Testicular protein Spag5 has similarity to mitotic spindle protein Deepest and binds outer dense fiber protein Odf1.

Outer dense fibers (ODF) and the fibrous sheath (FS) are major cytoskeletal structures in the mammalian sperm tail. The molecular mechanisms underlying their morphogenesis along the axoneme or their function are poorly understood. Recently, we reported the cloning and characterization of Odf2, a major ODF protein, and Spag4, an axoneme-binding protein, by virtue of their strong interaction with Odf1, the 27 kDa major ODF protein. We proposed a crucial role for leucine zippers in molecular interactions during sperm tail morphogenesis. Here we report the cloning and characterization of a novel gene, Spag5, which encodes a 200 kDa testicular protein that interacts strongly with Odf1. Spag5 is transcribed and translated in pachytene spermatocytes and spermatids. It bears 73% similarity with the mitotic spindle protein Deepest of unknown function. We identified two putative leucine zippers in the C-terminal part of the Spag5 protein, the downstream one of which is involved in interaction with Odf1. Interestingly, these motifs are present in Deepest. These results highlight the importance of the leucine zipper in sperm tail protein interactions. Mol. Reprod. Dev. 59: 410-416, 2001.

Germ cell specific promoter drives ectopic transgene expression during embryogenesis.

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.

Kinesin light-chain KLC3 expression in testis is restricted to spermatids.

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 heptad 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.

Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme.

Outer dense fibers are structures unique to the sperm tail. No definite function for these fibers has been found, but they may play a role in motility and provide elastic recoil. Their composition had been described before, but only two of the fiber proteins, Odf1 and Odf2, are cloned. We cloned Odf2 by virtue of its functional and specific interaction with Odf1, which, we show, is mediated by a leucine zipper. Further work demonstrated that the 84-kDa Odf2 protein localizes to both the cortex and the medulla of the fibers, whereas the 27-kDa Odf1 protein is present only in the medulla. Here we report the cloning and characterization of a new Odf1-interacting protein, Spag4. Spag4 mRNA is spermatid specific, and the 49-kDa Spag4 protein complexes specifically with Odf1, but not Odf2, mediated by a leucine zipper. It also self-associates. In contrast to Odf1 and Odf2, Spag4 protein localizes to two microtubule-containing spermatid structures. Spag4 is detectable in the transient manchette and it is associated with the axoneme in elongating spermatids and epididymal sperm. Our data suggest a role for Spag4 in protein localization to two major sperm tail structures.

Testis-specific TTF-D binds to single-stranded DNA in the c-mos and Odf1 promoters and activates Odf1.

We recently identified testis-specific nuclear factor binding sites in the testis-specific promoters of the c-mos gene and the Odf1 gene, which are 80% identical. Here we characterize a testis-specific nuclear factor, TTF-D, which is able to complex with both binding sites and stimulates Odf1 promoter activity. TTF-D is detectable in mouse testis as early as day 11 postpartum and contains three peptides of 22, 25, and 35 kDa in size. Surprisingly, TTF-D binds specifically to its cognate double-stranded DNA binding site as well as to its single-stranded DNA binding site. Both double-stranded and single-stranded binding site oligonucleotide DNA can specifically repress Odf1 promoter activity. Our results suggest that TTF-D is involved in positive transcription regulation of a pre-meiotic and a post-meiotic gene in the testis.

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.

Developmental expression of the 84-kDa ODF sperm protein: localization to both the cortex and medulla of outer dense fibers and to the connecting piece.

