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Absence of ribosomal DNA amplification in a meroistic polytrophic ovary
Printed in Sweden Copyright @ 1976 by Acudemic Press, Inc. raewed All rights of reproducriorr in any
form
Experimental
ABSENCE
Cell Research 101 (1976) 23-30
OF RIBOSOMAL
A MEROISTIC
DNA AMPLIFICATION
POLYTROPHIC
IN
OVARY
M. D. CAVE and J. SIXBEY Department of Anatomy, University of Arkansas College of Medicine, Little Rock, AR 72201, USA
SUMMARY The relative proportion of the genome coding for rRNA was determined in somatic and gametogenic tissues of the silkworm moth Bombyi mori. The nuclei of nurse cells, which account for more than 90% of the ovarian DNA, are highly - __polyploid, each containing hundreds of nucleoli. RNA-DNA hybridization analysis indicates that approx. 0.025-0.03% of the DNA from male and female gametogenic tissues and from somatic tissues hybridizes with Xenopus laevis rRNA. The fact that DNA hybridizing with I-RNA comprises approximately the same proportion of the total DNA extracted from ovarioles, testis, and somatic tissues indicates that rDNA is not amplified in the nurse cells of B. mori. Polyploidization of the entire nurse cell genome rather than amplification of a small part of it accounts for increasing the amount of rDNA in the ovary.
Amplification of DNA coding for ribosomal not established. The work of Vincent et al. RNA (rDNA) occurs in the ovaries of a [28] suggests that rDNA is amplified wide variety of organisms. During the in multinucleolate oocytes, but not in amplification process the genes coding for uninucleolate oocytes. Dawid & Brown 18s and 28s ribosomal RNA and an asso- [7] have reported amplification of rDNA ciated “spacer” DNA are replicated many in the uninucleolate oocyte of Urechis times in the absence of replication of the caupo. remainder of the genome [3, 121. This is The oocytes of insects show extensive highly advantageous to the organism. In diversity in regard to the mechanisms which the frog Xenopus luevis a single oocyte can contribute to oocyte development. In synthesize ribosomal RNA (rRNA) at a rate panoistic ovaries, which are characteristic comparable to an equal volume of liver of the older orders of insects, the oocyte tissue containing about 200000 nuclei [2]. nucleus actively synthesizes RNA and the If the oocytes of X. laevis contained the chromosomes demonstrate a well-denormal somatic rDNA complement and veloped lampbrush type condition. Large synthesized rRNA at a rate equal to rapidly numbers of active nucleoli develop, and in growing X. laevis cells in culture it would many cases the nucleoli are associated with take some 400 years for them to synthesize the presence of large extrachromosomal the amount of rRNA found in a mature DNA containing bodies [6]. Multiple oocyte [21]. Whether or not amplification of nucleoli and extrachromosomal DNA rDNA is a universal phenomenon as- bodies appear to be manifestations of rDNA sociated with oogenesis in all organisms is amplification [4, 261. Exp Cd/ RPS 101 (1976)
24
Cave and Sixbe,
In meroistic ovaries the chromosomes of the oocyte nucleus are condensed into a small heteropyknotic karyosphere and demonstrate little if any RNA synthesis. The nucleolar apparatus itself is inactive. The RNA synthetic activities of the oocyte are taken over by nurse cells or trophocytes. In the telotrophic meroistic ovary the nurse cells are localized at the apical end of each ovariole and communicate with the oocyte via long nutritive cords through which RNA synthesized by the nurse cells is transported to the oocyte. In the polytrophic meroistic ovary each oocyte is associated with 1, 3, 7, or 15 nurse cells. The nurse cells are derived from the same cell that gave rise to the oocyte. The nurse cells, which are highly polyploid, are active in RNA synthesis and may develop many active nucleoli. Intercellular channels provide for transport of RNA from nurse cells to oocyte [18]. In order to determine whether rDNA is amplified in the nurse cells or oocytes of such a polytrophic ovary, the relative amount of rDNA in ovarioles of the oriental was moth Bombyx mori silkworm compared with the relative amount of rDNA in testis and in various somatic tissues.
MATERIALS
AND METHODS
Eggs of the oriental silkworm moth, Bombvx mori, w&h were obtained from Turtox Biological Supply House (Chicago. Ill.) were hatched at 25°C. The young larvae were reared on fresh mulberry leaves in Petri dish moisture chambers for one week. Subsequently, the animals were moved to large screencovered pans where they received 14 h of light and 10 h of dark, and were supplied with fresh water and mulberry leaves daily. Various tissues were collected from ureuunae - -. larvae and IO-dav-old nuuae with the aid of a drssecting microscope. The-tissues were either frozen on drv ice and stored at -70°C (eviscerated carcasses and total silk glands) or placed in 70% ethanol and stored at -20°C (testis, ovarioles, posterior and anterior silk glands). Exp Cell Res 101 (1976)
Preparation
of DNA
Tissues treated with 70% ethanol were extracted for 1 h, 37°C. in 2 x SSC (SSC=O.15 M NaCI. 0.015 M sodium citrate, pH 7.0) containing 200 pg/ml pamylase (Sigma Chemical Co, St Louis, MO), and then for 2 h, 37°C in 2 x SSC containing 100 Fg/ml RNase-A (Worthington Biochemicals. Freehold, N. J.) which had been heated to 90°C for IO min. The tissues were subsequently digested for 2 h at 37°C with 100 pg/ml Pronase (Calbiochem, San Diego, Calif.) which had been allowed to autodigest for 2 h at 37°C. The 2 x SSC containing the enzymes was gently removed and tissues were washed with several changes of 2 x SSC. Then the tissues were suspended in I ml of 0.1 M Tris buffer. 0.05 M EDTA, 0.5% SDS PH 7.5. After 1 h thev were homogenized for 3 min at high speed in a Sorvall omnimixer (DuPont Co., Newtown, Corm.). The homogenate was centrifuged at 1900 g for 30 min. The supernate was mixed with 4 ml saturated CsCl solution and the DNA purified by isopycnic banding in CsCI. DNA was extracted from frozen tissue by a modification of the technique described by Gage [I I]. Frozen tissues were homogenized for 5 min in SSC containing 0.1 M Tris, 0.05 M EDTA, 0.5% SDS, pH 7.5, at high speed in the Sorvall omnimixer. The homogenate was made 1 mglml autodigested pronase and incubated for 3 h at 37°C on a magnetic stirrer. The pronase digest was extracted for 30 min with an equal volume of water saturated phenol. The aqueous phase was re-extracted with phenol, made 0.2 M sodium acetate, and the nucleate precipitated by the addition of 2 vol of absolute ethanol. After washing with ethanol, the nucleate was dissolved in 2 x SSC. digested at 37°C for 1 h with P-amylase (200 pg/ml), 2 h with RNase A(100 &ml), and 1 h with nronase (100 pg/ml). The ‘digest-was extracted twice with water-saturated phenol and DNA precipitated from the aqueous phase with ethanol. It was further purified on CsCl gradients. DNA was suspended in CsCl solution, the final density of which was 1.70, and centrifuged for 60 h, 2o”C, 115695 g in the Spinco SW-50.1-rotor of the Beckman L-2 65B preparative ultracentrifuge (Spinco Division, Beckman Instruments, Palo Alto, Calif.). The gradients were fractionated and the DNA containing tubes identified by ultraviolet spectrophotometry (260 nm). Tubes containing DNA were pooled, and dialyzed overnight against 0.1 X SSC. The DNA was stored at high concentration (I-10 mglml) in 0.1 X SSC.
