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Evidence of receptor/ligand interactions that regulate testis cell function was sought in order to broaden the current understanding of the molecular basis of testis cell function. Using reverse transcription and the polymerase chain reaction, we have obtained novel evidence for the expression of three mRNAs encoding receptor tyrosine kinases in the adult rat testis: the platelet-derived growth factor type A receptor (PDGF-RA), the basic fibroblast growth factor receptor (flg), and fetal liver kinase 1 (Flk-1). A 6.8 kb transcript encoding the PDGF-RA was observed in RNA prepared from testes of rats aged day 5 through adult, with a decline in relative abundance with increasing age after day 17. Analysis of mRNA from isolated cell preparations (day 21 Sertoli cells, adult Leydig cells, round spermatids, and primary spermatocytes) and testes depleted of specific cell types [ethane dimethane sulfonate (EDS)-treated and cryptorchid] indicated that the Leydig cell was the predominant source of this mRNA in the adult testis. The addition of PDGF-BB to cultures of highly purified adult rat Leydig cell preparations resulted in a 40% increase in LH-stimulated testosterone production, confirming a role for this growth factor in regulation of Leydig cell function. These data indicate that the Leydig cell is a principal site of action of PDGF in the testis.

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta-galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both ~70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.

Screening of a rat testis expression library with an antiserum specific for an outer dense fibre (ODF) has led to the identification of a gene encoding for a putative protein previously unknown as a component of the sperm tail. This gene has been designated tpx-1 by virtue of its homology with the mouse and human gene of the same name (79 and 73%, respectively). The tpx-1-like gene encoded a 1.6-kb mRNA and a 243-amino-acid protein that had significant homology with members of the cysteine-rich secretory protein (CRISP) family and partial homology with several venom/allergen proteins from both plants and insects. During rat spermatogenesis, the tpx-1-like transcript was first detected by in situ hybridization in low levels in late pachytene spermatocytes. Low but detectable levels of expression continued up to step 5 round spermatids, after which expression levels increased dramatically to a maximum in step 11-12 spermatids. Progressively decreasing levels of expression were detected in up to step 17 elongating spermatids. Testicular somatic cells did not contain detectable tpx-1-like transcript. This pattern of expression is consistent with published data on the development of the ODF in spermatogenesis and, when taken together with a comparison of the predicted amino acid sequence of tpx-1 with the animo acid analysis of a 29-kDa rat ODF protein, suggests that the tpx-1-like gene may encode for this protein.

Evidence of receptor/ligand interactions that regulate testis cell function was sought in order to broaden the current understanding of the molecular basis of testis cell function. Using reverse transcription and the polymerase chain reaction, we have obtained novel evidence for the expression of three mRNAs encoding receptor tyrosine kinases in the adult rat testis: the platelet-derived growth factor type A receptor (PDGF-RA), the basic fibroblast growth factor receptor (flg), and fetal liver kinase 1 (Flk-1). A 6.8 kb transcript encoding the PDGF-RA was observed in RNA prepared from testes of rats aged day 5 through adult, with a decline in relative abundance with increasing age after day 17. Analysis of mRNA from isolated cell preparations (day 21 Sertoli cells, adult Leydig cells, round spermatids, and primary spermatocytes) and testes depleted of specific cell types [ethane dimethane sulfonate (EDS)-treated and cryptorchid] indicated that the Leydig cell was the predominant source of this mRNA in the adult testis. The addition of PDGF-BB to cultures of highly purified adult rat Leydig cell preparations resulted in a 40% increase in LH-stimulated testosterone production, confirming a role for this growth factor in regulation of Leydig cell function. These data indicate that the Leydig cell is a principal site of action of PDGF in the testis.

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta-galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both ~70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.

Screening of a rat testis expression library with an antiserum specific for an outer dense fibre (ODF) has led to the identification of a gene encoding for a putative protein previously unknown as a component of the sperm tail. This gene has been designated tpx-1 by virtue of its homology with the mouse and human gene of the same name (79 and 73%, respectively). The tpx-1-like gene encoded a 1.6-kb mRNA and a 243-amino-acid protein that had significant homology with members of the cysteine-rich secretory protein (CRISP) family and partial homology with several venom/allergen proteins from both plants and insects. During rat spermatogenesis, the tpx-1-like transcript was first detected by in situ hybridization in low levels in late pachytene spermatocytes. Low but detectable levels of expression continued up to step 5 round spermatids, after which expression levels increased dramatically to a maximum in step 11-12 spermatids. Progressively decreasing levels of expression were detected in up to step 17 elongating spermatids. Testicular somatic cells did not contain detectable tpx-1-like transcript. This pattern of expression is consistent with published data on the development of the ODF in spermatogenesis and, when taken together with a comparison of the predicted amino acid sequence of tpx-1 with the animo acid analysis of a 29-kDa rat ODF protein, suggests that the tpx-1-like gene may encode for this protein.