Germ-cell Specific Transcription Factor SOHLH1 Is Crucial In Folliculogenesis

ChapterⅠ.Screening Downstream Targets of Sohlh1OBJECTIVE:Sohlh1(spermatogenesis and oogenesis specific basic helix-loop-helix transcription factor 1),an oocyte-specific helix-loop-helix(HLH)gene,plays a critical role during early folliculogenesis.Females lacking of Sohlh1 in mice appear to have normal embryonic gonadogenesis,but form imperfect primordial follicles that not progress to primary follicles.The objective of this study is to investigate if meiosis perturbations account for the rapid oocytes loss and also to identify the downstream targets of Sohlh1.METHODS:RT-PCR and immunofulorescence were carried out to detect meiosis genes Dmc1,Spo11,Msh5,Rec8 and Scp3 expression in Sohlh1 null mice ovaries or oocytes.Then we compared the gene expression difference of newborn mouse ovaries between wild type and Sohlh1 knockout using Affymetrix 430 2.0 microarray platform.The microarray transcripts profiles from Sohlh1,Lhx8 and Nobox null mice were combined and analyzed to find the common downstream target genes.RESULTS:The relative levels of Dmc1,Msh5,Spo11,or Rec8 transcripts were not significantly different between the wild type and Sohlh1 null ovaries.Also no appreciable differences are noted with staining of Scp3 between wild type and Sohlh1 null oocytes. Further we compared Sohlh1 deficient and wild-type ovaries using Affymetrix 430 2.0 microarray platform.494 genes were down-regulated and 165 gene were up-regulated more than 2-fold in Sohlh1 null ovaries.Combining of Lhx8 and Nobox null ovary arrays,we identified a common set of 73 transcripts whose expression level were affected in all Sohlh1, Lhx8 and Nobox deficient mice;and among them,33(45.2%)were preferentially expressed in oocytes with more than 5 fold change.By exploring functional annotation,gene expression patterns analysis and cis-acting sites screening,we discoved a subset of target genes were regulated in the SOHLH1-LHX8-NOBOX pathway.CONCLUSION:These results indicate that meiotic components currently known to disrupt early oogenesis are not affected by Sohlh1 deficiency and suggest that oocyte differentiation can be governed by factors independent of meiosis.Downstream transcripts like LHX8, NOBOX,GDF9,PADI6,NALP and OAS1 famlily should be account for the failure in maintenance and differentiation of the oocyte during early folliculargenesis in Sohlh1 dificient mice. ChapterⅡ.Sohlh1 Transgenic MouseOBJECTIVE:Mammalian oocytes have been proposed to have important roles in the orchestration of ovarian follicular development and fertility and Sohlh1 is one of the oocyte-specific genes expressed maily in early stage of follicles.To determine whether over-expressed Sohlh1 have functions after the transition from primordial follicles to primary follicles,we generated a Sohlh1 transgenic(Sohlh1^tg)mouse model.MATERIALS AND METHODS:The full coding region of mouse Sohlh1 cDNA was subcloned into the SmaI site of a routine Zp3 promoter-BGH poly-A expression vector.After injection into the male pronucleus of one cell zygotes from F2 of C57 mice,the embryos were transferred to oviducts of foster mothers and correct transgenic mice were indentified by PCR genotyping.Subsequent studies were carried out to investigate the phenotype of Sohlh1 transgenic mice.RESULTS:By RT-PCR and immunohistochemistry probed by anti-Sohlh1 antibodies, transgenic active Sohlh1 is detected through all stages of ovarian follicles.Sohlh1^tgfemales can fertilze and Sohlh1^tgoocytes were matured and ovulated early than wild type,or, alternatively,the pups from Sohlh1^tgfemales are preferrecially carring Sohlh1^tg,especially in female pups.CONCLUSION:We successfully constructed mZp3-Sohlh1 transgenic mouse strain.The results from the current study indicate that Sohlh1 overexpression during the late follicle stages can promote ovulation. ChapterⅢ.Germ-cell Specific Genes-SOHLH1,FIGLA and GDF9 Mutations in Women with Premature Ovarian FailureSectionⅠ.Mutation analysis of SOHLH1 in Caucasian Women with POFOBJECTIVE:Sohlh1 deficiency in female mice leads to unfertility because of postnatal oocyte loss and failure in transition from primordial to primary follcles.The phenotype mimics human premature ovarian failure(POF)well.The aim of this study is to investigate whether SOHLH1 mutation is associated with human POF.MATERIALS AND METHODS:We have directly sequenced SOHLH1 in a cohort of 96 Caucasian POF patients presenting with