The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells.

STUDY QUESTION: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? SUMMARY ANSWER: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. WHAT IS KNOWN ALREADY: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. STUDY DESIGN, SIZE, DURATION: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. LIMITATIONS, REASONS FOR CAUTION: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. WIDER IMPLICATIONS OF THE FINDINGS: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

Analysis of spatial and temporal expression patterns of bone morphogenetic protein family members in the rat uterus over the estrous cycle.

Recent studies have demonstrated that bone morphogenetic proteins (BMPs) play fundamental roles in female fertility. This is particularly evident in terms of the ovary. One major question that is just beginning to be addressed is the role of BMPs in the non-pregnant uterus. To help fill this gap, we used in situ hybridization to investigate the expression of BMP family members in the rat uterus over the estrous cycle. We found that the endometrial/uterine cycle is accompanied by the expression of several components of the BMP pathway - including ligands, receptors and antagonists. The mRNAs encoding BMP receptors are expressed in the epithelial (BMP-RIA, -RIB and -RII), periluminal stroma (BMP-RIA and -RII) and smooth muscle cells (BMP-RIA and -RII). The expression of all three receptors showed clear cyclic variations. The mRNAs encoding BMP ligands were highly expressed in the periluminal stroma (BMP-2 and -7) and glandular epithelium (BMP-7). The expression of BMP-2, but not BMP-7, was cyclical. Notably, the periluminal stroma expressed noggin mRNA. In the blood vascular system, BMP-4, -6 and -RII mRNAs were expressed in myometrial endothelial cells. Interestingly, follistatin, noggin, and BMP-4, -6 and -7 mRNAs were expressed in eosinophilic leukocytes, suggesting unexpected roles for eosinophil-derived BMPs in uterine function. We conclude that BMP ligands, receptors and antagonists are expressed in spatially and temporally restricted patterns that are consistent with a physiological role for these regulatory molecules in promoting uterine cellular processes including cell proliferation, differentiation and apoptosis during the cycle.

The role of bone morphogenetic proteins in ovarian function.

Bone morphogenetic proteins (BMPs) represent the largest subclass of growth factors in the transforming growth factor beta (TGF-beta) superfamily. BMPs have proven to be multifunctional regulators of a wide variety of biological processes in numerous types of cell and tissue. The role of inhibins, activins and TGF-betas (which also belong to the TGF-beta superfamily) in reproduction has been studied extensively over the last 15 years. However, there were no reports on the role of BMPs in the mammalian ovary until 1999 when we reported an intrinsic ovarian BMP system replete with BMP ligands, receptors and novel biological functions. Since this report it has become clear that the BMP system plays an important role in the regulation of ovarian function, evidenced by the ability of BMPs to control granulosa cell proliferation and cytodifferentiation, as well as oocyte development. The physiological relevance of the BMP system has recently been highlighted by the discovery that genetic mutations in the BMP-15 ligand or the BMP type IB receptor lead to critical aberrations in folliculogenesis and ovulation. This review provides a current overview of the rapidly emerging field of the BMP system in mammalian ovarian function.

Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells.

A major concept in mammalian ovarian physiology is that follicle-stimulating hormone (FSH) activates the granulosa cells (GCs) in the Graafian follicle to selectively produce estradiol, but not progesterone, during the follicular phase of the menstrual or estrous cycle. However, given the fact that FSH can induce production of both estradiol and progesterone by GCs cultured in vitro, it has been postulated for a long time that there is a factor present in the ovary that selectively prevents FSH-induced progesterone production. Here, we provide evidence that two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase-1 and -2 (ERK1/2) can differentially regulate FSH-stimulated estradiol and progesterone production. Using primary rat GCs from early antral follicles cultured in serum-free medium for 48 h, we found that the addition of a specific inhibitor of ERK1/2 activation, U0126, caused the attenuation or enhancement of FSH-induced progesterone or estradiol production, respectively, in a dose-dependent manner. Throughout the 48-h culture period in this culture system ERK1/2 molecules in their activated state (phospho-ERK1/2) were clearly detectable in GCs exposed to FSH. The addition of U0126 caused a decrease in the levels of phosphorylated but not unphosphorylated ERK1/2 which was maintained throughout the 48-h culture, suggesting that U0126 was continuously active to inhibit the phosphorylation of ERK1/2. The divergent regulation of FSH-induced progesterone and estradiol synthesis by U0126 was further supported by demonstrating that U0126 inhibits and stimulates FSH-induced mRNA levels of steroidogenic acute regulatory protein and P450 aromatase, respectively. Collectively, this study clearly identified ERK1/2 as the first intracellular signaling molecules that differentially regulate FSH-induced progesterone and estradiol synthesis in GCs.

