218.FGF9 stimulates ovarian progesterone production

2004 ◽  
Vol 16 (9) ◽  
pp. 218
Author(s):  
A. E. Drummond ◽  
M. Dyson ◽  
J. K. Findlay
Keyword(s):  
Granulosa Cells ◽  
Side Chain ◽  
Immature Rat ◽  
Progesterone Production ◽  
Chain Cleavage ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rat Ovary ◽  
The Impact ◽  
Formalin Fixed

FGF9, a member of the fibroblast growth factor family (FGF), is known to be a male sex-determining factor involved in testicular cord formation (1). FGF9 knockout males are sex-reversed (2). However, nothing is known about FGF9's role in folliculogenesis because these mice die at birth (2). We previously reported the presence of FGF9 mRNA and protein in the immature rat ovary (3). In these studies we investigated: (1) the presence of FGF9 receptors (FGFR3) on granulosa cells (GC); and (2) the impact of FGF9 on GC progesterone production. GC isolated from 21 day old diethylstilboestrol (DES)-treated rats were cultured for either 2 hours (RNA) or 2 days (progesterone) in McCoys 5C with FGF9 (0.1-50ng/ml) � FSH (100ng/ml). Progesterone was measured in conditioned media by radioimmunoassay. RNA was extracted from the granulosa cells and reverse-transcribed for PCR. Specific primers for P450 side chain cleavage (SCC) amplified a 329�bp cDNA fragment. GAPDH was used for data normalisation. The FGF9 receptor FGFR3, was immunolocalised on formalin-fixed, paraffin-embedded sections of immature rat ovary. FGFR3 protein was localised only to GC of the ovary. Progesterone production by cultured GC was significantly elevated by FGF9, consistent with the presence of FGFR3. Relative to a maximally stimulating dose of FSH, FGF9 increased progesterone production 10- fold. In preliminary studies, FGF9 increased the expression of P450 SCC mRNA by cultured GC revealing a mechanism by which FGF9 increases progesterone production. These data suggest a role for FGF9 not just in testicular formation, but in the regulation of ovarian steroidogenesis. Supported by the NH&MRC of Australia (Regkey 241000 & 198705). (1) Cotinot et al. (2002) Semin. Reprod. Med. 20, 157. (2) Colvin et al. (2001) Cell 104, 875. (3) Drummond et al. (2003) SRB Abstract 90.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals A correlative chemical and histochemical study of the O-acetylated sialic acids of human colonic epithelial glycoproteins in formalin fixed paraffin embedded tissues.

1978 ◽  
Vol 26 (12) ◽  
pp. 1033-1041 ◽  
Author(s):  
P E Reid ◽  
C F Culling ◽  
W L Dunn ◽  
M G Clay ◽  
C W Ramey
Keyword(s):  
Epithelial Cells ◽  
Histochemical Study ◽  
Sialic Acids ◽  
Side Chain ◽  
Isolation And Identification ◽  
New Information ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Human Large Intestine ◽  
Formalin Fixed

Procedures are described for the isolation and identification of epithelial glycoproteins from formalin fixed, paraffin embedded specimens of human large intestine. The side chain O-acetylation patterns of the sialic acids of these glycoproteins were surprisingly similar to those of purified glycoproteins prepared from epithelial cells obtained from the same tissue before fixation. These results were consistent with those obtained by histochemical procedures performed on representative sections taken from the same tissue blocks. The methodology described permits a direct correlation of chemical and histochemical results obtained from the study of colonic epithelial glycoproteins of both normal and diseased tissues. It eliminates some of the difficulties associated with interpretation of the results by either discipline and may provide new information which would be unavailable by either chemistry or histochemistry alone.

scholarly journals Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells

Reproduction ◽  
2009 ◽  
Vol 138 (6) ◽  
pp. 961-969 ◽  
Author(s):  
P L Rangel ◽  
A Rodríguez ◽  
S Rojas ◽  
P J Sharp ◽  
C G Gutierrez
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Passive Immunization ◽  
Side Chain ◽  
Lh Receptor ◽  
Side Chain Cleavage ◽  
Progesterone Production ◽  
Chain Cleavage ◽  
Chicken Ovary ◽  
In Cells

