scholarly journals Establishment of a Human Nonluteinized Granulosa Cell Line that Transitions from the Gonadotropin-Independent to the Gonadotropin-Dependent Status

Endocrinology ◽  
2012 ◽  
Vol 153 (6) ◽  
pp. 2851-2860 ◽  
Author(s):  
Bayasula ◽  
Akira Iwase ◽  
Tohru Kiyono ◽  
Sachiko Takikawa ◽  
Maki Goto ◽  
...  
Keyword(s):  
Cell Line ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Early Stage ◽  
Regulatory Protein ◽  
Cell Types ◽  
Mrna Levels ◽  
Steroidogenic Cell ◽  
Morphogenetic Protein ◽  
Steroidogenic Acute Regulatory

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.

scholarly journals A Novel Antagonistic Effect of the Bone Morphogenetic Protein System on Prolactin Actions in Regulating Steroidogenesis by Granulosa Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5506-5518 ◽  
Author(s):  
Eri Nakamura ◽  
Fumio Otsuka ◽  
Kenichi Inagaki ◽  
Tomoko Miyoshi ◽  
Ryutaro Yamanaka ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Regulatory Protein ◽  
Antagonistic Effect ◽  
Janus Kinase ◽  
Progesterone Production ◽  
Morphogenetic Protein ◽  
High Concentrations ◽  
Steroidogenic Acute Regulatory

To investigate the mechanism by which prolactin (PRL) regulates follicular steroidogenesis in the ovary, we examined the functional roles of PRL in steroidogenesis using rat oocyte/granulosa cell coculture and focusing on the bone morphogenetic protein (BMP) system. The expression of long and short forms of PRL receptor (PRLR) were detected in both oocytes and granulosa cells, and PRL effectively up-regulated PRLR expression in granulosa cells in the presence of FSH. PRL suppressed FSH-induced estradiol production and increased FSH-induced progesterone production in granulosa cells. The PRL effects on FSH-induced progesterone were blocked by coculture with oocytes, implying roles of oocyte-derived factors in suppression of progesterone production in PRL-exposed granulosa cells. In accordance with the data for steroids, FSH-induced aromatase expression was suppressed by PRL, whereas FSH-induced steroidogenic acute regulatory protein, P450scc (P450 side-chain cleavage enzyme), and 3β-hydroxysteroid dehydrogenase type 2 levels were amplified by PRL. However, forskolin- and N6,O2-dibutyryl cAMP-induced steroid levels and FSH- and forskolin-induced cAMP were not affected by PRL, suggesting that PRL action on FSH-induced steroidogenesis was not due to cAMP-protein kinase A regulation. Treatment with a BMP-binding protein, noggin, facilitated PRL-induced estradiol reduction, and noggin increased PRL-induced progesterone production in FSH-treated granulosa cells cocultured with oocytes, suggesting that endogenous BMPs reduce progesterone but increase estradiol when exposed to high concentrations of PRL. PRL increased the expression of BMP ligands in oocyte/granulosa cell coculture and augmented BMP-induced phosphorylated mothers against decapentaplegic 1/5/8 signaling by reducing inhibitory phosphorylated mothers against decapentaplegic 6 expression through the Janus kinase/signal transducer and activator of transcription (STAT) pathway. In addition to STAT activation, PRL enhanced FSH-induced MAPK phosphorylation in granulosa cells, in which ERK activation was preferentially involved in suppression of FSH-induced estradiol. Furthermore, noggin treatment enhanced PRLR signaling including MAPK and STAT. Considering that BMPs suppressed PRLR in granulosa cells, it is likely that the BMP system in growing follicles plays a key role in antagonizing PRLR signaling actions in the ovary exposed to high concentrations of PRL.

