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The uterine epithelium of Ascaris lumbricoides as a model system for the study of polyploidy
Experimental
Cell Research 56 (1969) 275-280
THE UTERINE EPITHELIUM OF ASCARIS LUMBRICOIDES AS A MODEL SYSTEM FOR THE STUDY OF POLYPLOIDY A. D. FLOYD’
and F. J. SWARTZ
Department of Anatomy, University of Louisville, Louisville, Ky 40202, USA
SUMMARY For several reasons Ascaris uterine epithelium is useful for the study of polyploidy. It shows a range of polyploid values which, when expressed as average nuclear DNA, correlate with variations in cytoplasmic ribonucleoprotein. The arrangement of the uterine epithelial cells in a single sheet consisting of large cells makes possible detailed analysis of cell function from end to end of the uterus. This possibility, coupled with the ability to study the uterine epithelium in both differentiating and adult ammals, permits study of the function of single cells or discrete populations of cells during the development of polyploidy. Thus use of Ascaris uterine epithelium as an experimental system may yield important information concerning the significance of polyploidy in biological systems.
Localized tissue polyploidy is a biological phenomenon of widespread occurrence [19, 201, yet its functional significance has eluded detection. Although occurring in various mammalian tissues [2, 9, 10, 11, 12, 221,localized tissue polyploidy has received most extensive investigation in mammalian liver [14]. These studies have yielded little insight into the functional significance of polyploidy however. One of the basic problems in elucidating the significance of polyploidy has been the lack of an adequately characterized systemfor investigation. Cultured cell lines [5] have permitted some comparisons between diploid and heteroploid cells and study of plant tissues has indicated certain differences or differentiations between polyploid and diploid cells [ 18, 211. In invertebrate systems polyploidy has a protective value for cells exposed to ionizing radiations [23]. The study of polytene systems has provided much data which has been extrapolated to polyploid systems. This extrapolation is based on the similarity of polyploidy and polyteny in terms 1 Present address: Department of Anatomy, University of Michigan, Ann Arbor, Mich. 48104, USA. 18-691803
of total nuclear DNA and may or may not be a valid comparison. The relationship of polyploidy to differentiation has been suggested for the past 20 years. Huskins states “The implication from work by Bushnell & Geitler is that nuclei which have become endopolyploid may be highly active in some metabolic processes. . .” [7]. The regular occurrence of polyploidy in highly secretory insect tissues and such active mammalian tissues as liver has also suggested that polyploidy is related to functional differentiation. Painter [15] seems convinced that polyploid cells have an increased capability for polyribosome production. A cell system with well defined functional parameters and nuclear DNA variations would be useful for study of the functional significance of polyploidy. The amount of nuclear DNA varies in uterine epithelial cells of Ascaris Zumbricoides [17] and it will be demonstrated in this study that these variations fall into polyploid classes. Further, it will be shown that these polyploid classes are arranged in linear fashion
from one end of the uterus to the other
and are correlated with variations in cell morExptl Cell Res 56
276 A. D. Floyd h F. J. Swartz
phology and cytoplasmic RNA content. The uterine epithelial cells are of adequate size for studies of cytoplasmic specialization and are arranged in a manner that facilitates correlation with cell function. Although changes in nuclear DNA occurring during differentiation have not been worked out in detail, it can be demonstrated that the polyploid gradient of the uterine epithelium is correlated with the differentiation or maturation of the animal. MATERIALS AND METHODS Live specimens of Ascaris lumbricoides were collected at a local abbatoir along with swine intestinal contents. The worms were washed with and maintained in a balanced salt solution [I]. As soon after collection as possible the parasites were decapitated and the uteri removed. These tissues were fixed for 2 h in Carnoy’s fluid and embedded in Tissuemat. The uterus of Ascaris reaches 10 to 12 cm in length in mature animals. It was arbitrarily divided into segments of equal length and each segment sectioned at 5 to 15 p. A rigidly controlled Feulaen nrocedure was emuloved with-a hydrolysis time of? n& as reported by SW&z et al. 1171for tissues of Ascaris. These DNA urenarations were quantitated using a Barr & Stroud integrating microdensitometer [4]. Feulgen microspectrophotometry requiries measurement of whole or uncut nuclei, but in Ascaris uterus, even in 15 ,u sections, many nuclei extend through more than one section. To obtain individual nuclear values serial sections were prepared and nuclei mapped and numbered. After measuring each nuclear slice in each section, it was possible to reconstruct cut nuclei and obtain a reasonable