. PCR analysis was always performed at least twice to ensure reproducibility of the results.

Immunochemistry

The normal or tumorous ovarian tissue samples were fixed in formalin and embedded in paraffin. The 5-µm paraffin whole tissue or microarray sections were immunohistochemically stained as described previously (18) with a primary antibetaglycan antibody (sc-6199, Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution. Each tumor was represented on the microarrays by four cores. Omission of the primary antibody was used as a negative control. For immunocytochemistry, granulosa-luteal cells were cultured on two-well chamber plastic slides in the conditions described above. After treatment, the cells were washed three times in PBS, fixed in 4% paraformaldehyde with 5% acetic acid and 0.9% NaCl, washed in PBS again, and rehydrated through an ethanol series. The slides were then immunostained as described previously (18).

Progesterone measurement

Progesterone was measured by a competitive enzyme immunoassay purchased from Diagnostic Systems Laboratories, Inc. (Webster, TX), according to the manufacturer’s instructions. The detection limit of the assay was considered at 1 nM. The intra- and interassay coefficients of variation were 7.5% and 9.4%, respectively.

Statistics

The differences in the betaglycan mRNA levels and progesterone concentrations in different treatment groups in vitro were assessed by the Mann-Whitney test. The level of significance chosen was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of betaglycan in normal ovaries and ovarian sex cord-stromal tumors

Betaglycan transcript was detectable in normal human ovaries as well as in all ovarian compartments (granulosa, thecal, and stroma cells) by RT-PCR (Fig. 1Go). The granulosa-luteal cells freshly collected during IVF or cultured for 1–8 d without gonadotropin stimulation also expressed betaglycan mRNA. Besides ovaries, betaglycan mRNA was also detectable by both RT-PCR and Northern analysis in other human steroidogenic tissues, including testes, a Leydig cell tumor, adrenals, and adrenocortical tumors (data not shown), and in the adrenocortical cell line NCI-H295R (Fig. 1Go). Immunohistochemical staining of normal human ovaries with the betaglycan antibody demonstrated moderate immunoreactivity in the thecal cells of antral and preovulatory follicles. Granulosa cells of the late stage follicles showed only weakly positive staining for betaglycan (Fig. 2AGo). Strong positive staining of betaglycan was detected in the peripheral areas of corpora lutea (Fig. 2BGo), representing the thecal-luteal cells (28). Weakly positive staining was detected in some ovarian stroma and vascular cells. In contrast, primordial, primary, preantral, and early antral follicles and ovarian surface epithelial cells were negative. To study whether ovarian sex cord-stromal tumors (granulosa cell tumors, thecomas, and fibromas) express the betaglycan gene, we immunohistochemically stained microarray sections of ovarian tumors. Consistent with normal ovaries, tumor cells were only weakly stained (16 of 29) or were not stained in granulosa cell tumors. The fibrothecomatous stroma tissues separating the granulosa cells and some vascular cells were moderately or strongly positive for betaglycan immunostaining in 27 of the 29 granulosa cell tumors (Fig. 2Go, D and E). All 42 thecomas and fibromas expressed betaglycan with moderate to strong immunoreactivities. Positively stained cells were generally evenly spread in the vast majority of these tumors (Fig. 2FGo).



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  		FIG. 1. RT-PCR analysis of betaglycan mRNA in different compartments of human ovary and in granulosa-luteal cells (GL) from IVF patients. Total RNA was reverse transcribed and then amplified by PCR. The agarose-gel electrophoresis bands show the mRNA expression (374 bp in size) after 40 PCR cycles. The negative control (H2O) was from PCR amplification without any RT template. Genomic DNA and positive RNA controls were from human adrenocortical NCI-H295R cells (H295R).

 


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  		FIG. 2. Immunochemical staining of normal human ovary, sex cord-stromal ovarian tumors, and cultured granulosa-luteal cells with a betaglycan antibody. A, Moderate immunoreactivity was seen in thecal cells (Tc) of an antral follicle. Granulosa cells (Gc) show weakly positive staining. B, The peripheral area of a corpus luteum (Cl) was positively stained. C, Betaglycan was not detectable in a primary follicle (Pf). D and E, Strong positive staining in fibrothecomatous tissues and weak positivity in granulosa cells of granulosa cell tumors. F, Diffuse immunostaining in a thecoma. G, Weak staining in cultured granulosa-luteal cells without treatment. After treatment with FSH (H) or LH (I) at a concentration of 100 IU/liter for 24 h, immunopositivity was clearly increased. Original magnification: A and G–I, x200; B–F, x100.

