Changing expression and subcellular distribution of karyopherins during murine oogenesis

Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined (Kpna1, Kpna2, Kpna3, Kpna4, Kpna6, Kpna7, Kpnb1, Ipo5 and Xpo1), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1, Kpna2, Kpna4, Kpna6 and Ipo5 and down-regulation of Kpnb1, Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs – KPNA1, KPNA2 and IPO5 – displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development.

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RNA Sequencing in Comparison to Immunohistochemistry for Measuring Cancer Biomarkers in Breast Cancer and Lung Cancer Specimens

Biomedicines ◽

10.3390/biomedicines8050114 ◽

2020 ◽

Vol 8 (5) ◽

pp. 114

Author(s):

Maxim Sorokin ◽

Kirill Ignatev ◽

Elena Poddubskaya ◽

Uliana Vladimirova ◽

Nurshat Gaifullin ◽

...

Keyword(s):

Breast Cancer ◽

Lung Cancer ◽

Rna Sequencing ◽

Expression Profiles ◽

Correlation Study ◽

Cancer Tissue ◽

Transcriptional Level ◽

Tissue Samples ◽

Protein Levels ◽

Formalin Fixed Paraffin Embedded

RNA sequencing is considered the gold standard for high-throughput profiling of gene expression at the transcriptional level. Its increasing importance in cancer research and molecular diagnostics is reflected in the growing number of its mentions in scientific literature and clinical trial reports. However, the use of different reagents and protocols for RNA sequencing often produces incompatible results. Recently, we published the Oncobox Atlas of RNA sequencing profiles for normal human tissues obtained from healthy donors killed in road accidents. This is a database of molecular profiles obtained using uniform protocol and reagents settings that can be broadly used in biomedicine for data normalization in pathology, including cancer. Here, we publish new original 39 breast cancer (BC) and 19 lung cancer (LC) RNA sequencing profiles obtained for formalin-fixed paraffin-embedded (FFPE) tissue samples, fully compatible with the Oncobox Atlas. We performed the first correlation study of RNA sequencing and immunohistochemistry-measured expression profiles for the clinically actionable biomarker genes in FFPE cancer tissue samples. We demonstrated high (Spearman’s rho 0.65–0.798) and statistically significant (p < 0.00004) correlations between the RNA sequencing (Oncobox protocol) and immunohistochemical measurements for HER2/ERBB2, ER/ESR1 and PGR genes in BC, and for PDL1 gene in LC; AUC: 0.963 for HER2, 0.921 for ESR1, 0.912 for PGR, and 0.922 for PDL1. To our knowledge, this is the first validation that total RNA sequencing of archived FFPE materials provides a reliable estimation of marker protein levels. These results show that in the future, RNA sequencing can complement immunohistochemistry for reliable measurements of the expression biomarkers in FFPE cancer samples.

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Value of serum and follicular fluid Sirt1,Sirt2 protein levels in predicting the outcome of assisted reproduction

10.21203/rs.2.23749/v1 ◽

2020 ◽

Author(s):

Lingnv Yao ◽

Wenqin Lin ◽

Nan Jiang ◽

Chuyan Li ◽

Haifeng Cao ◽

...

Keyword(s):

Assisted Reproduction ◽

Follicular Fluid ◽

Ovarian Reserve ◽

Antral Follicle ◽

Antral Follicle Count ◽

Advanced Age ◽

Control Group ◽

Age Group ◽

Protein Levels ◽

Study Population

Abstract Background: To explore whether serum and follicular fluid Sirt1, Sirt2 can reflect ovarian reserve and predict the outcome of assisted reproduction. Methods: The study population comprised 125 patients , 39 in OPOI（occult form of premature ovarian insufficiency）group, 49 in advanced age group , and 37 in control group. The levels of serum Sirt1, Sirt2 were measured on the 2nd to 5th day of menstruation (bSirt1,bSirt2) and HCG day. Follicular fluid Sirt1 (FFSirt1) and Sirt2 (FFSirt2), were determined on OPU (oocyte pick up) day. Results: The level of FFSirt2 in the advanced age group was significantly lower than those in other two groups. FFSirt 2 and Sirt 2 (HCG day ) were negatively correlated with age (r=-0.35, r=-0.19), but there were no value of them for assessing DOR (diminished ovarian reserve). The level of bSirt2 in (cumulative) pregnant group was significantly higher (r=0.24, P=0.00). Conclusions: This was the first study to show that FFSirt2 and Sirt2 (HCG day) might be negatively correlated with age and antral follicle count (AFC). bSirt2 could predict cumulative pregnancy outcome together with anti-Mullerian hormone (AMH), AFC and age.

