mRNA In Granulosa Cells Research Articles

Changes in brain ribonuclease (BRB) messenger RNA in granulosa cells (GCs) of dominant vs subordinate ovarian follicles of cattle and the regulation of BRB gene expression in bovine GCs

Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1–5 mm; pooled per ovary), medium (5–8 mm; individually collected), or large (8.1–17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs was 8.6- to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66), and large (n = 33) subordinate E2-inactive (FFL E2 < P4) follicles than in large (n = 16) dominant E2-active (FFL E2 > P4) follicles. In the largest 4 follicles, GCs BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = −0.65) and E2:P4 ratio (r = −0.46). In experiment 2, GCs from large (8–22 mm diameter) and small (1–5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P > 0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P < 0.05), whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GCs. Treatment of small-follicle GCs with recombinant human RNase1 increased (P < 0.05) GCs numbers and E2 production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GCs and may regulate E2 production during follicular growth in cattle.

The domestic chicken: Causes and consequences of an egg a day

The domestic laying chicken has been intensely selected to be a persistent ovulator. That is, the tendency for broodiness has been nearly eliminated and, given the appropriate lighting and nutrition, many strains of laying hens produce an egg on almost every day. The regulatory mechanisms involved in coordination of neuroendocrine and ovarian events have been well studied and described. In spite of this, there has been little attention focused on the oocyte itself. Recent findings in mammals have indicated that the oocyte produces several oocyte-specific factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15), which influence the surrounding cells and follicular development. Our studies indicate that GDF9 is present in the hen oocyte and influences granulosa cell proliferation. Additionally,Bmp15 mRNA is most abundant in oocytes of small follicles and stimulates an increase in follicle stimulating hormone (FSH) receptor mRNA in granulosa cells. BMP15 also enhances yolk uptake in growing follicles by decreasing tight junctions between granulosa cells. These studies indicate that the oocyte likely contributes to follicle development. Commercial laying hens also spontaneously develop ovarian cancer at a high rate, and susceptibility to this disease has been associated with ovulatory events in women. Studies have shown that ovulation, or events associated with ovulation, increase the prevalence of ovarian cancer in hens. Inhibition of ovulation in hens through a hormonal strategy mimicking oral contraceptives results in a decrease of ovarian cancer incidence. Recent studies in women have suggested that some ovarian tumors may arise from the distal oviduct. Gene expression profiles in very early stage tumors from hens show a high expression of oviduct-related genes, supporting the possibility of oviduct origin for some ovarian tumors. Genetic selection for high productivity in commercial laying hens has generated an efficient and valuable food source as well as an important animal model for human ovarian cancer.

Insulin-like growth factor-1 regulates the expression of luteinizing hormone receptor and steroid production in bovine granulosa cells.

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin-like growth factor-1 (IGF-1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF-1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF-1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF-1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non-luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid-sized follicles (6-8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF-1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF-1 (0.1-100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF-1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF-1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF-1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.

Wilms' tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells.

BackgroundThe important role of WT1 in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of inhibin-α and FSH receptor. There is also considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage.MethodsGCs were obtained from small preantral follicles of immature rat ovary. Bax and bcl-2 mRNA and protein levels in GCs transfected with WT1 (−KTS) or WT1 (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with caspase 3/7 activity and TUNEL assay. The mechanism by which WT1 regulates Bax expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs.ResultsHere, we showed that WT1 (−KTS) suppressed endogenous Bax transcript and protein expression, and this inhibition resulted from direct binding of WT1 in the Bax promoter region in vivo. In addition, anti-apoptotic effects of WT1 (−KTS) were demonstrated based on MTT assays, a sensitive bioluminescence caspase 3/7 assay and TUNEL assays. On the other hand, WT1 has no role on bcl-2 expression in GCs.ConclusionThese findings suggest that activation of WT1 is necessary for maintenance of GC survival during early stage of follicles and WT1 can play a role in protecting apoptosis through the regulation of upstream activator (Bax), as well as through regulation of downstream effecter (caspases 3 and 7).

