Changes in the Sequence Diversity of Polyadenylated Cytoplasmic RNA during Testis Differentiation in Rainbow Trout (Salmo gairdnerii)

Transcription

1 Eur. J. Biochem. 74,61-67 (1977) Changes in the Sequence Diversity of Polyadenylated Cytoplasmic RNA during Testis Differentiation in Rainbow Trout (Salmo gairdnerii) Beatriz LEVY W. and Gordon H. DIXON Division of Medical Biochemistry, Faculty of Medicine, University of Calgary (Received September 6, 1976) We have compared the sequence diversity of polyadenylated cytoplasmic RNA derived from naturally maturing trout testis at three different stages of spermatogenesis, by performing hybridization experiments between cdna synthesized on a template of polyadenylated RNA and a vast excess of polyadenylated RNA. Polyadenylated RNA from early testis has a base sequence complexity of 7.2 x lo9 daltons. As testis maturation proceeds, there is a decrease in the complexity of polyadenylated mrna sequences in testis cells and the relative abundance of individual mrnas varies over a more narrow range. Heterologous hybridization reactions demonstrate that a substantial fraction of polyadenylated RNA sequences present in early testis cytoplasm is absent from late testis cytoplasm. Nevertheless, all the sequences of late testis mrna are represented in the population of early testis mrna molecules. Spermatogenesis is a complex process by which diploid spermatogonial stem cells become differentiated into haploid spermatozoa. While in most nonhibernating mammals spermatozoa are produced continuously, in fish there is a yearly cycle of spermatogenesis. This cycle takes a period of several months, during which time the testes grow rapidly and increase in weight fold [l]. Because of the seasonal nature of this phenomenon in fish, the development of the testis is relatively synchronous and, although at any time during the development of the testis all cell types are usually present [2,3 1, one cell type normally predominates and is indicative of the particular stage of maturation. In addition to the observed morphological changes (such as attenuation of the cytoplasm and condensation of the nucleus [4]), biochemical changes also occur during spermatogenesis. For instance, both in trout and salmon testis, the RNA content of the maturing spermatids progressively decreases and RNA synthesis becomes undetectable in late testis [5]. In addition, a characteristic feature of the late stages of spermatogenesis is the appearance of the protamines (sperm-specific chromosomal proteins), which eventually replace histones and give rise to highly condensed nucleoprotamine in the mature sperm Abbreviations. cdna, complementary DNA; mrna, polyadenylated cytoplasmic RNA. nucleus [4,6]. Furthermore, a marked decrease in template activity of chromatin for added Escherichia coli polymerase [4] and a reduction in the amount of non-histone chromosomal proteins [4] are also characteristic features of the later stages of testis maturation. In the present report we are concerned with the variations of sequence complexity and diversity that occur in the population of cytoplasmic polyadenylated RNAs as testis maturation proceeds. MATERIALS AND METHODS Trout Testis Testes were collected at different stages of maturation (October 1973, August 1974 and September 1974) from freshly killed trout (Dantrout, Brande, Denmark), immediately frozen on solid COZ and stored at - 70 "C. Preparation of Cytoplasmic Polyadenylated RNA Cytoplasmic extracts were prepared from testis at different stages of maturation as described above. The cytoplasmic supernatants were adjusted to 0.1 M

2 62 Cytoplasmic Poly(A)-Containing RNA NaC1, 10 mm Tris-HC1, ph 7.5, 1 mm EDTA and 0.5 % sodium dodecylsulfate and RNA was extracted as described previously [8]. Polyadenylated RNA was obtained by chromatography on poly(u)-sepharose as described earlier [8]. Synthesis of Complementary DNA The conditions for synthesis of cdna for purified protamine mrna were as follows. The complete reaction mixture, 100 pl, contained the following final concentrations of reagents: 50 mm Tris-HC1, ph 8.3, 10 mm dithiotreitol, 6.5 mm MgClz, 45 mm KC1, 10 mm NaCl, 100 pg/ml of actomycin D (Boehringer- Mannheim), 100 pg/ml bovine serum albumin, 0.6 mm of datp, dttp and dgtp, 200 pci/ml of [3H]dCTP (Schwartz-Mann, S.A. 25 Ci/mmol), 9 pg/ml of (dt)12-18, 10 pg/ml of polyadenylated RNA and 160 units/ml of avian myeloblastosis viral reverse transcrip tase (kindly supplied by Dr J. W. Beard). Incubation was for 40 min at 45 "C. After