 
Regulation of betaglycan expression by gonadotropins and protein kinase A and C modulators

We immunostained cultured granulosa-luteal cells with the antibetaglycan antibody and detected weak staining in some cells (Fig. 2GGo). Treatment with rhFSH (100 IU/liter) and rhLH (100 IU/liter) for 24 h induced positive staining for betaglycan in most cells (Fig. 2Go, H and I, respectively). In addition, betaglycan mRNA expression was detectable by Northern blotting in cultured granulosa-luteal cells (Fig. 3AGo). rhFSH and rhLH treatments (both 100 IU/liter for 24 h) increased the accumulation of betaglycan mRNA to more than 3-fold of the control level (both P < 0.05). The stimulatory effects of rhFSH and rhLH on betaglycan gene expression were dose dependent in a concentration range of 1–100 IU/liter (Fig. 3BGo; rhLH data not shown). The effect of rhFSH was already detectable after 3 h of treatment (Fig. 3AGo), reached its maximum at 24 h of treatment, and was maintained at the same level until at least the 48 h point. rhLH had no significant effect on betaglycan mRNA levels during the short 3-h treatment (Fig. 3AGo). Interestingly, the regulation of progesterone secretion by gonadotropins followed the same dose- and time-dependent pattern as that of betaglycan expression. Both FSH and LH increased progesterone production during 24-h treatment (29), but in 3-h incubations rhLH (100 IU/liter) had no effect, whereas rhFSH (100 IU/liter) increased it to 153%, and (Bu)2cAMP (1 mM) increased it to 165% of the basal value. This suggests that LH signaling in these cells differs from that of FSH (30).



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  		FIG. 3. The effects of rhFSH, rhLH, and (Bu)2cAMP on betaglycan mRNA accumulation in primary cultures of granulosa-luteal cells. The dispersed cells were allowed to grow for 7 d and then were treated with the indicated agents. Cytoplasmic RNA was extracted from the cells, and the Northern blots were prepared with 10 µg total RNA in each lane. The filters were sequentially hybridized with 32P-labeled betaglycan oligonucleotide and 28S ribosomal RNA cDNA probes. A, A representative Northern blot showing the effects of rhFSH (100 IU/liter), rhLH (100 IU/liter), and (Bu)2cAMP (1 mM) treatments for 3 h on betaglycan (ßG) mRNA expression. The migration of 28S and 18S ribosomal RNA is indicated. The dose-dependent effect of rhFSH (B) and (Bu)2cAMP (C) for 24 h on betaglycan gene expression was analyzed by Northern blotting. Each point in B and C represents the mean ± SEM of three experiments (from different patients), with the control levels adjusted to 100. *, P < 0.05 vs. control.

 
As the protein kinase A pathway is the main signal transduction system in the induction of steroidogenesis by gonadotropins in human granulosa-luteal cells, we examined whether the stimulatory effect of rhFSH and rhLH on betaglycan mRNA accumulation involves protein kinase A activation. As shown in Fig. 3AGo, the short-term effect of rhFSH on betaglycan gene expression was mimicked by (Bu)2cAMP treatment, suggesting that protein kinase A pathway is probably involved in the stimulatory effect of FSH. Immunocytochemical analysis demonstrated increased betaglycan immunostaining in cultured granulosa-luteal cells after 24-h treatment with 1 mM (Bu)2cAMP (data not shown). We also studied the effects of protein kinase C modulators on betaglycan expression, as the protein kinase C activator TPA regulates progesterone secretion in cultured granulosa-luteal cells (29). Treatment for 24 h with the protein kinase C inhibitor staurosporine (50 nM) increased the expression of betaglycan mRNA (P < 0.05; Fig. 4AGo). The stimulatory effect of staurosporine was time and dose dependent and was detectable during the short-term (3-h) treatment and at a concentration of 1 nM, with a maximal effect in the concentration range of 30–50 nM. However, staurosporine had no significant effect on progesterone secretion (P > 0.05; n = 8). In contrast, the protein kinase C activator TPA had no effect on the expression of betaglycan mRNA (Fig. 4BGo), even though it increased progesterone secretion as reported previously (29). Treatment with activin A (100 ng/ml) or etomidate (10 µg/ml), an inhibitor of steroidogenesis, for 24 h had no significant effect on basal or (Bu)2cAMP-induced betaglycan mRNA expression.