Preparation
of labeled RNA
Difficulties in preparing labeled RNA of sufficient specific activity for hybridization experiments precluded the use of B. mori rRNA. Previous studies Figs Z4. Light microscope sections of B. mori ovary stained according to the Feulgen procedure (fig. 1) or with azure B according to the procedure of Gabruscwvcz-Garcia & Kleinfetd IlO1 (figs 2, 3, 4). Arrows indicate nucleoli within the nuclei-of oocytes (figs 1,2,4) and nurse cells (fig. 3). x750.
rDNA amplification
in a polytrophic
ovary
25
2
x
* -..j
26
Cave and Sixbe) RNase A in 2xSSC on a wrist action shaker. Subsequently, the filters were washed with 2xSSC and the amount of RNA bound to each filter was determined by liquid scintillation counting. The amount of DNA on each filter was determined by measuring the amount of A,, absorbing material in a 5% perchloric acid hydrolysate (70°C) of the filters.
RESULTS I
: 2
: 3
: 4
J 5
6
5. Abscissa: vol. of cvst x IO8 (urn?: ordinate: (left) no. follicle cell nucleijcyst; (rig&) “0. nucleoli/ nurse cell nucleus. Regression lines for the number of didoid follicle cell &lei (-); and the number of nucleoli in a nurse cell nucleus (an indication of ploidy level) (- --) in ovarian cysts of different size classes. The number of follicle cells associated with 130 ovarian cysts ranging in size from 0.5-6.0~ 106 pm3 was estimated f;om counts made on Feulgen-stained sections of B. mori ovaries. The number of nucleoli in nuclei of 168 nurse cells associated with cysts of the same size range was estimated from counts made on azure B stained sections. The regression lines were plotted from the equation Y=a+b (X). The correlation coefficients for the number of follicle cell nuclei/cyst and the number of nucleoli/nurse cell nucleus were 0.9171 and 0.5576, respectively. Fk.
have demonstrated extensive homologies between the rRNA of a wide variety of organisms [25]. The rRNA of the South African clawed toad, Xeno~us laevis, has been utilized extensively in such hybridization experiments. Labeled 18s and 28s RNA was prepared by growing a cell line of X. hevis kidney cells (line CCL 102A, obtained from the American Type Culture Collection, Rockville, Md) in modified Leibovitz L-15 medium with 15% fetal calf serum. Cells were grown for 3-5 days in medium containing 25 /.&i/ml r5JHluridine (spec. act 29.2 Ci/mMole. New England Nic1ea.r Co:,-Boston, Mass.) and then for an additional 24 h in 2 changes of fresh medium containing unlabeled uridine. Subsequently, RNA was extracted from the cells by a modification of the phenol procedure and 18s and 28s RNA isolated as described elsewhere [4]. rRNA was reconstituted by combining 1 part 18s to 2 parts 28s RNA.
