Rainbow Trout Testis Research Articles

Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells.

Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.

Steroidogenesis in the gonads of rainbow trout fry (Salmo gairdneri) before and after the onset of gonadal sex differentiation

Gonadal homogenates of rainbow trout from D(ay) 60, D100 and D200 after fertilization have been incubated in vitro in the presence of dehydroepiandrosterone-3H to demonstrate 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and delta 5,4-isomerase activity, of pregnenolone-3H to assess the synthesis of "progestins" and of androstenedione-3H to determine oestrogen synthesis in the ovaries and the formation of androgens in the testes. Histologically indifferent gonads from D50 contained 3 beta-HSD, delta 5,4-isomerase and 17 alpha-hydroxylase, indicating a capacity to synthesize progestins. Ovaries from D100 possessed the same enzymes as D50 gonads; those from D200 had, in addition, 17 beta-HSD and aromatase, indicative of oestrogen synthesis. Unlike the D50 gonads, the D100 testes also contained 17 alpha, 20-desmolase and 11 beta-hydroxylase, showing a capacity to synthesize androgens. 17 beta-HSD was only demonstrated in D200 testes. The possible role of androgens and progestins in the regulation of gonadal sex differentiation in rainbow trout has been discussed. Ultrastructural and enzymecytochemical investigations have demonstrated stroma (Leydig) cells as sources of steroidogenesis in rainbow trout testis from D100 onwards. The appearance of big mitochondria with many tubular cristae in those cells synchronized with the appearance of 11 beta-hydroxylase activity. Obvious steroidogenic sites could not be demonstrated in younger gonads and developing ovaries.

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis

Spermatogenesis is a complex developmental process which sequentially generates several different germ cell types. These cell types from rainbow trout ( Salmo gairdnerii) testis were separated by sedimentation in serum albumin gradients and charaterized on the basis of their physical properties, chronological appearance, and protein synthesis. The rate of RNA synthesis, the types of RNA made, and the RNA polymerase activities present were determined for each cell type. The rate of RNA synthesis decreased from a high level in spermatogonia and spermatocytes to a low level in early spermatids and was absent in late spermatids and mature spermatozoa. Newly synthesized RNA in spermatogonia and spermatocytes consisted of a variety of molecular weight species, including 18 S and 28 S ribosomal RNAs. The synthesis of high molecular weight RNAs, especially ribosomal RNAs, decreased drastically in early spermatids, leading to the synthesis of only small molecular weight RNAs. RNA polymerase I and II were present in all cell types but the activities of both showed large decreases between spermatocytes and middle spermatids. Both RNA polymerase activities were almost absent from spermatozoa. The activities of RNA polymerase I and II from unfractionated testis cells at different stages of hormone-induced spermatogenesis were quantitated by fractionation of the solubilized extract on DEAE-cellulose. Both polymerases showed major decreases in activity which began near the chronological mid-point of development. For polymerase I the decrease in activity was over 400 fold, for polymerase II over 200 fold. The number of RNA polymerase II molecules per testis cell, quantitated by the binding of [ 3H]amanitin to cell extracts, also decreased markedly during spermatogenesis. The reduction in polymerase II activity was accompanied by a parallel 200-fold decrease in [ 3H]amanitin binding. The reduction in polymerase activity appears, therefore, to be due to an actual reduction in the cellular content of RNA polymerase II molecules. These results suggest that transcription in maturing testes is regulated, at least in part, by the concentrations of the RNA polymerases.

The Complete Amino‐Acid Sequence of a Trout‐Testis Non‐histone Protein, H6, Localized in a Subset of Nucleosomes and Its Similarity to Calf‐Thymus Non‐histone Proteins HMG‐14 and HMG‐17

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.

Sequences of large T1 ribonuclease-resistant oligoribonucleotides from protamine mRNA: the overall architecture of protamine mRNA.

Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's.

Number and frequency of protamine genes in rainbow trout testis.

Number and frequency of protamine genes in rainbow trout testis.

Protamine messenger RNA from rainbow trout testis contains the nucleotide sequence A-A-U-A-A-A in an untranslated region

Full-length, complementary DNAs were prepared to rainbow trout protamine mRNA using reverse transcriptase and were labelled during synthesis by the replacement of dATP by [α- 32 P] dATP or dTTP by [α 32 P] dTTP . The 32P-labelled protamine complementary DNAs were digested with T 4 endonuclease IV. Fragments from the digests were separated in two dimensions, and those discrete fragments which could be identified from both the A-labelled and T-labelled complementary DNAs were subjected to sequence analysis. The sequences described here all arise from the non-coding region. One pentadecanucleotide contained the sequence A-A-U-A-A-A which has been reported by Proudfoot and Brownlee ((1976) Nature 263, 211–214) to occur in the non-coding regions of six other eukaryotic mRNAs.