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  		FIG. 4. Two representative Northern blots showing the effects of (Bu)2cAMP (A and B), staurosporine (A), and TPA (B) on betaglycan (ßG) mRNA accumulation in primary cultures of granulosa-luteal cells. The dispersed cells were cultured for 7 d and treated with (Bu)2cAMP (1 mM), staurosporine (ST; 50 nM), and TPA (160 nM) for 3 h. RNA analysis was similar to that in Fig. 3Go. The experiment was repeated three times, and the results were comparable.

 
Induction of betaglycan expression by PGE2

As PGs have previously been reported to regulate inhibin/activin system in granulosa-luteal cells (23, 29), we tested their effect on betaglycan mRNA expression. Within 24 h of treatment, PGE2 up-regulated betaglycan mRNA expression in a dose-dependent manner in cultured granulosa-luteal cells (Fig. 5Go). This stimulatory effect was also time dependent, with a detectable increase in the steady state levels of the betaglycan mRNA after 1-h stimulation, and maximal induction achieved by 6 h. Thereafter, the stimulatory effect of PGE2 declined slowly, but remained significant at the 24 h point. Immunocytochemistry confirmed increased betaglycan immunopositivity after 24-h treatment with PGE2 at a concentration of 1 µM (data not shown). The effect of PGE2 was mimicked by butaprost, a selective agonist of PGE2 receptor EP2 subtype (Fig. 5AGo). This effect was dose dependent after 24-h treatment. Similarly to PGE2, butaprost dose-dependently increased progesterone secretion, with a 2-fold increase at a concentration of 1 µM after 24-h treatment. In contrast, PGF2{alpha} had no significant effect on betaglycan mRNA accumulation, even though it increased progesterone secretion by 100% after 24 h of treatment at concentrations of 1 and 10 µM.



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  		FIG. 5. Northern blot analysis of betaglycan mRNA expression regulated by PGE2 and butaprost in cultured granulosa-luteal cells. The culture conditions and Northern analysis were the same as in Fig. 3Go. A, Representative Northern blot showing the effects of PGE2 (1 µM) and butaprost (1 µM) for 24 h on betaglycan (ßG) mRNA expression. B, Dose-dependent effect of PGE2 on betaglycan gene expression analyzed by Northern blotting. Each point represents the mean ± SEM of three experiments (from different patients), with the control levels adjusted to 100. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we showed that human ovarian late stage follicular granulosa and thecal cells express the betaglycan gene, confirming a previous report on rat ovaries (15). The localization of positive betaglycan immunostaining in these cells is consistent with the expression of inhibin {alpha} in ovarian follicles (31), suggesting that betaglycan may function as an inhibin coreceptor in ovarian follicular development. Besides ovarian cells, we detected betaglycan mRNA in other human steroidogenic tissues, including testes and adrenals, where the expression of inhibins also occurs. In addition, our data show that betaglycan is present in the vast majority of ovarian sex cord-stromal tumors, suggesting that betaglycan protein may contribute to the phenotype of these tumors. It was previously shown that betaglycan mRNA expression detected by RT-PCR was variable in individual granulosa cell tumors (16). This discrepancy may be explained by variable fibrothecomatous stroma proportion in the tumor tissues used for extracting RNA, as by immunohistochemistry betaglycan expression is mostly localized to the fibrothecomatous stroma of granulosa cell tumors. This is in contrast to the situation for inhibin {alpha}-subunit, which is diffusely expressed in granulosa cells of these tumors (32). In cultured human granulosa-luteal cells, betaglycan mRNA accumulation and immunopositivity were up-regulated by FSH and LH, parallel to the stimulation of progesterone secretion and steroidogenic enzyme gene expression (21, 33). This in vitro finding is in agreement with the induction of betaglycan immunopositivity to the granulosa cells during the gonadotropin-induced follicular development. It is well known that the actions of FSH and LH are mediated principally through the activation of adenylate cyclase and the subsequent increase in intracellular cAMP levels. Consistently, treatment with (Bu)2cAMP efficiently increased the expression of the betaglycan gene, suggesting that the protein kinase A pathway is involved in the stimulatory effect of gonadotropins on betaglycan gene expression in cultured granulosa-luteal cells. As the protein kinase C signal transduction pathway regulates follistatin-related gene (FLRG; an activin-binding protein) mRNA expression and steroidogenesis (29), we also studied the effects of protein kinase C modulators on betaglycan expression. Although the protein kinase C activator TPA had no effect on betaglycan mRNA accumulation, the effect of the protein kinase C inhibitor staurosporine suggests that protein kinase C may be involved in the regulation of betaglycan gene expression in human ovaries.