RNA-DNA
hybridization
CsCl banded DNA was denatured in 0.2 N NaOH, neutralized, and bound to Millipore HAWP nitrocellulose membrane filters (Millipore Corp., Bedford, Mass.). The filters were dried for 2 h at 25°C and then for 2 h at 80°C in vacua, and stored over desiccant. Filters were hvbridized for 18 h at 68°C in RNA solutions conta&ng 4xSSC and 0.4% SDS. After hybridizatiqn the filters were washed in several changes of 2xSSC and treated for 1 h with 100 pg/ml Exp Cell Res IO1 (1976)
The ovarioles of prepupae are encased in a connective tissue sheath. There are four ovarioles in each ovary. In each ovariole there is a repeating sequence of nurse cellsoocyte. Each oocyte is associated with a cluster of nurse cells (fig. 2). The nuclei of the nurse cells are large and intensely Feulgen positive reflecting a high degree of polyploidy (fig. 1). Each nurse cell nucleus demonstrates a large number of nucleoli which stain purple violet with azure B (figs 2, 3). The germinal vesicle of the oocyte is Feulgen negative (fig. 1). The oocyte nucleus contains a single nucleolus which stains purple violet with azure B (fig. 4). For hybridization analysis DNA was extracted from ovarioles from which the ovarian sheath had been stripped, thus reducing contamination of nurse celloocyte DNA with somatic cell DNA. The relative contribution of nurse cell DNA to the total ovarian DNA extract, was assessed by cytological means. The relative ploidy level can be estimated from nucleolar number [8]. Follicle cell nuclei have a single nucleolus and contain approximately the diploid amount of DNA. Nurse cell nuclei are several orders of magnitude larger than follicle cell nuclei, are intensely Feulgen positive and contain hundreds of nucleoli. These three factors reflect a high degree of polyploidy. As the size of the ovarian cyst increases so does the number of follicle cells associated with it. A similar increase occurs in the number of nucleoli present in the nurse cell nucleus
t-DNA amplification
in a polytrophic
ovary
27
of this single band. There are no detectable differences between DNA extracted from o0 I,,.. somatic tissues (fig. 7d, e) and gametogenic O”o 0 0 o 0.’ ,69.. tissues (fig. 7 b, c) in the proportion of DNA sedimenting in the region of the gradients where the genes are coding for rRNA sediment. --200 RNA-DNA hybridization analysis indicates that 0.025 to 0.030% of the DNA extracted from ovarioles, testis, silk gland and eviscerated carcass hybridizes with X. ..I00 laevis rRNA (fig. 8). The fact that there are no detectable differences between female and male gametogenic tissues and somatic 30 tissues in the relative amount of DNA Fig. 6. Abscissa: tube no.; ordinate: (ZOWU left) sedimenting in the region of the gradient extinction 260 nm, 0-O; (upper left) density g/cm3, where rDNA sediments, nor in the per0 0; (lower right) radioactivity 3H-cpm, 0- --0. Den&v of 8. mori DNA hvbridizine with rRNA. cent of DNA hybridizing with rRNA at
I 7L0 o 0
DNA isdlated from the testis-of B. iori was sedimented to equilibrium in CsCl and the gradients fractionated. The density of each fraction was determined from the relationship between density and refractive index [16]. The amount of DNA in each fraction was determined by ultraviolet spectrophotometry and the DNA in each fraction was denatured and bound to nitrocellulose filters. The filters were hybridized with 3H-labeled X. laevis 18s and 28s rRNA (spec. act. 250000 cpm/pg).
(fig. 5). The follicle cell number and the number of nurse cell nucleoli increase in a parallel manner; the ratio, nurse cell nucleolar number: follicle cell number, remains about 2: 1. Since there are seven nurse cells in each cyst the data indicate that more than 90% of the DNA extracted from the ovary is derived from the nurse cells. B. mori DNA hybridizing with rRNA has a buoyant density of 1.706 g/cm3 while bulk DNA has a density of 1.699 (fig. 6). In the analytical ultra centrifuge B. mori DNA demonstrates a single band with a buoyant density of 1.699 g/cm3 (fig. 7a). NO satellite bands are detectable, the DNA hybridizing with rRNA sedimenting on the denser side
,731
I699
Fig. 7. Abscissa: Density g/cm3; ordinate: extinction 260 nm. Isopycnic CsCl density gradient patterns of B. mori DNA in neutral CsCI. DNA was mixed with CsCl solution, brought to a density of 1.7 and centrifuged at 44000 rpm for 20 h at 20°C in the An-GTi rotor of a Spinco model E ultracentrifuge equipped with a monochromometer, multiplexer, and photoelectric scanner. Scans were made at a wavelength of 260 nm (a) 0.2 AZ60units of DNA from testis of B. mori with Micrococcus lysodeiktius DNA (0.05 AzeOunits, Q= 1.731 &m3) as an internal density marker; (b-e) 0.6 A,, units of DNA from (b) testis; (c) prepupae ovaries; (d) silk glands; and (e) carcasses. Exp Cell Res 101 (1976)
28
Cave and Sixhey
2
1<.+-
I>
9t
+-++-~ .+J I? 25
8. Abscissa: pg/ml RNA; ordinate: 5% DNA hybridized. Filters containing DNA isolated from silk glands (0); carcasses (M): ovaries (0); and testis (0~ were hybridized with increasing concentrations 3H-labeled X: luevis 18s and 28S-RNA (spec. act. 250000 cpmli4. Fig.
saturation, indicates that amplification of rDNA does not occur in the ovarioles of B. mori.
Comparison of the data from these experiments with the studies of Gage [ 1I] in which B. mori DNA was hybridized with B. mori rRNA indicate that X. laevis rRNA hybridizes with B. mori rDNA about 20% as efficiently as B. mori does. The size of the B. mori haploid genome is 0.5 X lo-l2 g DNA [22]. Assuming that the molecular weight of B. mori rRNA is 2.2 X lo6 D (the combined molecular weight of X. laevis 18S+28S RNA), one would expect that there be 170-200 genes for rRNA per haploid genome. DISCUSSION Amplification of DNA has been detected in oocytes within the polytrophic ovary of certain Dytiscidae (Coleoptera) where the process is associated with the presence of a large extrachromosomal DNA body “Giardini’s mass” [13]. Unlike the oocytes of most species of insects having polytrophic ovaries the germinal vesicles of the Dytiscid water beetles actively synthesize RNA and develop many active nucleoli [27]. Similar extrachromosomal DNA bodies have been found in oocytes within Exp CellRrs 101 (1976)
