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Title Effect of Maternal Age on Transcriptome of Granulosa Cells from Bovine Dominant Follicles
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University/Publisher University of Saskatchewan
Abstract Advanced maternal age has been shown to influence follicular and luteal dynamics in bovine ovary resulting in reduced fertility. The overall objective of the four studies presented in this thesis is to identify the maternal age-associated transcriptional changes in granulosa cells of the dominant follicles during follicle development. In the first study, mRNA expression levels of housekeeping genes were measured by real–time quantitative PCR (RT-qPCR) in granulosa cells of dominant follicles and FSH-stimulated follicles to select and validate suitable reference genes for relative gene expression analyses during maternal and follicular aging. Stability of six reference genes (GAPDH, ACTB, EIF2B2, UBE2D2, SF3A1 and RNF20) was analyzed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined based on these programs. Geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) was more appropriate reference control than individual genes for the comparison of relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies. In the second study, maternal age-associated changes in the transcriptome of granulosa cells recovered at the time of selection of the dominant follicle from aged (n=3) and young cows (n=3) were determined by EmbryoGENE bovine oligo-microarrays (EMBV3, Agilent Technology). The mRNA expression of five transcripts (CYP19A1, PCNA, GJA1, TPM2, and VNN1) was confirmed in a different set of granulosa cell samples by RT-qPCR to validate microarray data. A total of 169 genes/isoforms were differentially expressed (≥ 2-fold-change; P ≤ 0.05) in aged cows vs. young cows. These transcripts revealed inefficient 1) control of gonadotropins, and gonadotropin-induced changes in the cytoskeleton and extracellular matrix, 2) lipid metabolism and steroidogenesis 3) cell proliferation, cell cycle control and intercellular communication, and 4) higher oxidative stress responses in aged cows vs. young cows. In the third study, changes in the transcriptome of granulosa cells of the preovulatory follicle 24 h after LH treatment from aged (n= 3) and young (n=3) were determined. A total of 1340 genes were expressed differentially (≥ 2-fold change; P ≤ 0.05) in aged cows vs. young cows. The mRNA expression of five transcripts (RGS2, PTGS2, TNFAIP6, VNN1, NR5A2 and GADD45B) was confirmed in a different set of granulosa cell samples to validate microarray data. These transcripts were related to delayed 1) response to LH treatment 2) cellular differentiation and luteinization and 3) progesterone synthesis. Intra-follicle levels of progesterone were lower (P < 0.05) in aged cows compared to young and mid-aged cows. The fourth study compared the aged-associated changes in the transcriptome of granulosa cells during follicle development from the time of dominant follicle selection to preovulatory stage (24 h after LH). In comparison to young cows, aged cows expressed fewer differentially expressed genes/isoforms (1206 vs. 2260, respectively)…
Subjects/Keywords Maternal age, Granulosa cells, Dominant follicle at selection, Preovulatory follicle, Reference genes, Microarrays, RT-qPCR, Bovine, Luteinizing hormone, Ovariectomy, Follicular aspiration, Transcriptome analysis, Ingenuity Pathway Analysis, Upstream regulators, Estradiol, Progesterone, Follicular fluid, Ultrasonography, Ovulation
Contributors Singh, Jaswant; Adams, Gregg P.; Sirard, Marc A.; Misra, Vikram; Anzar, Muhammad
Language en
Country of Publication ca
Record ID handle:10388/ETD-2014-01-1364
Other Identifiers TC-SSU-2014011364
Repository sask
Date Retrieved
Date Indexed 2020-07-20

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…SELECTION OF STABLE REFERENCE GENE FOR GENE EXPRESSION ANALYSES OF BOVINE GRANULOSA CELLS....................................... 50 3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.4 Introduction…

…68 CHAPTER 4: MATERNAL AGE RELATED CHANGES IN TRANSCRIPTOME OF BOVINE GRANULOSA CELLS OF DOMINANT FOLLICLE AT THE TIME OF SELECTION…

…OF BOVINE GRANULOSA CELLS OF PREOVULATORY FOLLICLE 24 H AFTER LH TREATMENT ........................................................................................................................... 103 5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6…

…transcriptome of granulosa cells of dominant follicle at selection.............................................................................................................. 188 8.3 Maternal age related changes in transcriptome of bovine granulosa cells of…

…51 Materials and Methods .......................................................................................... 53 Collection of Granulosa cells .............................................................................. 53 Granulosa cells RNA…

…TRANSCRIPTOME OF GRANULOSA CELLS BETWEEN THE TIME OF DOMINANT FOLLICLE SELECTION AND 24 H POST LH IN YOUNG VS. AGED CATTLE ................................................................................................................ 139 6.1 Introduction…

…177 CHAPTER 8: GENERAL CONCLUSIONS.......................................................................... 188 8.1 Selection of stable reference gene for granulosa cells. ....................................... 188 8.2 Maternal age related changes in…

…preovulatory follilce 24 h after LH treatment. ................................................................... 189 8.4 Changes in transcriptome of granulosa cells between the time of dominant follicle selection and 24 h post LH in young vs. aged cattle…

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