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

2013 ◽  
Vol 27 (12) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hsun-Ming Chang ◽  
Jung-Chien Cheng ◽  
Christian Klausen ◽  
Peter C. K. Leung
Keyword(s):  
Granulosa Cells ◽  
Critical Role ◽  
Regulatory Protein ◽  
Granulosa Cell Tumor ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Progesterone Production ◽  
Steroidogenic Acute Regulatory

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.

scholarly journals Novel Action of Activin and Bone Morphogenetic Protein in Regulating Aldosterone Production by Human Adrenocortical Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 639-649 ◽  
Author(s):  
Jiro Suzuki ◽  
Fumio Otsuka ◽  
Kenichi Inagaki ◽  
Masaya Takeda ◽  
Toshio Ogura ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Regulatory Protein ◽  
Mapk Signaling ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mrna Levels ◽  
Type Ii ◽  
Ang Ii ◽  
Morphogenetic Protein ◽  
Aldosterone Production ◽  
Steroidogenic Acute Regulatory

Abstract We have uncovered a functional bone morphogenetic protein (BMP) and activin system complete with ligands (BMP-6 and activin βA/βB), receptors (activin receptor-like kinase receptors 2, 3, and 4; activin type-II receptor; and BMP type-II receptor), and the binding protein follistatin in the human adrenocortical cell line H295R. Administration of activin and BMP-6 to cultures of H295R cells caused concentration-responsive increases in aldosterone production. The mRNA levels of steroidogenic acute regulatory protein or P450 steroid side-chain cleavage enzyme, the rate-limiting steps of adrenocortical steroidogenesis, were enhanced by activin and BMP-6. Activin and BMP-6 also activated the transcription of steroidogenic acute regulatory protein as well as the late-step steriodogenic enzyme CYP11B2. Activin enhanced ACTH-, forskolin-, or dibutyryl-cAMP- but not angiotensin II (Ang II)-induced aldosterone production, whereas BMP-6 specifically augmented Ang II-induced aldosterone production. Activin and ACTH but not BMP-6 increased cAMP production. Follistatin, which inhibits activin actions by binding, suppressed basal and ACTH-induced aldosterone secretion but failed to affect the Ang II-induced aldosterone level. Furthermore, MAPK signaling appeared to be involved in aldosterone production induced by Ang II and BMP-6 because an inhibitor of MAPK activation, U0126, reduced the level of aldosterone synthesis stimulated by Ang II and BMP-6 but not activin. In addition, Ang II reduced the expression levels of BMP-6 but increased that of activin βB, whereas ACTH had no effect on these levels. Collectively, the present data suggest that activin acts to regulate adrenal aldosterone synthesis predominantly by modulating the ACTH-cAMP-protein kinase A signaling cascade, whereas BMP-6 works primarily by modulating the Ang II-MAPK cascade in human adrenal cortex in an autocrine/paracrine fashion.

scholarly journals Oxysterols regulate expression of the steroidogenic acute regulatory protein

2004 ◽  
Vol 32 (2) ◽  
pp. 507-517 ◽  
Author(s):  
SR King ◽  
AA Matassa ◽  
EK White ◽  
LP Walsh ◽  
Y Jo ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Direct Conversion ◽  
The Body ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Steroid Synthesis ◽  
Star Protein ◽  
Protein Levels ◽  
Steroidogenic Cell ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

Rosiglitazone increases expression of steroidogenic acute regulatory protein and progesterone production through PPARγ–EGFR–ERK1/2 in human cumulus granulosa cells

2019 ◽  
Vol 31 (11) ◽  
pp. 1647
Author(s):  
Kristina Pogrmic-Majkic ◽  
Gordana Kosanin ◽  
Dragana Samardzija Nenadov ◽  
Svetlana Fa ◽  
Bojana Stanic ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Regulatory Protein ◽  
Growth Factor Receptor ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Progesterone Production ◽  
Extracellular Signal ◽  
Epidermal Growth ◽  
Steroidogenic Acute Regulatory

The mechanism by which rosiglitazone (ROSI: a thiazolidinedione (TZD)) affects steroid production in undifferentiated human granulosa cells is not known. In this study, cultured human cumulus granulosa cells were exposed to ROSI and pharmacological inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2), epidermal growth factor receptor (EGFR) and peroxisome proliferator-activated receptor gamma (PPARγ) signalling pathways. Expression of progesterone biosynthetic enzymes, PPARγ and PPARα, progesterone production and ERK1/2 activation were analysed. After 48h, 30μM ROSI increased STAR, 3βHSD and PPARγ mRNA and elevated progesterone production in human cumulus granulosa cells. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the ROSI-induced STAR mRNA expression and progesterone production after 48h. Inhibition of PPARγ, but not EGFR or ERK1/2, decreased the PPARγ mRNA levels induced by ROSI in human cumulus granulosa cells after 48h. On the other hand, U0126 and GW9662 prevented the ROSI-induced increase in PPARγ transcripts after 6h. Western blot analysis showed that ROSI induced a rapid ERK1/2 activation, which was prevented by inhibition of ERK1/2, EGFR and PPARγ in human cumulus granulosa cells. Overall, these data suggested that PPARγ, EGFR and ERK1/2 were involved in the stimulatory effect of ROSI on STAR expression and progesterone production in undifferentiated human cumulus granulosa cells.