the polytrophic ovary of many species of Tipulidae (Diptera) the oocyteh of which actively synthesize RNA [ 11. Cytological observations suggest that in addition to the increased transcription capacity provided for by polyploidization of the nurse cell chromosomes, DNA within the nurse cell nucleolar organizer is also amplified. In Tip&a oleracea, a large extrachromosomal DNA body similar to that found in the oocytes is present within the nucleolar apparatus of some nurse cells [ 191. Large numbers of nucleoli develop within the nurse cell nuclei of Calliphora erythrocephala (Diptera) [24]. In specific strains which show polytenization of the nurse cell chromosomes, the nucleoli are seen to lie free of the chromosomes. In other tissues the nucleoli are attached to a specific chromosome loci. The free nucleoli synthesize RNA, and contain small DNA particles which can be visualized by various histochemical procedures. Therefore, in addition to polyploidization of the chromosomes, the nurse cells appear to form additional copies of the nucleolar organizer DNA. RNA-DNA hybridization experiments indicate that DNA extracted from the polytrophic ovary of Rhynchosciara angelae (Diptera) contains twice as much DNA complementary to rRNA as does DNA from salivary glands [14]. This difference appears to be due to underreplication of rDNA in the polytene chromosomes of the salivary gland rather than amplification of rDNA in the ovary [15]. DNA extracted from the polytrophic ovaries of Drosophila melanogaster (Diptera) and Rhynchosciara americana (Diptera) contains the same proportion of DNA hybridizing with rRNA as does DNA extracted from whole animals or carcasses [15, 201. These results are complicated by the fact that whole animals or carcasses
rDNA amplification
contain polytene cells which are known to underreplicate their rDNA. Underreplication of rDNA has been detected in the ovaries of D. virilis, D. hydei, and Sarcophaga barbata (Diptera) [9, 231. A low level of rDNA is present in spermatogonia of X. laevis. The amplified copies of rDNA disappear prior to the onset of meiosis [17]. Cytological analysis of B. mori prepupae testis reveals cells in various stages of meiosis up to the mature spermatozoa. In the lo-day pupae testis all of the cells have entered spermiogenesis. In both prepupae and lo-day-old pupae testis approx. 0.02% of the DNA hybridizes with rRNA. There appears to be no amplification of DNA in testis at this time. In B. mori there is extensive diversity in the degree of polyploidy expressed by cells of different tissues. Silk glands are believed to contain up to 1000000 times the amount of DNA found in the gamete [22]. Most of the nuclei from carcass fall in the size class of diploid cells and probably contain the diploid amount of DNA [ 111. The ovarian nurse cells display a high level of ploidy as indicated by their nuclear volume, intense Feulgen reaction, and large number of nucleoli. The fact that the proportion of the genome coding for rRNA is the same for silk gland, the cells of which are highly polyploid; for carcass, most of the cells of which are diploid; for testis, which contains few if any premeiotic cells; and for isolated ovarioles, in which more than 90% of the DNA is nurse cell DNA indicates that rDNA is not amplified in the nurse cells. Thus the increased number of nucleoli in the nurse cells reflects polyploidization of the entire genome rather than specific amplification of genes coding for rRNA. The fact that the proportion of the genome coding for rRNA in silk glands is the same as that of whole
in a polytrophic
ovary
29
carcass DNA indicates that the ribosomal genes are not amplified nor are they underreplicated in this highly polyploid tissue. These observations are in agreement with the observations of Gage [l l] who found that the relative proportions of repetitive and non-repetitive sequence DNA as well as the relative proportions of rDNA were the same in DNA extracted from silk glands and carcasses. Therefore, uniform replication of the entire genome without specific loss or amplification of any DNA fraction accounts for the increase in DNA content of the silk gland cells. The genes for rRNA are not amplified in nurse cells within the telotrophic ovary of Oncopeltus fasciatus (Hemiptera) [5]. If they are amplified in the oocyte this represents less than a 50-fold enrichment in rDNA. In 0. fasciatus most of the nurse cells contain between 32 and 64 times the haploid amount of DNA (E. Rasch, personal communication). A low level of amplification by the oocytes of B. mori would not be detected in the present study. The present studies as well as those made on 0. fasciatus indicate that in the typical meroistic ovary (telotrophic and polytrophic) it is polyploidization of the nurse cells and not rDNA amplification which accounts for increasing the number of genes coding for rRNA. We gratefully acknowledge the kindness of Drs G. Dalrymple and J. Moss, Department of Radiology, Little Rock Veterans Administration Hospital, for putting their preparative and analytical ultracentrifuges at our disposal. Mr Larry Hulsey provided technical assistance. This work was supported by USPHS grant lRO1 GM-21446. One of us (J. S.) was partially supported by a work study grant from NIH.
REFERENCES 1. Bayreuther, K, Naturwiss 54 (1%7) 189. 2. Brown, D D, Current topics in developmental biology (ed A Monroy & A A Moscona) vol. 2, p. 47. Academic Press, New York (1966). Exp Cell Res I01 (1976)
30
Cuve and Sixhe)
3. Brown, D D & Dawid. 1 B, Science 160 (1968) 272. 4. Cave. M D. J cell biol 55 (1972) 310. 5. - Ibid 66 (1975) 461. 6. Cave, M D & Allen. E R. Exo cell res 58 (1969) 201. 7. Dawid, I B & Brown, D D. Dev biol 22 (1970) 1. 8. Dear&, W H, J morph01 56 (1934) 157. 9. Endow, S A & Gall, J G, Chromosoma 50 (1975) 175. 10. Gabrusewycz-Garcia, N & Kleinfeld, R, J cell biol 29 (1966) 347. 11. Gage L P, J mol bio186 (1974) 97. 12. Gall, J G, Proc natl acad sci US 60 (1968) 533. 13. Gall, J G Macgregor, H C & Kidston, M E, Chromosoma 26 (1969) 169. 14. Gambarini, A G & Meneghini, R, J cell biol 54 (1972) 421. 15. Gambarini, A G & Lara, F J S, J cell biol 62 (1974) 215. 16. Ifft, J B, Voet, D H & Vinograd, J, J physiol them, 65 (1961) 1138. 17. Kalt, M, J cell biol 62 (1974) 460.
Exp Cell RPS 101 (1976)
18. King. R C, J morph01 I21 (1967) 55. 19. Lima-de-Faria, A & Moses. M J, J cell biol 30 (1966) 177. 20. Mohan, J & Ritossa, F M, Dev biol 22 (1970) 495. 21. Perkowska, E. Macgregor, H C & Birnstiel, M, Nature 217 (1968) 649. 22. Rasch, E M, Chromosoma 45 (1974) 1. 23. Renkawitz, R & Kunz, W. Chromosoma 53 (1975) 131. 24. Ribbert, D & Bier, K, Chromosoma 27 (1969) 178. 25. Sinclair, J H & Brown, D D. Biochemistry IO (1971) 2761. 26. Ullman, D S, Lima-de-Faria, A, Jaworska, H & Bryngelsson, Hereditas 74 ( 1973) 13. 27. Urbani, E & Russo-&a, S, Rendiconti inst sci univ Camerino 5 (1964) 19. 28. Vincent, W S, Halvorson, H 0, Chen, H R & Schin, D, Exp cell res 57 (1969) 240. Received February 24, 1976 Accepted March 4, 1976.