Primary cultures of human granulosa-luteal cells from IVF are a widely used in vitro model for studying biochemical changes occurring during luteinization in vivo. We compared the regulation pattern of betaglycan gene expression in granulosa-luteal cells after 1-d preculture (allowing the cells to attach to the dishes) with our standard (5–10 d) preculture method. Although the stimulatory effects of (Bu)2cAMP and PGE2 were similar in both culture systems, the effects of FSH and LH were not consistent. As expected, the effects of FSH and LH on progesterone secretion were not significant in the 1-d preculture system (data not shown). Therefore, we chose the same long-term preculture model we have used previously for investigating the effects of gonadotropins on inhibin/activin ßB-subunit and FLRG expression (17, 29). Because FSH and LH are essential physiological hormones regulating granulosa cell function, their stimulatory effects on betaglycan expression in cultured granulosa-luteal cells suggest a role for betaglycan in follicular growth and development. Cultured human granulosa-luteal cells and freshly isolated preovulatory granulosa cells express the specific mRNAs for all currently known serine/threonine kinase activin receptors, i.e. activin receptors types I, IB, II, and IIB (11, 13). It has been hypothesized that the follicular environment changes from activin- to inhibin-dominant during folliculogenesis on the basis that granulosa cells exhibit an increase in the inhibin/activin expression ratio toward the end of the follicular phase in vivo or after gonadotropin stimulation in vitro (9, 34). We speculate that progressive gonadotropin stimulation will also sensitize the granulosa cells to inhibin influence by increasing betaglycan expression.

PGE2 is found in significant amounts in human follicular fluid and corpus luteum tissue extracts. PGE2 exerts its luteotropic effects through binding to cell surface EP receptors. Human granulosa-luteal cells express functional EP1 and EP2 PG receptors, both of which bind PGE2 with high affinity (35). Our present data demonstrate that betaglycan gene expression is up-regulated by PGE2 in cultured granulosa-luteal cells. The induction of betaglycan expression by PGE2 is mimicked by an EP2-selective agonist, butaprost. It is known that the activation of EP2 receptors is associated with an increase in cAMP concentrations (35). Therefore, it is likely that PGE2 regulated betaglycan expression through the cAMP-dependent protein kinase A pathway. Previous reports show that PGE2 is a potent inducer of dimeric inhibin A and follistatin secretion in cultured granulosa-luteal cells (23, 36). It is interesting that FLRG expression is increased by PGE2 too, but probably through the EP1 receptor and an increase in the intracellular Ca2+ concentration (29). FLRG protein has high affinity for activin and is able to inhibit activin-induced transcriptional responses (37, 38). Therefore, PGE2 may dictate inhibin/activin functions through three coincident changes: induction of inhibin A synthesis to reduce the activin/inhibin ratio, increase in follistatin and FLRG production to reduce activin bioavailability, and stimulation of betaglycan expression to strengthen inhibin bioactivity. The high concentration of PGE2 in luteinizing granulosa cells after the gonadotropin surge (39) may thus facilitate the inhibin-dominant environment.

In summary, we found that the betaglycan gene is expressed in human ovarian granulosa and thecal cells, sex cord-stromal ovarian tumors, and cultured granulosa-luteal cells. Thecal cells or fibrothecomatous tissues were the major expressing site for betaglycan in our study. The accumulation of betaglycan mRNA and immunopositivity in cultured granulosa-luteal cells was up-regulated by FSH, LH, and PGE2, mainly through the protein kinase A pathway. The specific expression and regulation pattern of the betaglycan gene are likely to have a role in the functional antagonism of inhibins in activin-induced signal transduction.


   Acknowledgments
 
Ms. Merja Haukka and Helena Kemiläinen are thanked for their skillful technical assistance. rhFSH and LH for in vitro experiments were generously provided by Serono-Nordic (Vantaa, Finland).


   Footnotes
 
This work was supported by the Jalmari and Rauha Ahokas Foundation, the Academy of Finland, the Sigrid Juselius Foundation, and Kuopio University Hospital.

Abbreviations: ActRII, Activin receptor type II; BMP, bone morphogenetic protein; (Bu)2cAMP, dibutyryl cAMP; EP2, prostaglandin E receptor subtype 2; FLRG, follistatin-related gene; IVF, in vitro fertilization; PG, prostaglandin; rh, recombinant human; TPA, 12-O-tetradecanoyl phorbol 13-acetate.

Received April 21, 2003.

Accepted July 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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