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Testis |
a Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
b Laboratories for Reproductive Biology, Departments of Cell and Developmental Biology and Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7090
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, sperm motility and transport, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testis brain RBP was initially identified in the ribonucleoprotein particle fraction of mouse testicular postmitochondrial supernatants, where it bound to the 3' untranslated region (UTR) of translationally regulated mRNAs encoding protamine 2, protamine 1, and transition protein 1 [5, 6]. Testis brain RBP is the mouse ortholog of translin, a human DNA-binding protein, proposed to bind to consensus sequences at breakpoint junctions of chromosomal translocations in lymphoid tumors and at recombination hot spots during meiosis in germ cells [7–11]. Testis brain RBP accumulates in the nuclei of male germ cells during meiosis, suggesting that it could play a role in DNA recombination, repair, and/or metabolism [3, 10]. As a RNA-binding protein, TB-RBP binds to conserved Y and H elements of many testis and brain mRNAs, including those for protamine 1 and 2, AKAP4, myelin basic protein, 
calmodulin kinase II, and tau protein [12–14]. The ability of TB-RBP to link translationally suppressed mRNAs to microtubules and the release of bound mRNAs following disassociation of microtubules suggest that TB-RBP may function in the storage and transport of mRNAs [13, 14]. Testis brain RBP has been detected moving between nuclei and the cytoplasm and through intercellular bridges in male germ cells, suggesting a transport function for the intracellular and intercellular distribution of specific mRNAs in haploid male germ cells [3]. Recently, combining in situ hybridization and immunoelectron microscopy, multimeric complexes of TB-RBP and the Ter ATPase with Akap4 mRNA or protamine 2 mRNA have been found in nuclei and cytoplasm before the mRNAs undergo translation [4]. The dissociation of these RNA-protein complexes in later stage spermatids at the times the mRNAs are translated indicates an in vivo involvement of TB-RBP in the movement, stability, and/or translational suppression of specific mRNAs in male germ cells [4]. In hippocampal neurons, blocking the binding of TB-RBP to specific transported mRNAs disrupts mRNA sorting [15]. Although TB-RBP is expressed in many somatic tissues, its RNA-binding activity appears mainly limited to the brain and testis [12].
Testis brain RBP functions as a multimer and oligomerization of TB-RBP is required for either DNA or RNA binding. Studies with truncated and mutated forms of TB-RBP have identified several essential domains for nucleic acid binding [9, 16–18]. A leucine zipper in the C-terminus is required for protein dimerization, which is stabilized by a disulfide bond involving cysteine 225. Two basic domains in the N-terminus are essential for either DNA or RNA binding and TB-RBP contains a GTP-binding domain [19, 20]. The addition of GTP or its poorly metabolized analogue, GTP
S, to recombinant TB-RBP suppresses its binding to RNA but not to DNA in vitro. These binding changes induced by GTP also dissociate TB-RBP from its target mRNAs in extracts from brain or testis [20].
Glyceraldehyde 3-phosphate dehydrogenase-s (Gapds) encodes a glycolytic isozyme that is present only in condensing spermatids and spermatozoa in mice [21]. GAPD2, the human ortholog of Gapds, is also expressed only in the testis [22]. In both species, the GAPDS protein in spermatogenic cells is 
70% homologous to the somatic GAPD isozyme and contains a novel proline-rich sequence at the N-terminus. Gapds appears to be translationally regulated during mouse spermiogenesis because the steady-state level of Gapds mRNA is maximal in step 9 spermatids [23] and GAPDS protein is not detected by immunohistochemistry until steps 12–13 [24]. In mouse and human spermatozoa, GAPDS is localized in the principal piece, the longest segment of the flagellum [22, 24]. The GAPDS protein is tightly bound to the fibrous sheath, a cytoskeletal element that defines the limits of the principal piece, suggesting a role in the regulation of sperm glycolysis and motility [24].