form
Experimental
ABSENCE
Cell Research 101 (1976) 23-30
OF RIBOSOMAL
A MEROISTIC
DNA AMPLIFICATION
POLYTROPHIC
IN
OVARY
M. D. CAVE and J. SIXBEY Department of Anatomy, University of Arkansas College of Medicine, Little Rock, AR 72201, USA
SUMMARY The relative proportion of the genome coding for rRNA was determined in somatic and gametogenic tissues of the silkworm moth Bombyi mori. The nuclei of nurse cells, which account for more than 90% of the ovarian DNA, are highly - __polyploid, each containing hundreds of nucleoli. RNA-DNA hybridization analysis indicates that approx. 0.025-0.03% of the DNA from male and female gametogenic tissues and from somatic tissues hybridizes with Xenopus laevis rRNA. The fact that DNA hybridizing with I-RNA comprises approximately the same proportion of the total DNA extracted from ovarioles, testis, and somatic tissues indicates that rDNA is not amplified in the nurse cells of B. mori. Polyploidization of the entire nurse cell genome rather than amplification of a small part of it accounts for increasing the amount of rDNA in the ovary.
Amplification of DNA coding for ribosomal not established. The work of Vincent et al. RNA (rDNA) occurs in the ovaries of a [28] suggests that rDNA is amplified wide variety of organisms. During the in multinucleolate oocytes, but not in amplification process the genes coding for uninucleolate oocytes. Dawid & Brown 18s and 28s ribosomal RNA and an asso- [7] have reported amplification of rDNA ciated “spacer” DNA are replicated many in the uninucleolate oocyte of Urechis times in the absence of replication of the caupo. remainder of the genome [3, 121. This is The oocytes of insects show extensive highly advantageous to the organism. In diversity in regard to the mechanisms which the frog Xenopus luevis a single oocyte can contribute to oocyte development. In synthesize ribosomal RNA (rRNA) at a rate panoistic ovaries, which are characteristic comparable to an equal volume of liver of the older orders of insects, the oocyte tissue containing about 200000 nuclei [2]. nucleus actively synthesizes RNA and the If the oocytes of X. laevis contained the chromosomes demonstrate a well-denormal somatic rDNA complement and veloped lampbrush type condition. Large synthesized rRNA at a rate equal to rapidly numbers of active nucleoli develop, and in growing X. laevis cells in culture it would many cases the nucleoli are associated with take some 400 years for them to synthesize the presence of large extrachromosomal the amount of rRNA found in a mature DNA containing bodies [6]. Multiple oocyte [21]. Whether or not amplification of nucleoli and extrachromosomal DNA rDNA is a universal phenomenon as- bodies appear to be manifestations of rDNA sociated with oogenesis in all organisms is amplification [4, 261. Exp Cd/ RPS 101 (1976)
24
Cave and Sixbe,
In meroistic ovaries the chromosomes of the oocyte nucleus are condensed into a small heteropyknotic karyosphere and demonstrate little if any RNA synthesis. The nucleolar apparatus itself is inactive. The RNA synthetic activities of the oocyte are taken over by nurse cells or trophocytes. In the telotrophic meroistic ovary the nurse cells are localized at the apical end of each ovariole and communicate with the oocyte via long nutritive cords through which RNA synthesized by the nurse cells is transported to the oocyte. In the polytrophic meroistic ovary each oocyte is associated with 1, 3, 7, or 15 nurse cells. The nurse cells are derived from the same cell that gave rise to the oocyte. The nurse cells, which are highly polyploid, are active in RNA synthesis and may develop many active nucleoli. Intercellular channels provide for transport of RNA from nurse cells to oocyte [18]. In order to determine whether rDNA is amplified in the nurse cells or oocytes of such a polytrophic ovary, the relative amount of rDNA in ovarioles of the oriental was moth Bombyx mori silkworm compared with the relative amount of rDNA in testis and in various somatic tissues.
MATERIALS
AND METHODS
Eggs of the oriental silkworm moth, Bombvx mori, w&h were obtained from Turtox Biological Supply House (Chicago. Ill.) were hatched at 25°C. The young larvae were reared on fresh mulberry leaves in Petri dish moisture chambers for one week. Subsequently, the animals were moved to large screencovered pans where they received 14 h of light and 10 h of dark, and were supplied with fresh water and mulberry leaves daily. Various tissues were collected from ureuunae - -. larvae and IO-dav-old nuuae with the aid of a drssecting microscope. The-tissues were either frozen on drv ice and stored at -70°C (eviscerated carcasses and total silk glands) or placed in 70% ethanol and stored at -20°C (testis, ovarioles, posterior and anterior silk glands). Exp Cell Res 101 (1976)
Preparation
of DNA
Tissues treated with 70% ethanol were extracted for 1 h, 37°C. in 2 x SSC (SSC=O.15 M NaCI. 0.015 M sodium citrate, pH 7.0) containing 200 pg/ml pamylase (Sigma Chemical Co, St Louis, MO), and then for 2 h, 37°C in 2 x SSC containing 100 Fg/ml RNase-A (Worthington Biochemicals. Freehold, N. J.) which had been heated to 90°C for IO min. The tissues were subsequently digested for 2 h at 37°C with 100 pg/ml Pronase (Calbiochem, San Diego, Calif.) which had been allowed to autodigest for 2 h at 37°C. The 2 x SSC containing the enzymes was gently removed and tissues were washed with several changes of 2 x SSC. Then the tissues were suspended in I ml of 0.1 M Tris buffer. 0.05 M EDTA, 0.5% SDS PH 7.5. After 1 h thev were homogenized for 3 min at high speed in a Sorvall omnimixer (DuPont Co., Newtown, Corm.). The homogenate was centrifuged at 1900 g for 30 min. The supernate was mixed with 4 ml saturated CsCl solution and the DNA purified by isopycnic banding in CsCI. DNA was extracted from frozen tissue by a modification of the technique described by Gage [I I]. Frozen tissues were homogenized for 5 min in SSC containing 0.1 M Tris, 0.05 M EDTA, 0.5% SDS, pH 7.5, at high speed in the Sorvall omnimixer. The homogenate was made 1 mglml autodigested pronase and incubated for 3 h at 37°C on a magnetic stirrer. The pronase digest was extracted for 30 min with an equal volume of water saturated phenol. The aqueous phase was re-extracted with phenol, made 0.2 M sodium acetate, and the nucleate precipitated by the addition of 2 vol of absolute ethanol. After washing with ethanol, the nucleate was dissolved in 2 x SSC. digested at 37°C for 1 h with P-amylase (200 pg/ml), 2 h with RNase A(100 &ml), and 1 h with nronase (100 pg/ml). The ‘digest-was extracted twice with water-saturated phenol and DNA precipitated from the aqueous phase with ethanol. It was further purified on CsCl gradients. DNA was suspended in CsCl solution, the final density of which was 1.70, and centrifuged for 60 h, 2o”C, 115695 g in the Spinco SW-50.1-rotor of the Beckman L-2 65B preparative ultracentrifuge (Spinco Division, Beckman Instruments, Palo Alto, Calif.). The gradients were fractionated and the DNA containing tubes identified by ultraviolet spectrophotometry (260 nm). Tubes containing DNA were pooled, and dialyzed overnight against 0.1 X SSC. The DNA was stored at high concentration (I-10 mglml) in 0.1 X SSC.