scholarly journals GATA4 Reduction Enhances 3′,5′-Cyclic Adenosine 5′-Monophosphate-Stimulated Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid and Progesterone Production in Luteinized Porcine Granulosa Cells

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5557-5567 ◽  
Author(s):  
Yvonne Y. Hui ◽  
Holly A. LaVoie
Keyword(s):  
Granulosa Cells ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mrna Levels ◽  
Down Regulation ◽  
Lh Surge ◽  
Progesterone Production ◽  
Steroidogenic Acute Regulatory ◽  
Camp Induction ◽  
Star Gene

Previous studies with cultured granulosa cells implicated GATA4 in gonadotropin regulation of the steroidogenic acute regulatory protein (STAR) gene. Caveats to these prior studies exist. First, GATA4 levels are reduced in granulosa-luteal cells after the LH surge when GATA6 expression is relatively high. Second, STAR mRNA expression is negligible in granulosa cells until after the LH surge. Both exogenous GATA4 and GATA6 can transactivate STAR gene promoter constructs. We used an RNA interference (RNAi) approach to determine the contributions of GATA4 and GATA6 to cAMP analog regulation of the endogenous STAR gene in luteinizing granulosa cells. STAR mRNA was stimulated by cAMP under control RNAi conditions. Surprisingly, GATA4 reduction by its respective RNAi approximately doubled the cAMP induction of STAR mRNA. At 24 h cAMP treatment, this augmentation was abolished by co-down-regulation of GATA4+GATA6. GATA6 down-regulation by itself did not alter STAR mRNA levels. GATA4+GATA6 co-down-regulation elevated basal CYP11A mRNA at 24 h treatment but did not affect its induction by cAMP. Basal levels of HSD3B mRNA were reduced by GATA4 RNAi conditions leading to a greater fold induction of its mRNA by cAMP. Fold cAMP-stimulated progesterone production was enhanced by GATA4 down-regulation but not by GATA4+GATA6 co-down-regulation. These data implicate GATA6 as the facilitator in cAMP-stimulated STAR mRNA and downstream progesterone accumulation under reduced GATA4 conditions. Data also demonstrate that basal levels of GATA4/6 are not required for cAMP induction of the STAR gene. The altered ratio of GATA4 to GATA6 after ovulation may allow GATA6 to enhance STAR mRNA accumulation.

scholarly journals Establishment of a stable steroidogenic porcine granulosa cell line

1998 ◽  
Vol 20 (2) ◽  
pp. 287-292 ◽  
Author(s):  
PJ Chedrese ◽  
MR Rodway ◽  
CL Swan ◽  
C Gillio-Meina
Keyword(s):  
Primary Culture ◽  
Cell Line ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Early Stage ◽  
Fold Increase ◽  
Stable Cell Line ◽  
Porcine Granulosa Cells ◽  
Progesterone Synthesis ◽  
Porcine Granulosa Cell

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.

scholarly journals BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (11) ◽  
pp. 4269-4280 ◽  
Author(s):  
Han Zhang ◽  
Christian Klausen ◽  
Hua Zhu ◽  
Hsun-Ming Chang ◽  
Peter C. K. Leung
Keyword(s):  
Granulosa Cells ◽  
Small Interfering Rna ◽  
Regulatory Protein ◽  
Human Granulosa Cells ◽  
Progesterone Production ◽  
Morphogenetic Protein ◽  
Rna Targeting ◽  
Successful Establishment ◽  
Steroidogenic Acute Regulatory ◽  
Interfering Rna

Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

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