Characterization Of Oocyte-Specific Transcriptional Factors NOBOX, NANOS3 And LHX8 In Premature Ovarian Failure

Chapter I. Microarray analysis of newborn mouse ovaries lacking NoboxOBJECTIVE: Nobox (newborn ovary homeobox gene), an oocyte-specific homeobox gene, plays a critical role during early folliculogenesis and represents a candidate gene for non-syndromic ovarian failure. The objective of this study is to investigate the downstream targets of Nobox.METHODS: We compared the gene expression difference of newborn mouse ovaries between wild type and Nobox knockout using Affymetrix 430 2.0 microarray platform. We also quantified 10 genes that were potentially important during oogenesis using real-time PCR and discussed the possible roles of these genes in oocyte maturation.RESULTS: Genes whose proteins were predicted to be secreted including Jagged 1 (Jag1) and respondin 2 (Rspo2) were down regulated in Nobox knockouts. Nalp4c, a gene that encoded protein involved in apoptotic and inflammatory signaling pathways, was down-regulated in Nobox^-/- ovaries. In addition, pluripotency associated genes, Oct4 and Sall4 were drastically down-regulated while stimulated by retinoic acid gene 8 (Stra8) which was involved in the retinoic acid metabolism, was among few up-regulated genes expressed in oocytes. Testes determining genes, including Dmrt1, Tekt2 and 1700019D03Rik were over-expressed in Nobox deficient ovaries.CONCLUSION: Our findings indicate that Nobox is likely regulator of oocyte-specific gene expression, and suggest that transcription factors preferentially expressed in oocytes may repress male determining gene expression. Chapter II. NOBOX Homeobox Mutations Cause Premature Ovarian FailureOBJECTIVE: NOBOX (Newborn Ovary Homeobox gene), an oocyte-specific homeobox gene, plays a critical role during early folliculogenesis. Disruption of the murine Nobox gene causes non- syndromic ovarian failure in Nobox^-/- females. We investigate whether NOBOX has mutations causing premature ovarian failure (POF).METHODS: We sequenced NOBOX gene in 96 Caucasian women with POF, followed by electrophoretic mobility shift assay (EMSA) to verify the effect of novel mutations on protein-DNA.RESULTS: Seven known SNPs and 4 novel variations were discovered. Two of the latter, p. Arg355His and p. Arg360Gln, resides in the highly conserved homeodomain region. Using EMSA, we confirmed that mutation p. Arg355His in mouse decreases the affinity of homeodomain binding to the Nobox DNA binding elements, producing a dominant negative effect that disabled the DNA binding activity of wild type NOBOX.CONCLUSION: Our findings suggest that NOBOX mutations contribute to a subset of women with POF. Chapter III. Mutation analysis of NANOS3 and LHX8 in Womenwith Premature Ovarian FailurePart I. Mutation Analysis of NANOS3 in 80 Chinese and 88 Caucasian Women with Premature Ovarian FailureBACKGROUND AND OBJECTIVE: NANOS3 encodes an RNA binding protein and has a conserved function in germ cell development. Our objective was to investigate whether mutations in NANOS3 are present in Chinese and Caucasian women with premature ovarian failure (POF).METHODS: Mutation Analysis of NANOS3 in 80 Chinese and 88 Caucasian women with POF were performed using denaturing high-performance liquid chromatography (DHPLC), followed by sequencing and restriction fragment length polymorphism (RFLP).RESULTS: A known synonymous single nucleotide polymorphism (SNP) (rs 2016163) in exon 1 was identified through sequencing 80 Chinese and 88 Caucasian women with POF. No additional SNPs or mutations were found in exons encoding for NANOS3.CONCLUSION: Our findings suggest that mutations in NANOS3 exons are rare in both Chinese and Caucasian women with POF. Part II. Perturbations of LHX8 Do not Explain Caucasian Women with Premature Ovarian FailureOBJECTIVE: LHX8 (LIM homeobox 8) encodes a LIM homeodomain transcriptional regulator preferentially expressed in germ cells and is a critical regulator of mammalian oogenesis. We investigated whether nucleotide changes are present in the LHX8 gene of Caucasian women with premature ovarian failure (POF) as compared to control women.METHODS: Mutation of LHX8 was studied in 95 Caucasian women with POF and 94 controls by seqencing.RESULTS: Two novel single nucleotide polymorphisms (SNPs) were discovered in intron 3 (c.769+10G>T) and 3' untranslated region (c.1787A>G) of the LHX8 gene. These polymorphisms were also found in controls with frequencies that were not statistically different from POF women.CONCLUSION: Mutations in the LHX8 exons are uncommon in Caucasian women with POF.