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11–14 h than 1–4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1–100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1–4, or 11–14 h after ovulation. Testosterone (0–4000 ng/ml) alone or in combination with LH (0–100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1–4 and 11–14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

scholarly journals Fibroblast Growth Factor-9, a Local Regulator of Ovarian Function

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3711-3721 ◽  
Author(s):  
Ann E. Drummond ◽  
Marianne Tellbach ◽  
Mitzi Dyson ◽  
Jock K. Findlay
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Corpora Lutea ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Immature Rat ◽  
Progesterone Production ◽  
Rat Ovary

Fibroblast growth factor 9 (FGF9) is widely expressed in embryos and fetuses and has been shown to be involved in male sex determination, testicular cord formation, and Sertoli cell differentiation. Given its male gender bias, the ovary has not been reported to express FGF9, nor has a role in ovarian function been explored. We report here that FGF9 mRNA and protein are present in the rat ovary and provide evidence that supports a role for FGF9 in ovarian progesterone production. FGF9 mRNA levels as determined by real-time PCR were high in 4-d-old rat ovaries, thereafter declining and stabilizing at levels approximately 30% of d 4 levels at d 12–25. Levels of FGF9 mRNA in the ovary were significantly higher than that present in adult testis, at all ages studied. The FGF9 receptors FGFR2 and FGFR3 mRNAs were present in postnatal and immature rat ovary and appeared to be constitutively expressed. FGF9 protein was localized to theca, stromal cells, and corpora lutea and FGFR2 and FGFR3 proteins to granulosa cells, theca cells, oocytes, and corpora lutea, by immunohistochemistry. Follicular differentiation induced by gonadotropin treatment reduced the expression of FGF9 mRNA by immature rat ovaries, whereas the estrogen-stimulated development of large preantral follicles had no significant effect. In vitro, FGF9 stimulated progesterone production by granulosa cells beyond that elicited by a maximally stimulating dose of FSH. When the granulosa cells were pretreated with FSH to induce LH receptors, FGF9 was found not to be as potent as LH in stimulating progesterone production, nor did it enhance LH-stimulated production. The combined treatments of FSH/FGF9 and FSH/LH, however, were most effective at stimulating progesterone production by these differentiated granulosa cells. Analyses of steroidogenic regulatory proteins indicate that steroidogenic acute regulatory protein and P450 side chain cleavage mRNA levels were enhanced by FGF9, providing a mechanism of action for the increased progesterone synthesis. In summary, the data are consistent with a paracrine role for FGF9 in the ovary.

Effect of high-density lipoprotein on bovine granulosa cells: progesterone production in newly isolated cells and during cell culture

1990 ◽  
Vol 124 (2) ◽  
pp. 255-260 ◽  
Author(s):  
P. J. O'Shaughnessy ◽  
S. Pearce ◽  
M. A. Mannan
Keyword(s):  
High Density Lipoprotein ◽  
Granulosa Cells ◽  
Density Lipoprotein ◽  
High Density ◽  
Luteal Cell ◽  
Side Chain ◽  
Serum Free ◽  
Side Chain Cleavage ◽  
Progesterone Production ◽  
Chain Cleavage

ABSTRACT It has been proposed that changes in steroidogenesis which occur during early development of the corpus luteum may be due to increased availability of lipoproteins. Bovine follicular fluid, however, contains significant amounts of high-density lipoprotein (HDL), and granulosa cells are exposed to this lipoprotein before ovulation. To determine whether bovine granulosa cells can utilize HDL the effects of this lipoprotein on freshly isolated, non-luteinized granulosa cells and on granulosa cells undergoing luteinization in serum-free culture were examined. Cells were isolated from non-atretic, antral follicles and cultured for 12 h in 10% (v/v) lipoprotein-deficient serum to allow cell attachment. After this time cells were cultured in serum-free medium. During culture the cells underwent functional luteinization as assessed by an increase in basal progesterone output (9·6-fold in 7 days) which was associated with a marked increase in activity of cholesterol side-chain cleavage and loss of aromatase activity. Dibutyryl cyclic AMP (dbcAMP) increased basal production of progesterone about twofold but HDL alone had no effect. Addition of HDL plus dbcAMP, in contrast, caused a very marked stimulation (up to ten times) of basal steroidogenesis. This trophic effect of HDL and dbcAMP lasted at least 2 weeks. Activity of cholesterol side-chain cleavage was stimulated (threefold over basal) by dbcAMP during culture but HDL was without effect, alone or with dbcAMP. Addition of HDL (in the presence or absence of dbcAMP) to freshly isolated granulosa cells had no significant stimulatory effect on progesterone production over 12 h in six experiments, and in two of these experiments a significant inhibitory effect was seen. Incubation with 22R-hydroxycholesterol, in contrast, caused a marked stimulation of progesterone production, indicating that the steroidogenic capacity of the cells was not already saturated. Results presented here suggest that bovine granulosa cells are able to utilize HDL for steroidogenesis only after luteinization. The massive secretion of progesterone by luteinized granulosa cells which occurs in the presence of HDL suggests that this lipoprotein is very important in the development and maintenance of luteal cell function in cattle. Journal of Endocrinology (1990) 124, 255–260