incubation, the cdna was treated with NaOH as described before [8] and further purified as described by Iatrou and Dixon [17]. Hybridization Reactions Hybridization reactions were carried out in 0.5 M NaCl, 10 mm Tris-HC1, ph 8.0, 1 mm EDTA, 0.01 sodium dodecyl sulfate. Samples (in triplicate), containing about 1000 counts/min of cdna and various amounts of RNA were sealed in 5-pl capillaries, boiled for 10 min and incubated at 70 "C for various time periods. At the end of each incubation period, samples were diluted with 1 ml of 0.3 M NaCl, 0.03 M NaOAc, 3 mm ZnCL, ph 4.5 and digested with S1 nuclease (Miles Laboratories) for 40 min at 45 "C. After incubation, the samples were precipitated with 20 % trichloroacetic acid filtered onto glass fibre filters and counted. Zero time control samples were included in every experiment. The amount of 3H remaining after S1 nuclease digestion of this control (representing self annealing of the cdna) was routinely subtracted from each experimental point. The extent of self annealing varied between 5-15% for the different cdnas. Fractionation of cdna Fractionated cdna probes were obtained from middle-late testis by partial reaction of middle-late cdna with middle-late RNA to an rot value of 1.2 moll-' s. The cdna hybridized at this point (representing abundant mrna sequences) was separated from the unreacted cdna (representative of the rare sequences) by hydroxyapatite chromatography at 70 "C as described previously [8]. RESULTS Preparation of Polyadenylated RNA and cdna Polyadenylated cytoplasmic RNA was prepared from three different batches of trout testis collected at different stages of maturation. For simplicity, we shall call them early, middle-late and late. In the absence of a precise method to ascertain the exact stages of testis development and to determine more accurately the stages of development of our three batches of testes studied, we performed an analysis of their basic chromosomal proteins by electrophoresis on starch gels [7], chromatography on CM-cellulose (CM-52) columns [9] and gel filtration on Bio-Gel P60 columns [lo] (data not shown). By comparison of our results with those of Marushige and Dixon [4], we estimate that our early preparation would be equivalent to a stage between 3 and 4, our middle-late would range between stages 4 and 5 and our late testis batch would be equivalent to developmental stage 5 as reported by these authors. The three mrna populations were sized by sedimentation on sucrose gradients after denaturation with dimethyl sulfoxide to prevent aggregation [ll] (and Levy & Dixon, unpublished results). In all three cases, a major fraction of the polyadenylated RNA sedimented with a mean value of about 6.0 S, but some material sedimenting between 18 S and 28 S was present in all preparations of RNA. This material represented about 10-20% of the mass of late testis polyadenylated mrnas (middle-late and late) and some % of the early testis mrna (Fig.1). The possibility of fragmentation of the mrnas upon treatment with dimethyl sulfoxide cannot be ruled out, since in the absence of the denaturing agent the proportion of RNA molecules sedimenting faster than 18 S was always higher in all three mrna populations studied. For this reason, it is very difficult to estimate accurately a mean sedimentation value for each of our mrna populations. All three RNA populations were used as templates for the synthesis of complementary DNA by reverse transcriptase. Reactions were totally dependent upon the addition of oligo (dt) primer, implying transcription of sequences adjacent to poly(a). The efficiencies of the three RNA preparations as templates for the viral enzyme were similar. The cdna probes displayed a very broad size distribution on alkaline sucrose gradients, with a major proportion (greater in late RNAs) representing full copies of the templates (data not shown). Complexity of Polyadenylated RNA,from Testis at Dij'erent Stages of Spermatogenesis cdna transcribed on either early, middle-late or late testis cytoplasmic polyadenylated RNA was hy-