estimate of total nuclear DNA. To eliminate errors due to possible loss of DNA from sectioned nuclei and avoid any error as the result of the reconstruction process, uterine whole mounts were prepared. These whole mounts were obtained by slitting uteri lengthwise while immersed in balanced salt solution and then gently flushing out the eggs with a jet of the immersing fluid from a small syringe. This process results in an epithelial sheet because the uterine wall of Ascaris consists only of an epithelium on a thick basement membrane with a very sparse muscle layer. This muscle layer is so sparse as to cause no problems in subsequent quantitation. The epithelial sheets were placed on slides previously coated with albumin and were briefly dried on a 50°C hot plate and subsequently fixed in Carnoy’s fluid. These whole mounts were stained by the Feulgen reaction and quantitated as described above. Variations in epithelial cell cytoplasmic ribonucleic acid from end to end of the uterus were quantitated using cresyl violet staining as reported by Ritter et al. [16]. Control sections were subiected to a two hour incubation with 0.5 mg/ml ribonuclease (Worthington B&hem. Corp., Freehold, N.J.) in water adjusted to pH 6.8 at 60°C. RNA was evaluated with the Barr & Stroud microdensitometer employed as an absorption photometer. Transmittance at 590 nm through three snots 6 .U in diameter was determined in the apical cytopfasm of each cell measured. Each cell value was derived from the average of the three spots. Thirty cells were measured at Exptl Cell Res 56
each uterine level. Transmittance was converted into absorbance (extinction) by the relationship: Absorbance = - log Transmittance.
RESULTS General observations
The simple epithelium of Ascaris uterus varies morphologically from one end of the uterus to the other although all of the epithelial cells are basically similar in shape. These differentiations are expressed as size variations and differences in types of inclusion bodies [8]. In general, cells in the middle of the uterus are larger than those at either end; a typical miduterine cell measured 100 x 25 x 100,u. The epithelial cells are normally binucleate but some may contain four or more nuclei. Uninucleate cells are occasionally seen. “Amitotic” figures are frequent and no mitoses were encountered. These nuclei were not included in the DNA measurements. Complete end to end analysis of any parameter of the uterine epithelium of Ascaris is technically difficult because of the length of the uterus. The data reported in this study are based on analyses of individual animals and these data have been shown to be representative by numerous selected area analysis of a great number of animals. Total body length is pertinent in these results as this is the only criterion for age of individuals collected. Normally fertilized eggs are only found in females over 20 cm in length. Maximal lengths which have been observed are approx. 36 cm. In this study, animals between 30 and 36 cm in length have been arbitrarily classified as “mature”, and females of this size make up the bulk of any collection. DNA quantitation. Using mapped serial sections of the uterus of a mature worm (30 cm body length) peak nuclear DNA values, expressed as average DNA per nucleus in a given level of the uterus, were found in miduterus with lower values being obtained at both ends (fig. 1). Uterine DNA values were determined similarly in a small female. Although this specimen was only 24 cm in body length, fertilized eggs were present in the uterus. The smaller worm also
Polyploidy model system
yielded high DNA average values per nucleus in miduterus with lower values at both ends (fig. 1) but maximal values were lower in the smaller than in the larger animal. Further, there was a noticeable plateau of values throughout the middle region of the animal as compared to the same area of the larger animal. As seen in the histogram plot of values from the 30 cm animal (fig. 2), nuclear DNA varied widely at a given level, with the variation approaching that expected of nuclear polyploid classes. However, the many intermediate values gave considerable class overlap. In order to eliminate possible errors in the serial section approach, quantitation of nuclear DNA variability was carried out on whole mounts. As described above there was again a great difference between average DNA per nucleus values at miduterus and each end and the peak value was very high. When the whole mount values were plotted as a histogram (fig. 3) there was a definite grouping into nuclear classeswith a marked reduction in class spread compared to the results from the serial sections approach. The increase in average nuclear DNA in the miduterus was the result of increases in ploidy level of the nuclei involved and changesin the proportions of the various ploidy classes,as can be seenfrom the data plotted in fig. 3.
z
r
0 t -.ot!rq
II
217
2 f f a* XSX~ L..-
Abscissa: Relative DNA units. Fig. 2. Distribution of nuclear DNA content of serial sections of the uterus of a 30 cm specimen of Ascaris. Vertical bars represent number of nuclei of particular DNA content.
