Gene Expression In Ovarian Cells Research Articles

Molecular cloning, expression profile and transcriptional modulation of two splice variants of very low density lipoprotein receptor during ovarian follicle development in geese (Anser cygnoide)

Very low density lipoprotein receptor (VLDLR)-mediated endocytosis of plasma lipoproteins into the ovary is essential for ovarian follicle development. Two splice variants of VLDLR have been identified in several species, yet little is known about their distinctive roles in ovarian developing follicles. In the present study, the full-length cDNAs of two splice isoforms of VLDLR were obtained from geese (Anser cygnoide) ovaries using the RACE method. The longer isoform (TypeI VLDLR) is 3141bp and contains five conserved structural domains, while the other (TypeII VLDLR) lacks 90bp encoding for the O-linked sugar domain. TypeII VLDLR was predominantly expressed in the ovary, with greater amounts of mRNA in theca and granulosa cells from early stages of follicle development but decreased during vitellogenesis. However, there was minimal expression of the TypeI VLDLR gene in theca cells and expression was almost undetectable in granulosa cells throughout follicle development. Yolk VLDL concentrations decreased as stage of development advanced while yolk triglyceride and cholesterol concentrations increased in a follicular size-dependent manner. The significant correlations between transcripts of TypeII VLDLR and yolk lipids supported its important role on yolk lipid deposition. In addition, in vitro experiments suggested that exogenous cholesterol, 25-hydroxycholesterol and mevinolin (a highly potent competitive inhibitor of HMG-CoA) treatment could significantly alter TypeII VLDLR gene expression in granulosa cells from both pre-hierarchical and pre-ovulatory follicles. Collectively, data from the present study indicate that TypeII VLDLR is more important for the transport of plasma lipoproteins into developing follicles than TypeI VLDLR, and provide new evidence about the influence of steroids in modulating VLDLR gene expression in ovarian cells.

Abstract A71: 4-HPR trial: A new phase III chemoprevention trial with fenretinide in high-risk women. Rationale, design and implementation

Abstract Retinoids have been studied as chemopreventive compounds because of their role in regulating cell growth, differentiation and apoptosis in preclinical models. Induction of apoptosis is a unique feature of fenretinide (4-hydroxyphenylretinamide, 4-HPR) the most studied retinoid in clinical trials of breast cancer (BC) prevention for its selective accumulation in the breast tissue and its low toxicity. Fenretinide is effective in inhibiting the growth of BRCA1 mutated BC cell lines. Recent studies showed that it modulates gene expression in ovarian cells, with an up-regulation of expression of proapoptotic genes in OVCA433 cells. The fifteen-year follow up of a randomized phase III trial of fenretinide to prevent second BC indicates that it induced a 17% reduction of second BC incidence. More importantly, when stratified by menopausal status, the analysis showed a 38%, statistically significant reduction of second BC in premenopausal women and this effect persisted for up to 15 years. Interestingly, the younger were the women (≤ 40 years), the greater was the trend of benefit by fenretinide. When considering the protective activity of fenretinide on second BC and a similar trend on ovarian cancer (OC) it appears that young women at high risk for both diseases such as carriers of germline BRCA1 and BRCA2 mutations or those with a high family risk may be ideal candidates for further investigation on this retinoid. Since a reduction of second BC might be a surrogate marker of primary prevention, a favorable effect of fenretinide provides strong rationale for a primary prevention trial in unaffected women at high-risk for BC. The European Institute of Oncology (IEO, Milan, Italy) has promoted a multi-centric (15 centres nationwide), randomized phase III placebo-controlled study with fenretinide in healthy young women: 758 healthy women, 25-44 years old, at increased BC risk (BRCA1/2 mutation carriers or subjects with mutation probability ≥20% acc. to BRCAPRO), will be randomized to 4-HPR 200 mg/day vs placebo for 5 years followed by a 10 years follow up period. We will perform a clinical examination every 6 months, and annual breast ultrasound and breast MRI. For subjects ≥ 35 years old also an annual mammography is requested. All subjects will undergo a transvaginal ultrasound at least once a year. At each visit, safety tests (β-HCG, AST, ALT, blood count, creatinine, glycemia, lipid profile, CA-125), and blood samples for biomarkers evaluation at baseline, 12, 36, and 60 months. Enrollment started at the IEO in December 2009. The other centers are expected to start within early 2011. The primary endpoint of the trial is to assess the efficacy of fenretinide in reducing the incidence of invasive BC and ductal intraepithelial neoplasia (DIN). Secondary endpoints are the incidence of non-invasive breast disorders, OC, other cancers and the study of various risk biomarkers. In particular, we will evaluate the change in circulating biomarkers of the IGF system, androgens, retinol binding protein (RBP-4), insulin, blood glucose and VEGF. Genotyping of single nucleotide polymorphisms (SNPs) linked to BC risk (MTHFR, COMT, GH, IGFBP-3, AR, TGF-β genes), degree of methylation of RASSAF1 and RARβ and circulating progranulin will be assessed. The results will be correlated with mammographic instrumental measurements, plasma and tissue biomarkers after 1 year treatment. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A71.