Identification and isolation of protamine messenger ribonucleoprotein particles from rainbow trout testis.

Treatment of rainbow trout testis polyribosomes with ethylenediaminetetraacetic acid released polyadenylated protamine messenger RNA in the form of a ribonucleoprotein (mRNP) particle. This mRNP particle which sedimented at 12-14 S could be identified by hybridization to [3H]poly(U) and was partially purified by two successive sucrose gradient sedimentations. When RNA was extracted from the mRNP particle and used as a template in the wheat germ cell-free protein synthesizing system the sole product of translation was protamine. Of this RNA, 30% contained poly(A) sequences and was shown to comigrate with polyadenylated protamine messenger RNA during polyacrylamide gel electrophoresis. When the proteins of the mRNP particle were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two prominent polypeptides with apparent molecular weights of 73 000 and 29 000 appeared reproducibly. Treatment of trout testis polyribosomes with puromycin in the presence of 0.5 M KCl released a smaller (8-10S) mRNP particle which was similarly shown to contain protamine messenger RNA. Trout testis postribosomal supernatant fraction possessed 16-18S mRNP particles containing polyadenylated RNA which cosedimented with protamine messenger RNA when the particles were dissociated with sodium dodecyl sulfate.

The purification, characterization and partial sequence determination of a trout testis non-histone protein, HMG-T.

A specific non-histone chromatin-associated protein, having a high content of both acidic and basic amino acids has been isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells. The protein has been prepared by the extraction of chromatin with 0.35 M sodium chloride and purified by chromatography on carboxymethyl-cellulose with a gradient of lithium chloride at pH 9.0. The amino acid sequence of the first 29 residues of the amino-terminal region has been determined using an automatic protein sequencer. The primary structure of this protein differs from that of any of the histones yet sequenced and, therefore, cannot be a degradation produce of any of them. Moreover, the N-terminal amino acid sequence shows considerable similarity to the HMG-1 and HMG-2 chromosomal proteins described by Goodwin et al. [Eur. J. Biochem. 38, 14-19 (1973)] whose-N-terminal sequences were also determined in this laboratory.

Renaturation kinetics of cDNA complementary to cytoplamic polyadenylated RNA from rainbow trout testis. Accessibility of transcribed genes to pancreatic DNase.

We have determined the fraction of polyadenylated cytoplasmic RNA from trout testis complementary to unique and repetitive DNA. Some 21% of the cDNA probe representative of this RNA population renatures with rapid kinetics, characteristics of repetitive sequences. The major proportion of the cDNA renatures with unique sequence DNA. Experiments with fractionated cDNA probes allow us to conclude that, in trout testis, the most abundant polyadenylated mRNAs are not preferentially transcribed from repetitive DNA, as it has shown to be the case in two eukaryotic cell lines. Treatment of trout testis nuclei with DNase I, under conditions in which 10% of the total DNA is digested, preferentially depletes the DNA of sequences being transcribed into polyadenylated mRNA. These data confirm the results of H. Weintraub and M. Groundine [(1976) Science 193, 848-856] and those of A. Garel and R. Axel [(1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3966-3970] and suggest that the conformation of DNA in the active genes of chromatin is such that it is more susceptible to digestion by DNaseI.

Purification and properties of biologically active rainbow trout testis protamine mRNA.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.

Fish Gonadotropin(s). III. Evidence for More Than One Gonadotropin in Chum Salmon Pituitary Glands

Two proteins with gonadotropin activity have been isolated from a highly purified chum salmon (Oncorhynchus keta) gonadotropin preparation (G-75 Fraction II) by chromatography on DEAE Bio Gel A. These gonadotropins exhibited distinct behaviour in polyacrylamide gel electrophoresis, chromatography on Sephadex G-75 superfine, and ratios of cAMP stimulation in immature rainbow trout ovaries and testes. Rechromatography of G-75 Fraction II on Sephadex G-75 superfine gave a symmetrical protein peak with a coincident cAMP activity profile. Repeated freezing and thawing elicited a shift in the cAMP activity profile toward the trailing edge of the protein peak. Data are discussed in terms of two gonadotropin molecules which respond differently to phase changes. Charge polymorphism was exhibited by isoelectric focusing in polyacrylamide gels of one of the DEAE fractions. Five UV absorbing bands were observed which stimulated cAMP production in immature rainbow trout gonads. Three of these bands increased adenyl cyclase activity in trout ovaries and testes. One of the bands stimulated cAMP production primarily in trout testes and the other stimulated trout ovaries, providing evidence for two gonadotropins, each of which is sex specific.