In studies seeking to identify mRNAs that interact with and may be regulated by TB-RBP in the testis, we found Gapds mRNA selectively precipitated with TB-RBP as a protein mRNA complex. The aim of this study was to determine the functional association between TB-RBP protein and Gapds mRNA. In vitro translation assays indicate that wild-type recombinant TB-RBP can inhibit Gapds mRNA translation in cell-free translation assays, whereas mutant forms of TB-RBP that do not bind RNA show no suppression. Similarly, constructs of Gapds mRNA lacking a TB-RBP binding element are not translationally suppressed in vitro. The Gapds mRNA is the first mRNA identified where TB-RBP appears to affect translation in vitro by interaction with a binding sequence in its 5' UTR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testis extracts from sexually mature CD-1 mice (60–80 days old) were prepared as described by Wu et al. [11] with the modification that RNasin (100 U/ml) was added to the homogenization buffer. To immunoprecipitate TB-RBP-RNA complexes, testis extracts (10 mg) were precleared with protein A agarose beads (100 µl) and with preimmune serum (10 µl) in 5 ml of TKM-T buffer (20 mM Tris, pH 7.6, 40 mM KCl, 1.5 mM MgCl2, 0.1% Tween-20, 0.1% Empigen BB) containing 100 U/ml of RNasin at 4°C for 2 h. After centrifugation at 2000 rpm for 2 min, the supernatants were incubated overnight at 4°C with protein A agarose beads (40 µl) and affinity-purified antibody to TB-RBP (15 µg) in the presence or absence of 0.5 mM GTPS. The mixtures were centrifuged at 2000 rpm for 5 min, and the pellets were washed four times with TKM-T (5 ml). The pellets were suspended in 250 µl of TriRegent (Sigma Chemical Co., St. Louis, MO), followed by phenol-chloroform extraction. After ethanol precipitation, the RNA pellets were dissolved in dethylpyrocarbonate-treated water.
Assay of RNAs from Immunoprecipitations by Reverse Transcription Polymerase Chain Reaction
Immunoprecipitated RNA was reverse transcribed with an oligo(dT) NotI unidirectional primer (Invitrogen, Carlsbad, CA) with the RETROscript reverse transcription kit (Ambion, Austin, TX). Polymerase chain reaction (PCR) was performed using an oligo(dT) NotI primer and random decamers using 33 cycles at 94°C for 50 sec, 41°C for 1.5 min, and 72°C for 1.5 min. One millimole of dATP was added to the reaction mixture at 72°C for 10 min. The PCR products were precipitated and cloned into Topo TA vectors (Invitrogen). Colonies containing inserts greater than 100 nt were identified, amplified, and the inserts sequenced.
The PCR primers used for amplification of specific mouse genes are as follows: protamine 1, 5'-atggccagataccgatgctgccgc-3' and 5'-ggacttgctattctgtgcatc-3'; protamine 2, 5'-atggttcgctaccgaatgaggagcc-3' and 5'-ttagtgatggtgcctcctacatttcc-3'; GAPDS, 5'-acctttggctggcatccttg-3' and 5'-gttacttctctgggctaaac-3'; TB-RBP, 5'-gatggaattctgtgagcgagatcttcgtg-3' and 5'-ctgcggatcctatttttcaccacaagccgc-3'; and clusterin, 5'-tatgggcctccgagcttccac-3' and 5'-acctctgagtcagaggaatgg-3'. All primers were designed to bridge introns to detect amplification of contaminating DNA. The PCR reactions were carried out at 94°C for 50 sec, 58°C for 50 sec, 72°C for 50 sec, and up to 32 cycles. The PCR products were analyzed on 1.2% agarose gels, and the DNA products were detected with ethidium bromide staining.
Polysomal Gradient Fractionation and Analysis
Testes from eight adult CD-1 mice (60–80 days old) were decapsulated and homogenized on ice with a motor-driven Teflon homogenizer in 3 ml of homogenization buffer (20 mM HEPES, pH 7.6, 40 mM KCl, 1.5 mM MgCl2). Following homogenization, the postmitochondrial extracts were centrifuged at 5000 rpm for 10 min at 4°C. The supernatants were layered over 15–40% sucrose gradients (10 ml) over a 60% sucrose cushion (0.5 ml). Following centrifugation at 28 000 rpm for 3 h at 4°C in a SW28 rotor, the gradients were fractionated into 20 tubes. One third of each tube was extracted with Tri Reagent (Sigma), and the purified RNAs were used for Northern blotting with 32P-labeled Gapds or protamine 2 cDNAs [4]. Protein extracts were prepared from the remainder of each tube for use in Western blotting [11].
