Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis

To discover germ cell-specific genes, we used in silico subtraction and identified testis expressed gene 14 (Tex14). Mouse Tex14 contains an open reading frame encoding a 1450-amino-acid protein, which shares 64% amino acid identity with the predicted human TEX14 protein. The predicted TEX14 amino acid sequence consists of three ankyrin repeats, a protein kinase domain, and a leucine zipper dimerization motif. Northern blot analysis and in situ hybridization show that Tex14 mRNA is expressed specifically in the testis, with highest levels observed in pachytene, diplotene, and meiotically dividing spermatocytes. Two 5' splice variants of mouse Tex14 were discovered by sequencing 5'-RACE polymerase chain reaction products. TEX14 is predicted to be localized to the nucleus, suggesting that it may play a key role in regulating gene expression or modulating nuclear events during mammalian spermatogenesis.     

Cloning of type 1 cannabinoid receptor in Rana esculenta reveals differences between genomic sequence and cDNA

The endocannabinoid system is a conserved system involved in the modulation of several physiologic processes, from the activity of the central nervous system to reproduction. Type 1 cannabinoid receptor (CNR1) cDNA was cloned from the brain and testis of the anuran amphibian, the frog Rana esculenta. Nucleotide identity ranging from 62.6% to 81.9% is observed among vertebrates. The reading frame encoded a protein of 462 amino acids (FCNR1) with all the properties of a membrane G-coupled receptor. Alignments of FCNR1 with those of other vertebrates revealed amino acid identity ranging from 61.9% to 88.1%; critical domains for CNR1 functionality were conserved in the frog. As nucleotide differences of cnr1 cDNA were observed in brain and testis, the genomic sequence of the cnr1 gene was also determined in the same tissue preparations. Nucleotide changes in codons 5, 30, 70, 186, 252 and 408 were observed when cDNA and genomic DNA were compared; the nucleotide differences did not affect the predicted amino acid sequences, except for changes in codons 70 and 408. Interestingly, the predicted RNA folding was strongly affected by different nucleotide sequences. Comparison of cnr1 mRNA sequences available in GenBank with the corresponding genomic sequences revealed that also in human, rat, zebrafish and pufferfish, nucleotide changes between mRNA and genomic sequences occurred. Furthermore, amino acid sequences deduced from both mRNA and the genome were compared among vertebrates, and also in pufferfish the nucleotide changes corresponded to modifications in the amino acid sequence. The present results indicate for the first time that changes in nucleotides may occur in cnr1 mRNA maturation and that this phenomenon might not be restricted to the frog.     

Appearance of thymosin alpha 1 in supernatants of monocytes incubated with prothymosin alpha.

Prothymosin alpha, a polypeptide of 109 to 111 amino acid residues, contains the entire thymosin alpha 1 sequence (residues 1-28) at its amino terminal. Human peripheral blood monocytes incubated with prothymosin alpha release thymosin alpha 1 in the culture supernatants. In addition total RNA is found to increase. The production of thymosin alpha 1 involves de novo protein synthesis as shown by the kinetics of this release and its inhibition by actinomycin D and cycloheximide. Thymosin alpha 1 release, possibly in association with HLA-DR, stimulates the proliferation of the T cell population.     

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B).

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.     

Identification and characterization of evolutionarily conserved pufferfish, zebrafish, and frog orthologs of GASZ

We previously identified Gasz (a germ cell-specific gene encoding a protein containing four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper) in six mammalian species. Here, we report GASZ orthologs in pufferfish (Fugu rubripes), zebrafish (Danio verio), and frog (Xenopus laevis). Sequences of the three Gasz cDNAs were determined by database mining and 5'- and 3'-rapid amplification of cDNA ends (RACE) followed by sequencing. The three orthologous vertebrate genes encode proteins structurally similar to mammalian GASZ and contain the characteristic four ankyrin repeats (ANKs) and sterile-alpha motif (SAM). Their ANK and SAM domains share 55- 74% and 38-55% amino acid identity with those in human GASZ, respectively. Similar to human and mouse Gasz genes, pufferfish Gasz is composed of 13 exons, spanning approximately 12 kilobases, and flanked by Cftr at its 5'-end and Wnt2 at its 3'-end. Northern and Western blot analyses detect frog Gasz expression only in testis and ovary. In situ hybridization and immunohistochemical analyses show that frog Gasz mRNA and protein expression is confined to pachytene spermatocytes in the testis and to oocytes in the ovary. In frog oocytes, GASZ protein appears to localize to a cytoplasmic structure resembling the Balbiani body, a postulated mRNA transport organizer in the cytoplasm. The high evolutionary conservation and germ cell specificity suggest that GASZ plays an essential role in gametogenesis. The data presented here are important for future studies of the physiological roles of GASZ using fish and amphibians as animal models.     

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.