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.

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 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.

Follicular somatic cell factors and follicle development

2011 ◽  
Vol 23 (1) ◽  
pp. 32 ◽  
Author(s):  
J. Buratini ◽  
C. A. Price
Keyword(s):  
Somatic Cell ◽  
Granulosa Cell ◽  
Bone Morphogenetic Proteins ◽  
Granulosa Cells ◽  
Primordial Follicle ◽  
Antral Follicle ◽  
Follicle Development ◽  
Follicle Growth ◽  
Theca Cells ◽  
Secreted Factors

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

scholarly journals RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Wang ◽  
Yan-long Jia ◽  
Yi-chun Li ◽  
Chang-qin Jing ◽  
Xiao Guo ◽  
...  
Keyword(s):  
Cho Cells ◽  
Transgene Expression ◽  
Recombinant Protein Expression ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Vector System ◽  
Gene Copy ◽  
Elongation Factor 1Α ◽  
Cmv Promoter ◽  
Expression Levels

Abstract In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

scholarly journals Oocyte control of ovarian follicular development and function in mammals

Reproduction ◽  
2001 ◽  
pp. 829-838 ◽  
Author(s):  
JJ Eppig
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Primordial Follicle ◽  
Dominant Factor ◽  
Ovarian Follicular Development ◽  
New Perspective ◽  
Regulatory Loop ◽  
Sphere Of Influence ◽  
And Function

A new perspective on ovarian follicular development has emerged over the last decade. Whereas the oocyte was previously considered only a passive recipient of developmental signals from oocyte-associated granulosa cells, it is now clear that communication between oocytes and granulosa cells is bidirectional. A complex interplay of regulatory factors governs the development of both types of cell. This interplay is essential not only for oocyte development but also for follicular development, beginning with the initial assembly of the primordial follicle and continuing throughout ovulation. The existence of an oocyte-granulosa cell regulatory loop, essential for normal follicular differentiation as well as for the production of an oocyte competent to undergo fertilization and embryogenesis, is proposed. Although gonadotrophins are essential for driving the differentiation of granulosa cell phenotypes, within its sphere of influence, the oocyte is probably the dominant factor determining the direction of differentiation and the function of the granulosa cells associated with it.

scholarly journals Effect of 3-nitropropionic acid inducing oxidative stress and apoptosis of granulosa cells in geese

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Bo Kang ◽  
Xinxing Wang ◽  
Qilin Xu ◽  
Yongsheng Wu ◽  
Xiaohui Si ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Caspase 3 ◽  
B Cell Lymphoma ◽  
Vital Role ◽  
Ros Production ◽  
Expression Levels ◽  
Nitropropionic Acid ◽  
3 Nitropropionic Acid

The mechanism of action by which oxidative stress induces granulosa cell apoptosis, which plays a vital role in initiating follicular atresia, is not well understood. In the present study, the effect of 3-nitropropionic acid (3-NPA) on oxidative stress and apoptosis in granulosa cells in geese was investigated. Our results showed that treatment with 3-NPA at 5.0 mmol/l for 24 h increased intracellular reactive oxygen species (ROS) production by 25.4% and decreased granulosa cell viability by 45.5% (P<0.05). Catalase and glutathione peroxidase gene expression levels in granulosa cells treated with 3-NPA were 1.32- and 0.49-fold compared with those of the control cells, respectively (P <0.05). A significant decrease in the expression level of B-cell lymphoma 2 (Bcl-2) protein and remarkable increases in the levels of Bax, p53 and cleaved-Caspase 3 proteins and the ratio of Bax/Bcl-2 expression in granulosa cells treated with 3-NPA were observed (P<0.05). Furthermore, a 38.43% increase in the percentage of early apoptotic cells was also observed in granulosa cells treated with 3-NPA (P<0.05). Moreover, the expression levels of NF-κB, Nrf2, Fhc, Hspa2 and Ho-1 in granulosa cells treated with 3-NPA were elevated 4.36-, 1.63-, 3.62-, 27.54- and 10.48-fold compared with those of the control cells (P<0.05), respectively. In conclusion, the present study demonstrates that treatment with 3-NPA induces ROS production and apoptosis and inhibits the viability of granulosa cells in geese. Furthermore, 3-NPA triggers increases in the expression of cleaved-Caspase 3 protein and the ratio of Bax/Bcl-2 expression, and induces the early apoptosis of granulosa cells.

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.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Sign in / Sign up

Export Citation Format

Share Document