Preparation
of labeled RNA
Difficulties in preparing labeled RNA of sufficient specific activity for hybridization experiments precluded the use of B. mori rRNA. Previous studies Figs Z4. Light microscope sections of B. mori ovary stained according to the Feulgen procedure (fig. 1) or with azure B according to the procedure of Gabruscwvcz-Garcia & Kleinfetd IlO1 (figs 2, 3, 4). Arrows indicate nucleoli within the nuclei-of oocytes (figs 1,2,4) and nurse cells (fig. 3). x750.
rDNA amplification
in a polytrophic
ovary
25
2
x
* -..j
26
Cave and Sixbe) RNase A in 2xSSC on a wrist action shaker. Subsequently, the filters were washed with 2xSSC and the amount of RNA bound to each filter was determined by liquid scintillation counting. The amount of DNA on each filter was determined by measuring the amount of A,, absorbing material in a 5% perchloric acid hydrolysate (70°C) of the filters.
RESULTS I
: 2
: 3
: 4
J 5
6
5. Abscissa: vol. of cvst x IO8 (urn?: ordinate: (left) no. follicle cell nucleijcyst; (rig&) “0. nucleoli/ nurse cell nucleus. Regression lines for the number of didoid follicle cell &lei (-); and the number of nucleoli in a nurse cell nucleus (an indication of ploidy level) (- --) in ovarian cysts of different size classes. The number of follicle cells associated with 130 ovarian cysts ranging in size from 0.5-6.0~ 106 pm3 was estimated f;om counts made on Feulgen-stained sections of B. mori ovaries. The number of nucleoli in nuclei of 168 nurse cells associated with cysts of the same size range was estimated from counts made on azure B stained sections. The regression lines were plotted from the equation Y=a+b (X). The correlation coefficients for the number of follicle cell nuclei/cyst and the number of nucleoli/nurse cell nucleus were 0.9171 and 0.5576, respectively. Fk.
have demonstrated extensive homologies between the rRNA of a wide variety of organisms [25]. The rRNA of the South African clawed toad, Xeno~us laevis, has been utilized extensively in such hybridization experiments. Labeled 18s and 28s RNA was prepared by growing a cell line of X. hevis kidney cells (line CCL 102A, obtained from the American Type Culture Collection, Rockville, Md) in modified Leibovitz L-15 medium with 15% fetal calf serum. Cells were grown for 3-5 days in medium containing 25 /.&i/ml r5JHluridine (spec. act 29.2 Ci/mMole. New England Nic1ea.r Co:,-Boston, Mass.) and then for an additional 24 h in 2 changes of fresh medium containing unlabeled uridine. Subsequently, RNA was extracted from the cells by a modification of the phenol procedure and 18s and 28s RNA isolated as described elsewhere [4]. rRNA was reconstituted by combining 1 part 18s to 2 parts 28s RNA.
RNA-DNA
hybridization
CsCl banded DNA was denatured in 0.2 N NaOH, neutralized, and bound to Millipore HAWP nitrocellulose membrane filters (Millipore Corp., Bedford, Mass.). The filters were dried for 2 h at 25°C and then for 2 h at 80°C in vacua, and stored over desiccant. Filters were hvbridized for 18 h at 68°C in RNA solutions conta&ng 4xSSC and 0.4% SDS. After hybridizatiqn the filters were washed in several changes of 2xSSC and treated for 1 h with 100 pg/ml Exp Cell Res IO1 (1976)
The ovarioles of prepupae are encased in a connective tissue sheath. There are four ovarioles in each ovary. In each ovariole there is a repeating sequence of nurse cellsoocyte. Each oocyte is associated with a cluster of nurse cells (fig. 2). The nuclei of the nurse cells are large and intensely Feulgen positive reflecting a high degree of polyploidy (fig. 1). Each nurse cell nucleus demonstrates a large number of nucleoli which stain purple violet with azure B (figs 2, 3). The germinal vesicle of the oocyte is Feulgen negative (fig. 1). The oocyte nucleus contains a single nucleolus which stains purple violet with azure B (fig. 4). For hybridization analysis DNA was extracted from ovarioles from which the ovarian sheath had been stripped, thus reducing contamination of nurse celloocyte DNA with somatic cell DNA. The relative contribution of nurse cell DNA to the total ovarian DNA extract, was assessed by cytological means. The relative ploidy level can be estimated from nucleolar number [8]. Follicle cell nuclei have a single nucleolus and contain approximately the diploid amount of DNA. Nurse cell nuclei are several orders of magnitude larger than follicle cell nuclei, are intensely Feulgen positive and contain hundreds of nucleoli. These three factors reflect a high degree of polyploidy. As the size of the ovarian cyst increases so does the number of follicle cells associated with it. A similar increase occurs in the number of nucleoli present in the nurse cell nucleus
t-DNA amplification
in a polytrophic
ovary
27
of this single band. There are no detectable differences between DNA extracted from o0 I,,.. somatic tissues (fig. 7d, e) and gametogenic O”o 0 0 o 0.’ ,69.. tissues (fig. 7 b, c) in the proportion of DNA sedimenting in the region of the gradients where the genes are coding for rRNA sediment. --200 RNA-DNA hybridization analysis indicates that 0.025 to 0.030% of the DNA extracted from ovarioles, testis, silk gland and eviscerated carcass hybridizes with X. ..I00 laevis rRNA (fig. 8). The fact that there are no detectable differences between female and male gametogenic tissues and somatic 30 tissues in the relative amount of DNA Fig. 6. Abscissa: tube no.; ordinate: (ZOWU left) sedimenting in the region of the gradient extinction 260 nm, 0-O; (upper left) density g/cm3, where rDNA sediments, nor in the per0 0; (lower right) radioactivity 3H-cpm, 0- --0. Den&v of 8. mori DNA hvbridizine with rRNA. cent of DNA hybridizing with rRNA at
I 7L0 o 0
DNA isdlated from the testis-of B. iori was sedimented to equilibrium in CsCl and the gradients fractionated. The density of each fraction was determined from the relationship between density and refractive index [16]. The amount of DNA in each fraction was determined by ultraviolet spectrophotometry and the DNA in each fraction was denatured and bound to nitrocellulose filters. The filters were hybridized with 3H-labeled X. laevis 18s and 28s rRNA (spec. act. 250000 cpm/pg).