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

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 4h after human chorionic gonadotrophin (hCG) treatment. In situ hybridisation analysis demonstrated that Clmp mRNA expression was stimulated in theca cells at 4h after hCG and remained elevated until 12h. 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.

Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: evidence for premature luteinization.

Premature luteinization is a possible cause of infertility in women. It is currently unknown whether environmental chemicals can induce changes associated with premature luteinization. Using rat granulosa cells (GC) in vitro, we demonstrated that exposure to atrazine (ATR), a widely used herbicide, causes GC phenotype that resembles that of human premature luteinization. At the end of the 48-h stimulation with FSH, ATR-exposed GC showed (1) higher levels of progesterone, (2) overexpression of luteal markers (Star and Cyp11a1), and (3) an increase in progesterone:estradiol ratio above 1. Mechanistic experiments were conducted to understand the signaling events engaged by ATR that lead to this phenotype. Western blot analysis revealed prolonged phosphorylation of protein kinase B (AKT) and cAMP response element-binding protein (CREB) in ATR- and FSH-exposed GC. An increased level of ERK1/2-dependent transcriptional factor CCATT/enhancer-binding protein beta (CEBPB) was observed after 4 h of ATR exposure. Inhibitors of PI3K (wortmannin) and MEK (U0126) prevented ATR-induced rise in progesterone level and expression of luteal markers in FSH-stimulated GC. Atrazine intensified AKT and CEBPB signaling and caused Star overexpression in forskolin-stimulated GC but not in epidermal growth factor (EGF)-stimulated GC. In the presence of rolipram, a specific inhibitor of phosphodiesterase 4 (PDE4), ATR was not able to further elevate AKT phosphorylation, CEBPB protein level, and Star mRNA in FSH-stimulated GC, suggesting that ATR inhibits PDE4. Overall, this study showed that ATR acts as a FSH sensitizer leading to enhanced cAMP, AKT, and CEBPB signaling and progesterone biosynthesis, which promotes premature luteinization phenotype in GC.

MRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.

Elevated levels of FMR1 mRNA in granulosa cells are associated with low ovarian reserve in FMR1 premutation carriers.

AimTo assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD).DesignCase control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR.ResultsIn FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80–120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R2 linear = 0.231, P = 0.02).ConclusionWe suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80–120 repeats).

Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age.

Is the activity of sirtuin 3 (SIRT3) altered in granulosa and cumulus cells from young women with reduced ovarian reserve or women of advanced maternal age? SIRT3 mRNA and active protein in granulosa and cumulus cells were decreased in women with reduced ovarian reserve and advanced maternal age. Young women with reduced ovarian reserve or women of advanced maternal age have reduced oocyte viability, possibly due to altered granulosa and cumulus cell metabolism. The mitochondrial SIRT3 protein may be implicated in these processes as it is able to sense the metabolic state of the cell and alter mitochondrial protein function post-translationally. This is a prospective cohort study, in which women (n = 72) undergoing routine IVF/ICSI were recruited and allocated to one of three cohorts based on age and ovarian reserve (as assessed by serum anti-Mullerian hormone level). Women were classified as young (≤35 years) or of advanced maternal age (≥40 years). Granulosa and cumulus cells were collected. SIRT3 mRNA and protein levels and protein activity was analysed in granulosa and cumulus cells via quantitative PCR, immunohistochemistry and western blotting, and deacetylation activity, respectively. Activity of the glutamate dehydrogenase (GDH) enzyme, a known target of SIRT3, was assessed, and acetylated proteins in mitochondria isolated from granulosa and cumulus cells were separated by immunoprecipitation and acetylation of GDH assessed by western blotting. Data for women with good prognosis (young women with normal ovarian reserve) were compared with those from young women with reduced ovarian reserve and those of advanced maternal age. SIRT3 mRNA and active protein were present in granulosa and cumulus cells and co-localized to the mitochondria. SIRT3 mRNA in granulosa cells was decreased in young women with reduced ovarian reserve and advanced maternal age versus young women with normal ovarian reserve (P < 0.05). SIRT3 mRNA in cumulus cells was decreased in women of advanced maternal age versus young women with normal ovarian reserve only (P < 0.05). Granulosa cell GDH activity was decreased in young women with reduced ovarian reserve and in women of advanced maternal age (P < 0.05), whereas cumulus cell GDH activity was reduced in the advanced maternal age group only (P < 0.05). The acetylation profile of GDH in mitochondria revealed increased acetylation of GDH in granulosa and cumulus cells from women of advanced maternal age (P < 0.05) while young women with reduced ovarian reserve had increased GDH acetylation in granulosa cells only (P < 0.05). Although patients were allocated to groups based on maternal age and ovarian reserve and matched for BMI, other maternal factors may also alter the 'molecular health' of ovarian cells. Our data suggest that SIRT3 post-translational modification of mitochondrial enzymes in human granulosa and cumulus cells may regulate GDH activity, thus altering the metabolic milieu surrounding the developing oocyte. Owing to the association between the decline in oocyte quality and pregnancy rates in women of advanced maternal age and the possible association with reduced ovarian reserve, knowledge of perturbed SIRT3 function in granulosa and cumulus cells may lead to novel therapies to improve mitochondrial metabolism in the oocyte and follicular cells in women undergoing IVF treatment. No conflicts of interest to declare. Research was funded by an NHMRC project grant.

Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle

Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation? Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation. Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows. Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbæk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration. Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay. Eleven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Müllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested--besides LH--TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response. The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle. These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers. Grants from the Research Fund of Region Sjælland are gratefully acknowledged. None of the authors declared any conflict of interest. Not applicable.

Evidence that the dominant follicle of the first wave is more active than that of the second wave in terms of its growth rate, blood flow supply and steroidogenic capacity in cows

To clarify the different characteristics of the dominant follicle (DF), the DF in first ovarian follicular wave (DF-1) after spontaneous ovulation and DF in second follicular wave (DF-2) and after induced ovulation of the first-wave DF by GnRH were examined in non-lactating Holstein cows. Follicular maturation of DF-1 and DF-2 were induced by PGF2α and GnRH treatment on Day 6 and 8 (Day 0=Day of follicular wave emergence), respectively. Follicular growth and blood flow (BF) in the follicular wall of DF-1 and DF-2 were examined. To analyze sex steroids in follicular fluid (FF) and amount of mRNA in granulosa cells, DF-1 and DF-2 were aspirated on Day 8 or 9 in different estrous cycle. Diameter in DF-1 was larger than DF-2 on Day 8 and 9. From Day 8 to 9, BF area (BFA) and percentage of the follicular wall with BF, which represents the degree of distribution of BF, increased in DF-1 but not in DF-2. BFA per length of follicle circumference with BF, which represents the thickness of BF, was not different between DF-1 and DF-2. Concentrations of 17β-estradiol (E2) in plasma, E2 and androstendione in FF and amounts of LH receptor mRNA were greater in the DF-1 on Day 8. Gene expression for steroidogenesis, prostaglandin synthesis and angiogenesis did not differ between DF-1 and DF-2. These results indicated that DF-1 were more active than DF-2 in growth, BF supply and steroidogenesis. The greater BFA observed in the DF-1 may be derived from as a result of the greater vascularity in the follicular wall.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers

BackgroundHeifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.ResultsGranulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.ConclusionsThese results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Transcription Factors GATA-4 and GATA-6: Molecular Characterization, Expression Patterns and Possible Functions During Goose (&lt;i&gt;Anser cygnoides&lt;/i&gt;) Follicle Development

