A number of genes have been associated with variation in human spermatogenesis related to fertility. One of these, the Deleted in Azoospermia (DAZ) gene, exists as copies on two chromosomes, 3 and Y. The autosomal copy, DAZ-like (DAZL), has one RNA recognition motif (RRM) and is homologous to the DAZL gene found throughout the vertebrate lineage. There are four copies of DAZ on the Y chromosome with a pair at each of two sites. One pair contains a single RRM and the other has three RRMs. Human DAZ is homologous to genes in old world primates and ape Y chromosomes. Both DAZ and DAZL bind messenger RNAs at U-rich sequences near the poly-A tail in a manner that facilitates translation. Both are expressed in spermatogonia during the transition from mitotic cellular expansion through meiotic chromosomal reduction and during spermiogenesis. This study examined genomic variation in DAZ and DAZL, including deletion of DAZ from individuals with various levels of sperm cell production and mutations of DAZL in male partners of infertile couples. Deletions in DAZ are not as common in azoospermic men from central Texas as compared to other reports. Single nucleotide polymorphisms (SNPs) were identified in anonymous infertility patients, but were not located in the exons of the RRM. Proteins produced from transcripts encoded by genes from human DAZL, DAZL with SNPs within and outside the RRM, and a DAZ with single RRM were identified. Binding activity of DAZL to mRNA was confirmed using a microarray method, and mRNA from human testes was screened to identify at least 1,313 mRNA potential targets for DAZL. These targets were involved in ribosome construction, pyruvate metabolism, cell cycle control, and proteasome function. Variations in binding of protein to a high and a low bound target mRNA were demonstrated between protein constructs of DAZL, DAZL with mutations, and DAZ. Binding of DAZL to mRNA was also confirmed using electrophoretic mobility shift assays. With materials and procedures developed during this study, comparisons of genetic variants of DAZ and DAZL can be performed to identify mechanisms responsible for structural and functional differences in control of spermatogenesis.

Gametogenesis is a highly complex process that requires stringent control of gene expression, in which translational regulation plays an essential role. Deleted in Azoospermia-like (DAZL) belongs to the DAZ family of RNA-binding proteins, which are restricted to germ cells, and regulate mRNA translation. Importantly, loss of function of these proteins results in infertility in both males and females in a wide variety of organisms. A model for the mechanism by which DAZL stimulates translation has been proposed based on work in Xenopus laevis (X. laevis) oocytes. In this model, DAZL functions by recruiting the translation initiation factor poly(A)-binding protein (PABP) to the 3’ untranslated region (UTR) of messenger RNAs. Simultaneous binding of PABP to Dazl and factors at the 5’ end confers a “closed-loop” mRNA conformation, which promotes translation initiation. To examine whether DAZL plays a similar role in mammals, co-expression of Dazl and PABP family members was investigated in fetal and adult mouse gonads. In contrast to X. laevis, mammals encode four cytoplasmic PABPs which share a similar domain organisation: PABP1, tPABP, ePABP and PABP4, of which PABP1 and PABP4 appear to be expressed in a wide range of tissues. Immunohistochemistry revealed that Dazl, Pabp1 and Pabp4 are all expressed in primordial germ cells (PGCs) but these show different expression patterns following germ cell sex differentiation. In adult testes Dazl is expressed in spermatogonia and spermatocytes, coinciding with the peak of Pabp4 expression. In contrast, the peak of Pabp1 expression occurs later than that of Dazl, with these proteins only being co-expressed in late pachytene and secondary spermatocyte phases. In adult ovaries, Pabp1, Pabp4 and Dazl are all expressed in the oocytes of primordial and primary follicles. Since both PABP family members are co-expressed with Dazl, the ability of DAZL to interact with PABP1 and PABP4 was investigated in vitro and in vivo. Surprisingly, these studies showed that DAZL discriminates between different PABP family members, only interacting with PABP1, providing the first report of a PABP-specific protein partner. Several putative DAZL mutations have been identified in patients with impaired fertility. Two of these mutations, I37A and R115G, are located in the RNA recognition motif (RRM), a domain which is found in many RNA-binding proteins and mediates both RNA and protein interactions. Thus, the role of these mutations in the ability of DAZL to stimulate translation was investigated. To this end, a translational target of human DAZL (hDAZL) was sought. The 3’UTR of growth differentiation factor 9 (hGDF9) mRNA was found to confer regulation by hDAZL and thus the ability of mutant DAZLs to stimulate reporter mRNAs containing this 3’UTR was examined. This revealed that both mutations compromised the ability of hDAZL to stimulate hGDF9 translation, suggesting a causative effect. These results were further confirmed in assays in which hDAZL is artificially tethered to mRNAs. The ability of mutant hDAZLs to stimulate translation in this assay was compromised suggesting that loss of function is, at least in part, due to impaired protein-protein interactions rather than altered RNA-binding. This work provides insights into the molecular mechanism by which DAZL stimulates the translation of specific mRNAs during mammalian gametogenesis and provides evidence that this function may play an important physiological role in human reproduction.

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The proper expression of a gene to a protein is a complicated process with many steps. One of the major steps is translation, the process of decoding a messenger RNA signal and the building of a protein from its component parts. The control of translation is one of the major steps for the overall control of gene expression and its dysregulation is associated with a wide variety of human diseases including neurological, metabolic and reproductive disorders. Dazl family proteins are germ cell restricted RNA binding proteins that contain a motif characteristic of this family, the DAZ domain. Whilst humans encode all three family members DAZ, DAZL and BOULE, flies only possess the boule gene. The members of this family have an essential conserved role in gametogenesis in a wide variety of organisms from worm to man with loss of function resulting in phenotypes ranging from male or female infertility or both. However, little is known about the molecular role of these proteins in germ cell development. A previous study within the laboratory showed that several vertebrate Dazl family members can stimulate translation of a reporter gene in Xenopus laevis oocytes, suggesting a conserved role in mRNA specific translational control. This is consistent with studies in invertebrates. It was proposed that Dazl proteins fulfil this function through an interaction with a translation initiation factor, poly(A) binding protein, PABP. The aim of this thesis was to further refine this model of action. The work presented here investigates several fundamental questions regarding the mechanism of Dazl-mediated stimulation. First, it investigated the step of translation initiation that Dazl acts upon and explored the initiation factors that may be required. Second, it addressed in more detail the requirements for an interaction between Dazl and the poly(A) binding protein, PABP. Third, it examined the potential role of another factor, DAZ associated protein 1, DAZAP1, in Dazl-mediated stimulation. The role of multi-protein complexes containing Dazl bound to the 3’UTR that localise, repress and stimulate translation of specific mRNAs at defined times during gametogenesis are discussed.

Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.

No description available.

No description available.