(fig. 5). The follicle cell number and the number of nurse cell nucleoli increase in a parallel manner; the ratio, nurse cell nucleolar number: follicle cell number, remains about 2: 1. Since there are seven nurse cells in each cyst the data indicate that more than 90% of the DNA extracted from the ovary is derived from the nurse cells. B. mori DNA hybridizing with rRNA has a buoyant density of 1.706 g/cm3 while bulk DNA has a density of 1.699 (fig. 6). In the analytical ultra centrifuge B. mori DNA demonstrates a single band with a buoyant density of 1.699 g/cm3 (fig. 7a). NO satellite bands are detectable, the DNA hybridizing with rRNA sedimenting on the denser side
,731
I699
Fig. 7. Abscissa: Density g/cm3; ordinate: extinction 260 nm. Isopycnic CsCl density gradient patterns of B. mori DNA in neutral CsCI. DNA was mixed with CsCl solution, brought to a density of 1.7 and centrifuged at 44000 rpm for 20 h at 20°C in the An-GTi rotor of a Spinco model E ultracentrifuge equipped with a monochromometer, multiplexer, and photoelectric scanner. Scans were made at a wavelength of 260 nm (a) 0.2 AZ60units of DNA from testis of B. mori with Micrococcus lysodeiktius DNA (0.05 AzeOunits, Q= 1.731 &m3) as an internal density marker; (b-e) 0.6 A,, units of DNA from (b) testis; (c) prepupae ovaries; (d) silk glands; and (e) carcasses. Exp Cell Res 101 (1976)
28
Cave and Sixhey
2
1<.+-
I>
9t
+-++-~ .+J I? 25
8. Abscissa: pg/ml RNA; ordinate: 5% DNA hybridized. Filters containing DNA isolated from silk glands (0); carcasses (M): ovaries (0); and testis (0~ were hybridized with increasing concentrations 3H-labeled X: luevis 18s and 28S-RNA (spec. act. 250000 cpmli4. Fig.
saturation, indicates that amplification of rDNA does not occur in the ovarioles of B. mori.
Comparison of the data from these experiments with the studies of Gage [ 1I] in which B. mori DNA was hybridized with B. mori rRNA indicate that X. laevis rRNA hybridizes with B. mori rDNA about 20% as efficiently as B. mori does. The size of the B. mori haploid genome is 0.5 X lo-l2 g DNA [22]. Assuming that the molecular weight of B. mori rRNA is 2.2 X lo6 D (the combined molecular weight of X. laevis 18S+28S RNA), one would expect that there be 170-200 genes for rRNA per haploid genome. DISCUSSION Amplification of DNA has been detected in oocytes within the polytrophic ovary of certain Dytiscidae (Coleoptera) where the process is associated with the presence of a large extrachromosomal DNA body “Giardini’s mass” [13]. Unlike the oocytes of most species of insects having polytrophic ovaries the germinal vesicles of the Dytiscid water beetles actively synthesize RNA and develop many active nucleoli [27]. Similar extrachromosomal DNA bodies have been found in oocytes within Exp CellRrs 101 (1976)
the polytrophic ovary of many species of Tipulidae (Diptera) the oocyteh of which actively synthesize RNA [ 11. Cytological observations suggest that in addition to the increased transcription capacity provided for by polyploidization of the nurse cell chromosomes, DNA within the nurse cell nucleolar organizer is also amplified. In Tip&a oleracea, a large extrachromosomal DNA body similar to that found in the oocytes is present within the nucleolar apparatus of some nurse cells [ 191. Large numbers of nucleoli develop within the nurse cell nuclei of Calliphora erythrocephala (Diptera) [24]. In specific strains which show polytenization of the nurse cell chromosomes, the nucleoli are seen to lie free of the chromosomes. In other tissues the nucleoli are attached to a specific chromosome loci. The free nucleoli synthesize RNA, and contain small DNA particles which can be visualized by various histochemical procedures. Therefore, in addition to polyploidization of the chromosomes, the nurse cells appear to form additional copies of the nucleolar organizer DNA. RNA-DNA hybridization experiments indicate that DNA extracted from the polytrophic ovary of Rhynchosciara angelae (Diptera) contains twice as much DNA complementary to rRNA as does DNA from salivary glands [14]. This difference appears to be due to underreplication of rDNA in the polytene chromosomes of the salivary gland rather than amplification of rDNA in the ovary [15]. DNA extracted from the polytrophic ovaries of Drosophila melanogaster (Diptera) and Rhynchosciara americana (Diptera) contains the same proportion of DNA hybridizing with rRNA as does DNA extracted from whole animals or carcasses [15, 201. These results are complicated by the fact that whole animals or carcasses
rDNA amplification