Preparation of Gapds Transcripts
For the in vitro translation assays, a 1451 nucleotide Gapds cDNA containing most of the open reading frame (30 nucleotides were deleted but the open reading frame was in frame) was PCR amplified and subcloned into the XbaI and SmaI sites of pSP64 poly (A) vector [21]. The primers 5'-tgctctagaagtaacaccacggaggggggccaaggcagccaggccatgagatc-3' and 5'-tcccccgggtggaagccgaagtcaggaac-3' were used for the full length Gapds mRNA amplification. The vector generates a 30-nucleotide poly A tail with the transcribed mRNAs. Capped Gapds mRNA was synthesized in vitro with the mMessage mMachine kit (Ambion) according to the supplier's instructions. The capped and polyadenylated Gapds mRNAs were purified with the Oligodtex mRNA kit (Qiagen, Valencia, CA), quantitated, and stored at -80°C until use. Gapds cDNAs, in which 21 nucleotide fragments containing the H or Y elements were deleted, were prepared by PCR amplification without altering the open reading frame of Gapds mRNA. To create the H-element-deleted construct, the primers 5'-tgctctagaagtaacaccacggaggggggccaaggcagccatgtcgagacgtgacgtggtc-3' and 5'-tcccccgggtggaagccgaagtcaggaac-3' were used. To create the Y-element-deleted construct, the primers 5'-atgccatggcctacagccttggcagcc-3' and 5'-ggccatgggaagctaacaggaatggc-3' were used. Because the Y element is located in the middle of the Gapds mRNA, we ligated two fragments 5' and 3' to the Y element to create the full length Gapds mRNA lacking 21 nt of the Y element. These cDNAs were cloned, and mRNAs were transcribed as described above.
In Vitro Translation of Gapds mRNA
In vitro translations were carried out with wheat germ extracts (Ambion). Purified, capped, and polyadenylated Gapds mRNA (0.5 µg) was used as template for protein synthesis. Each reaction mixture (25 µl) contained 12.5 µl of wheat germ extract and 10 µCi of [35S]methionine (Amersham Biosciences, Piscataway, NJ). After a 60-min incubation at 27°C, the synthesized protein products were analyzed by 10% SDS-PAGE. Following drying, GAPDS was visualized by autoradiography. In assays where TB-RBP protein was added, increasing amounts of recombinant wild-type TB-RBP or mutant TB-RBP were incubated with Gapds mRNA in RNA-protein binding buffer (20 mM Hepes, pH 7.6, 3 mM MgCl2, 40 mM KCl, 2 mM DTT, 10 U RNasin, and 5% glycerol) for 15 min at room temperature before being added to the wheat germ extract. The recombinant wild-type and mutant TB-RBP proteins were prepared as described [16, 19]. (The TB-RBP 1-204 mutant is described by Wu et al. [16].)
35S-Labeled GAPDS was detected and quantitated by PhosphorImaging (Molecular Dynamics, Sunnyvale, CA). The protein amounts synthesized by capped and polyadenylated full length or Y- or H-deleted Gapds mRNAs in the absence of TB-RBP were set at 100%. The effects of wild-type or mutant TB-RBP on translation were evaluated by comparing the amount of protein synthesized in the presence of TB-RBP to the control translations.
To insure that changes in the amount of GAPDS synthesized were not due to mRNA degradation, a low level (5 µCi) of [32P]UTP was added to the Gapds mRNA transcription mixture. Radiolabeled mRNAs were synthesized and purified and used as templates in the translation assays identical to those described above. After 1 h of translation, the template RNAs from each reaction tube were purified with Tri Reagent (Sigma), dissolved in water, and separated on formaldehyde-denatured agarose gels. The gels were dried and exposed for RNA analysis.
Procedures involving animals were approved in advance by the Institutional Animal Care and Use Committee.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify mRNAs that are specifically bound by TB-RBP, affinity-purified antibody to TB-RBP was used to immunoprecipitate TB-RBP-mRNA complexes from testis extracts. Following precipitation and RNA purification, cDNAs were prepared and sequenced. One clone out of the first group of 20 contained a fragment of the Gapds mRNA.
To establish the specificity of the immunoprecipitation, primers were designed to assay for mRNAs that bind TB-RBP and therefore are predicted to be immunoprecipitated and those that do not bind TB-RBP [4]. The mRNAs encoding protamine 1 and protamine 2 represent positive controls because the genes are translationally regulated and the mRNAs contain Y and H elements in their 3' UTRs that are recognized by TB-RBP. Negative control mRNAs that lack Y or H elements include TB-RBP mRNA itself and the mRNA encoding the Sertoli cell-expressed gene, clusterin. Gapds, protamine 1, and protamine 2 mRNAs were immunoprecipitated in TB-RBP-RNA complexes with antibody to TB-RBP (Fig. 1, lane 2). Preimmune serum did not precipitate any of the mRNAs (Fig. 1, lane 1) and TB-RBP mRNA or clusterin mRNA were not precipitated by either preimmune serum (Fig. 1, lane 1) or antibody to TB-RBP (Fig. 1, lane 2), although they could be readily detected in total testis RNA (Fig. 1, lane 4).