DNA analysis of 5 ,u sections of the uterus of a 6.5 cm worm (these results are confined to this one animal) indicated variation in average nuclear DNA content from end to end of the uterus but the pattern was distinctly different
500 .
450 400./’
350 -
‘\
.’ 300 250 -
.
/ *-.
0 Anterior
Pormrior
Abscissa: Uterine levels; ordinate: relative DNA units. Fig. 1. Average value of DNA per nucleus in various levels of the uterus of three specimens of Ascaris as determined by Feulgen microphotometry. 0 - 0, Sections 30 CM animal; x - x , sections 24 CM animal; e- 8, whoiemount 30 CM animal.
Abscissa: Relative DNA units. Fig. 3. Distribution of nuclear DNA content in a whole mount preparation of the uterus of Ascaris. Vertical bars represent number of nuclei of particular DNA content. Exptl Cell Res 56
278 A. D. Floyd & F. J. Swartz
Table 1. Cresyl violet RNA quantitation Uterus of 30 cm animal. Uterine levels
% Trans.
Extinction
s.E.*
Anterior A B C D E
73 62 35 32 21
0.138 0.231 0.467 0.499 0.584
: H Posterior I
27 18 31 38
0.581 0.763 0.563 0.429
0.016 0.019 0.018 0.016 0.016 0.018 0.019 0.017 0.014
RNA quantitation. With cresyl violet staining Abscissa:Uterinelevels;ordinate: relativeDNA units. highest absorbanceswere found slightly posterior Fig. 4. Averagevaluesof DNA per nucleusin various to miduterus (fig. 5). These determinations relevels of the uterusof a 6.5 cm specimenof Ascuris. vealed a large variation in cytoplasmic RNA concentration from end to end of the uterus. from that seenin reproductively mature animals Standard error of the mean calculated for the (fig. 4). Nuclei in this animal were small and the various levels quantitated demonstrated the highest absorbances measured were only one- constancy of the error envolved in these measurefifth that of the lowest values found in uteri ments, irrespective of the actual absorbancies containing fertilized eggs. Highest values in the being measured (table 1). To verify that cresyl violet staining in Ascuris small specimenwere found in the anterior uterus could be attributed to RNA, adjacent sections near the vagina with a continuous decline in to those quantitated were subjected to RNAase average nuclear DNA proceeding posteriorly. extraction. After enzyme treatment all cytoSince many of the fields measured in this small animal contained more than one nucleus, only plasmic and nucleolar staining was abolished. average values were obtained and histogram Non-nucleolar areas of nuclei still retained the plots of nuclear distributions and possible poly- stain. Posterior
Ant*rior
ploid classeswere not obtainable. 0.9 O.S-
r
0.7 0.6 0.5 0.40.3 0.2 0.1 Anterior
Post.rior
Abscissa: Uterinelevels;ordinate: extinction. Fig. 5. Distribution of the dyecresylviolet from end to end of the uterusof an adult specimenof Ascaris. Vertical bars represent S.E.M. Exptl Cell Res 56
DISCUSSION Classically, localized tissue polyploidy is defined as a multiple of the haploid chromosome number in individual nuclei. In current usage the term polyploidy is often applied to multiples of the diploid DNA value in individual nuclei. In the mammalian liver, where chromosome counts can be correlated with DNA values, this usage has proven valid. However, DNA values alone cannot distinguish between polyploidy and polyteny. No evidence of polyteny has been seen in the interphase nuclei of Ascaris uterus and the DNA variations reported here are considered to be a reflection of polyploidy. As mitotic figures were not seen in Ascaris uterine epithelium and
Polyploidy model system
have not been reported in any somatic tissue of the nematode, actual chromosome counts are not possible. A further complication is the process of chromatin diminution which occurs during Ascaris embryogenesis [3]. As this process has not been quantitated, the possibility exists that the diminished “diploid” DNA value for nuclei of various somatic tissues of the worm is not constant. Uncertainty concerning the constancy of diminution from tissue to tissue and lack of quantitative data regarding it negate attempts to describe the absolute levels of polyploidy attained in adult tissues [17]. Regardless of this limitation, the various polyploid classes in Ascaris uterus may be described as 2kC, 4kC, 8kC, etc., where k is an unknown constant. The excellent fit of measured nuclei into polyploid classessupports