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138 Relationships Between Antral Follicle Count, Ovarian Volume, Pre-Antral Follicle Number, and Oocyte Quality

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab138 ◽

2018 ◽

Vol 30 (1) ◽

pp. 209

Author(s):

G. L. Vasconcelos ◽

R. Maculan ◽

N. Alves ◽

A. L. A. P. L. Ribeiro ◽

A. W. B. Silva ◽

...

Keyword(s):

Ovarian Follicle ◽

Primordial Follicle ◽

Antral Follicle ◽

Oocyte Quality ◽

Antral Follicle Count ◽

Primary Follicle ◽

Ovarian Volume ◽

Primordial Follicles ◽

Antral Follicles ◽

Grade 3

The objective was to evaluate the possible relationships between AFC, ovarian volume, ovarian follicle reserve and oocyte quality in abattoir-derived ovaries (experiment 1) and in cows (experiment 2) submitted to OPU. Antral follicle counts of ≥25, 16 to 24, and ≤ 16 were used to define AFC classes as high (HAFC), intermediate (IAFC), and low (LAFC) in both experiments. In experiment 1, after antral follicles were aspirated, abattoir ovaries (n = 10 per AFC class) were processed by conventional histology and pre-antral follicles were counted within primordial, primary, secondary, and tertiary classes and classified as either healthy or degenerate under regular microscopy (Cushman et al. 1999). In experiment 2, HAFC (n = 42), IAFC (n = 34), and LAFC (n = 29) cows were submitted to OPU and oocytes classified as grades 1, 2, and 3 or degenerate (IETS, 2010). Antral follicles (≥3 mm in diameter) were counted by ultrasonography. Data were analysed by GENMOD and GLM procedures of SAS (SAS Institute Inc., Cary, NC, USA) after transformations, when required. In experiment 1, mean normal primordial follicle number was higher (P < 0.001) in HAFC (137.0 ± 1.6)a compared with IAFC (52.6 ± 1.9)b and LAFC (20.2 ± 5.3)c ovaries. However, the mean number of degenerate primordial follicles was lower (P < 0.001) in low count ovaries (2.4 ± 0.6) compared with HAFC (19.0 ± 4.7) and IAFC (16.4 ± 1.5, P < 0.001). Normal primary follicle number was higher in the HAFC compared with IAFC and LAFC ovarian classes (86.2 ± 7.0a v. 34.6 ± 5.1b and 14.4 ± 3.3c, respectively; P < 0.01). Degenerate primary follicles were higher in the HAFC compared with LAFC ovarian class (16.8 ± 6.5 v. 5.2 ± 2.64; P < 0.05). Normal secondary follicle number was also higher in the HAFC compared to LAFC ovarian classes (25.2 ± 7.67 v. 2.4 ± 0.8; P < 0.05). The number of degenerate secondary follicles differed (P < 0.01) only between the IAFC and the LAFC ovarian classes (0.6 ± 0.4 and 7.2 ± 2.4, respectively), which were similar (P > 0.5) to the HAFC class (3.8 ± 1.0). In experiment 2, grade 1, 2, and 3 oocytes, viable oocytes, and ovarian volume (mm3) were higher (P < 0.001) in HAFC compared with IAFC and LAFC cows (grade 1: 7.9 ± 0.6a, 4.9 ± 0.7b and 3.3 ± 0.7c; grade 2: 4.0 ± 0.4a, 2.8 ± 0.4b and 1.2c; grade 3: 2.1 ± 0.4a, 2.5 ± 0.4a and 1.3 ± 0.5b, respectively; viable oocytes: 16.3 ± 1.1a, 13.1 ± 1.2b, and 8.1 ± 1.3c, respectively; (volumes: 12.6 ± 0.7a, 10.1 ± 0.8b, and 8.1 ± 0.9c, respectively). In conclusion, high AFC is linked to a higher follicular reserve, oocyte quality, and ovarian volume. It is safe to apply AFC in the selection of bovine females without compromising oocyte or pre-antral follicular population qualities.