Histone IIb1 from Rainbow Trout: COMPARISON IN AMINO ACID SEQUENCE WITH CALF THYMUS IIB1

Histone IIb1 from immature testes of rainbow trout has been highly purified and then succinylated or maleylated and treated with trypsin. Thirteen tryptic peptides have been purified, with a total composition accounting for all amino acid residues in intact trout IIb1. Eleven of these peptides have been sequenced completely, and partial sequences have been obtained for the other two peptides. Intact histone IIb1 was also treated with chymotrypsin and with N-bromosuccinimide. Eleven peptides were isolated in pure or nearly pure form. Compositional and sequence information from these peptides allowed all but two of the tryptic peptides to be placed in unambiguous order, and these two peptides were placed by homology with the recently published sequence of calf histone IIb1 (Y eoman , L. C., O lson , J. O. J., S ugano , N., J ordan , J. J., T aylor , C. W., S tarbuck , W. C., and B usch , H. (1972) J. Biol. Chem. 247, 6018–6023). Rainbow trout histone IIb1 and calf IIb1 appear to differ in primary sequence by four point substitutions and four deletions. The NH2-terminal half of IIb1 is highly conserved in sequence, with the most striking evolutionary changes seen near the COOH terminus of the molecule. The highly regular spacing of basic residues as well as the existence of partially acetylated and phosphorylated residues in the NH2-terminal region of IIb1 suggests a specific role for this region of the molecule for interaction with each of the two phosphate backbones forming the major groove of the DNA double helix in eukaryotic chromosomes.

Protein kinase from rainbow-trout-testis ribosomes. Partial purification and characterization.

A protein kinase specifically associated with the ribosomal fraction has been isolated from rainbow trout testis. The enzyme was extracted from isolated trout‐testis ribosomes in 0.6 M KCl, and was separated from trout testis protamine kinase by hydroxylapatite chromatography. The ribosomal protein kinase is a Mg2+‐dependent enzyme that will transfer the terminal phosphoryl group from ATP into O‐phosphoseryl linkages of the substrate. The enzyme will catalyze the phosphorylation of several basic proteins, and the slightly lysine‐rich histone IIb2 is most readily phosphorylated followed by protamine and ribosomal proteins. Histones I, IIb1 and III are phosphorylated at an intermediate rate, while the arginine‐rich histone IV and an acidic protein such as phosvitin are poor substrates. The enzyme is stimulated by the addition of dithiothreitol, and cyclic AMP enhances enzyme activity between 10 and 25%.

Acetylation of Trout Testis Histones in Vivo: SITE OF THE MODIFICATION IN HISTONE IIb1

Abstract Extensive acetylation of four of the five major histones occurs during spermatogenesis in rainbow trout testis. At least one site of acetylation can be detected in histone IIb1, two sites in IIb2, two sites in histone III, and four sites in histone IV. No acetylation of histone I has been observed. The major site of acetylation in IIb1 is at lysyl residue 5. This region of the IIb1 molecule is homologous to the corresponding region in histone IV.

Intracellular distribution and substrate specificity of steroid 11 -hydroxylase in testes of rainbow trout (Salmo gairdneri).

Subcellular fractions of testes of rainbow trout (Salmo gairdneri) were prepared by a differential centrifugation, examined under an electron microscope, and incubated with 4-14C-labeled androstenedione, testosterone, 11-deoxycorticosterone, or 11-deoxycortisol at 16°C for 60 min under an aerobic condition. After identification of the metabolites obtained, activities of 11β-hydroxylase, 17α-hydroxylase, and 17β-hydroxysteroid dehydrogenase per unit weight of protein were estimated from the quantities of the metabolites. Among the subcellular fractions, the activity of 11β-hydroxylase upon testosterone, 11-deoxycorticosterone, and 11-deoxycortisol was found concentrated in the mitochondrial fraction, while the activities of 17α-hydroxylase upon 11-deoxycorticosterone and 17β-hydroxysteroid dehydrogenase upon androstenedione and testosterone were localized mostly in the microsomal fraction. Moreover, the 11β-hydroxylase in the rainbow trout testes preferred 11-deoxycorticosterone most and 11-deoxycortisol next as the substrate, whereas testosterone was hydroxylated at 11β-position in low yield and androstenedione was not. By raising incubation temperature from 16°C to 37°C, 11β-hydroxylation of testosterone was not enhanced at all, while the 17β-hydroxysteroid dehydrogenase activity was increased more than two times as much as the activity at 16°C. From the results obtained, the biosynthetic pathway of 11-oxygenated androgens in the teleostean testes were discussed.