scholarly journals Analysis of Ovarian Gene Expression in Follicle-Stimulating Hormone β Knockout Mice*

Endocrinology ◽  
2001 ◽  
Vol 142 (7) ◽  
pp. 2742-2751 ◽  
Author(s):  
Kathleen H. Burns ◽  
Changning Yan ◽  
T. Rajendra Kumar ◽  
Martin M. Matzuk
Keyword(s):  
Cell Cycle ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Knockout Mice ◽  
Marker Genes ◽  
Cyclin D2 ◽  
Glycoprotein Hormone ◽  
Lh Receptor ◽  
Receptor Mrna ◽  
Deficient Mice

Abstract FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHβ knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHβ knockout mice develop a well organized thecal layer, which is positive for P450 17α-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that implicate FSH signaling cascades in the expression of these genes. 3) In contrast to the thecal layer, granulosa cell populations in FSHβ knockout mice do not accumulate LH receptor mRNA. This suggests that although the granulosa cells have a block in proliferation at the antral follicle stage in the absence of FSH, they do not initiate programs of terminal differentiation as seen in luteinizing cells of wild-type ovaries. 4) Ovaries of FSH-deficient mice demonstrate a modest decrease in cyclin D2 mRNA, without up-regulation of cell cycle inhibitor mRNAs associated with luteinization (i.e. p15, p27, and p21). Although components of the FSH null phenotype may be caused by partial cyclin D2 loss of function, these findings indicate that the mechanisms of granulosa cell cycle arrest in FSHβ knockout mice are distinct from those of cycle withdrawal at luteinization. Underscoring the usefulness of the FSH-deficient mouse model, this study clarifies aspects of gonadotropin-dependent folliculogenesis, thecal layer development, cycle control in granulosa cells, and luteinization.

scholarly journals Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 929-935 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
K. M. J. Menon
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ampk Activation ◽  
Cyclin D2 ◽  
Cell Cycle Inhibitor ◽  
Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.

scholarly journals Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells

2000 ◽  
Vol 25 (1) ◽  
pp. 53-61 ◽  
Author(s):  
M Hattori ◽  
K Takesue ◽  
N Nishida ◽  
Y Kato ◽  
N Fujihara
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Inhibitory Effect ◽  
Immature Stage ◽  
Mrna Levels ◽  
Lh Receptor ◽  
Receptor Mrna ◽  
Immature Cells

The present study investigated the effect of retinoic acid (RA) on the differentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and further treated for 24 h with LH in order to induce their transformation into luteal cells. After the cells had been exposed to 1 microM retinoids (RA, retinal and retinol) for 87 h, analysis of the LH receptor mRNA expression, an indicator of granulosa cell differentiation, was carried out by using semiquantitative RT-PCR. The results showed that there was a decrease in LH receptor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stage (before the addition of FSH) or the early stage of development (0-24 h after the addition of FSH), expression of LH receptor mRNA was greatly diminished. When the immature cells were cultured for 15 h with RA, then washed and cultured for 48 h with FSH and for 24 h with LH, the expression of LH receptor mRNA was not reversed. In the differentiated cells (24 h after the addition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to RA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transcription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a potent inhibitor in the immature stage of porcine granulosa cell differentiation, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.

scholarly journals Protein Kinase B Is Obligatory for Follicle-Stimulating Hormone-Induced Granulosa Cell Differentiation

Endocrinology ◽  
2003 ◽  
Vol 144 (9) ◽  
pp. 3985-3994 ◽  
Author(s):  
Anthony J. Zeleznik ◽  
Deeksha Saxena ◽  
Lynda Little-Ihrig
Keyword(s):  
Cell Differentiation ◽  
Protein Kinase ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Intracellular Signaling ◽  
Protein Kinase B ◽  
Mrna Levels ◽  
Lh Receptor ◽  
Receptor Mrna ◽  
Constitutively Active

Abstract Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human Gsα Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active Gsα Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and Gsα signaling pathways are required.

scholarly journals AMPK Activation by Dihydrotestosterone Reduces FSH-Stimulated Cell Proliferation in Rat Granulosa Cells by Inhibiting ERK Signaling Pathway

Endocrinology ◽  
2012 ◽  
Vol 153 (6) ◽  
pp. 2831-2838 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
K. M. J. Menon
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Maximum Effect ◽  
Ampk Activation ◽  
Cyclin D2 ◽  
Compound C ◽  
Erk Phosphorylation

We have previously reported that 5α-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P < 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2′-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.