Follistatin inhibits the function of the oocyte-derived factor BMP-15.

Recent studies have highlighted the importance of a novel oocyte-derived growth factor, bone morphogenetic protein-15 (BMP-15) in the regulation of proliferation and differentiation of granulosa cells in the ovary. Namely, BMP-15 stimulates granulosa cell mitosis and inhibits follicle-stimulating hormone (FSH) receptor mRNA expression in granulosa cell, thereby playing a critical role in the elaborate mechanism controlling ovarian folliculogenesis. At present, however, nothing is known about molecules which may regulate the biological activity of BMP-15. Here we demonstrate evidence that follistatin can form an inactive complex with BMP-15, through which follistatin inhibits BMP-15 bioactivities. The binding of follistatin to BMP-15 was directly demonstrated by a surface plasmon resonance biosensor, and the ability of follistatin to inhibit BMP-15 functions was determined by established BMP-15 bioassays using primary rat granulosa cells. Specifically, follistatin attenuated BMP-15 stimulation of granulosa cell proliferation and reversed BMP-15 inhibition of FSH receptor mRNA expression leading to the suppression of FSH-induced progesterone synthesis. This is the first demonstration of the biochemical interaction and biological antagonism of follistatin and BMP-15, which may be involved in the complex yet well-controlled mechanism of the regulation of follicle growth and development.

The physiology of folliculogenesis: the role of novel growth factors.

OBJECTIVE: To assess major physiological events underlying folliculogenesis, including FSH-dependent dominant follicle (DF) formation, LH/hCG signaling, and the role of novel regulatory molecules in these developmental processes. DESIGN: Review of some of the past and recent advances in ovarian biology, focusing attention on [1] two novel oocyte-derived growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic protein (BMP-15); and [2] a recently discovered follicular insulin-like growth factor binding protein-4 (IGFBP-4) protease, pregnancy-associated plasma protein-A (PAPP-A), that can degrade the FSH antagonist IGFBP-4. RESULT(S): Oocyte-derived GDF-9 and BMP-15 are obligatory for folliculogenesis and female fertility in laboratory animals through their ability to stimulate granulosa cell proliferation and modulate FSH-dependent cytodifferentiation. The expression of these growth factors in human primary oocytes supports the hypothesis that GDF-9 and BMP-15 could be involved in ovary function in women. Pregnancy-associated plasma protein-A is a marker for the human dominant follicle and its product the corpus luteum, raising the possibility that this putative FSH antagonist might regulate FSH bioactivity during folliculogenesis and luteogenesis. CONCLUSION(S): Oocyte-derived and granulosa-derived regulatory proteins perform very important functions in FSH-dependent folliculogenesis. The current challenges are to understand the role of these novel proteins in ovary physiology and pathophysiology in women.

Effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat.

We have previously established the presence of a functional bone morphogenetic protein (BMP) system in the ovary by demonstrating the expression of BMP ligands and receptors as well as novel cellular functions. Specifically, BMP-4 and BMP-7 are expressed in theca cells, and their receptors by granulosa cells. These BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. To investigate the underlying mechanism of the differential regulation, we analyzed mRNA levels for key regulators in the steroid biosynthetic pathways by RNase protection assay. BMP-7 enhanced P450 aromatase (P450(arom)) but suppressed steroidogenic acute regulatory protein (StAR) mRNAs induced by FSH, whereas mRNAs encoding further-downstream steroidogenic enzymes, including P450 side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase, were not significantly altered. These findings suggest that BMP-7 stimulation and inhibition of P450(arom) and StAR mRNA expression, respectively, may play a role in the mechanisms underlying the differential regulation of estradiol and progesterone production. To establish the physiological relevance of BMP functions, we investigated the in vivo effects of injections of recombinant BMP-7 into the ovarian bursa of rats. Ovaries treated with BMP-7 had decreased numbers of primordial follicles, yet had increased numbers of primary, preantral, and antral follicles, suggesting that BMP-7 may act to facilitate the transition of follicles from the primordial stage to the pool of primary, preantral, and antral follicles. In this regard, we have also found that BMP-7 caused an increase in DNA synthesis and proliferation of granulosa cells from small antral follicles in vitro. In contrast to the stimulatory activity, BMP-7 exhibited pronounced inhibitory effects on ovulation rate and serum progesterone levels. These findings establish important new biological activities of BMP-7 in the context of ovarian physiology, including folliculogenesis and ovulation.

Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary.

The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-beta superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P(4)) but not estradiol (E(2)) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P(4) and E(2) production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P(4) production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action.

Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression.

We have recently reported that oocyte-derived bone morphogenetic protein-15 (BMP-15) can directly modulate follicle-stimulating hormone (FSH) action in rat granulosa cells. Here, we investigate underlying mechanisms of this BMP-15 effect. Treatment with BMP-15 alone exerted no significant effect on the basal expression of mRNAs encoding steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3beta-hydroxysteroid dehydrogenase, luteinization hormone receptor, and inhibin/activin subunits. However, BMP-15 markedly inhibited the FSH-induced increases in these messages. In striking contrast, BMP-15 did not change the forskolin-induced levels of these transcripts. Thus, the inhibitory effect of BMP-15 on FSH action must be upstream of cAMP signaling. We next examined changes in FSH receptor mRNA expression. Interestingly, BMP-15 severely reduced the levels of FSH receptor mRNA in both basal and FSH-stimulated cells. To determine whether this effect was at the level of FSH function, we investigated the effect of BMP-15 on FSH bioactivity. Consistent with the mRNA data, BMP-15 inhibited the biological response of FSH, but not that of forskolin. Based on these results, we propose that BMP-15 is an important determinant of FSH action through its ability to inhibit FSH receptor expression. Because FSH plays an essential role in follicle growth and development, our findings could have new implications for understanding how oocyte growth factors contribute to folliculogenesis.

Difference between follistatin isoforms in the inhibition of activin signalling: activin neutralizing activity of follistatin isoforms is dependent on their affinity for activin.

We demonstrate the difference between the follistatin isoforms (FS-288 and FS-315), two activin-binding proteins, in the neutralizing activity for activin signalling. Transcriptional reporter assay using 3TP-Lux, an activin-responsive reporter construct, showed that the inhibitory effect of FS-288 on activin-induced transcriptional response is more potent than that of FS-315. The potency was not influenced by the presence of heparan sulfates, by which FS, in particular FS-288, associates with cell surfaces at a high affinity. Furthermore, FS-288 inhibited the binding of activin to its type II receptor more markedly than did FS-315, as evidenced by surface plasmon resonance and affinity cross-linking experiments. Moreover, the Kd of FS-288 and FS-315 for activin A was estimated to be 46.5+/-0.37 pM and 432+/-26 pM, respectively, by surface plasmon resonance experiments. These results indicate that the different potency between the two FS isoforms in the inhibition of activin activities depends on their affinity for activin A.

Bone morphogenetic protein-15. Identification of target cells and biological functions.

In developing ovarian follicles, the regulation of cell proliferation and differentiation is tightly coordinated. Precisely how this coordination is achieved is unknown, but recent observations have suggested that molecules emitted by the oocyte are involved in the process. The newly discovered oocyte-specific growth factor, bone morphogenetic protein-15 (BMP-15), is one such molecule. At present, nothing is known about the target cells and biological functions of BMP-15. To fill this gap in our knowledge, recombinant BMP-15 and its antibody were produced and used to determine BMP-15 expression and bioactivity. BMP-15 mRNA and protein were shown to be co-expressed in oocytes throughout folliculogenesis, supporting the idea that BMP-15 is a physiological regulator of follicle cell proliferation and/or differentiation. To test this, we used primary cultures of rat granulosa cells (GCs). We found that BMP-15 is a potent stimulator of GC proliferation, and importantly, the mitogenic effect was follicle-stimulating hormone (FSH)-independent. By contrast, BMP-15 alone had no effect on steroidogenesis. However, it produced a marked decrease in FSH-induced progesterone production, but had no effect on FSH-stimulated estradiol production. This result indicates that BMP-15 is a selective modulator of FSH action. In summary, this study identifies GCs as the first target cells for BMP-15. Moreover, it identifies the stimulation of GC proliferation and the differential regulation of two crucial steroid hormones as the first biological functions of BMP-15. Significantly, BMP-15 is the first growth factor that can coordinate GC proliferation and differentiation in a way that reflects normal physiology.

Characterization of follistatin isoforms in early Xenopus embryogenesis.

Follistatin is expressed in Spemann's organizer in the Xenopus gastrula and mimics the activity of the organizer, inducing a neural fate directly in the ectoderm. We have previously shown that follistatin inhibits BMP activity through a direct interaction. In this study, we have characterized the localization and function of two follistatin isoforms to examine the functional differences between them. One notable difference, previously described, is that the shorter form (xFSS or xFS319) but not the C-terminally extended long form (xFSL) associates with cell-surface matrices. Here, we show that the spatial-temporal expression pattern of xFSL and xFSS is indistinguishable. Interestingly, however, xFSS was found to have a more potent inhibitory activity against BMP-4 than xFSL. Furthermore, using a surface plasmon resonance biosensor, xFSS was shown to have a higher binding capacity for BMP subtypes. The diffusion rates of xFSS and xFSL ectopically expressed in Xenopus embryos were similar. Taken together, our results suggest that the difference in BMP-inhibiting activity of the two follistatin isoforms is mainly attributable to a difference in their BMP binding properties rather than to their diffusion rates.

The role of the oocyte in folliculogenesis.

Novel regulatory proteins have been identified within oocytes that are crucially involved in folliculogenesis. One of the most exciting oocyte signaling molecules is a novel member of the transforming growth factor beta (TGF-beta) superfamily, growth differentiation factor 9 (GDF-9). Loss-of-function studies have established that GDF-9 is obligatory for proper folliculogenesis and fertility in female mice. The current challenges are to understand how oocyte morphogens regulate folliculogenesis and how their actions and interactions are integrated into the overall processes of physiology and pathophysiology. Who would have thought that oocyte morphogens would be so crucial for reproduction?

Reliability and validity of a rating scale for post-stroke psychiatric symptoms. SKETCH Study Group.

Reliability and validity of a rating scale for post-stroke psychiatric symptoms were examined by the videotape method. The scale comprised 10 items categorized into two symptom domains of decreased spontaneity and impaired emotion. Also, two items for global assessment of the two symptom domains were added. Each item had seven anchor points from 0, representing no impairment, to 6, corresponding to complete impairment. Face validity of the scale was confirmed through the questionnaire survey. Nine neurologists independently assessed psychiatric symptoms in 30 videotaped post-stroke patients. Weighted kappa coefficients of more than 0.5 were noted for all the items except for one item. Data from three cerebral metabolism enhancers trials were used to examine the validity. Changes in severity in the Global Change Scale (GCS) from the baseline to the final assessment was assessed by raters' impression in these trials. Factorial validity of the scale was confirmed by the factor analysis. GCS in the three trials were considerably related to the summed scores of the items in the two categories. Namely, in the box plot figures, boxes of the middle 50% of data well differentiated the adjacent categories of GCS. However, overlap from vertical bars was observed. These results suggest reliability and validity of the scale.

A functional bone morphogenetic protein system in the ovary.

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo.

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

Activin-induced inhibin alpha-subunit production by rat granulosa cells requires endogenous insulin-like growth factor-I.

Inhibin-alpha subunit (Inh-alpha) gene expression is important for granulosa cell (GC) differentiation and prevention of ovarian tumorigenesis. Studies on Inh-alpha regulation have implicated activin and insulin-like growth factor-I (IGF-I) in the mechanisms of expression. Here we present evidence that endogenously produced IGF-I plays an obligatory role in activin-induced Inh-alpha production. Primary cultures of rat GC were incubated with increasing concentrations of various regulatory molecules, and the levels of Inh-alpha protein and its mRNA were measured in conditioned medium and cells, respectively. Recombinant activin A stimulated Inh-alpha expression, and the effects were dose- and time-dependent. The receptor tyrosine kinase inhibitor tyrphostin A23 caused a dose-dependent inhibition of activin-dependent Inh-alpha expression, whereas the inactive isomer, A63, had no effect. The stimulatory effect of activin was also blocked in a dose-dependent manner by added IGF binding protein-4 or -5, and the effects were reversed by IGF-I. Moreover, increasing concentrations of an anti-IGF-I antibody had a similar inhibitory effect on activin-stimulated Inh-alpha expression. Collectively, these results suggest, for the first time, that endogenously produced IGF-I is required for activin stimulation of Inh-alpha expression in cultured rat GC.

Endogenous insulin-like growth factor-I is obligatory for stimulation of rat inhibin alpha-subunit expression by follicle-stimulating hormone.

Insulin-like growth factor-I (IGF-I) is essential for FSH-dependent steroidogenesis by rat granulosa cells (GC), but whether IGF-I is required for other FSH-dependent functions is unknown. To investigate the role of IGF-I in the mechanisms of FSH-stimulated inhibin alpha-subunit (Inh-alpha) production, rat GC were cultured with FSH, IGF-I, insulin-like growth factor-binding protein (IGFBP)-4, IGFBP-5, and/or anti-IGF-I antibody. Inh-alpha protein and mRNA levels were measured in conditioned medium and cells by Western immunoblotting and Northern analysis, respectively. Inh-alpha expression was increased by FSH (3.5-fold) and IGF-I (2.5-fold), and the effects were dose and time dependent. FSH stimulation of Inh-alpha was attenuated by IGFBP-4 or -5 in a dose-dependent fashion, and the effects were reversed by IGF-I. Anti-IGF-I antibody mimicked the inhibitory effects of IGFBP-4 and -5. Forskolin, cholera toxin, and 8-bromo-cAMP increased Inh-alpha production approximately 3.5-fold, and the effects were blocked by IGFBP-4 or -5. Increases in Inh-alpha by FSH, IGF-I, forskolin, cholera toxin, and 8-bromo-cAMP were totally blocked by the protein tyrosine kinase inhibitor, tyrphostin A23. In summary, these results suggest that the stimulation of Inh-alpha expression by FSH requires activation of protein tyrosine kinases by endogenously produced IGF-I. We propose that the IGF-I signaling is obligatory for FSH stimulation of Inh-alpha expression in rat GC.

Transforming growth factor-alpha stimulates insulin-like growth factor binding protein-4 (IGFBP-4) expression and blocks follicle-stimulating hormone regulation of IGFBP-4 production in rat granulosa cells.

The ability of TGF-alpha to regulate insulin-like growth factor binding protein-4 (IGFBP-4), was investigated. Primary cultures of rat granulosa cells (GC) were grown in serum-free medium with rat (r) TGF-alpha and/or rFSH, and secreted IGFBP-4 protein and its steady state mRNA levels were measured by Western immunoblotting and Northern blotting, respectively. Control (untreated) cells secreted IGFBP-4 spontaneously, and the levels were increased by rTGF-alpha in a dose- and time-dependent manner. rTGF-alpha abolished FSH-induced IGFBP-4 protease activity and suppressed FSH-dependent effects on IGFBP-4 production. IGFBP-4 mRNA levels were decreased and increased by FSH and TGF-alpha, respectively, and TGF-alpha blocked the FSH effects. These results demonstrate that TGF-alpha is a potent stimulator of IGFBP-4 expression in rat GC and can overcome the regulatory effects of FSH on IGFBP-4 production. The consequence of these TGF-alpha effects is a marked, sustained increase in the levels of IGFBP-4 in the microenvironment.

A novel role of follistatin, an activin-binding protein, in the inhibition of activin action in rat pituitary cells. Endocytotic degradation of activin and its acceleration by follistatin associated with cell-surface heparan sulfate.

There are two types of the activin-binding protein follistatin (FS), FS-288 and FS-315. These result from alternative splicing of mRNA. FS-288 exhibits high affinity for cell-surface heparan sulfate proteoglycans, whereas FS-315 shows low affinity. To understand the physiological role of cell-associated FS, we investigated the binding of activin to cell-associated FS and its behavior on the cell surface using primary cultured rat pituitary cells. Affinity cross-linking experiments using 125I-activin A demonstrated that activin bound to rat pituitary cells via FS as well as to their receptors on the cell surface. FS-288 promoted the binding of activin A to the cell surface more markedly than FS-315. When the cells were incubated with 125I-activin A in the presence of FS-288, significant degradation of activin A was observed, and this was dependent on the FS-288 concentration. This activin degradation was abolished by heparan sulfate, chloroquine, and several lysosomal enzyme inhibitors. Moreover, FS-288 stimulated cellular uptake of activin A, whereas chloroquine suppressed lysosomal degradation following internalization, as demonstrated by microscopic autoradiography. These results suggest that cell-associated FS-288 accelerates the uptake of activin A into pituitary cells, leading to increased degradation by lysosomal enzymes, and thus plays a role in the activin clearance system.