the constancy of k. Since high and low values of DNA content were included in these measurements, there was some loss of linearity of the DNA classes at high levels of DNA content. This is felt to be an expression of instrumental errors caused by the extreme range of concentrations covered in this study. The gradient of polyploid values in the uterine epithelium will permit comparison of cell function with polyploid level. Previous reports of the secretory function of the uterine epithelium [6] and description of morphologically distinctive cytoplasmic “secretory” materials within various segments of the epithelium [8] indicate that functional gradients may also be present in this organ. The potential of this system for investigation of the significance of polyploidy is thus apparent. The patterns of nuclear differentiation observed in this study suggest that Ascaris uterus may provide answers to the role of quantitative chromosomal variations in differentiation and maturation. Data from the immature animal reported in this study suggest that initially the uterus differentiates in an anterior to posterior direction. An anterior-posterior nucleic acid gradient has been described in the tapeworm [ 131 but not in Ascaris. In the case of adult Ascaris, this early gradient is lost as the uterus itself differentiates. Although animals have not
279
been examined in all stages of development, the evidence suggests that polyploidy is initiated before the initiation of uterine function and that further nuclear differentiation continues as the animal grows and develops. As eggsfrom young females are viable, the young uterus is functional. (Ascaris eggs are not viable unless the secondary egg coat, which is a product of the uterine secretion, is complete.) Thus the further differentiation of the uterus in older animals may be related to increased eggproduction which necessitatesincreased uterine secretion. The general occurrence of high levels of cytoplasmic RNA in secretory tissues, coupled with the extreme basophilia of the ascarid uterine epithelium prompted quantitation of cytoplasmic RNA concentrations. As the small spots through which transmissions were taken were kept constant throughout the measurements, the data represent the actual variation of RNA concentration per unit area within individual cells. Any distributional error which may have been involved due to the plug type measurements employed should have remained relatively constant throughout the measurements, thus the values given are valid relative to one another but are not indicative of absolute RNA concentration. The values reported represent RNA concentrations, not total cell RNA. Therefore, as cells in miduterus have a greater volume than those at either end of the uterus and also a higher concentration of RNA per unit area, the actual amount of RNA per cell in miduterus relative to the cells at either end of the uterus would be much greater than that shown in the RNA concentration curve. The higher concentration of ribonucleoprotein supports Painter’s hypothesis of increased RNA synthetic ability in polyploid nuclei although it certainly doesnot demonstrate either a causeor effect relationship. This work was submitted by the Senior author in partial fulfilment of the requirements for the Doctor of Philosophy degree, University of Louisville Graduate School, Louisville, Ky. Supported by NSF research grant GB 4490 and by USPHS training grant 5 TO1 GM-01356. Exptl Cell Res 56
280 A. D. Floyd & F. J. Swartz
9. Kompmann, M, Paddags, I & Sandritter, W, Arch path01 82 (1966) 303. LaCour, L F, Proc roy sot Edinburgh B 62 (1944) 73. :;: Laiham, L & Johnstone, M A, Arch neurol9 (1963)
13. Lee, T W & Campbell, J W, Nature 203 (1964) 661, 14. Naora, H, J biophys biochem cytol 3 (1957) 949. 15. Painter, T S & Biesele, J J, Proc natl acad sci US 56 (1966) 1920. 16. Ritter, P, DiStefano, H S & Farah, A, J histochem cytochem 9 (1961) 97. 17. Swartz, F J, Henry, M & Floyd, A, J exptl zoo1 164 (1967) 297. 18. Torrey, J G, Science 128 (1958) 1148. 19. Tschermak-Woess, E, Protoplasma 46 (1956) 798. 20. White, M J D, Animal cytology and evolution, 2nd ed. Cambridge University Press (1954). 21. Witsch, H & Flugel, A, Z Botan 40 (1952) 281. 22. Wodsedalek, J E, Anat ret 81 (1941) 79. 23. Zirkel, R E & Tobias, C E, Arch biochem biophys 42 (1953) 2.
