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Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG.

ABSTRACT: Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.We conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.

SUBMITTER: Lussier JG

PROVIDER: S-EPMC5670713 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG.

Lussier Jacques G JG Diouf Mame N MN Lévesque Valérie V Sirois Jean J Ndiaye Kalidou K

Reproductive biology and endocrinology : RB&E 20171103 1

<h4>Background</h4>Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.<h4>Methods</h4>Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of huma ...[more]

PMID: 29100496

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Project description:BACKGROUND: The major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm). RESULTS: Principal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-? and tumour necrosis factor-? pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-? signalling and were up regulated. CONCLUSIONS: Small healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Project description:At later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small (< 5 mm; n = 10) and large (> 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets).Principal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues.Our data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells.

Project description:The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the pre-ovulatory LH-surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal and granulosa cell type-specific biological functions and signaling pathways, large dominant bovine follicles were collected before and 21 hrs after an exogenous GnRH induced LH surge. Because LH receptor density varies within the granulosa cell populations, antral granulosa (aGC; those aspirated by follicular puncture) and membrane associated granulosa (mGC; those scraped from the follicular wall) were compared to thecal cell expression profiles determined by mRNA microarrays. Thecal cell gene expression was less affected in the peri-ovulatory follicle when compared to granulosa cells, as evidenced by only 2% versus 25% of the ~11,000 genes expressed changing in response to the LH surge, respectively. The majority of the 203 LH-regulated thecal genes were also LH regulated in granulosa cells, leaving a total of 58 genes as LH-regulated theca cell specific genes. Most of the 58 genes (i.e., 74%) thecal specific genes including several known thecal markers (CYP17A1, NR5A1) were downregulated, while most genes identified are new to theca. Many of the newly identified upregulated thecal genes (e.g., PTX3, RND3, PPP4R4) were also upregulated in granulosa. Minimal expression differences were observed between aGC and mGC, however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) predominated these differences. We also identified large numbers of unknown LH-regulated granulosa cell genes and discuss their putative roles in ovarian function. The single dominant ovarian follicle was collected from each cow before the LH surge or 22 hours after GnRH (used to induce LH surge). RNA was extracted from three independent cells within each follicle and there were hybridized on Affymetrix microarrays.

Dataset Information

Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG.

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Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG.

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