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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Abstract not available

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The sex-determining gene on the Y chromosome, named Sry, initiates differentiation of gonadal somatic cells into testes, which in turn regulate the development of male phenotype. / The B6.YTIR sex-reversed mouse provides a good model for studying sex-determining mechanisms. We proposed a hypothesis that the testis-determining pathway is impaired downstream of Sry transcription in the B6.YTIR fetus. / The current study aimed to determine the hierarchical order of Sry, Sox9, Pn1, and Mis by examining their expression in B6.YTIR gonads as compared to normal B6.XY gonads by RT-PCR. / Results. Sry expression was comparable between B6.Y TIR and B6.XY gonads, with its onset between 10.5 and 11.5 dpc, a peak at 11.5 dpc, and downregulation thereafter. Sox9 expression was detectable in both B6.XX and B6.XY gonads at 11.5 dpc at comparable levels, but was then downregulated in B6.XX gonads at 12.5 dpc, by which stage testicular cord formation had began in B6.XY gonads. Pn1 was expressed in both B6.XX and B6.XY gonads at comparable levels at 11.5 dpc and was upregulated in B6.XY gonads at 12.5dpc. Mis expression in B6.Y TIR gonads was low at 10.5 and 11.5 dpc with a peak at 12.5dpc and higher levels only in ovotestes at 14.5dpc. / These results indicate that all Sox9, Pn1, and MIS genes follow a sexually dimorphic pattern of expression associated with development of testicular cords. Therefore, these genes are placed downstream of Sry in the fetal mouse gonad. Furthermore, we conclude that the testis-determining pathway is impaired upstream of Sox9 and Pn1 and Mis in the B6.YTIR gonad. (Abstract shortened by UMI.)

Methylenetetrahydrofolate reductase (MTHFR), an important enzyme in folate metabolism, mediates the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which serves as the carbon donor for the conversion of homocysteine to methionine. It is also inhibited by S-adenosylmethionine which has shown to be actively demethylated to form S-adenosylhomocysteine, which is hydrolysed to homocysteine. MTHFR deficiency exhibits well-documented clinical and biochemical symptoms. The human MTHFR cDNA was isolated by Goyette et al (1994), and fifteen mutations have been identified at this locus. / An animal model would prove to be useful for designing therapeutic approaches for understanding the pathogenesis of this genetic disease at the molecular level. The mouse MTHFR gene and cDNA have been isolated and partially characterized. Four genomic clones were isolated by library screening. One of these clones (clone 3) contained the 5$ sp prime$ end of the gene and was completely characterized. The clone was shown to have no rearrangements and is to be used to design targeting vectors for 'knockout mice' and mice carrying a common mutation which has been postulated to be a genetic risk factor for cardiovascular disease. The other three clones contain the remaining 3$ sp prime$ portion of the gene. The coding portion has approximately 90% homology with the human cDNA and also shows a similar gene structure. / A 2.2 Kb mouse MTHFR cDNA was isolated by library screening and was found to contain a 320 base pair extension at the 5$ sp prime$ end which has not been found in the human cDNA. The cDNA contains exons -1 -3, but also contains two possibly unspliced introns. A portion of this cDNA can however still be used to rescreen libraries to isolate a full length cDNA. The above research is the first genetic data on the mouse MTHFR gene and provides the basis for future research involving mouse models of MTHFR deficiency.

Sry on the Y-chromosome triggers the fetal gonad to begin differentiation into testis in mammals. Mutation or absence of Sry results in development of ovaries and the female phenotype. However, XY sex reversal in the presence of wild-type Sry exists in mice and man. One such example is the B6-YTIR mouse, whose autosomes and X-chromosome are of the C57BL6/J mouse (Mus musculus molossinus) whereas the Y-chromosome is from a mouse originating in Tirano, Italy (Mus musculus domesticus). B6-YTIR mice develop only ovaries or ovotestes in fetal life. The objective of my thesis was to identify the mechanism of sex reversal in the B6-YTIR mouse. The results indicate that onset of Sry transcription in B6-YTIR gonads is comparable to control B6 XY gonads. On the other hand, onset of Mis, 17alpha-HA, 3beta-HSD (testicular cell products), p450arom as well as inactivation of Sry transcription are delayed or absent in the sex reversed gonads. It has been suggested that low levels of Sry transcription may account for aberrant testis differentiation in B6-YTIR mice. We observed relatively low levels of Sry transcripts not only in B6-YTIR but also in B6 mice. However, levels in normal B6-YSJL mice were significantly greater. On the SJLB6F1 background, where no sex reversal occurs, Sry transcript levels of the TIR allele increased while those of B6 and SJL alleles remained the same as in the B6 background. Thus, low levels of Sry transcript from the B6 allele are sufficient whereas the levels from TIR and SJL alleles (both DOM type) appear to be critical for testis determination. We then compared the levels of endogenous Sry proteins. A combination of immunoprecipitation and immunoblotting succeeded in detecting a protein band whose expression profile and molecular size are consistent with those of the predicted Sry. Sry protein levels in B6-Y TIR gonads were roughly two fold greater than in B6 XY gonads. We hypothesize that the Sry protein of the TIR/SJL alleles is less efficient

TGFbeta signaling peptides have been shown to play increasingly diverse roles in metazoan development and tissue homeostasis. Negative regulation of TGFbeta ligands such as Nodal can be achieved by physical interactions with inhibitory molecules. Dante is a recently identified putative member of DAN/Cerberus family of TGFbeta inhibitors. Previously shown to be unilaterally expressed on the right side of the mouse node, Dante has been suggested to play a role in Left-Right axis formation possibly by inhibition of Nodal molecules. The aim of this study was to further characterize Dante and to determine whether it can physically interact with Nodal. First, a longer Dante cDNA was isolated in an attempt to clone the full-length transcript. Furthermore, Dante expression pattern was analyzed during murine development and adult tissues. Lastly, co-immunoprecipitation experiments demonstrated that Dante is able to physically interact with Nodal, providing further support for its potential role as a Nodal inhibitor.

Although mRNAs encoding the histone proteins are among the most abundant mRNAs in mammalian oocytes, the mechanism regulating their translation in these cells has not been identified. Most histone mRNAs are not polyadenylated but instead carry in their 3'-utr a highly conserved stem-loop structure. In somatic cells, the stem-loop binding protein (SLBP) is expressed during S-phase of the cell cycle and associates with the stem-loop of histone mRNAs promoting their processing and translation and thereby coordinating their expression to DNA replication. As histone mRNAs are abundant in immature oocytes which are in G2 of the cell cycle and in ovulated or mature oocytes which are in M-phase, I examined the expression and the regulation of histone mRNAs in immature and maturing mouse oocytes. First, I described SLBP expression during oogenesis and pre-implantation embryonic development. I showed that SLBP is present at low levels in the nucleus of the immature oocyte and accumulates significantly during maturation of the oocyte. At both stages, SLBP is the only stem-loop binding activity present. I showed that SLBP meiotic accumulation correlates with the adenylation of SLBP mRNA and is mediated by the presence of a cytoplasmic polyadenylation element in SLBP 3'-utr. Also, I demonstrated that histones are synthesized in the immature and mature oocyte and that the translation of a reporter mRNA bearing the histone 3'-utr increases dramatically during oocyte maturation consistent with the accumulation of SLBP. I specifically blocked SLBP accumulation using RNA interference and observed that both translation of the reporter mRNA and endogenous histone synthesis are significantly reduced. Moreover, SLBP-depleted eggs display a significant decrease in pronuclear size and in the total amount of histones detectable on their chromatin. Finally, I also showed that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translatio

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