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GEDS: A Gene Expression Display Server for mRNAs, miRNAs and Proteins

Cells ◽

10.3390/cells8070675 ◽

2019 ◽

Vol 8 (7) ◽

pp. 675 ◽

Cited By ~ 5

Author(s):

Xia ◽

Liu ◽

Zhang ◽

Guo

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Gene Expression Data ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cancer Cell Line ◽

Tissue Expression ◽

The Cancer Genome Atlas ◽

Expression Data ◽

Protein Levels

High-throughput technologies generate a tremendous amount of expression data on mRNA, miRNA and protein levels. Mining and visualizing the large amount of expression data requires sophisticated computational skills. An easy to use and user-friendly web-server for the visualization of gene expression profiles could greatly facilitate data exploration and hypothesis generation for biologists. Here, we curated and normalized the gene expression data on mRNA, miRNA and protein levels in 23315, 9009 and 9244 samples, respectively, from 40 tissues (The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GETx)) and 1594 cell lines (Cancer Cell Line Encyclopedia (CCLE) and MD Anderson Cell Lines Project (MCLP)). Then, we constructed the Gene Expression Display Server (GEDS), a web-based tool for quantification, comparison and visualization of gene expression data. GEDS integrates multiscale expression data and provides multiple types of figures and tables to satisfy several kinds of user requirements. The comprehensive expression profiles plotted in the one-stop GEDS platform greatly facilitate experimental biologists utilizing big data for better experimental design and analysis. GEDS is freely available on http://bioinfo.life.hust.edu.cn/web/GEDS/.

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The Emerging Role of Galectins and O-GlcNAc Homeostasis in Processes of Cellular Differentiation

Cells ◽

10.3390/cells9081792 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1792 ◽

Cited By ~ 1

Author(s):

Rada Tazhitdinova ◽

Alexander V. Timoshenko

Keyword(s):

Cancer Biology ◽

Cellular Differentiation ◽

Current Knowledge ◽

Expression Profiles ◽

Cell Types ◽

Innovative Strategies ◽

Protein Levels ◽

Differentiated Cells ◽

Independent Functions

Galectins are a family of soluble β-galactoside-binding proteins with diverse glycan-dependent and glycan-independent functions outside and inside the cell. Human cells express twelve out of sixteen recognized mammalian galectin genes and their expression profiles are very different between cell types and tissues. In this review, we summarize the current knowledge on the changes in the expression of individual galectins at mRNA and protein levels in different types of differentiating cells and the effects of recombinant galectins on cellular differentiation. A new model of galectin regulation is proposed considering the change in O-GlcNAc homeostasis between progenitor/stem cells and mature differentiated cells. The recognition of galectins as regulatory factors controlling cell differentiation and self-renewal is essential for developmental and cancer biology to develop innovative strategies for prevention and targeted treatment of proliferative diseases, tissue regeneration, and stem-cell therapy.

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Comparative Transcriptome Combined with Proteome Analyses Revealed Key Factors Involved in Alfalfa (Medicago sativa) Response to Waterlogging Stress

International Journal of Molecular Sciences ◽

10.3390/ijms20061359 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1359 ◽

Cited By ~ 4

Author(s):

Ningbo Zeng ◽

Zhijian Yang ◽

Zhifei Zhang ◽

Longxing Hu ◽

Liang Chen

Keyword(s):