Outer dense fibers (ODF) are specialized cytoskeletal elements of the mammalian sperm tail which are composed of several prominent proteins. We previously reported the isolation of a cDNA (111-450) encoding a putative 84-kDa ODF protein. Here we demonstrate by independent cDNA isolations and by translational/immunoprecipitation of testicular mRNAs using anti-ODF 84 antibodies that 111-450 cDNA encodes the 84-kDa protein. We then analyzed the testicular expression of the ODF 84 mRNA and protein. Riboprobes generated from the clones recognized four testicular-specific transcripts of 1.6, 2.2, 2.4, and 2.8 kb in both rat and bull of which the immunoprecipitable product of the 2.4-kb mRNA comigrates with ODF 84 protein. Developmental Northerns indicated that the 2.2- and 2.4-kb mRNAs are first transcribed during meiotic prophase while the other two species are first expressed in round spermatids. The levels of all the transcripts steadily increased up to elongated spermatids. Immunocytochemistry revealed that the anti-84 reactive ODF proteins were synthesized and assembled in the cytoplasm of elongated spermatids (steps 9-18) with peak activity occurring in step 16 of spermiogenesis. Immunogold labeling was selective to the assembling ODF and connecting piece of the tail and to granulated bodies of the cytoplasmic lobe. Both the striated collar and capitulum of the connecting piece were immunolabeled as well as the basal plate of the implantation fossa. A combination of pre- and postembedding immunogold labeling provided evidence that the 84-kDa ODF protein is localized to both the cortex and medulla of the ODF in contrast to the sole medullary localization of the major 27-kDa ODF protein. Thus the 84-kDa ODF protein, encoded by the 2.4 transcript, is translationally regulated, packaged after synthesis into granulated bodies, assembled in a proximal to distal direction along the axoneme and may interact by means of leucine zippers specifically with the 27-kDa ODF protein during assembly. Its localization to both the cortex and medulla of the ODF, as opposed to exclusive medullary localization of the 27-kDa ODF protein, and the presence of two leucine zippers, only one of which interacts with the 27-kDa ODF, suggests that it could act as a link between proteins of the two regions of the ODF.

Human outer dense fiber gene, ODF2, localizes to chromosome 9q34.

We have isolated the human homolog of the rat Odf2 gene. In rat, Odf2, the 84-kDa major outer dense fiber protein, interacts strongly and specifically with Odf1, the 27-kDa major outer dense fiber protein. The interaction is mediated by leucine zippers during ODF assembly along the sperm axoneme. We compared homology and genomic structure to rat and mouse Odf2 genes. Using fluorescence in situ hybridization, we mapped the human Odf2 gene (ODF2) to chromosome 9q34.

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.

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.

Self-interaction of the major 27-kilodalton outer dense fiber protein is in part mediated by a leucine zipper domain in the rat.

The RT7 gene is exclusively expressed in spermatids and encodes the 27-kDa major outer dense fiber (ODF) protein ODF27. Analysis of its amino acid structure had indicated the presence of a putative leucine zipper dimerization motif in the N-terminus and the presence of PCX repeats in the C-terminus. We had previously shown that the ODF27 N-terminal fragment can interact with full-length ODF27. We have used two different methods to analyze this interaction further. First we used fusion proteins between glutathione S-transferase (GST) and ODF27-derived fragments to show that the N-terminal half of ODF27 as well as the first 100 amino acids can interact with ODF27. A fusion protein consisting of GST and the ODF27 leucine zipper did not interact with ODF27. We found that the ODF27 C-terminal half can also interact with ODF27. The yeast two-hybrid method was next employed to analyze these interactions in vivo. We found that 1) N-terminal fragments containing the leucine zipper interact with the ODF27 N-terminus, but not with its C-terminus, 2) deletion of the leucine zipper abolished this interaction, and 3) the PCX repeats are involved in the self-interaction of the ODF27 C-terminus. The detected self-associations are weak. To analyze the molecular weight of in vitro-translated ODF27, we carried out gel filtration experiments. They show that at low concentrations, a fraction of ODF27 proteins exists as multimers while the rest are monomers whose shape deviates considerably from that of globular proteins. Our results identify regions in the N- and C-termini of ODF27 involved in self-interactions and suggest that in ODF, where high protein concentrations prevail, ODF27 can self-interact.

Cell interactions in testis development: overexpression of c-mos in spermatocytes leads to increased germ cell proliferation.

