Transforming growth factor beta (TGFβ) superfamily members are involved in controlling mammalian fertility. The largest subset of the TGFβ superfamily are the bone morphogenetic proteins (BMP). BMP ligands signal through the type I and II BMP receptors and utilise the Smads1/5/8 phosphorylation cascade to control gene expression in the cell nucleus. Although BMPs act through the same pathway, they have the ability to activate unique sets of genes dependant on the identity of the ligand. In this study, HEK293T cells were challenged with BMP ligands for four hours and gene expression profiles were compared using microarray technology. The genes upregulated in the presence of BMP2, BMP4, BMP6 and BMP7 play roles in cellular proliferation and differentiation. These functions are critical stages in the successful development of an ovarian follicle whilst undergoing folliculogenesis. All of the BMP ligands investigated in this study were also observed to upregulate the expression of a small group of common genes indicating that a shared regulatory pattern occurs within the BMP pathway. Of these genes, Smad6 and Smad7, inhibitor of DNA binding proteins 1-4 (ID 1-4), and msh homeobox homolog 2 (MSX2) were previously known BMP target genes. However, none of the remaining genes upregulated by all BMPs were previously shown to be BMP targets.
The results from the microarray experiment were used as founding data for the in silico mining of novel genes not present on the array that may be differentially expressed in response to these ligands. The expression levels of several of the novel genes identified by in silico mining were then measured in vitro, however the results showed no differential expression in the HEK293T cells.
To apply the knowledge of the microarray studies to the tissue of interest, eight genes were selected for assessment in ovine granulosa cells. Four of the genes upregulated in response to BMP6 in HEK293T cells were also differentially expressed in primary ovine granulosa cell cultures in response to BMP6 addition.
The identification of several sheep breeds with mutations in TGFβ superfamily members has enabled investigations into the roles that specific TGFβ components play in controlling fertility. The highly fertile Booroola sheep has a substitution mutation in the type IB BMP receptor that results in an additive effect on ovulation rate. The Booroola mutation causes precocious maturation of ovarian follicles with fewer granulosa cells surrounding an enlarged oocyte, and carriers of the mutation have higher levels of circulating follicle stimulating hormone (FSH). BMPs have previously been shown to influence the regulation of FSH synthesis and secretion in the pituitary gland. In this study, primary pituitary cells were harvested and cultured from homozygous Booroola ewes and from wildtype ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with bone morphogenetic proteins (BMP2, BMP4, BMP6), growth and differentiation factor-9 (GDF9), transforming growth factor β1 (TGFβ1), activin-A and gonadotropin releasing hormone (GnRH). The levels of FSH and luteinising hormone (LH) were measured by radioimmunoassay and compared to the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than wildtype cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The BMPRIB receptor however, was not found to co-localise with gonadotroph cells in either Booroola or wildtype pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6, and GDF9 cannot be due to the direct action of the BMPRIB mutant Booroola receptor in the cells that synthesize FSH. The alternative type I BMP receptor to BMPRIB that can act in BMP signal transduction is BMPRIA. This receptor was also not found in gonadotroph cells of wildtype orBooroola ewes This is in contrast to findings in other flocks which have been shown to express BMPRIA in gonadotroph cells.
This study has identified unique sets of differentially regulated genes in response to BMP-2, 4, 6, and 7 as well as TGFβ1 in a human HEK293T cell culture system. Among the differentially expressed genes, a common set of 12 genes were upregulated by all BMP ligands. None of these genes were present in the TGFβ1 set. Selected genes were validated in ovine primary granulosa cell cultures, showing that the human cell culture system functions similarly to cells of biologial relevance in fertility. Within the pituitary gland, BMPs are shown to influence FSH secretion. The presence of the Booroola mutation enhances the BMP effects on gonadotroph cells, however the lack of BMPRIB on gonadotroph cells indicates that the effects are indirect.
| Identifer | oai:union.ndltd.org:ADTP/211303 |
| Date | January 2008 |
| Creators | Young, Julia, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| 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 Julia Young |
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