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As a second indicator of immunoprecipitation specificity, GTP was added to the extracts. Testis brain RBP is a DNA/RNA-binding protein with a GTP-binding site in its C-terminus [20]. GTP, especially its poorly hydrolyzed analogue GTPS, reduces TB-RBP binding to specific mRNAs by 90% in gel shift and immunoprecipitation assays [20]. When GTPS was added to immunoprecipitation buffer, a substantial reduction in the amount of protamine 1 and 2 and Gapds mRNAs in the precipitates was seen (Fig. 1, lane 3), indicating that TB-RBP is not adventitiously bound to Gapds mRNA in testis extracts.
Gapds mRNA and TB-RBP Protein Colocalize in Ribonucleoprotein Particles
To determine whether TB-RBP is bound to polysomal or nonpolysomal Gapds mRNA, a postmitochondrial testicular extract was separated into polysomal and nonpolysomal fractions following sucrose gradient centrifugation. Using mouse protamine 2 cDNA to calibrate the gradient, we detected the characteristic distribution of the fully adenylated protamine 2 mRNA in the nonpolysomal fractions with the partially deadenylated mRNAs in polysomes (Fig. 2B). Gapds mRNA showed a similar distribution pattern as mouse protamine 2 mRNA, with the majority of Gapds mRNA in the nonpolysomal fractions (Fig. 2A, fractions 1–8) and the remainder in polysomes (Fig. 2A, fractions 12–18). Western blot analyses of the proteins from the same fractions revealed that TB-RBP was concentrated in fractions 1–6 (Fig. 2C). Following RNase digestion of the protein extract, TB-RBP is primarily in fractions 1 and 2 (data not shown). This movement of TB-RBP in the sucrose gradient fractions suggests it exists as a free protein and also associates with nonpolysomal mRNAs. These data suggest that Gapds mRNA is one of the target molecules of TB-RBP and may be stored and/or transported by TB-RBP in the form of a RNA-protein complex.
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TB-RBP Protein Inhibits Gapds mRNA Translation In Vitro
To begin to define the significance of TB-RBP binding to Gapds mRNA, we have used capped Gapds mRNA as template for cell-free translation in the presence of recombinant TB-RBP or forms of recombinant TB-RBP that have altered RNA-binding properties. Figure 3 shows a schematic diagram of putative TB-RPB binding sites in Gapds (Fig. 3A) and the location of the two basic domains of TB-RBP that are required for RNA or DNA binding (Fig. 3B). Previous studies have demonstrated that the Cb mutation in the second basic domain or a C-terminal truncation of TB-RBP (amino acids 1–204) that disrupts the leucine zipper domain abolishes RNA binding, whereas the Nb mutation decreases but does not abolish RNA binding [19]. Translation of 0.5 µg of capped Gapds mRNA generated GAPDS with an apparent molecular weight of 69 000 (Fig. 4). When increasing amounts of TB-RBP protein were added to the cell-free translation assays, a reduction in GAPDS synthesis was seen (Fig. 4A, lanes 1–4). Quantitation with ImageQuant software revealed that 0.2 and 0.4 µg of TB-RBP protein decreased synthesis of GAPDS to about 28% and 9%, respectively (Fig. 4B). A similar reduction was seen with the TB-RBP-Nb protein (Fig. 4A, lanes 5–8). In contrast, no significant difference in GAPDS synthesis was detected when identical amounts of TB-RBP-Cb protein (Fig. 4A, lanes 9–12) or the truncated C-terminal-deleted TB-RBP (1–204aa) was added (Fig. 4A, lanes 13–16). In fact, more than 80% of the control GAPDS was synthesized in the presence of recombinant TB-RBP-Cb protein or when C-terminal-deleted TB-RBP replaced full-length TB-RBP in the Gapds mRNA translation reactions. We conclude that recombinant TB-RBP proteins capable of binding to mRNAs inhibit cell-free translation of Gapds mRNA, whereas TB-RBP protein that does not bind RNA does not substantially affect the translation assay.