12. Lapham, L W, Excerpta med intern congr 100 (1965) 445.
Received December 19, 1968
REFERENCES Baldwin, E & Moyle, V, J exptl biol 23 (1947) 277. 1: Beams, H W & King, R L, Anat ret 83 (1942) 281. 3. Bonnevie, K, Jena Z 29 (1901) 275. 4. Deeley, E M, J sci instr 32 (1955) 263. 5. Eagle, H, Science 148 (1965) 42. Fairbairn, D, Exptl parasitol 6 (1957) 491. f : Huskins, C L & Steinitz, L M, J heredity 39 (1948) K & Yanagisawa, T, Jap j med sci bio17 (1954) 8. r3s5dii,
95.
Exptl Cdl Res 56
Cell Research 56 (1969) 275-280
THE UTERINE EPITHELIUM OF ASCARIS LUMBRICOIDES AS A MODEL SYSTEM FOR THE STUDY OF POLYPLOIDY A. D. FLOYD’
and F. J. SWARTZ
Department of Anatomy, University of Louisville, Louisville, Ky 40202, USA
SUMMARY For several reasons Ascaris uterine epithelium is useful for the study of polyploidy. It shows a range of polyploid values which, when expressed as average nuclear DNA, correlate with variations in cytoplasmic ribonucleoprotein. The arrangement of the uterine epithelial cells in a single sheet consisting of large cells makes possible detailed analysis of cell function from end to end of the uterus. This possibility, coupled with the ability to study the uterine epithelium in both differentiating and adult ammals, permits study of the function of single cells or discrete populations of cells during the development of polyploidy. Thus use of Ascaris uterine epithelium as an experimental system may yield important information concerning the significance of polyploidy in biological systems.
Localized tissue polyploidy is a biological phenomenon of widespread occurrence [19, 201, yet its functional significance has eluded detection. Although occurring in various mammalian tissues [2, 9, 10, 11, 12, 221,localized tissue polyploidy has received most extensive investigation in mammalian liver [14]. These studies have yielded little insight into the functional significance of polyploidy however. One of the basic problems in elucidating the significance of polyploidy has been the lack of an adequately characterized systemfor investigation. Cultured cell lines [5] have permitted some comparisons between diploid and heteroploid cells and study of plant tissues has indicated certain differences or differentiations between polyploid and diploid cells [ 18, 211. In invertebrate systems polyploidy has a protective value for cells exposed to ionizing radiations [23]. The study of polytene systems has provided much data which has been extrapolated to polyploid systems. This extrapolation is based on the similarity of polyploidy and polyteny in terms 1 Present address: Department of Anatomy, University of Michigan, Ann Arbor, Mich. 48104, USA. 18-691803
of total nuclear DNA and may or may not be a valid comparison. The relationship of polyploidy to differentiation has been suggested for the past 20 years. Huskins states “The implication from work by Bushnell & Geitler is that nuclei which have become endopolyploid may be highly active in some metabolic processes. . .” [7]. The regular occurrence of polyploidy in highly secretory insect tissues and such active mammalian tissues as liver has also suggested that polyploidy is related to functional differentiation. Painter [15] seems convinced that polyploid cells have an increased capability for polyribosome production. A cell system with well defined functional parameters and nuclear DNA variations would be useful for study of the functional significance of polyploidy. The amount of nuclear DNA varies in uterine epithelial cells of Ascaris Zumbricoides [17] and it will be demonstrated in this study that these variations fall into polyploid classes. Further, it will be shown that these polyploid classes are arranged in linear fashion
from one end of the uterus to the other
and are correlated with variations in cell morExptl Cell Res 56
276 A. D. Floyd h F. J. Swartz
phology and cytoplasmic RNA content. The uterine epithelial cells are of adequate size for studies of cytoplasmic specialization and are arranged in a manner that facilitates correlation with cell function. Although changes in nuclear DNA occurring during differentiation have not been worked out in detail, it can be demonstrated that the polyploid gradient of the uterine epithelium is correlated with the differentiation or maturation of the animal. MATERIALS AND METHODS Live specimens of Ascaris lumbricoides were collected at a local abbatoir along with swine intestinal contents. The worms were washed with and maintained in a balanced salt solution [I]. As soon after collection as possible the parasites were decapitated and the uteri removed. These tissues were fixed for 2 h in Carnoy’s fluid and embedded in Tissuemat. The uterus of Ascaris reaches 10 to 12 cm in length in mature animals. It was arbitrarily divided into segments of equal length and each segment sectioned at 5 to 15 p. A rigidly controlled Feulaen nrocedure was emuloved with-a hydrolysis time of? n& as reported by SW&z et al. 1171for tissues of Ascaris. These DNA urenarations were quantitated using a Barr & Stroud integrating microdensitometer [4]. Feulgen microspectrophotometry requiries measurement of whole or uncut nuclei, but in Ascaris uterus, even in 15 ,u sections, many nuclei extend through more than one section. To obtain individual nuclear values serial sections