Possible functions of the c-mos proto-oncogene during spermatogenesis were investigated through perturbations of its expression in transgenic mice. Two promoters, one from the pre-meiotic male germ cell-specific mouse phosphoglycerate kinase 2 gene, and the other from the post-meiotic male germ cell-specific rat RT7 gene were used to direct expression of c-mos. Northern blot analysis of testis RNA from transgenic PGK-c-mos mice indicated elevated levels of c-mos RNA in spermatocytes and spermatids compared to controls. No transgene expression was detected in any other tissue examined, suggesting that the mouse PGK2 promoter, like the previously used human PGK2 promoter, confers correct cell-specific expression onto c-mos. The promoter from a newly characterized rat gene, RT7, was shown to direct expression specific to post-meiotic spermatids. Transgenic mice carrying an RT7-lacZ construct displayed immunoreactive bacterial beta-galactosidase as well as enzyme activity in round spermatids. The cellular specificity for beta-galactosidase expression observed in RT7-lacZ transgenic animals was in agreement with endogenous RT7 transcript expression. Northern blot analysis of testis RNA of RT7-c-mos transgenic mice showed elevated levels of c-mos in spermatids, but not in other cells or tissues examined. Western blot analysis demonstrated elevated levels of p43c-mos in spermatids of both PGK-c-mos and RT7-c-mos transgenic animals, but only PGK-c-mos transgenics had increased p43c-mos levels in spermatocytes. Both RT7-c-mos and PGK-c-mos transgenic mice are fertile and show no tendency toward transformation. RT7-c-mos mice have no discernible phenotype associated with the c-mos overexpression in spermatids. However, PGK-c-mos transgenic males exhibited a significant increase in germ cell number, as determined by cell counts using total germ cells and germ cells fractionated by centrifugal elutriation. Because mitotic divisions of germ cells occur prior to PGK-c-mos transgene expression, our observations suggest that c-mos overexpression in spermatocytes causes an alteration in cell-cell interactions.

Testis-specific RT7 protein localizes to the sperm tail and associates with itself.

The RT7 gene recently cloned by us is expressed as an abundant RNA in round spermatids. In vitro transcription-translation showed that the RT7 gene encodes a protein of 26-27 kDa on SDS-polyacrylamide gels. Here we report the development of monoclonal antibodies (mAbs) raised against a peptide from the predicted N-terminal amphipathic alpha-helix of the rat RT7 protein. All mAbs recognize RT7 protein or N-terminal parts of it. To investigate RT7 in vivo, mAbs were used in immunofluorescence microscopy and confocal laser immunofluorescence microscopy. Several mAbs recognize RT7 protein in elongating spermatids: the observed staining pattern suggests a nonrandom localization in these cells. Two mAbs recognize the protein only in sperm tails. Using co-immunoprecipitation assays, we found that RT7 can form stable complexes with itself that are associated through a region located in the N-terminal half of RT7. Our results identify the RT7 protein as a major sperm tail component and suggest that it may be a structural component of sperm tail outer dense fibers (ODF).

Identification of the testis c-mos promoter: specific activity in a seminiferous tubule-derived extract and binding of a testis-specific nuclear factor.

The c-mos proto-oncogene is predominantly expressed in male and female germ cells and is involved in the regulation of meiosis. To investigate the mechanism of testis-specific regulation of c-mos transcription, I set out to identify the rat testis c-mos promoter. This was achieved by characterization of the rat testis c-mos transcription start site by primer extension and sequence analysis of cDNAs obtained by polymerase chain reaction amplification of 5' ends of c-mos RNA. The rat testis c-mos transcription start site is located 0.56 kb upstream of the coding region. A fragment containing the rat testis c-mos promoter directs transcription in a nuclear extract derived from rat seminiferous tubules, but not in a liver nuclear extract. DNAase I footprint analysis and gel-retardation assays showed binding of a novel testis-specific nuclear factor to the rat testis c-mos promoter at a site homologous to the testis-specific cis-acting element identified in the promoter of the RT7 gene, which is specifically expressed in haploid male germ cells.

Simultaneous determination of free catecholamines and epinine and estimation of total epinine and dopamine in plasma and urine by high-performance liquid chromatography with fluorimetric detection.