secondary amenorrhea and high FSH levels and in a control population including 96 women with regular menstrual cycles who had at least one child.RESULTS:We have identified 16 novel heterozygous variants.One alteration c.423A＞G(K120R)in the downstream of HLH domain is conserved among vertebrate species and also not presented in control samples.On the other hand,several variants were detected in the 3'UTR region,including some only appeared in POF samples and some rare SNPs in both POF and controls.CONCLUSION:We propose c.423A＞G(K120R)mutation may be involved in POF; nonetheless,a bunch of SNPs identified in SOHLH1 3'UTR region may impact the binding of mircoRNAs which have potential roles in regulating m RNA translation and protein inhibition. SectionⅡ.Mutation analysis of FIGLA in Chinese Women with POFOBJECTIVE:Premature Ovarian Failure(POF)is a common cause of hypergonadotropic ovarian failure and infertility,affecting 1-2%of women.POF is a genetically heterogenous disease,with few known causative genes.We utilized a candidate gene approach to study if FIGLA,a transcriptional regulator preferentially expressed in the ovary,is mutated in Chinese women with POF.MATERIALS AND METHODS:100 Chinese POF women,as well as 304 control age-matched women,were recruited for this study.The coding regions of FIGLA gene were amplified using polymerase chain reaction(PCR)with 4 pairs of specific primers. Sequencing was performed after PCR amplification on ABI Prism Sequencer 3130XL (Applied Biosystems).To further test whether the missense or deleted(c.11C＞A and c.419-421delAAC)alleles affect FIGLA's ability to dimerize with itself or TCF3,we employed a yeast two-hybrid strategy to study protein-protein interactions.RESULTS:Three novel variants were identified in four POF individuals:c.11C＞A(p.A4E), c.15-36del(p.G6fsX66)and c.419-421delACA(p.140delN).The c.15-36delta causes a frameshift mutation with haploinsufficiency.Functional analyses by the yeast two-hybrid assay demonstrated that p.140delN mutation disrupted FIGLA binding to the TCF3 HLH domain.The c.15-36 delta and c.419-421delACA mutations were not present in the 304 control women.CONCLUSION:Our findings show that a subset of Chinese women with sporadic premature ovarian failure harbor mutations in FIGLA.Haploinsufficiency in transcription factors is known to cause many human Mendelian disorders,and c.15-36delta frameshift results essentially in haploinsufficiency by terminating FIGLA open reading frame immediately after the first five amino acids.Moreover,we show that the c.419-421delACA disrupts FIGLA's interaction with TCF3.High throughput sequencing of genes involved in the FIGLA pathway will be useful to detect other genes that play critical roles in premature ovarian aging. SectionⅢ.Mutation analysis of GDF9 in Chinese Women with POFOBJECTIVE:Growth differentiation factor 9(GDF9)is a germ cell specific growth factor secreted by oocytes and crucial for folliculogenesis.Our goal was to determine if perturbations in the GDF9 gene occur in Chinese women having Premature Ovarian Failure (POF).MATERIALS AND METHODS:100 POF women and 96 control women,were recruited in the Reproductive Medical Center,Shandong Provincial Hospital,China.Inclusion criteria were defined as twice serum follicle stimulating hormone concentrations greater than 20IU/ml before age of 40 years,and normal karyotype.After genomic DNA was extracted from blood samples,the coding regions of GDF9 were amplified using polymerase chain reaction(PCR)with 3 pair of primers.Heteroduplexes were detected using denaturing high-performance liquid chromatography(DHPLC).Samples which demonstrated heteroduplex formation on DHPLC were then sequenced directly after PCR amplification on an automated sequencer.RESULTS:In the POF group,we found 3 nonsynonymous SNPs in the GDF9 gene: c.436C＞T(p.Arg146Cys),c.712A＞G(p.Thr238Ala)and c.1283G＞C(p.Ser428Thr).Novel mutations c.436C＞T and c.1283G＞C were also discovered in the controls.The only nonsynonymous SNP(c.712A＞G)detected in POF group but not in controls was present in a 30-year-old woman with secondary amenorrhea.In addition,we found 3 synonymous(silent) mutations in the POF group,two also present in controls.CONCLUSION:Although 4 novel SNPs and 2 additional known mutations were found in the POF cases,all except one(c.712A＞G)was either found in controls or was silent.712A＞G mutation was not present in controls.712A＞G mutation in GDF9 may be associated with POF in our study population.