4-HPR modulates gene expression in ovarian cells

4-HPR modulates gene expression in ovarian cells

4‐HPR modulates gene expression in ovarian cells

Ovarian cancer has a high rate of recurrence and subsequent mortality following chemotherapy despite intense efforts to improve treatment outcomes. Recent trials have suggested that retinoids, especially 4-(N-hydroxyphenyl) retinamide (4-HPR), play an important role as a chemopreventive agent and are currently being used in clinical trials for ovarian cancer chemoprevention as well as treatment. This study examines the mechanism of its activity in premalignant and cancer cells. We investigated the modulation of gene expression by 4-HPR in immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer (OVCA433) cells with DNA microarray. Real time RT-PCR and western blotting were used to confirm the microarray results and metabolic changes were examined with optical fluorescence spectroscopy. 4-HPR resulted in an up-regulation of expression of proapoptotic genes and mitochondrial uncoupling protein in OVCA433 cells and modulation of the RXR receptors in IOSE cells, and down-regulation of mutant BRCA genes in both IOSE and OVCA433 cells. 4-HPR had a larger effect on the redox in the 433 cells compared to IOSE. These findings suggest that 4-HPR acts through different mechanisms in premalignant ovarian surface cells and cancer cells, with a preventive effect in premalignant cells and a treatment effect in cancer cells.

A Novel Functional Interaction between the Sp1-like Protein KLF13 and SREBP-Sp1 Activation Complex Underlies Regulation of Low Density Lipoprotein Receptor Promoter Function

Cholesterol homeostasis is regulated by a family of transcription factors designated sterol regulatory element-binding proteins (SREBPs). Precise control of SREBP-targeted genes requires additional interactions with co-regulatory transcription factors. In the case of the low density lipoprotein receptor (LDLR), SREBP cooperates with the specificity protein Sp1 to activate the promoter. In this report, we describe a novel pathway in LDLR transcriptional regulation distinct from the SREBP-Sp1 activation complex involving the Sp1-like protein Krueppel-like factor 13 (KLF13). Using a combination of RNA interference, electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays, deletion, and site-directed mutagenesis, we demonstrated that KLF13 mediates repression in a DNA context-selective manner. KLF13 repression of LDLR promoter activity appears to be needed to keep the receptor silent, a state that can be antagonized by Sp1, SREBP, and inhibitors of histone deacetylase activity. Chromatin immunoprecipitation assay confirmed that KLF13 binds proximal LDLR DNA sequences in vivo and that exogenous oxysterol up-regulates such binding. Together these studies identify a novel regulatory pathway in which gene repression by KLF13 must be overcome by the Sp1-SREBP complex to activate the LDLR promoter. Therefore, these data should replace a pre-existent and more simple paradigm that takes into consideration only the induction of the activator proteins Sp1-SREBP as necessary for LDLR promoter drive without including default repression, such as that by KLF13, of the LDLR gene.

Expression and functional analysis of liver receptor homologue 1 as a potential steroidogenic factor in rat ovary.

