Human granulosa cells play an important role in the follicle, providing the oocyte with nutrients and growth factors to ensure successful ovulation. Normal granulosa cell functioning is thus crucial to human fertility. By studying their transcriptome, the mechanisms underpinning follicle development and infertility will be better understood.
In this study, granulosa cells were retrieved at the time of oocyte removal for in vitro fertilization (IVF). Cells were purified through a combination of a Percoll gradient to remove red blood cells and positive selection of granulosa cell aggregations. On average, contamination by white blood cells was 2% as measured by FACS analysis using specific white blood cell markers. Histological and electron microscopy of granulosa cell aggregations did not detect evidence of resident ovarian white blood cells. This technique provided a good source of pure, healthy granulosa cells for RNA extraction and subsequent gene expression studies.
The construction of a human granulosa cell SAGE library derived 1689 SAGEtags and 1289 discrete mRNA transcripts. SAGEtags for a number of well recognized granulosa cell genes (FSH receptor, follistatin, connexin 43) were found in addition to hormone receptors, multiple kinases, structural genes, apoptosis related genes and secreted proteins. A variety of other SAGE libraries were downloaded from SAGEmap (two ovary epithelium, ovary, white blood cell, brain, cerebellum, heart, liver, lung, kidney, pancreas and universal human reference) and compared to the human granulosa cell library. This was based on two measures: a gene specificity score (GSS) and a tissue specificity score (TSS). Three SAGEtags were identified with high levels of expression in granulosa cells, but no or low expression in the other libraries. These were retinol binding protein 1 (RBP 1), scavenger receptor class B member 1 (SCARB 1) and hydroxysteroid (11-beta) dehydrogenase 1 (11-β-HSD). Library comparisons were validated by real time RT-PCR. The TSS score revealed granulosa cells were most similar to the universal reference library and least similar to liver.
Granulosa cell samples were collected from woman undergoing IVF for a range of infertility disorders. These included polycystic ovary syndrome (PCOS), tubal disease, endometriosis and idiopathic (unknown). Gene expression was compared between these groups using real time RT-PCR. Candidate genes included RBP 1, 11-β-HSD, SCARB 1, FSH receptor, follistatin, decidual protein induced by progesterone, and progesterone membrane receptor component 1 and 2. Granulosa cell gene expression was significantly different (p<0.05) from human white blood cells. No differences were found in gene expression levels between the infertility disorders. Analysis of each patient, however, revealed individuals with marked over-expression of selected genes.
The technique of Generation of Longer cDNA fragments for Gene Identification (GLGI) was used to investigate seven SAGEtags that did match known genes or ESTs. Although no novel genes were characterized, a further 14 granulosa cell transcripts were identified by this technique.
This thesis used an integrated approach to the study of human granulosa cell gene expression. This has involved development of a purification method, the use of a high-throughput technique (SAGE), bioinformatics tools to identify candidates genes, real time RT-PCR to investigate gene expression of particular genes in infertility disorders and finally a technique with the potential to characterize unknown SAGEtags (GLGI). This systematic approach has advanced the understanding of gene expression in human granulosa cells and identified avenues for future research into folliculogenesis and human infertility.
| Identifer | oai:union.ndltd.org:ADTP/217591 |
| Date | January 2005 |
| Creators | Quinn, Michael Corwin James, n/a |
| Publisher | University of Otago. Department of Anatomy & Structural Biology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Michael Corwin James Quinn |
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