Structure and expression of rodent genes encoding the testis-specific cytochrome c. Differences in gene structure and evolution between somatic and testicular variants

Mammalian testis contains two forms of cytochrome c, one identical to the form found in somatic tissues and a second that is expressed in a stage-specific manner during spermatogenic differentiation. We have isolated both rat and mouse cDNA clones and the rat gene encoding the testis-specific cytochrome c and determined their DNA sequences. The testicular variant displays a number of notable differences with its somatic counterpart. 1) In contrast to the multipseudogene family derived from mammalian somatic cytochrome c genes, the testis gene is single-copy in genomic DNA with no detectable pseudogenes. 2) The rat testis gene is approximately 7 kilobases (kb) long with three introns totaling nearly 6.5 kb whereas the two introns dividing the 2.1-kb somatic gene occupy only 0.9 kb. Introns differ in position as well as size. 3) The testicular variant has a longer 5'-untranslated leader (230 versus 70 base pairs for the somatic gene) with an upstream open reading frame of 129 base pairs beginning with an AUG in a favorable translational context. 4) A single polyadenylation site in the testicular mRNA (approximately 900 nucleotides) contrasts with the three functionally equivalent sites observed in rat somatic messages. 5) Finally, rat and mouse testis cytochromes c differ at 4 amino acid residues as opposed to the complete sequence identity found in the somatic proteins suggesting a shorter unit evolutionary period for these molecules. These observations are consistent with a duplication of an ancestral cytochrome c gene leading to the emergence of novel structural features and regulatory properties likely associated with the striking tissue specificity of the testicular cytochrome c.     

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.     

大鼠睾丸发育过程中促甲状腺激素释放激素受体mRNA的表达

为研究促甲状腺激素释放激素受体(TRHR)在大鼠睾丸组织中的表达规律和在生殖发育调节中的作用,依据大鼠垂体中的TRH-RcDNA设计引物,采用RT-PCR法从大鼠睾丸组织中获得了TRH-R的cDNA克隆,测序表明其核苷酸序列与大鼠垂体中的TRH-RcDNA序列完全一致.应用非放射性原位杂交(NR-ISH)技术观察TRH-RmRNA在大鼠睾丸中的定位,结果显示杂交信号集中在间质细胞中,生精细胞无杂交信号.利用实时动态定量RT-PCR法观察了TRH-R在不同发育阶段大鼠睾丸中的表达变化,发现在睾丸间质细胞发育的初期阶段(第8天),没有TRH-R的表达,但从第15天起能观察到TRH-R的表达,并且表达量在20天、35天、60天、90天逐渐增加.这些结果表明,大鼠睾丸组织间质细胞能特异性表达TRH-R,并且表达量与发育过程相关.     

Differential expression of protein kinase C alpha and delta in testes of mouse at various stages of development

Protein kinase C (PKC) describes a family of serine/threonine protein kinases, and multiple isoforms are expressed in various mammalian tissues. In the present study, we examined the expression of PKC-alpha and PKC-delta at protein and mRNA level in mouse testis by Western blotting and RT-PCR. We also examined the expression of both PKC isoenzymes in the developing mouse testis. In testes of mouse at various developmental stages, both the protein and the mRNA of PKC-alpha were uniformly distributed; but PKC-delta expression occurred in the testes of 3-week-old mice, perhaps even at a relatively late stage in spermatid development. The results suggest that each isoenzyme may have different functional roles in processing and modulating physiological cellular responses of spermatogenesis.