3 B. Levy W. and G. H. Dixon E 0.1 c 0 L m $ 0 m e z: bottom Fraction number to P Fig. 1. Six rlisirrhuiiori of curly arid lute testis ~y~opi~~.srni(. polyudenyhted RNA. Early and middle-late polyadenylated RNAs were prepared as described in Methods. A sample of each RNA was incubated for 5 min at 60 "C in 80% dimethyl sulfoxide and then analyzed on a 15-30"/, sucrose gradient in 0.1 M NaCI, 10 mm Tris-HCI, ph 7.5, 1 mm EDTA, 0.5% sodium dodecyl sulfate. Centrifugation was for 19 h at rev./min in a Beckman SW-41 rotor at 22 'C. 0.5-ml fractions were collected and the absorbance at 260 nm of each fraction was determined in a Beckman Acta VI M spectrophotometer. The arrows, from left to right, denote the positions of 28-S, 18-S amd 4-S trout ribosomal RNA markers run on a parallel gradient. (0-0) Early mrna; (M----M) middle-late mrna bridized to a vast excess of the RNA used as a template. The results of these experiments are shown in Fig.2. Reproducibility was checked by performing each experiment twice using a different preparation of cdna and RNA each time. From Fig.2 we observe that, in all three cases, the reaction kinetics extend over several log units of rot indicating, threfore, the existence of RNA molecules present at widely different concentrations in the cell [8, Abundant polyadenylated RNA hybridizes more rapidly to its cdna than is the case for rare species. To calculate the number of different sequences in each of the three testis mrna populations, we applied the following equation [15]: M, of unknown RNA = rotli2 of unknown RNA rotli2 of standard RNX x M, of standard RNA. We used as a standard the reaction between purified ovalbumin mrna of M, and its cdna. Under our hybridization conditions, we obtained a rgtli2 value of moll-' s for this reaction. This rot (mol ~-'s) Fig. 2. Coni,ule.yity oj curl)., rnitlt/lc~-lute und lute testis cy~oplu,sn~ic. polyudenylated RNA. cdna was synthesized on a template of either early, middle-late or late mrna and hybridized with a vast excess of the RNA used as a template as described in Methods. ( ) Early mrna reaction; ( L M ) middle-late mrna reaction; (A----A) late mrna reaction value is in good agreement with values obtained by others [16]. When semilog rot plots are complex, the resolution of individual kinetic components may be achieved through the use of a plot linear in rot [8], or through the use of a computer program designed to obtain best fit of the data [8,11,13]. Both methods give essentially the same results [8,11]. In this report, we have chosen the former alternative (whose only pitfall is the somewhat subjective extrapolation of an asymptotic plot) since our main interest is a comparison of the three mrna populations and not an exact determination of the number of sequences in each case. Fig.3, 4 and 5 represent linear plots of the data of Fig. 2. Three kinetic components are evident in the early mrna reaction and two components are observed in the reactions of the late (middle-late and late) mrnas. Each of these kinetic components corresponds to one frequency class of mrnas. However, it should be stated that the resolution of complex semilog rot plots into individual components represents only an approximation which facilitates the interpretation of the data, since it is much more likely that such curves represent a continuous spectrum of a large number of frequency classes in which RNAs hybridizing at the highest rot values contribute the most to the overall complexity of the mrna population. In addition, there is no reason to assume that all molecules in each lo4

4 64 Cytoplasmic Poly(A)-Containing RNA -40rT, c 0 m d n Or 20 30b 100 ' rot (mol ills) Fig. 3. Linear plots of' hybridi:ation between early mrna and its cdna rot (mol I-'s) Fig. 5. Linear plots qj lijynydi;ation betn,een late mrna and its cdna 0 c - c " E 60 - [L '8 0.b8 Oj16 O(24 0: L most frequent sequences is similar in both late mrnas 20 " rot (rnol IPS) Fig. 4. Linear plots of' hybridization between middle-late mrna and its cdna mrna is about 7.2 x lo9 daltons, that of middle-late mrna about 3.6 x lo9 daltons while late mrna, has a complexity of about 1.5 x lo9 daltons. We conclude, therefore, that a significant reduction in the complexity of polyadenylated RNA sequences occurs in the cytoplasm of testis cells as spermatogenesis proceeds. In addition, the kinetics of the reaction indicate that the relative concentrations of these mrnas also changes during testis differentiation. For example, Fig.2 shows that, the most abundant mrnas of early testis (rot < 0.2 moll-' s) are present at a much higher concentration in the cell than is the case for the most frequent mrnas of late testis (middle-late and late), as evidenced by a more rapid reaction rate in the early mrna reaction. Furthermore, the relative concentrations in the cell of these studied (middle-late and late). On the other hand, the concentration of the less abundant, more rare sequences is the highest for the late mrna, decreases in middle-late mrna, and it is even lower in early mrna (Fig. 2). frequency class are represented in exactly equal numbers. In Table 1, the data of Fig. 3-5 are summarized and show that the total complexity of early testis Degree of Sequence Overlap among Early and Late Testis Polyadenyluted RNA Populations The results in Table 1 demonstrated that early mrna had a complexity of about 7.2 x lo9 daltons