were prepared and nuclei mapped and numbered. After measuring each nuclear slice in each section, it was possible to reconstruct cut nuclei and obtain a reasonable estimate of total nuclear DNA. To eliminate errors due to possible loss of DNA from sectioned nuclei and avoid any error as the result of the reconstruction process, uterine whole mounts were prepared. These whole mounts were obtained by slitting uteri lengthwise while immersed in balanced salt solution and then gently flushing out the eggs with a jet of the immersing fluid from a small syringe. This process results in an epithelial sheet because the uterine wall of Ascaris consists only of an epithelium on a thick basement membrane with a very sparse muscle layer. This muscle layer is so sparse as to cause no problems in subsequent quantitation. The epithelial sheets were placed on slides previously coated with albumin and were briefly dried on a 50°C hot plate and subsequently fixed in Carnoy’s fluid. These whole mounts were stained by the Feulgen reaction and quantitated as described above. Variations in epithelial cell cytoplasmic ribonucleic acid from end to end of the uterus were quantitated using cresyl violet staining as reported by Ritter et al. [16]. Control sections were subiected to a two hour incubation with 0.5 mg/ml ribonuclease (Worthington B&hem. Corp., Freehold, N.J.) in water adjusted to pH 6.8 at 60°C. RNA was evaluated with the Barr & Stroud microdensitometer employed as an absorption photometer. Transmittance at 590 nm through three snots 6 .U in diameter was determined in the apical cytopfasm of each cell measured. Each cell value was derived from the average of the three spots. Thirty cells were measured at Exptl Cell Res 56
each uterine level. Transmittance was converted into absorbance (extinction) by the relationship: Absorbance = - log Transmittance.
RESULTS General observations
The simple epithelium of Ascaris uterus varies morphologically from one end of the uterus to the other although all of the epithelial cells are basically similar in shape. These differentiations are expressed as size variations and differences in types of inclusion bodies [8]. In general, cells in the middle of the uterus are larger than those at either end; a typical miduterine cell measured 100 x 25 x 100,u. The epithelial cells are normally binucleate but some may contain four or more nuclei. Uninucleate cells are occasionally seen. “Amitotic” figures are frequent and no mitoses were encountered. These nuclei were not included in the DNA measurements. Complete end to end analysis of any parameter of the uterine epithelium of Ascaris is technically difficult because of the length of the uterus. The data reported in this study are based on analyses of individual animals and these data have been shown to be representative by numerous selected area analysis of a great number of animals. Total body length is pertinent in these results as this is the only criterion for age of individuals collected. Normally fertilized eggs are only found in females over 20 cm in length. Maximal lengths which have been observed are approx. 36 cm. In this study, animals between 30 and 36 cm in length have been arbitrarily classified as “mature”, and females of this size make up the bulk of any collection. DNA quantitation. Using mapped serial sections of the uterus of a mature worm (30 cm body length) peak nuclear DNA values, expressed as average DNA per nucleus in a given level of the uterus, were found in miduterus with lower values being obtained at both ends (fig. 1). Uterine DNA values were determined similarly in a small female. Although this specimen was only 24 cm in body length, fertilized eggs were present in the uterus. The smaller worm also
Polyploidy model system
yielded high DNA average values per nucleus in miduterus with lower values at both ends (fig. 1) but maximal values were lower in the smaller than in the larger animal. Further, there was a noticeable plateau of values throughout the middle region of the animal as compared to the same area of the larger animal. As seen in the histogram plot of values from the 30 cm animal (fig. 2), nuclear DNA varied widely at a given level, with the variation approaching that expected of nuclear polyploid classes. However, the many intermediate values gave considerable class overlap. In order to eliminate possible errors in the serial section approach, quantitation of nuclear DNA variability was carried out on whole mounts. As described above there was again a great difference between average DNA per nucleus values at miduterus and each end and the peak value was very high. When the whole mount values were plotted as a histogram (fig. 3) there was a definite grouping into nuclear classeswith a marked reduction in class spread compared to the results from the serial sections approach. The increase in average nuclear DNA in the miduterus was the result of increases in ploidy level of the nuclei involved and changesin the proportions of the various ploidy classes,as can be seenfrom the data plotted in fig. 3.
z
r
0 t -.ot!rq
II
217
2 f f a* XSX~ L..-
Abscissa: Relative DNA units. Fig. 2. Distribution of nuclear DNA content of serial sections of the uterus of a 30 cm specimen of Ascaris. Vertical bars represent number of nuclei of particular DNA content.