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450arom transcripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450arom in both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450arom during early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450arom circumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450arom gene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.

Interleukin-8 gene and protein expression are up-regulated by interleukin-1β in normal human ovarian cells and a granulosa tumor cell line

Objective To evaluate the expression, regulation, and role of interleukin (IL)-8 in human ovary. Design Prospective study. Setting University hospital. Patient(s) Sixteen premenopausal women. Intervention(s) Follicular fluid and granulosa lutein cells (GLCs) were collected during IVF cycles. Ovarian stromal and theca cells were obtained from women underwent surgery. KGN cells, the human granulosa cell tumor cell line, were also used. Main outcome measure(s) The levels of IL-8 and IL-1β in follicular fluid and IL-8 protein production were determined using ELISA. Interleukin-8 and IL-8 receptor gene expression in ovarian cells and the effect of IL-8 on the proliferation of stromal cells were determined. The expression of pIκB was evaluated by Western blot, and the effect of NF-κB inhibitor APDC was examined by Northern blot analysis and ELISA in KGN cells. Result(s) The levels of IL-8 and IL-1β in follicular fluid; each concentration and the volume showed a positive correlation. Reverse transcription polymerase chain reaction showed the presence of IL-8 mRNA in all ovarian cells. In contrast, IL-8 receptor mRNA was only detected in stromal cells. The expression of IL-8 in GLCs and KGN cells was increased by addition of IL-1β and TNFα. Interleukin-8 increased the proliferation of ovarian stromal cells. The expression of pIκB in KGN cells was induced by IL-1β, and the effects were reduced by APDC. Conclusion(s) Interleukin 8 induced by IL-1β via activation of NF-κB in granulosa cells may have a role in the periovulatory period of follicular maturation.

A role for trkA nerve growth factor receptors in mammalian ovulation.

Several members of the neurotrophin (NT) family, including nerve growth factor (NGF), NT-3, and NT-4/5, are expressed in the mammalian ovary. As their respective receptor tyrosine kinases are also found in the gland, the possibility exists that NTs act directly on the gonads to exert effects unrelated to their support of the ovarian innervation. We now report that trkA, the NGF receptor tyrosine kinase, is involved in the acute activational process that leads to the first ovulation. The trkA gene becomes transiently expressed in periovulatory follicules at the time of the first preovulatory surge of gonadotropins at puberty; the increase in trkA messenger RNA (mRNA) content is dramatic ( > 100-fold), but transient (approximately 9 h). No such changes in trkB or trkC mRNA were observed; the abundance of these mRNAs, which encode the receptor tyrosine kinase for NT-4/5 and brain-derived neurotrophic factor, and NT-3, respectively, remained at very low levels throughout puberty. In vivo and in vitro experiments demonstrated that the activation of trkA gene expression is brought about by the proestrous discharge of LH. The increase in trkA mRNA levels is mainly localised to cells of the follicular wall and interstitial tissue of the ovary. NGF mRNA abundance also increases at proestrus, with peak values detected about 5 h before ovulation; as in the case of trkA mRNA, NGF mRNA was found in thecal-interstitial cells. Both trkA and NGF protein, detected by immunohistochemistry, were localized to this same ovarian compartment. Interleukin-1 beta (IL-1 beta), a putative mediator of LH action, enhances both trkA and NGF gene expression in ovarian cells, an effect prevented by IL-1ra, a natural IL-1 beta receptor antagonist. Il-1 beta also stimulates PGE2 release, and this effect was inhibited by both NGF antibodies and a trk receptor blocker, NGF antibodies administered in vivo attenuated the increase in ovarian PGE2 synthesis that antedates ovulation. Immunoneutralization of NGF action or pharmacological blockade of trk tyrosine kinase activity targeted to one ovary resulted in the ipsilateral inhibition of ovulation. The remarkably narrow time frame of trkA gene activation at the completion of follicular growth suggests that NGF acting as a neuroendocrinotrophic factor in a developmentally restricted manner contributes to the acute cytodifferentiation process that leads to the first ovulation in mammals.

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.