5 ~~ ~ - B. Levy W. and G. H. Dixon 65 Source of mrna cdna reacted rot1iz Complexity ~- ~ ~ ~ ~ - - ~~~ observed corrected daltons Early testis Middle-late testis Late testis ~ lo x lo x x lo x x lo x 109 I 80 I 60 -.' I' 70 t,.-!? c u $ o-~ t0-l too 10' 10' lo3 lo4 rot (mol IPS) Fig. 6. Hybridization between early cdna and early and middle-late mrnas. Hybridizations, in RNA excess, were performed as indicated in Methods. (O----O) Reaction of early cdna and early mrna; (-) reaction of early cdna and middle-late mrna and that the complexity of middle-late mrna was about 3.6 x lo9 daltons. Next, we proceeded to examine the degree of sequence overlap existing among these two RNA populations. To this effect, we performed heterologous hybridization reactions, using cdna from one stage of maturation and RNA from the other. Several experiments of this type were performed and some examples will be described. For instance, our data would predict that middle-late mrna would not react to completion with early cdna. When early cdna was annealed to an excess of middle-late mrna (Fig. 6), the reaction reached a plateau at a value significantly lower than that attained when the same cdna was annealed rot (mot Ps) Fig. 7. Hybridizations between ji.e4urnt und infrequent middle-lute testis cdna probes and early and middle-late testis mrna. Frequent and infrequent testis cdna probes were obtained as described in Methods, and hybridized with early and middle-late mrna. (-) Reaction of frequent middle-late cdna and middle-late mrna; (A-.-,-A) reaction of frequent middle-late cdna and early mrna; (@----*) reaction of infrequent middle-late cdna and middle-late mrna; ( ) reaction of infrequent middlelate cdna and early mrna with early mrna (Fig.6). Middle-late mrna only reacted with 74 % (48/65) of the early cdna, indicating that middle-late mrna was deficient in sequences present in early mrna. However, because we do not know precisely to which abundance class the unreacted cdna corresponds, we cannot say that 26% of the sequences present in early mrna were missing from middle-late mrna. The kinetics of the reaction further suggest that sequences of high abundance in early mrna are much less abundant in middle-late mrna,

6 66 Cytoplasmic Poly(A)-Containing RNA as indicated by a displacement of the reaction driven by middle-late mrna to higher rot values (Fig. 6). To decide whether all the sequences present in middle-late mrna were represented in the early mrna population, we performed the converse experiment, i.e. hybridization between middle-late cdna and early mrna. In this case, we chose to use cdna probes representing either the most frequent or the most infrequent sequences in middle-late testis mrna. A large batch ( lo6 counts/min) of middle-late testis cdna was fractionated into frequent and infrequent late cdna probes, by hybridization with middle-late testis mrna to an rot value of 1.2 moll-' s and further purification on hydroxyapatite as described in Methods. Successful fractionation was validated through hybridization of the fractionated cdna probes with late testis mrna. Frequent cdna reacted much faster than the infrequent cdna probe (Fig. 7). Early testis mrna reacted as well as middle-late testis mrna with both fractionated late testis cdna probes (Fig. 7), therefore proving that all of the sequences of middle-late mrna were also present in early mrna. In addition, the kinetics of these reactions demonstrated that the most abundant sequences of middle-late mrna were less abundant (more diluted) in early mrna (since the rot curve was displaced to the right Fig.7) and also that the concentration of the less frequent sequences of middle-late cdna was similar in both populations of RNA. DISCUSSION Determination of the complexity of polyadenylated RNA by hybridization of cdna prepared from it to an excess of RNA represents a new approach to the study of the diversity and sequence relationships between different populations of polyadenylated RNAs. The limitations inherent in this method have been thoroughly discussed [8, Complexity measurements obtained in this manner always represent minimum estimates