scholarly journals Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (1) ◽  
pp. 172-178 ◽  
Author(s):  
Jae-Sook Roh ◽  
Jonas Bondestam ◽  
Sabine Mazerbourg ◽  
Noora Kaivo-Oja ◽  
Nigel Groome ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Inhibin B ◽  
Inhibin A ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Ovarian Inhibin ◽  
Stimulation Of

Abstract Ovarian inhibin production is stimulated by FSH and several TGFβ family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFβ/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-α content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-α and inhibin-β subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-α gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-α promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-α gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-α promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.

scholarly journals Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

2000 ◽  
Vol 166 (2) ◽  
pp. 339-354 ◽  
Author(s):  
AE Drummond ◽  
M Dyson ◽  
E Thean ◽  
NP Groome ◽  
DM Robertson ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Inhibin B ◽  
Alpha Subunit ◽  
Inhibin A ◽  
Preantral Follicles ◽  
Subunit Mrna ◽  
Ovarian Cells ◽  
Old Rats

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

scholarly journals Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

2006 ◽  
Vol 189 (2) ◽  
pp. 329-339 ◽  
Author(s):  
L J Spicer ◽  
P Y Aad ◽  
D Allen ◽  
S Mazerbourg ◽  
A J Hsueh
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Promoter Activity ◽  
Calf Serum ◽  
Cell Numbers ◽  
Progesterone Production ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Igf I

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1–5 mm) and large (8–22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150–600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P<0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P<0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P<0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P>0.10). Treatment with 600 ng/ml GDF-9 increased (P<0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P<0.05) progesterone production (by 28%), increased (P<0.05) cell numbers (by 60%), and had no effect (P>0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13–48%) and estradiol (by 20–51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P<0.05) steroidogenesis by 27–42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.

206. Differential regulation of activin and inhibin production by interleukin 1 (IL1), transforming growth factor β1 (TGFβ1) and protein kinase C (PKC) in the Sertoli cell and granulosa cell

2008 ◽  
Vol 20 (9) ◽  
pp. 6 ◽  
Author(s):  
V. Eede ◽  
J. A. Muir ◽  
A. E. O. 'Connor ◽  
W. R. Winnall ◽  
A. E. Drummond ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Granulosa Cells ◽  
Sertoli Cells ◽  
Activin A ◽  
Inhibin B ◽  
Inhibin A ◽  
Α Subunit ◽  
Subunit Mrna ◽  
Subunit Expression

Activin and inhibin are gonadal regulatory proteins comprising an α-subunit and either a βA-subunit or βB-subunit (inhibin A or B), or two βA-subunits (activin A). Synthesis of the α-subunit, and the inhibins, is regulated by FSH via cAMP/protein kinase A. Regulation of the β-subunits in the gonads is less well defined, but the IL1/MAP kinase, TGFβ /Smad and PKC pathways have been implicated. Sertoli cells and granulosa cells were isolated from 18–22 day-old Sprague-Dawley rats under standard conditions and cultured with IL1, TGFβ1 and the PKC agonists, gonadotrophin releasing hormone (GnRH) or phorbol myristate acetate (PMA). Activin A, inhibin A and inhibin B were measured in culture medium (at 48h) by ELISA. Subunit mRNA expression was measured in cell extracts (at 4 h and 8h) using quantitative RT–PCR. IL1 stimulated βA-subunit and activin A production and inhibited α-subunit and βB-subunit expression and inhibin B production in Sertoli cells, but had no effect in granulosa cells. TGFβ1 stimulated activin A in both cell types, as well as the inhibins in granulosa cells. Surprisingly, TGFβ1 had no effect on Sertoli cell α-subunit or βA-subunit mRNA expression, but did cause a slight reduction of βB-subunit expression. GnRH increased activin A and inhibin A, but not inhibin B, production by granulosa cells and had no effect on Sertoli cells, which lack the GnRH receptor. However, direct activation of PKC by PMA stimulated βA-subunit mRNA expression and activin A production and decreased βB-subunit and inhibin B production by Sertoli cells, with marginal effects on inhibin A. These results indicate that activation of the TGFβ or PKC signalling pathways preferentially stimulates βA-subunit expression and/or translation, leading to increased activin A secretion by Sertoli cells and both activin A and inhibin A secretion by granulosa cells. The ability of IL1 to stimulate activin A is confined to the Sertoli cell.

scholarly journals A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep

Animals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 200
Author(s):  
Zhangyuan Pan ◽  
Xiangyu Wang ◽  
Ran Di ◽  
Qiuyue Liu ◽  
Wenping Hu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Methylation Level ◽  
Cpg Island ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Directed Mutation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Basal Transcriptional Activity

Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

scholarly journals Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Reproduction ◽  
2013 ◽  
Vol 145 (2) ◽  
pp. 127-135 ◽  
Author(s):  
Nazareth Loreti ◽  
Verónica Ambao ◽  
Luz Andreone ◽  
Romina Trigo ◽  
Ursula Bussmann ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Concanavalin A ◽  
Inhibin B ◽  
Inhibin A ◽  
Biological Responses ◽  
Immature Rats ◽  
Fold Stimulation ◽  
Recombinant Human Fsh

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

CXADR-like membrane protein (CLMP) in the rat ovary: stimulation by human chorionic gonadotrophin during the periovulatory period

2016 ◽  
Vol 28 (6) ◽  
pp. 742
Author(s):  
Feixue Li ◽  
Xiaoping Miao ◽  
Yonglong Chen ◽  
Thomas E. Curry
Keyword(s):  
Cell Adhesion ◽  
Protein Kinase ◽  
Membrane Protein ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Receptor Activation ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Rat Ovary ◽  
Stimulation Of

CXADR-like membrane protein (CLMP) is a novel cell–cell adhesion molecule. The present study investigated the spatiotemporal expression pattern of CLMP and its regulation in the rat ovary during the periovulatory period. Real-time polymerase chain reaction analysis revealed that Clmp mRNA was rapidly stimulated in intact ovaries by 4 h after human chorionic gonadotrophin (hCG) treatment. In situ hybridisation analysis demonstrated that Clmp mRNA expression was stimulated in theca cells at 4 h after hCG and remained elevated until 12 h. Clmp mRNA was also upregulated in granulosa cells and was present in forming corpora lutea. Our data indicate that the protein kinase A but not the protein kinase C pathway regulates the expression of Clmp mRNA in granulosa cells. Phosphatidylinositol 3 kinase and p38 kinase are also involved in regulating Clmp mRNA expression. The stimulation of Clmp mRNA by hCG requires new protein synthesis. Furthermore, inhibition of epidermal growth factor receptor activation significantly inhibited Clmp mRNA expression, whereas inhibition of prostaglandin synthesis or progesterone action had no effect. The stimulation of CLMP in the rat ovary may be important in cell adhesion events during ovulation and luteal formation such as maintaining the structure and communication of ovarian follicular and luteal cells.

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