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.

Effect of TNF-alpha on LH and IGF-I modulated chicken granulosa cell proliferation and progesterone production during follicular development

Reproduction ◽  
2000 ◽  
pp. 433-442 ◽  
Author(s):  
OM Onagbesan ◽  
J Mast ◽  
B Goddeeris ◽  
E Decuypere
Keyword(s):  
Cell Proliferation ◽  
Conditioned Medium ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Tnf Alpha ◽  
Dependent Manner ◽  
The Third ◽  
Progesterone Production ◽  
Igf I ◽  
Chicken Ovary

This study demonstrates the effects of recombinant human tumour necrosis factor a (rhTNF-alpha) and conditioned medium of the HD11-transformed chicken macrophage cell line on cultured chicken granulosa cells. Effects were studied on basal, IGF-I- and LH-stimulated progesterone production and cell proliferation. Recombinant human TNF-alpha stimulated basal progesterone production in a dose-dependent manner in the granulosa cells of the largest follicle but had no effect on cells from the third largest follicle. TNF-alpha stimulated and sometimes inhibited progesterone production stimulated by IGF-I and LH alone or in combination depending on the size of the follicle and the concentration of LH or IGF-I applied. However, the inhibitory effect of TNF-alpha was significantly more pronounced in cells from the third largest follicle when high concentrations of IGF-I, LH or a combination of both were applied. TNF-alpha had no effect on basal cell proliferation in both the largest and the third largest follicles, but regulated responses to IGF-I and a combination IGF-I and LH in the cells of the third largest follicle but not those of the largest follicle. The data indicate that the normal hierarchy of follicles is maintained in the chicken ovary through the regulation of the activity of IGF-I and its interaction with LH. Conditioned medium of LPS-activated HD11 macrophages mimicked the effects of TNF-alpha and its interaction with IGF-I and LH on progesterone production and cell proliferation. The observation that the HD11-conditioned medium contained TNF-alpha indicates that TNF-alpha produced by macrophages found in chicken follicles modulates granulosa cell growth and differentiation.

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.

Effect of growth hormone on steroidogenesis, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 production and DNA synthesis in cultured human luteinized granulosa cells

1994 ◽  
Vol 140 (2) ◽  
pp. 313-319 ◽  
Author(s):  
P Ovesen ◽  
H J Ingerslev ◽  
H Ørskov ◽  
T Ledet
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Thymidine Incorporation ◽  
Factor I ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Abstract Numerous clinical and experimental observations have suggested that GH is important in ovarian function. We have investigated the effect of GH alone and GH in combination with FSH on the secretion of oestradiol, progesterone, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) and on [3H]thymidine incorporation in cultured human luteinized granulosa cells. Granulosa cells from patients undergoing treatment for in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum. After this preincubation, the medium was removed and the cells were incubated with GH (1, 10 and 100 μg/l) with or without FSH in serum-free medium and in the presence of [3H]methylthymidine (2 μCi/ml). GH alone resulted in a significant dose-dependent increase of oestradiol (P<0·05) and in IGFBP-1 (P<0·002) in the medium. The release of IGF-I was undetectable and there was no increase in [3H]thymidine incorporation with GH alone. Neither GH nor FSH alone stimulated granulosa cell proliferation or progesterone release, while the combination induced increases (P<0·001) in both. The stimulatory effect of GH on steroidogenesis, IGFBP-1 production and granulosa cell proliferation supports a putative role for GH in the regulation of ovarian function. Journal of Endocrinology (1994) 140, 313–319

scholarly journals Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 481-487 ◽  
Author(s):  
Kenneth P McNatty ◽  
Jennifer L Juengel ◽  
Karen L Reader ◽  
Stan Lun ◽  
Samu Myllymaa ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Granulosa Cells ◽  
Progesterone Production ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Bone Morphogenetic Protein 15

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating 3H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating 3H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro

1994 ◽  
Vol 142 (3) ◽  
pp. 497-510 ◽  
Author(s):  
D Monniaux ◽  
C Pisselet ◽  
J Fontaine
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Growth ◽  
Proliferating Cells ◽  
Progesterone Secretion ◽  
Proliferation And Differentiation ◽  
Igf I

Abstract Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1–3 mm in diameter) and large follicles (5–7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro. IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P<0·001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P<0·001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P<0·001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone. Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected. Journal of Endocrinology (1994) 142, 497–510

scholarly journals Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 473-480 ◽  
Author(s):  
Kenneth P McNatty ◽  
Jennifer L Juengel ◽  
Karen L Reader ◽  
Stan Lun ◽  
Samu Myllymaa ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Granulosa Cells ◽  
Thymidine Incorporation ◽  
Progesterone Production ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Bone Morphogenetic Protein 15

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive α-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15

2016 ◽  
Vol 28 (4) ◽  
pp. 491 ◽  
Author(s):  
Karen L. Reader ◽  
David G. Mottershead ◽  
Georgia A. Martin ◽  
Robert B. Gilchrist ◽  
Derek A. Heath ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Bone Morphogenetic Protein ◽  
Granulosa Cell ◽  
Thymidine Incorporation ◽  
Signalling Pathways ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Bone Morphogenetic Protein 15 ◽  
Stimulation Of

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9 + BMP15) stimulated an increase in 3H-thymidine incorporation (P < 0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of 3H-thymidine incorporation by GDF9 + BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of 3H-thymidine incorporation by GDF9 + BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

scholarly journals Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles

Reproduction ◽  
2002 ◽  
pp. 891-898 ◽  
Author(s):  
PS Duggal ◽  
NK Ryan ◽  
KH Van der Hoek ◽  
LJ Ritter ◽  
DT Armstrong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Food Intake ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture Media ◽  
Ovarian Follicles ◽  
Meiotic Maturation ◽  
Feed Restriction ◽  
Preovulatory Follicles ◽  
Igf I

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

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