because of the difficulties in resolving individual components in the hybridization kinetics and in accurately determining the rotli2 value of the slowest component. A possible non-random behavior of reverse transcriptase would have the same effect. In this report, we have analyzed the distribution of the relative numbers of individual cytoplasmic poly (A)-containing RNAs within the populations of cells derived from testis at three different stages of spermatogenesis. Our results show that there are wide variations in the number of different polyadenylated RNAs present at different stages of testis maturation. Furthermore, the frequency distribution of individual mrnas differs considerably from one stage of maturation of the testis to the next. A significant reduction in the number of mrna sequences occurs as spermato- genesis proceeds. These findings are in agreement with those of Ando and Hashimoto [5] which indicate that the total RNA content of the testis sharply decreases during spermatogenesis in trout and with a reported reduction in chromatin template activity during testis maturation [4]. The results of experiments designed to measure the degree of overlap among the sequences present at different stages of spermatogenesis allow us to conclude that many sequences are lost as the tissue passes through the later stages of spermatogenesis. In addition, reactions performed with fractionated late testis cdna probes representative of the most frequent or most infrequent sequences of polyadenylated RNA, demonstrate that early testis mrna possesses all of the sequences also present in late testis mrna but at different concentrations. Chromosomal protein analysis was performed to characterize more precisely the state of maturation of each batch of testis employed. By comparison with the data of Marushige and Dixon [4], we estimate that the developmental stage of the testes used in the recent study vary from stages 3-5 as described by these authors. Unfortunately, none of the batches of testis used was representative of a very early stage or a very late stage of spermatogenesis. Furthermore, because each batch of testis is heterogenous in the sense that a population of naturally maturing fish was used without selection of size of the fish or testis weight, the actual developmental changes in mrna diversity are likely to be much larger than indicated here. Nevertheless, it seems clear that significant changes do occur in the populations of polyadenylated mrnas as testis development proceeds. Further experiments are underway involving preparation of mrnas from specific cell types, each representative of a well-defined stage in spermatogenesis (obtained by sedimentation in gradients of bovine serum albumin) [2] and should yield a much more precise description of the modulations in diversity and frequencies of mrna associated with the process of spermatogenesis in trout testis. We are indebted to Dr J. Beard, for supplying us with purified avian myeloblastosis viral reverse transcriptase. We thank Norman Wong for performing the starch gel electrophoresis analysis of the proteins and for his helpful advice. We also thank K. Iatrou for his comments and suggestions. This work was supported by a grant from the Medical Research Council of Canada. REFERENCES 1. Robertson, 0. H. & Rinfret, A. P. (1957) Endocrinology, 60, Louie, A. J. & Dixon, G. H. (1972) J. Biol. Chem. 247, Louie, A. J. & Dixon, G. H. (1972) J. Bid. Chem. 247, Marushige, K. & Dixon, G. H. (1969) Dev. Bid. 19, ,

7 B. Levy W. and G. H. Dixon Ando,T. & Hashimoto,C. (1958) J. Biochem. (Tokyo) 45, Ingles, C. J.,Trevithick, J. R., Smith, M. &Dixon, G. H. (1966) Biochem. Biophys. Res. Commun. 22, Sung, M. & Smithies, 0. (1969) Biopolymers, 7, Levy W., B. & McCarthy, B. J. (1975) Biochemistry, 14, Ling, V., Jergil, B. & Dixon, G. H. (1971) J. Bid. Chem. 246, Marushige, K. & Dixon, G. H. (1971)J. Bid. Chem. 246, Ryffel, G. U. (1976) Eur. J. Biochem. 62, Bishop, J. O., Morton, J. G., Rosbach, M. & Richardson, M. (1974) Nature (Lond.) 250, Axel, R., Feigelson, P. & Schutz, G. (1976) Cell 1, Young, B. D., Birnie, G. D. & Paul, J. (1976) Bioc/wmistry, 15, Lewin, B. (1974) in Gene Expression, vol. 2, pp. 243, John Wiley, New York. 16. Sullivan, D., Palacios, R., Stavnezer, J., Taylor, J. M., Faras, A. J., Kiely, M. L., Summers, N. M., Bishop, J. M. & Schimke, R. T. (1973) J. Biol. Chem. 248, Iatrou, K. & Dixon, G. H. (1977) CeN, in press. B. Levy W. and G. H. Dixon, Division of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, T2N 1N4