contain polytene cells which are known to underreplicate their rDNA. Underreplication of rDNA has been detected in the ovaries of D. virilis, D. hydei, and Sarcophaga barbata (Diptera) [9, 231. A low level of rDNA is present in spermatogonia of X. laevis. The amplified copies of rDNA disappear prior to the onset of meiosis [17]. Cytological analysis of B. mori prepupae testis reveals cells in various stages of meiosis up to the mature spermatozoa. In the lo-day pupae testis all of the cells have entered spermiogenesis. In both prepupae and lo-day-old pupae testis approx. 0.02% of the DNA hybridizes with rRNA. There appears to be no amplification of DNA in testis at this time. In B. mori there is extensive diversity in the degree of polyploidy expressed by cells of different tissues. Silk glands are believed to contain up to 1000000 times the amount of DNA found in the gamete [22]. Most of the nuclei from carcass fall in the size class of diploid cells and probably contain the diploid amount of DNA [ 111. The ovarian nurse cells display a high level of ploidy as indicated by their nuclear volume, intense Feulgen reaction, and large number of nucleoli. The fact that the proportion of the genome coding for rRNA is the same for silk gland, the cells of which are highly polyploid; for carcass, most of the cells of which are diploid; for testis, which contains few if any premeiotic cells; and for isolated ovarioles, in which more than 90% of the DNA is nurse cell DNA indicates that rDNA is not amplified in the nurse cells. Thus the increased number of nucleoli in the nurse cells reflects polyploidization of the entire genome rather than specific amplification of genes coding for rRNA. The fact that the proportion of the genome coding for rRNA in silk glands is the same as that of whole
in a polytrophic
ovary
29
carcass DNA indicates that the ribosomal genes are not amplified nor are they underreplicated in this highly polyploid tissue. These observations are in agreement with the observations of Gage [l l] who found that the relative proportions of repetitive and non-repetitive sequence DNA as well as the relative proportions of rDNA were the same in DNA extracted from silk glands and carcasses. Therefore, uniform replication of the entire genome without specific loss or amplification of any DNA fraction accounts for the increase in DNA content of the silk gland cells. The genes for rRNA are not amplified in nurse cells within the telotrophic ovary of Oncopeltus fasciatus (Hemiptera) [5]. If they are amplified in the oocyte this represents less than a 50-fold enrichment in rDNA. In 0. fasciatus most of the nurse cells contain between 32 and 64 times the haploid amount of DNA (E. Rasch, personal communication). A low level of amplification by the oocytes of B. mori would not be detected in the present study. The present studies as well as those made on 0. fasciatus indicate that in the typical meroistic ovary (telotrophic and polytrophic) it is polyploidization of the nurse cells and not rDNA amplification which accounts for increasing the number of genes coding for rRNA. We gratefully acknowledge the kindness of Drs G. Dalrymple and J. Moss, Department of Radiology, Little Rock Veterans Administration Hospital, for putting their preparative and analytical ultracentrifuges at our disposal. Mr Larry Hulsey provided technical assistance. This work was supported by USPHS grant lRO1 GM-21446. One of us (J. S.) was partially supported by a work study grant from NIH.
REFERENCES 1. Bayreuther, K, Naturwiss 54 (1%7) 189. 2. Brown, D D, Current topics in developmental biology (ed A Monroy & A A Moscona) vol. 2, p. 47. Academic Press, New York (1966). Exp Cell Res I01 (1976)
30
Cuve and Sixhe)
3. Brown, D D & Dawid. 1 B, Science 160 (1968) 272. 4. Cave. M D. J cell biol 55 (1972) 310. 5. - Ibid 66 (1975) 461. 6. Cave, M D & Allen. E R. Exo cell res 58 (1969) 201. 7. Dawid, I B & Brown, D D. Dev biol 22 (1970) 1. 8. Dear&, W H, J morph01 56 (1934) 157. 9. Endow, S A & Gall, J G, Chromosoma 50 (1975) 175. 10. Gabrusewycz-Garcia, N & Kleinfeld, R, J cell biol 29 (1966) 347. 11. Gage L P, J mol bio186 (1974) 97. 12. Gall, J G, Proc natl acad sci US 60 (1968) 533. 13. Gall, J G Macgregor, H C & Kidston, M E, Chromosoma 26 (1969) 169. 14. Gambarini, A G & Meneghini, R, J cell biol 54 (1972) 421. 15. Gambarini, A G & Lara, F J S, J cell biol 62 (1974) 215. 16. Ifft, J B, Voet, D H & Vinograd, J, J physiol them, 65 (1961) 1138. 17. Kalt, M, J cell biol 62 (1974) 460.
Exp Cell RPS 101 (1976)
18. King. R C, J morph01 I21 (1967) 55. 19. Lima-de-Faria, A & Moses. M J, J cell biol 30 (1966) 177. 20. Mohan, J & Ritossa, F M, Dev biol 22 (1970) 495. 21. Perkowska, E. Macgregor, H C & Birnstiel, M, Nature 217 (1968) 649. 22. Rasch, E M, Chromosoma 45 (1974) 1. 23. Renkawitz, R & Kunz, W. Chromosoma 53 (1975) 131. 24. Ribbert, D & Bier, K, Chromosoma 27 (1969) 178. 25. Sinclair, J H & Brown, D D. Biochemistry IO (1971) 2761. 26. Ullman, D S, Lima-de-Faria, A, Jaworska, H & Bryngelsson, Hereditas 74 ( 1973) 13. 27. Urbani, E & Russo-&a, S, Rendiconti inst sci univ Camerino 5 (1964) 19. 28. Vincent, W S, Halvorson, H 0, Chen, H R & Schin, D, Exp cell res 57 (1969) 240. Received February 24, 1976 Accepted March 4, 1976.