The transcription factors GATA-4 and GATA-6, members of the GATA family, play an important role in ovarian cell proliferation, differentiation and apoptosis. In this study, the full-length coding sequences of goose GATA-4 and GATA-6 were cloned and characterized. GATA-4 and GATA-6 consist of 1236 and 1104 nucleotides encoding proteins with 411 and 367 amino acids, respectively. The deduced amino acid sequences of both proteins include two adjacent zinc finger domains with the distinctive form (CVNC-X17-CNAC)-X29-(CANC-X17-CNAC) and share 84.76% identity within this domain. In silico prediction together with matching of the high affinity RRXS(T)Y motif revealed that the GATA-4 protein might be phosphorylated predominantly at S233, but no phosphorylation site was found in the GATA-6 protein. Real-time quantitative PCR analysis showed that GATA-4 and GATA-6 mRNAs were co-expressed in goose follicles, moderately expressed in granulosa cells and weakly expressed in theca cells. The expression level of GATA-4 mRNA in healthy follicles was significantly higher than in atretic follicles or postovulatory follicles (P<0.01), and the expression level of GATA-6 mRNA in healthy follicles was significantly lower than in atretic follicles or postovulatory follicles (P<0.01). The expression level of GATA-4 mRNA in granulosa cells was downregulated during follicle development; the peak of expression occurred in the 8-10 mm follicles, and the lowest expression was in the F1 follicles. GATA-6 was upregulated and reached its peak expression in the F1 follicles. These results indicate that the molecular structural differences in goose GATA-4 and GATA-6 may be related to their different roles during follicle development.

Transcriptional repression of the Ahr gene by LHCGR signaling in preovulatory granulosa cells is controlled by chromatin accessibility

Recent advances in establishing the role of the aryl hydrocarbon receptor (Ahr) in normophysiology have discovered its fundamental role, amongst others, in female reproduction. Considering previous studies suggesting the hormonal modulation of Ahr, we aimed to investigate whether in murine granulosa cells (GCs) the gonadotropins regulate Ahr expression and how this is mechanistically implemented. We found that the FSH-like substance – pregnant mare serum gonadotropin – led to stimulation of Ahr expression. More importantly hCG produced relatively rapid reduction of Ahr mRNA in GCs of preovulatory follicles. We show for the first time that LHCGR signaling in regulating the Ahr message involves protein kinase A pathway and is attributable to decreased transcription rate. Finally, we found that Ahr promoter accessibility was decreased by hCG, implicating chromatin remodeling in Ahr gene regulation by LH.

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.

Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer

ObjectiveTo investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer (IVF-ET) and to explore its mechanism. MethodsFifty-eight women with tubal infertility were randomized into two groups: 30 in an experimental group treated with Xiaoyao powder (Shugan) plus gonadotropin-releasing hormone analog (GnRHa)/follicle-stimulating hormone (FSH)/human chorionic gonadotropin (hCG) and 28 in the control group who were treated with GnRHa/FSH/hCG only. The total gonadotropin (Gn) doses required, endometrial thickness, oocyte numbers, high quality embryo production rate and pregnancy rate of the two groups were compared. The concentration of growth differentiation factor-9 (GDF-9) in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse transcription-polymerase chain reaction amplification. ResultsIn the experimental group, the Gn dose was significantly lower than that in the control group; the endometrial thickness, high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group (all P<0.05). ConclusionShugan treatment can improve the pregnancy rate of women with tubal infertility; its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.

Molecular Characterization and Transcriptional Regulation of a Disintegrin and Metalloproteinase With Thrombospondin Motif 1 (ADAMTS1) in Bovine Preovulatory Follicles

The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6 to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5'-deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.

Lhcgr Expression in Granulosa Cells: Roles for PKA-Phosphorylated β-Catenin, TCF3, and FOXO1

Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind β-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of β-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of β-catenin on Ser552 and Ser665. FSH activated the β-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed β-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-β-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.

The Influence of the Antiandrogen 2‐Hydroxyflutamide on the Androgen Receptor Expression in the Porcine Ovarian Follicles – An In Vitro Study

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48h. During the last 12 and 24h of culture, they were incubated in the presence of T (10(-7) m/ml), 2-Hf (1.7×10(-4) m) or both T and 2-Hf (T+2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6-8mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E(2)) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7-8 mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3 mm for 0, 0.1, and 1 μg/ml FGF10, respectively, at 72 h after treatment; P<0.05). In a third experiment, follicles were obtained 24 h after FGF10 (1 μg/ml) or PBS treatment through ovariectomy. In theca cells, FGF10 treatment did not affect mRNA encoding steroidogenic enzymes, LHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E(2) production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.