Heide A. Tarnasky

Interactional Cloning of the 84-kDa Major Outer Dense Fiber Protein Odf84 LEUCINE ZIPPERS MEDIATE ASSOCIATIONS OF Odf84 AND Odf27

The study of mammalian sperm tail outer dense fibers (ODF), a structure of unknown function, is hampered by the insoluble nature of ODF proteins and the availability of only one cloned component, Odf27. We report here the first use of the Odf27 leucine zipper as bait in a yeast two-hybrid screen to isolate a novel testis-specific protein whose interaction with Odf27 depends critically on the Odf27 leucine zipper. We find that the novel gene, 111-450, encodes a product that localizes to ODF as determined by fluorescence microscopy and immunoelectron microscopy and that the gene 111-450 product is identical to the major ODF protein, Odf84. Interestingly, Odf84 contains two C-terminal leucine zippers, and we demonstrate that all leucine residues in the upstream leucine zipper are required for interaction with Odf27, demonstrating the strategic validity of our approach. The use of the yeast screening approach to isolate leucine zipper containing proteins should be useful in other systems, and our findings have implications for ODF structural models.

A new rat gene RT7 is specifically expressed during spermatogenesis.

We report the isolation of a new rat male germ cell-specific gene, RT7, by differential cDNA cloning procedures. The RT7 cDNA nucleotide sequence is not homologous to any sequences present in the GenBank library. RT7 RNA is expressed at very high levels in rat early spermatids, while its expression is not detectable in any other organ or tissue examined. Mapping of the RT7 transcription start site by two independent procedures demonstrated that RT7 has two major and a number of upstream minor start sites for transcription. RT7 encodes a putative 90-amino acid protein, of which the N-terminus is predicted to fold as an amphipathic alpha helix with features resembling the leucine zipper structure found in a family of transcription factors. However, unlike the leucine zipper proteins the RT7 alpha helix is not preceded by a basic region. Analysis of the RT7 promoter sequence indicates that it contains a putative testis-specific regulatory sequence found in protamine P1 and P2 promoters, as well as binding sites for several other transcription factors.

Kinesin Light-Chain KLC3 Expression in Testis Is Restricted to Spermatids

Abstract Kinesins are tetrameric motor molecules, consisting of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) that are involved in transport of cargo along microtubules. The function of the light chain may be in cargo binding and regulation of kinesin activity. In the mouse, two KLC genes, KLC1 and KLC2, had been identified. KLC1 plays a role in neuronal transport, and KLC2 appears to be more widely expressed. We report the cloning from a testicular cDNA expression library of a mammalian light chain, KLC3. The KLC3 gene is located in close proximity to the ERCC2 gene. KLC3 can be classified as a genuine light chain: it interacts in vitro with the KHC, the interaction is mediated by a conserved heptade repeat sequence, and it associates in vitro with microtubules. In mouse and rat testis, KLC3 protein expression is restricted to round and elongating spermatids, and KLC3 is present in sperm tails. In contrast, KLC1 and KLC2 can only be detected before meiosis in testis. Interestingly, the expression profiles of the three known KHCs and KLC3 differ significantly: Kif5a and Kif5b are not expressed after meiosis, and Kif5c is expressed at an extremely low level in spermatids but is not detectable in sperm tails. Our characterization of the KLC3 gene suggests that it carries out a unique and specialized role in spermatids.

Gene trap mutation of murine Outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism

BackgroundOuter dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile.ResultsXL169 ES cells have a β-geo gene trap cassette inserted in the Odf2 gene. To determine possible underlying mechanisms resulting in infertility we analyzed epididymal sperm and observed that >50% displayed bent tails. We next performed ultrastructural analyses on testis of high percentage XL169 chimaeric mice. This analysis showed that high percentage XL169 chimaeric mice produce elongating spermatids that miss one or more entire outer dense fibers in their midpiece and principal piece. In addition, we observed elongating spermatids that show thinning of outer dense fibers. No other obvious abnormalities or defects are present in elongating spermatids. Spermatozoa from the caput and cauda epididymis of XL169 mice of high percentage chimaerism show additional tail defects, including absence of one or more axonemal microtubule doublets and bent tails. Sperm with bent tails display abnormal motility.ConclusionsOur results document the possible impact of loss of one Odf2 allele on sperm tail structure and function, resulting in a novel sperm tail phenotype.

Cux/CDP homeodomain protein binds to an enhancer in the rat c-mos locus and represses its activity.

The c-mos gene is transcribed in male and female germ cells, in differentiating myoblasts and in 3T3 cells from cell-specific promoters. We characterized the rat testis promoter, which contains a TATA-box and one binding site for a testis-specific transcription factor TTF-D, as well as a region which can act as enhancer, which is located approx. 2 kb upstream of the c-mos AUG start codon. It binds three factors at sites I, II and III as determined in DNAse I footprint assays. We demonstrated that a member of the NF-1/CTF family of transcription factors binds site II. Here we report the cloning of the protein that binds to enhancer site III. This protein is the rat homolog of human hCut/CDP, mouse Cux/CDP and canine Clox. hCut/Cux/CDP/Clox (hereafter called Cux/CDP), a 160 kDa protein containing multiple repeats and a homeodomain, negatively regulates the mammalian c-myc, gp91-phox and N-Cam genes. Using bacterially produced murine GST-Cux fusion proteins and GST-Cux deletion mutants, we find that Cux repeat CR3 and the homeodomain are both required for efficient binding to enhancer site III. Mouse lung and testis nuclear Cux/CDP bind to site III as determined in electrophoretic gel mobility supershift assays using two different anti-hCut specific monoclonal antibodies. Transfections of CAT constructs containing the enhancer fragment linked to a minimal promoter demonstrated that Cux/CDP represses c-mos enhancer activity.

Targeted Disruption of the Testicular SPAG5/Deepest Protein Does Not Affect Spermatogenesis or Fertility

ABSTRACT In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins.

A novel testis-specific gene, SPAG4, whose product interacts specifically with outer dense fiber protein ODF27, maps to human chromosome 20q11.2

The product of the spermatid-specific rat SPAG4 gene interacts specifically with the major 27-kDa outer dense fiber protein ODF27 in elongating spermatids. In this study we isolated the human homolog of the spermatid-specific rat SPAG4 gene and mapped it by fluorescence in situ hybridization to chromosome 20q11.2.

A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis

Abstract The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.