DNA analysis of 5 ,u sections of the uterus of a 6.5 cm worm (these results are confined to this one animal) indicated variation in average nuclear DNA content from end to end of the uterus but the pattern was distinctly different
500 .
450 400./’
350 -
‘\
.’ 300 250 -
.
/ *-.
0 Anterior
Pormrior
Abscissa: Uterine levels; ordinate: relative DNA units. Fig. 1. Average value of DNA per nucleus in various levels of the uterus of three specimens of Ascaris as determined by Feulgen microphotometry. 0 - 0, Sections 30 CM animal; x - x , sections 24 CM animal; e- 8, whoiemount 30 CM animal.
Abscissa: Relative DNA units. Fig. 3. Distribution of nuclear DNA content in a whole mount preparation of the uterus of Ascaris. Vertical bars represent number of nuclei of particular DNA content. Exptl Cell Res 56
278 A. D. Floyd & F. J. Swartz
Table 1. Cresyl violet RNA quantitation Uterus of 30 cm animal. Uterine levels
% Trans.
Extinction
s.E.*
Anterior A B C D E
73 62 35 32 21
0.138 0.231 0.467 0.499 0.584
: H Posterior I
27 18 31 38
0.581 0.763 0.563 0.429
0.016 0.019 0.018 0.016 0.016 0.018 0.019 0.017 0.014
RNA quantitation. With cresyl violet staining Abscissa:Uterinelevels;ordinate: relativeDNA units. highest absorbanceswere found slightly posterior Fig. 4. Averagevaluesof DNA per nucleusin various to miduterus (fig. 5). These determinations relevels of the uterusof a 6.5 cm specimenof Ascuris. vealed a large variation in cytoplasmic RNA concentration from end to end of the uterus. from that seenin reproductively mature animals Standard error of the mean calculated for the (fig. 4). Nuclei in this animal were small and the various levels quantitated demonstrated the highest absorbances measured were only one- constancy of the error envolved in these measurefifth that of the lowest values found in uteri ments, irrespective of the actual absorbancies containing fertilized eggs. Highest values in the being measured (table 1). To verify that cresyl violet staining in Ascuris small specimenwere found in the anterior uterus could be attributed to RNA, adjacent sections near the vagina with a continuous decline in to those quantitated were subjected to RNAase average nuclear DNA proceeding posteriorly. extraction. After enzyme treatment all cytoSince many of the fields measured in this small animal contained more than one nucleus, only plasmic and nucleolar staining was abolished. average values were obtained and histogram Non-nucleolar areas of nuclei still retained the plots of nuclear distributions and possible poly- stain. Posterior
Ant*rior
ploid classeswere not obtainable. 0.9 O.S-
r
0.7 0.6 0.5 0.40.3 0.2 0.1 Anterior
Post.rior
Abscissa: Uterinelevels;ordinate: extinction. Fig. 5. Distribution of the dyecresylviolet from end to end of the uterusof an adult specimenof Ascaris. Vertical bars represent S.E.M. Exptl Cell Res 56
DISCUSSION Classically, localized tissue polyploidy is defined as a multiple of the haploid chromosome number in individual nuclei. In current usage the term polyploidy is often applied to multiples of the diploid DNA value in individual nuclei. In the mammalian liver, where chromosome counts can be correlated with DNA values, this usage has proven valid. However, DNA values alone cannot distinguish between polyploidy and polyteny. No evidence of polyteny has been seen in the interphase nuclei of Ascaris uterus and the DNA variations reported here are considered to be a reflection of polyploidy. As mitotic figures were not seen in Ascaris uterine epithelium and
Polyploidy model system
have not been reported in any somatic tissue of the nematode, actual chromosome counts are not possible. A further complication is the process of chromatin diminution which occurs during Ascaris embryogenesis [3]. As this process has not been quantitated, the possibility exists that the diminished “diploid” DNA value for nuclei of various somatic tissues of the worm is not constant. Uncertainty concerning the constancy of diminution from tissue to tissue and lack of quantitative data regarding it negate attempts to describe the absolute levels of polyploidy attained in adult tissues [17]. Regardless of this limitation, the various polyploid classes in Ascaris uterus may be described as 2kC, 4kC, 8kC, etc., where k is an unknown constant. The excellent fit of measured nuclei