Australian Regenerative Medicine Institute

Category: Infertility

Association of the Mouse Infertility Factor: DISCUSSION(2)

While this report was in preparation, Maines and Was-serman reported that the Drosophila homologue of DAZL1—Boulc—controlledthetranslation ofTwine, a meiotic Cdc25-type phosphatase. They found that in boule mutants the translation, but not the transcription, of twine was significantly reduced, and that heterologous expression of Twine partially rescued the spermatogenic defect of bou-le mutants. Their results not…

Association of the Mouse Infertility Factor: DISCUSSION(1)

We present evidence for the association of DAZL1 with actively translating polyribosomes in testes. Our results suggest a role for DAZL1 in translational regulation of a subset of mRNAs in germ cells. Germ cell-specific translational regulation is well documented for transition proteins and protamines. Their mRNAs are synthesized in round spermatids and stored as cytoplasmic…

Association of the Mouse Infertility Factor: RESULTS(2)

Generation of Anti-DAZL1 Antibodies A polyclonal antiserum was generated against the last 76 amino acid residues of the human DAZL1 protein, which is 86% identical to the corresponding region of mouse DAZL1. On Western blots, the anti-DAZL1 antiserum detected a major band in mouse testicular extracts that comigrated with in vitro-synthesized DAZL1 and that was…

Association of the Mouse Infertility Factor: RESULTS(1)

Binding of DAZ and DAZL1 to RNA Molecules The abilities of DAZ and DAZL1 to bind RNA molecules were studied using an assay in which in vitro-synthe-sized 35S-labeled proteins were incubated with RNA homopolymers immobilized on agarose beads, and the bound proteins were analyzed by SDS-PAGE. As shown in Figure 1a, human DAZ and DAZL1,…

Association of the Mouse Infertility Factor: MATERIALS AND METHODS(3)

Inhibition of Western Detection of DAZL1 Unlabeled DAZL1 was synthesized in vitro using the TNT Coupled Reticulocyte Lysate System (Promega). T3 and T7 RNA polymerase transcribed the Dazll cDNA clone in the sense and antisense directions, respectively. Varying amounts of the in vitro synthesis mixtures were incubated with 2 ^l of anti-DAZL1 antiserum in a…

Association of the Mouse Infertility Factor: MATERIALS AND METHODS(2)

Generation of Deletion Constructs of DAZ and DAZL1 Deletion constructs of DAZL1 and DAZ were generated from cDNA clones DAZL1-f2 and DAZ-e11, respectively. Deletions were created either by using existing restriction sites within the coding regions or by polymerase chain reaction amplification of two regions followed by ligation. Correct joining at the junctions was verified…

Association of the Mouse Infertility Factor: MATERIALS AND METHODS(1)

Generation of Anti-DAZL1 Antibody A 229-base pair Sau3AI fragment encoding the last 76 amino acid residues of DAZL1 was isolated from a human DAZL1 cDNA clone f2 and cloned into the BamHI site of an expression vector pET32b (Novagen, Madison, WI). The construct was introduced into Escherichia coli to direct the synthesis of a fusion…

Association of the Mouse Infertility Factor: INTRODUCTION

A requirement of the Y chromosome long arm (Yq) for normal spermatogenesis was recognized more than two decades ago when six infertile men were found to have Yq deletions. More recent molecular screening has detected Yq microdeletions in 10-15% of males with idiopathic azoospermia. The DAZ (Deleted in AZoospermia) gene family was isolated from one…