Cell-specific expression of aromatase and LH receptor mRNAs in rat ovary

1992 ◽  
Vol 9 (3) ◽  
pp. 309-312 ◽  
Author(s):  
P.F. Whitelaw ◽  
C.D. Smyth ◽  
C.M. Howles ◽  
S.G. Hillier
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Direct Evidence ◽  
Specific Expression ◽  
Lh Receptor ◽  
Paracrine Regulation ◽  
Receptor Mrna ◽  
Rat Ovary ◽  
Per Se ◽  
Receptor Mrnas

ABSTRACT Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.

scholarly journals Cyclin D2 and p27 Are Tissue-Specific Regulators of Tumorigenesis in Inhibin α Knockout Mice

2003 ◽  
Vol 17 (10) ◽  
pp. 2053-2069 ◽  
Author(s):  
Kathleen H. Burns ◽  
Julio E. Agno ◽  
Piotr Sicinski ◽  
Martin M. Matzuk
Keyword(s):  
Cell Cycle ◽  
Sertoli Cell ◽  
Knockout Mice ◽  
Double Mutant ◽  
Adrenal Tumor ◽  
Tumor Development ◽  
Mutant Mice ◽  
Cyclin D2 ◽  
Double Knockout ◽  
Single Knockout

Abstract Inhibins are heterodimeric (α:βA and α:βB) endocrine, paracrine, and autocrine factors of the TGFβ superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin α (Inhα) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhα knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1 → S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhα mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhα knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhα lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin α single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin α knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhα double knockouts. After gonadectomy, 50% of cyclin D2, Inhα double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin α double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhα single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhα knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.

scholarly journals Mouse forkhead L2 maintains repression of FSH-dependent genes in the granulosa cell

Reproduction ◽  
2012 ◽  
Vol 144 (4) ◽  
pp. 485-494 ◽  
Author(s):  
Fang-Ting Kuo ◽  
Kenneth Fan ◽  
Ikuko Bentsi-Barnes ◽  
Gillian M Barlow ◽  
Margareta D Pisarska
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Knockout Mice ◽  
Cho Cells ◽  
Regulatory Protein ◽  
Transcriptional Repressor ◽  
Chinese Hamster ◽  
Primordial Follicle ◽  
Forkhead Transcription Factor ◽  
Expression Levels

The forkhead transcription factor forkhead box L2 (FOXL2) is expressed in granulosa cells of small and medium follicles in the mouse ovary.Foxl2female knockout mice exhibit primordial follicle depletion and primary ovarian failure, but evidence from adult female conditionalFoxl2knockout mice suggests that FOXL2 may also play a significant role in maintenance of ovarian differentiation at stages beyond the primordial follicle and initial wave of folliculogenesis. We previously showed that human FOXL2 functions as a transcriptional repressor of several key genes involved in granulosa cell proliferation and differentiation, including steroidogenic acute regulatory protein (STAR), P450aromatase (CYP19A1(CYP19)), P450scc (CYP11A1(CYP11A)), and cyclin D2 (CCND2). To elucidate the role of mouse FOXL2, we determined its role in transcriptional regulation in Chinese hamster ovary (CHO) cells and then confirmed our findings in mouse granulosa cells. We found that mouse FOXL2 represses the activities of the mouseStar,Cyp19a1,Cyp11a1promoters in CHO cells, but may not repress theCcnd2promoter, and identified the minimal mouseStar,Cyp19a1, andCyp11a1promoter regions responsive to FOXL2 regulation. We then knocked downFoxl2in mouse granulosa cells using siRNA, which resulted in significantly increased expression levels of mouseStar,Cyp19a1, andCyp11a1but notCcnd2. To increaseFoxl2expression levels, we generated a mouseFoxl2lentiviral construct and used it to infect mouse granulosa cells. Following lentiviral infection, the expression levels of mouseStar,Cyp19a1, andCyp11a1, but notCcnd2, decreased significantly. These data confirm that mouse FOXL2 functions as a transcriptional repressor of key granulosa cell genes that influence ovarian development.

Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†

2020 ◽  
Vol 103 (1) ◽  
pp. 60-69
Author(s):  
Changhuo Cen ◽  
Min Chen ◽  
Jingjing Zhou ◽  
Lianjun Zhang ◽  
Shuguang Duo ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Sertoli Cells ◽  
Cell Transformation ◽  
Ovary Development ◽  
Marker Genes ◽  
Cell Marker ◽  
Lineage Specification ◽  
Steroidogenic Cell ◽  
Steroidogenic Cells

Abstract Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1−/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3β-HSD-positive steroidogenic cells were detected in ovaries of Wt1−/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3β-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1−/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1−/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

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