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To confirm that the inhibition of translation was not due to Gapds mRNA degradation from contaminants in the added recombinant protein preparations, we synthesized Gapds mRNA with tracer amounts of [32P]UTP and used it for identical translation assays with and without added recombinant proteins as performed in Figure 4A. Following the 1-h in vitro translation assays, the Gapds mRNAs from each reaction tube were quite stable, showing little or no degradation whether recombinant wild-type or mutant TB-RBP proteins were added (Fig. 4C).
H Element in the 5' UTR of Gapds mRNA Is Necessary for the Inhibition of Translation by TB-RBP
TB-RBP recognizes a broad range of conserved H and Y elements in many translationally regulated mRNAs in testis and brain [13]. The Gapds mRNA contains an H element in its 5' UTR and a Y element in its open reading frame (Fig. 3A).
To determine whether TB-RBP binding to either of these putative binding sites is the cause of the reduction of GAPDS synthesis in the cell-free translation assay, we prepared Gapds mRNAs lacking the H or Y elements as templates for translation (Fig. 5). Aside from the deletion of 21 nucleotides for each putative binding element, the Gapds template mRNAs were identical to the control Gapds mRNAs. Translation of the Gapds mRNAs in the presence of wild-type recombinant TB-RBP decreased the amount of protein synthesized with control Gapds mRNA (Fig. 5A, Gapds mRNA lanes C, 1–4) and in constructs where the Y element was deleted from the Gapds mRNA (Fig. 5A, Gapds-Y- mRNA, lane C, 1–4). However, no significant decrease in GAPDS synthesis was seen with the Gapds RNA lacking the H element (Fig. 5A, Gapds-H- mRNA, lane C, 1–4), suggesting that the H element of the Gapds mRNA is essential for the inhibition of GAPDS synthesis by TB-RBP in cell-free translation assays.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fibrous sheath and outer dense fibers are cytoskeletal components of mammalian spermatozoa that surround the axoneme. Both structures are formed slowly during spermiogenesis and continue to be synthesized well after transcription ceases in midstage spermatids [26, 27]. Significant delays between the initiation of transcription and translation have been demonstrated for several fibrous sheath proteins, including GAPDS [24], AKAP4 [28, 29], and FS39 [30], and for outer dense fiber proteins Odf1 [31], Odf2 [32], and Spag4 [33]. The synthesis of these proteins after the termination of mRNA transcription necessitates posttranscriptional regulation of their mRNAs. For instance, in both mouse and rat, AKAP4 mRNAs are most abundant in midstage spermatids, although the peak amounts of protein are synthesized days later in late-stage spermatids [28, 29, 34]. Similarly, the steady-state level of Gapds mRNA is maximal at step 9 of mouse spermatogenesis, whereas GAPDS is not detected by immunohistochemistry until steps 12–13 [23, 24]. Sucrose density gradient analyses of testicular extracts indicate that the majority of Gapds and Akap4 mRNAs as well as mRNAs for other fibrous sheath proteins (rhophilin, ropporin, glutathione S-transferase) are present in the nonpolysomal fractions (data not shown).
Specific sequence elements and structural features in both the 5' and 3' untranslated regions of mRNAs have been implicated in translational regulation [25, 35]. Previous studies have not identified specific translational regulatory elements in any of the mRNAs that encode mammalian fibrous sheath or outer dense fiber proteins. Zhong et al. [36] have proposed that a translational control element (TCE) in the 3' untranslated region of Prm1 mRNA is essential for repression of protamine 1 synthesis. This element is not present in Gapds mRNA or several other spermatid mRNAs that show delayed translation, indicating that other regulatory mechanisms must operate during spermiogenesis.
Translational gene regulation also occurs during spermatogenesis in a variety of nonmammalian species. In Drosophila, a 12-base pair (bp) sequence (termed TCE but distinct from the Prm1 TCE) has been identified in the 5' UTR of mRNAs in the Mst(3)CPG gene family, which encodes structural proteins of the satellite fibers of the sperm tail [37]. The satellite fibers are analogous to mammalian outer dense fibers, and the MST(3)CPG proteins have sequence characteristics that are also found in Odf1, the major outer dense fiber protein [31, 38]. Sequences similar to this TCE have also been identified in other translationally regulated mRNAs [37]. A second translational control element (TRE) was recently identified in the Drosophila don juan gene, which encodes an H1 histone-like protein expressed only in spermatids [39]. RNA-binding proteins that interact with either the Drosophila TCE or TRE sequences have been identified and are thought to be involved in the translational repression of mRNAs during the meiotic and early postmeiotic stages of spermatogenesis [37, 39]. Don juan and MST(3)CPG-encoded proteins are synthesized at distinct stages of spermiogenesis, suggesting a coordinated program of translational repression and activation during germ cell differentiation that is mediated by distinct regulatory elements in the 5' UTR of these germ cell mRNAs.