Medicago Sativa ◽

Molecular Mechanism ◽

Crop Production ◽

Expression Profiles ◽

Resistance Breeding ◽

Ethylene Response Factor ◽

Proline Metabolism ◽

Comparative Transcriptome ◽

Waterlogging Stress ◽

Protein Levels

Alfalfa (Medicago sativa) is the most widely grown and most important forage crop in the world. However, alfalfa is susceptible to waterlogging stress, which is the major constraint for its cultivation area and crop production. So far, the molecular mechanism of alfalfa response to the waterlogging is largely unknown. Here, comparative transcriptome combined with proteomic analyses of two cultivars (M12, tolerant; M25, sensitive) of alfalfa showing contrasting tolerance to waterlogging were performed to understand the mechanism of alfalfa in response to waterlogging stress. Totally, 748 (581 up- and 167 down-regulated) genes were differentially expressed in leaves of waterlogging-stressed alfalfa compared with the control (M12_W vs. M12_CK), whereas 1193 (740 up- and 453 down-regulated) differentially abundant transcripts (DATs) were detected in the leaves of waterlogging-stressed plants in comparison with the control plants (M25_W vs. M25_CK). Furthermore, a total of 187 (122 up- and 65 down-regulated) and 190 (105 up- and 85 down-regulated) differentially abundant proteins (DAPs) were identified via isobaric tags for relative and absolute quantification (iTRAQ) method in M12_W vs. M12_CK and M25_W vs. M25_CK comparison, respectively. Compared dataset analysis of proteomics and transcriptomics revealed that 27 and eight genes displayed jointly up-regulated or down-regulated expression profiles at both mRNA and protein levels in M12_W vs. M12_CK comparison, whereas 30 and 27 genes were found to be co-up-regulated or co-down-regulated in M25_W vs. M25_CK comparison, respectively. The strongly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for co-up-regulated genes at mRNA and protein levels in M12_W vs. M12_CK comparison were ‘Amino sugar and nucleotide sugar metabolism’, ‘Arginine and proline metabolism’ and ‘Starch and sucrose metabolism’, whereas co-up-regulated protein-related pathways including ‘Arginine and proline metabolism’ and ‘Valine, leucine and isoleucine degradation’ were largely enriched in M25_W vs. M25_CK comparison. Importantly, the identified genes related to beta-amylase, Ethylene response Factor (ERF), Calcineurin B-like (CBL) interacting protein kinases (CIPKs), Glutathione peroxidase (GPX), and Glutathione-S-transferase (GST) may play key roles in conferring alfalfa tolerance to waterlogging stress. The present study may contribute to our understanding the molecular mechanism underlying the responses of alfalfa to waterlogging stress, and also provide important clues for further study and in-depth characterization of waterlogging-resistance breeding candidate genes in alfalfa.

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Post-ovulatory ageing of mouse oocytes affects the distribution of specific spindle-associated proteins and Akt expression levels

Reproduction Fertility and Development ◽

10.1071/rd13010 ◽

2014 ◽

Vol 26 (4) ◽

pp. 562 ◽

Cited By ~ 8

Author(s):

Sandra Cecconi ◽

Gianna Rossi ◽

Hamid Deldar ◽

Valerio Cellini ◽

Felice Patacchiola ◽

...

Keyword(s):

Histone H3 ◽

Meiotic Spindle ◽

Post Translational Modifications ◽

Expression Levels ◽

Acetylated Tubulin ◽

Protein Levels ◽

Histone 3 ◽

Phosphorylated Akt ◽

Associated Proteins

The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 post-translational modifications and on v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33 h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription–polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of γ-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively, and (3) a significant reduction in phosphorylation levels of serine 10 on histone 3. At 29 h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.

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NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib

Blood ◽

10.1182/blood.v128.22.2068.2068 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2068-2068

Author(s):

Xi Huang ◽

Enfan Zhang ◽

Xing Guo ◽

Jing Chen ◽

Xuanru Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Acquired Resistance ◽

Gene Array ◽

Cell Counting ◽

Control Group ◽

Protein Levels ◽

Significant Difference

Abstract Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM. Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells. Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated. Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments

Clinical Chemistry ◽

10.1373/clinchem.2005.062406 ◽

2006 ◽

Vol 52 (6) ◽

pp. 1161-1167 ◽

Cited By ~ 19

Author(s):

Jochen Wilhelm ◽

Jai Prakash Muyal ◽

Johannes Best ◽

Grazyna Kwapiszewska ◽

Maria Magdalena Stein ◽

...

Keyword(s):

Expression Profiles ◽

In Vitro Transcription ◽

Microarray Hybridization ◽

Rna Amplification ◽

Microarray Experiments ◽

Amplification Factors ◽

Total Rna ◽

Protein Levels ◽

Specific Sequences

Abstract Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. Methods: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART™ for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.

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Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

Biology of Reproduction ◽

10.1093/biolre/ioab193 ◽

2021 ◽

Author(s):

Emmalee A Ford ◽

Emily R Frost ◽

Emma L Beckett ◽

Shaun D Roman ◽

Eileen A McLaughlin ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Expression Profiles ◽

Primordial Follicle ◽

Differentially Expressed ◽

Primary Follicle ◽

Primordial Follicles ◽

Time Points ◽

Primordial Follicle Activation ◽

Follicle Activation

Abstract The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1 and ZFX, was investigated with in-situ immunolocalisation in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signalling pathway’s role in primordial follicle activation.

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