A Review of Preanalytical Factors Affecting Molecular, Protein, and Morphological Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue: How Well Do You Know Your FFPE Specimen?

2014 ◽  
Vol 138 (11) ◽  
pp. 1520-1530 ◽  
Author(s):  
B. Paige Bass ◽  
Kelly B. Engel ◽  
Sarah R. Greytak ◽  
Helen M. Moore
Keyword(s):  
Formalin Fixation ◽  
Research Database ◽  
Factors Affecting ◽  
Factors Associated ◽  
Paraffin Embedding ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Embedded Blocks ◽  
The Impact ◽  
Formalin Fixed

Context Formalin fixation and paraffin embedding is a timeless, cost-efficient, and widely adopted method of preserving human tissue biospecimens that has resulted in a substantial reservoir of formalin-fixed, paraffin-embedded blocks that represent both the pathology and preanalytical handling of the biospecimen. This reservoir of specimens is increasingly being used for DNA, RNA, and proteomic analyses. Objective To evaluate the impact of preanalytical factors associated with the formalin fixation and paraffin embedding process on downstream morphological and molecular endpoints. Data Sources We surveyed the existing literature using the National Cancer Institute's Biospecimen Research Database for published reports investigating the potential influence of preanalytical factors associated with the formalin fixation and paraffin embedding process on DNA, RNA, protein, and morphological endpoints. Conclusions Based on the literature evidence, the molecular, proteomic, and morphological endpoints can be altered in formalin-fixed, paraffin-embedded specimens by suboptimal processing conditions. While the direction and magnitude of effects associated with a given preanalytical factor were dependent on the analyte (DNA, RNA, protein, and morphology) and analytical platform, acceptable conditions are highlighted, and a summary of conditions that could preclude analysis is provided.

scholarly journals Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Sophia Rossouw ◽  
Hocine Bendou ◽  
Liam Bell ◽  
Jonathan Rigby ◽  
Alan Christoffels
Keyword(s):  
Polyethylene Glycol ◽  
Extraction Efficiency ◽  
Protein Extraction ◽  
Formalin Fixed Paraffin ◽  
Liquid Chromatography Tandem Mass ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Protein Selection ◽  
The Impact ◽  
Formalin Fixed

Background: Optimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues are not yet standardised and new techniques are continually developed and improved. The effect of polyethylene glycol (PEG) 20 000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction.Objective: The impact of PEG 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using FFPE colonic tissue via liquid chromatography tandem mass spectrometry analysis.Methods: This study was conducted from August 2017 to July 2019 using human FFPE colorectal carcinoma tissues from the Anatomical Pathology department at Tygerberg Hospital in South Africa. Samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using PEG 20 000 in the protein extraction solution. Data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories.Results: Polyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with PEG 20 000. However, no differences were observed with regard to protein selection bias. We found that higher protein concentrations ( 10 µg) compromised the function of PEG.Conclusion: This study indicates that protocols generating high protein yields from human FFPE tissues would benefit from the exclusion of PEG 20 000 in the protein extraction solution.

scholarly journals Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 24
Author(s):  
Hazem Ghebeh ◽  
Fatmah A. Mansour ◽  
Dilek Colak ◽  
Akram A. Alfuraydi ◽  
Amal A. Al-Thubiti ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immunohistochemical Detection ◽  
Breast Cancer Patients ◽  
Preservation Method ◽  
Detection Technology ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
The Impact ◽  
Formalin Fixed

Purpose: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. Methods: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. Results: In FFPE tissues, PD-L1 was positive in 7–10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20–30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13–20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. Conclusions: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.

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