into polyploid classessupports the constancy of k. Since high and low values of DNA content were included in these measurements, there was some loss of linearity of the DNA classes at high levels of DNA content. This is felt to be an expression of instrumental errors caused by the extreme range of concentrations covered in this study. The gradient of polyploid values in the uterine epithelium will permit comparison of cell function with polyploid level. Previous reports of the secretory function of the uterine epithelium [6] and description of morphologically distinctive cytoplasmic “secretory” materials within various segments of the epithelium [8] indicate that functional gradients may also be present in this organ. The potential of this system for investigation of the significance of polyploidy is thus apparent. The patterns of nuclear differentiation observed in this study suggest that Ascaris uterus may provide answers to the role of quantitative chromosomal variations in differentiation and maturation. Data from the immature animal reported in this study suggest that initially the uterus differentiates in an anterior to posterior direction. An anterior-posterior nucleic acid gradient has been described in the tapeworm [ 131 but not in Ascaris. In the case of adult Ascaris, this early gradient is lost as the uterus itself differentiates. Although animals have not
279
been examined in all stages of development, the evidence suggests that polyploidy is initiated before the initiation of uterine function and that further nuclear differentiation continues as the animal grows and develops. As eggsfrom young females are viable, the young uterus is functional. (Ascaris eggs are not viable unless the secondary egg coat, which is a product of the uterine secretion, is complete.) Thus the further differentiation of the uterus in older animals may be related to increased eggproduction which necessitatesincreased uterine secretion. The general occurrence of high levels of cytoplasmic RNA in secretory tissues, coupled with the extreme basophilia of the ascarid uterine epithelium prompted quantitation of cytoplasmic RNA concentrations. As the small spots through which transmissions were taken were kept constant throughout the measurements, the data represent the actual variation of RNA concentration per unit area within individual cells. Any distributional error which may have been involved due to the plug type measurements employed should have remained relatively constant throughout the measurements, thus the values given are valid relative to one another but are not indicative of absolute RNA concentration. The values reported represent RNA concentrations, not total cell RNA. Therefore, as cells in miduterus have a greater volume than those at either end of the uterus and also a higher concentration of RNA per unit area, the actual amount of RNA per cell in miduterus relative to the cells at either end of the uterus would be much greater than that shown in the RNA concentration curve. The higher concentration of ribonucleoprotein supports Painter’s hypothesis of increased RNA synthetic ability in polyploid nuclei although it certainly doesnot demonstrate either a causeor effect relationship. This work was submitted by the Senior author in partial fulfilment of the requirements for the Doctor of Philosophy degree, University of Louisville Graduate School, Louisville, Ky. Supported by NSF research grant GB 4490 and by USPHS training grant 5 TO1 GM-01356. Exptl Cell Res 56
280 A. D. Floyd & F. J. Swartz
9. Kompmann, M, Paddags, I & Sandritter, W, Arch path01 82 (1966) 303. LaCour, L F, Proc roy sot Edinburgh B 62 (1944) 73. :;: Laiham, L & Johnstone, M A, Arch neurol9 (1963)
13. Lee, T W & Campbell, J W, Nature 203 (1964) 661, 14. Naora, H, J biophys biochem cytol 3 (1957) 949. 15. Painter, T S & Biesele, J J, Proc natl acad sci US 56 (1966) 1920. 16. Ritter, P, DiStefano, H S & Farah, A, J histochem cytochem 9 (1961) 97. 17. Swartz, F J, Henry, M & Floyd, A, J exptl zoo1 164 (1967) 297. 18. Torrey, J G, Science 128 (1958) 1148. 19. Tschermak-Woess, E, Protoplasma 46 (1956) 798. 20. White, M J D, Animal cytology and evolution, 2nd ed. Cambridge University Press (1954). 21. Witsch, H & Flugel, A, Z Botan 40 (1952) 281. 22. Wodsedalek, J E, Anat ret 81 (1941) 79. 23. Zirkel, R E & