Hsun-Ming Chang

Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells.

In the ovary, connexin-coupled gap junctions in granulosa cells play crucial roles in follicular and oocyte development as well as in corpus luteum formation. Our previous work has shown that theca cell-derived bone morphogenetic protein (BMP)4 and BMP7 decrease gap junction intercellular communication (GJIC) activity via the down-regulation of connexin43 (Cx43) expression in immortalized human granulosa cells. However, the effects of oocyte-derived growth factors on Cx43 expression remain to be elucidated. The present study was designed to investigate the effects of oocyte-derived growth differentiation factor (GDF)9 and BMP15 on the expression of Cx43 in a human granulosa cell line, SVOG. We also examined the effect relative to GJIC activity and investigated the potential mechanisms of action. In SVOG cells, treatment with BMP15 but not GDF9 significantly decreased Cx43 mRNA and protein levels and GJIC activity. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by co-treatment with a BMP type I receptor inhibitor, dorsomorphin. Furthermore, knockdown of the central component of the transforming growth factor-β superfamily signaling pathway, Smad4, using small interfering RNA reversed the suppressive effects of BMP15 on Cx43 expression and GJIC activity. The suppressive effects of BMP15 on Cx43 expression were further confirmed in primary human granulosa-lutein cells obtained from infertile patients undergoing an in vitro fertilization procedure. These findings suggest that oocyte-derived BMP15 decreases GJIC activity between human granulosa cells by down-regulating Cx43 expression, most likely via a Smad-dependent signaling pathway.

Transforming Growth Factor-β1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein

Background: Transforming growth factor (TGF)-β1 treatment decreases human trophoblast invasion. Results: Smad-dependent up-regulation of Snail mediates TGF-β1-induced down-regulation of VE-cadherin. Conclusion: TGF-β1 decreases human trophoblast invasion by down-regulating VE-cadherin. Significance: Our results provide important insights into the molecular mechanisms mediating TGF-β1-induced down-regulation of VE-cadherin and decreased cell invasion in human trophoblast cells. Human trophoblast cells express transforming growth factor-β (TGF-β) and TGF-β receptors. It has been shown that TGF-β1 treatment decreases the invasiveness of trophoblast cells. However, the molecular mechanisms underlying TGF-β1-decreased trophoblast invasion are still not fully understood. In the current study, we demonstrated that treatment of HTR-8/SVneo human trophoblast cells with TGF-β1 decreased cell invasion and down-regulated the expression of vascular endothelial cadherin (VE-cadherin). In addition, the inhibitory effect of TGF-β1 on VE-cadherin was confirmed in primary cultures of human trophoblast cells. Moreover, knockdown of VE-cadherin using siRNA decreased the invasiveness of HTR-8/SVneo cells and primary cultures of trophoblast cells. Treatment with TGF-β1 induced the activation of Smad-dependent signaling pathways and the expression of Snail and Slug. Knockdown of Smads attenuated TGF-β1-induced up-regulation of Snail and Slug and down-regulation of VE-cadherin. Interestingly, depletion of Snail, but not Slug, attenuated TGF-β1-induced down-regulation of VE-cadherin. Furthermore, overexpression of Snail suppressed VE-cadherin expression. Chromatin immunoprecipitation analyses showed the direct binding of Snail to the VE-cadherin promoter. These results provide evidence that Snail mediates TGF-β1-induced down-regulation of VE-cadherin, which subsequently contributed to TGF-β1-decreased trophoblast cell invasion.

Oocyte–somatic cell interactions in the human ovary—novel role of bone morphogenetic proteins and growth differentiation factors

BACKGROUND Initially identified for their capability to induce heterotopic bone formation, bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β superfamily. Using cellular and molecular genetic approaches, recent studies have implicated intra-ovarian BMPs as potent regulators of ovarian follicular function. The bi-directional communication of oocytes and the surrounding somatic cells is mandatory for normal follicle development and oocyte maturation. This review summarizes the current knowledge on the physiological role and molecular determinants of these ovarian regulatory factors within the human germline-somatic regulatory loop. OBJECTIVE AND RATIONALE The regulation of ovarian function remains poorly characterized in humans because, while the fundamental process of follicular development and oocyte maturation is highly similar across species, most information on the regulation of ovarian function is obtained from studies using rodent models. Thus, this review focuses on the studies that used human biological materials to gain knowledge about human ovarian biology and disorders and to develop strategies for preventing, diagnosing and treating these abnormalities. SEARCH METHODS Relevant English-language publications describing the roles of BMPs or growth differentiation factors (GDFs) in human ovarian biology and phenotypes were comprehensively searched using PubMed and the Google Scholar database. The publications included those published since the initial identification of BMPs in the mammalian ovary in 1999 through July 2016. OUTCOMES Studies using human biological materials have revealed the expression of BMPs, GDFs and their putative receptors as well as their molecular signaling in the fundamental cells (oocyte, cumulus/granulosa cells (GCs) and theca/stroma cells) of the ovarian follicles throughout follicle development. With the availability of recombinant human BMPs/GDFs and the development of immortalized human cell lines, functional studies have demonstrated the physiological role of intra-ovarian BMPs/GDFs in all aspects of ovarian functions, from follicle development to steroidogenesis, cell–cell communication, oocyte maturation, ovulation and luteal function. Furthermore, there is crosstalk between these potent ovarian regulators and the endocrine signaling system. Dysregulation or naturally occurring mutations within the BMP system may lead to several female reproductive diseases. The latest development of recombinant BMPs, synthetic BMP inhibitors, gene therapy and tools for BMP-ligand sequestration has made the BMP pathway a potential therapeutic target in certain human fertility disorders; however, further clinical trials are needed. Recent studies have indicated that GDF8 is an intra-ovarian factor that may play a novel role in regulating ovarian functions in the human ovary. WIDER IMPLICATIONS Intra-ovarian BMPs/GDFs are critical regulators of folliculogenesis and human ovarian functions. Any dysregulation or variations in these ligands or their receptors may affect the related intracellular signaling and influence ovarian functions, which accounts for several reproductive pathologies and infertility. Understanding the normal and pathological roles of intra-ovarian BMPs/GDFs, especially as related to GC functions and follicular fluid levels, will inform innovative approaches to fertility regulation and improve the diagnosis and treatment of ovarian disorders.

BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

CONTEXT Aberrant regulation of ovulation is one of the major causes of infertility. In animal models, 3 epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), have been shown to be involved in ovulation by regulating cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. However, whether the same is true in humans remains largely unknown. OBJECTIVE Our objective was to investigate the effects of AREG, BTC, and EREG on COX-2 expression and PGE2 production in human granulosa cells. DESIGN AND SETTING SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effect of AREG, BTC, and EREG on ovulation-related functions at an academic research center. MAIN OUTCOME MEASURES Levels of mRNA and protein were examined by quantitative RT-PCR and Western blotting, respectively. The protein levels of PGE2 were measured by ELISA. RESULTS LH treatment upregulated AREG, BTC, EREG, and COX-2. Knockdown of EGF receptor (EGFR) attenuated LH-induced COX-2 expression and PGE2 production. Treatment with AREG, BTC, and EREG upregulated COX-2 expression and PGE2 production. The stimulatory effects of AREG, BTC, and EREG on COX-2 expression and PGE2 production were blocked by inhibition of EGFR activity and expression. AREG-, BTC-, and EREG-activated ERK1/2 signaling, but not Akt signaling, was required for AREG-, BTC-, and EREG-induced COX-2 expression and PGE2 production. CONCLUSION AREG, BTC, and EREG induced PGE2 production by upregulating COX-2 expression through ERK1/2 signaling in human granulosa cells.

TGF-β1 Induces COX-2 Expression and PGE2 Production in Human Granulosa Cells Through Smad Signaling Pathways

CONTEXT Cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production have been shown to play key roles in the regulation of ovulation. The TGF-β superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF-β1 and its receptors are expressed in human granulosa cells. However, to date, whether TGF-β1 can regulate COX-2 expression and PGE2 production, which in turn contribute to the process of ovulation, remains unknown. OBJECTIVE The objective of the study was to investigate the effects of TGF-β1 on COX-2 expression and PGE2 production in human granulosa cells. DESIGN SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with Simian virus 40 large T antigen. SVOG cells were used to investigate the effect of TGF-β1 on COX-2 expression and PGE2 production. SETTING The study was conducted at an academic research center. MAIN OUTCOME MEASURES mRNA and protein levels were examined by RT-quantitative real-time PCR and Western blotting, respectively. The concentrations of PGE2 in the culture medium were measured by an ELISA. RESULTS TGF-β1 treatment induced COX-2 expression and PGE2 production. The inductive effects of TGF-β1 on COX-2 and PGE2 were abolished by the inhibition of TGF-β type I receptor (TβRI). In addition, treatment with TGF-β1 activated phosphorylated mothers against decapentaplegic (Smad)-2 and Smad3 signaling pathways. Inhibition of the Smad signaling pathways by small interfering RNA-mediated approaches attenuated the TGF-β1-induced COX-2 expression and PGE2 production. CONCLUSION TGF-β1 induced PGE2 production by inducing the COX-2 expression through a Smad-dependent signaling pathway in human granulosa cells.

TGF-β1 Downregulates StAR Expression and Decreases Progesterone Production Through Smad3 and ERK1/2 Signaling Pathways in Human Granulosa Cells

CONTEXT Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. OBJECTIVE Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. DESIGN AND SETTING SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. MAIN OUTCOME MEASURES Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. CONCLUSION TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

Growth differentiation factor 8 down-regulates pentraxin 3 in human granulosa cells

Growth differentiation factor 8 (GDF8), also known as myostatin, is highly expressed in the mammalian musculoskeletal system and plays critical roles in the regulation of skeletal muscle growth. Though not exclusively expressed in the musculoskeletal system, the expression and biological function of GDF8 has never been examined in the human ovary. Pentraxin 3 (PTX3) plays a key role in the assembly of extracellular matrix, which is essential for cumulus expansion, ovulation and in vivo fertilization. The aim of this study was to investigate GDF8 expression and function in human granulosa cells and to examine its underlying molecular determinants. An established immortalized human granulosa cell line (SVOG), granulosa cell tumor cell line (KGN) and primary granulosa-lutein cells were used as study models. We now demonstrate for the first time that GDF8 is expressed in human granulosa cells and follicular fluid. All 16 follicular fluid samples tested contained GDF8 protein at an average concentration of 3 ng/ml. In addition, GDF8 treatment significantly decreased PTX3 mRNA and protein levels. These suppressive effects, along with the induction of SMAD2/3 phosphorylation, were abolished by co-treatment with the ALK4/5/7 inhibitor SB431542. Knockdown of ALK5, ACVR2A/ACVR2B or SMAD4 reversed the effects of GDF8-induced PTX3 suppression. These results indicate that GDF8 down-regulates PTX3 expression via ACVR2A/ACVR2B-ALK5-mediated SMAD-dependent signaling in human granulosa cells. These novel findings support a potential role for GDF8 in the regulation of follicular function, likely via autocrine effects on human granulosa cells.

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis.

Theca-Derived BMP4 and BMP7 Down-Regulate Connexin43 Expression and Decrease Gap Junction Intercellular Communication Activity in Immortalized Human Granulosa Cells

CONTEXT Connexin43 (Cx43)-coupled gap junctions in granulosa cells play important roles in follicular and oocyte development and may be modulated by theca cell-derived bone morphogenic protein (BMP) 4 and BMP7. OBJECTIVE The aim of this study was to examine the effects of BMP4 and BMP7 on Cx43 expression in human granulosa cells and its potential mediation by the Smad-dependent pathway. DESIGN An immortalized human granulosa (SVOG) cell was used to investigate Cx43 expression and gap junction intercellular communication (GJIC) activity after exposure to BMP4 and BMP7. A BMP type I inhibitor, dorsomorphin, and small interfering RNAs targeting Smad4 were used to verify the specificity of the effects. SETTING The study was conducted in an academic center. MAIN OUTCOME MEASURES Extracts were prepared from cultured cells, the Cx43 mRNA levels were examined using RT-quantitative real-time PCR, and the levels of Cx43 protein and phosphorylated Smad1/5/8 were assayed using Western blot analyses. GJIC activities between SVOG cells were evaluated using a scrape loading and dye transfer assay. RESULTS Treatment with BMP4 and BMP7 significantly decreased Cx43 mRNA and protein levels, as well as GJIC activities. These suppressive effects were attenuated by cotreatment with the BMP type I receptor inhibitor dorsomorphin. Furthermore, Smad4 knockdown reversed the effects of BMP4 and BMP7 on Cx43 expression. CONCLUSION Theca cell-derived BMP4 and BMP7 down-regulate Cx43 expression and decrease GJIC activity in human granulosa cells. Our findings indicate that this biological effect is most likely mediated by a Smad-dependent pathway.