Multiple cis-acting elements may also regulate translation during mammalian spermatogenesis [1]. As in Drosophila, some of these elements may be localized in 5' untranslated regions near the translation start site. Our in vitro translation experiments suggest that TB-RBP represses GAPDS synthesis by binding to the 5' UTR H element located just three nucleotides before the translation start site in Gapds mRNA. A RNA-binding protein has been shown to similarly modulate translation by binding to the 5' UTR of a testicular variant of a superoxide dismutase mRNA [40], and an alternative 5' UTR of cytochrome cS down-regulates cytochrome cS synthesis [41]. A number of proteins have been shown to bind to the 3' UTRs of protamine mRNAs and regulate their translation (reviewed in [1, 2]. Clearly, male germ cells utilize many strategies to regulate their posttranscriptional gene expression.
How might the interaction between TB-RBP and mRNAs encoding fibrous sheath proteins function in the testis? The binding and association of TB-RBP to AKAP4 mRNA [4] and to the specific site in the 5' untranslated region of Gapds mRNA described in this report suggests an involvement of TB-RBP in the regulation of at least two fibrous sheath proteins. Messenger RNAs encoding other fibrous sheath proteins such as rhophilin, ropporin, and a cAMP protein kinase also contain putative Y and H elements. Although the molecular mechanism of the assembly of the fibrous sheath is not known, electron microscopy autoradiography has demonstrated that the fibrous sheath forms in a distal to proximal orientation over a period of 15 days in the rat [26, 27]. Such assembly requires temporal and spatial regulation of a number of proteins in postmeiotic male germ cells. GAPDS contains a large proline-rich domain in its N terminus. Other proline-rich proteins such as collagen function as structural scaffolding proteins in somatic cells. The limited solubility of many of the fibrous sheath proteins may necessitate their translational delay and coordinate their expression and insertion into the growing fibrous sheath. Early unregulated expression of proteins such as GAPDS could lead to their incorrect cellular localization and interfere with male germ cell development, as has been seen with precocious expression of the DNA-binding protamines [42]. The association of AKAP4 mRNA with TB-RBP and the Ter ATPase in round spermatids raises the possibility that TB-RBP functions in the localization of mRNAs in male germ cells [4]. It is intriguing to consider the involvement of TB-RBP in the transport and/or localization of fibrous sheath mRNAs to specific cellular sites as a means to facilitate the lengthy multistep events needed for fibrous sheath formation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Norman B. Hecht, Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical School, 1310 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6142. FAX: 215 573 5408; e-mail: nhecht{at}mail.med.upenn.edu
Received: 6 August 2002.
First decision: 27 August 2002.
Accepted: 18 September 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calmodulin kinase II and protamines 1 and 2. Biol Reprod 2000 62:720-725This article has been cited by other articles:
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Y. S. Cho, N. Iguchi, J. Yang, M. A. Handel, and N. B. Hecht Meiotic Messenger RNA and Noncoding RNA Targets of the RNA-Binding Protein Translin (TSN) in Mouse Testis Biol Reprod, October 1, 2005; 73(4): 840 - 847. [Abstract] [Full Text] [PDF]
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Z. Yu, T. Raabe, and N. B. Hecht MicroRNA Mirn122a Reduces Expression of the Posttranscriptionally Regulated Germ Cell Transition Protein 2 (Tnp2) Messenger RNA (mRNA) by mRNA Cleavage Biol Reprod, September 1, 2005; 73(3): 427 - 433. [Abstract] [Full Text] [PDF]
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N. Iguchi, H. Tanaka, S. Yamada, H. Nishimura, and Y. Nishimune Control of Mouse hils1 Gene Expression During Spermatogenesis: Identification of Regulatory Element by Transgenic Mouse Biol Reprod, May 1, 2004; 70(5): 1239 - 1245. [Abstract] [Full Text] [PDF]
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V. Chennathukuzhi, C. R. Morales, M. El-Alfy, and N. B. Hecht The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells PNAS, December 23, 2003; 100(26): 15566 - 15571. [Abstract] [Full Text] [PDF]
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