Epinine (N-methyldopamine) is the pharmacologically active hydrolysis product of the prodrug ibopamine, which is currently being widely studied for the treatment of congestive heart failure. This paper reports a sensitive and reliable method for the simultaneous determination of free catecholamines and epinine in plasma and urine. The compounds are isolated from plasma or urine by a specific liquid-liquid extraction, derivatized with the selective fluorogenic agent 1,2-diphenylethylenediamine, and quantitated by high-performance liquid chromatography with gradient elution and fluorimetric detection. The limits of detection for the derivatized catecholamines and epinine are 0.3-0.6 pg of injected compound. Intra- and inter-assay coefficients of variation of all four compounds are good (1-8%), as are the accuracy and linearity. A method is also reported for the determination of total dopamine and epinine in plasma and urine based on the same principle. This method, in which deconjugation is accomplished by acid hydrolysis at 95 degrees C, also shows good sensitivity and reproducibility.

Factors involved in regulation of the RT7 promoter in a male germ cell-derived in vitro transcription system.

We recently cloned and characterized a rat male germ cell-specific gene, RT7. The RT7 promoter contains a TATA box as well as sequences with homology to binding sites for a number of transcription factors. To investigate the regulation of the RT7 promoter we developed an active in vitro transcription system derived from rat seminiferous epithelium, which, in contrast to total testis, consists mostly of male germ cells. Also, DNase I footprinting analysis and gel retardation experiments were performed to analyze RT7 promoter-protein interaction. The experiments demonstrate that nuclear extracts prepared from rat male germ cells support in vitro transcription and that the RT7 promoter is positively regulated by a testis-specific transcription factor, TTF-D, by a factor similar to the transcription factor CREB, and by a nuclear factor that binds immediately upstream of the RT7 transcription start site.

A 43 kD c-mos protein is only expressed before meiosis during rat spermatogenesis.

We have investigated the RNA and protein expression pattern of the rat c-mos proto-oncogene during spermatogenesis. In mouse testis a 43kD c-mos protein is expressed throughout spermatogenesis, which is in agreement with one report detecting a 1.7 kb c-mos RNA in pachytene spermatocytes and in early spermatids. However, several other reports show that the mouse 1.7 kb c-mos RNA is exclusively expressed in post-meiotic male germ cells. We report that in rat male germ cells three c-mos RNA species of 5, 3.6 and 1.7 kb are detectable by Northern blotting analysis both before and after meiosis, with highest levels in early spermatids. However, western immuno-blot analysis reveals the presence of a 43 kD c-mos protein in total testis and pachytene spermatocytes, but not in post-meiotic cells. These findings combined with those made in the mouse system strongly suggest that c-mos protein may be a regulator of meiosis during spermatogenesis.

Improved measurement of urinary catecholamines by liquid-liquid extraction, derivatization and high-performance liquid chromatography with fluorimetric detection.

We report a sensitive and reliable assay for the determination of the urinary catecholamines norepinephrine, epinephrine and dopamine, based on selective extraction by a liquid-liquid extraction procedure, followed by selective derivatization with the fluorigenic agent 1,2-diphenylethylenediamine and quantification by high-performance liquid chromatography with fluorimetric detection. Comparison with a method using electrochemical detection shows that interference of an unknown compound, most probably N-methylepinephrine, which is an often-overlooked problem with methods using electrochemical detection and results in falsely high epinephrine concentrations, does not occur with the described fluorimetric method.

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.

Ha-rasVal-12,Thr-59 activates S6 kinase and p34cdc2 kinase in Xenopus oocytes: evidence for c-mosxe-dependent and -independent pathways.

Treatment with insulin or progesterone or microinjection of the transforming protein product of Ha-rasVal-12,Thr-59 (p21) is known to induce germinal vesicle breakdown in Xenopus oocytes. We have investigated the effect of p21 on S6 kinase and the H1 histone kinase of maturation-promoting factor in the presence and absence of antisense oligonucleotides against the c-mosxe proto-oncogene. Injection of p21 led to a rapid increase in S6 phosphorylation, with kinetics similar to those previously observed with insulin. Microinjection of c-mosxe antisense oligonucleotides inhibited germinal vesicle breakdown induced by p21 and totally abolished S6 kinase activation by insulin or progesterone but only partially inhibited activation by p21. However, the activation of p34cdc2 protein kinase by all three stimuli was blocked by antisense oligonucleotides. The results suggest that in oocyte maturation c-mosxe functions downstream of p21 but upstream of p34cdc2 and S6 kinase activation, although not all p21-induced events require c-mosxe.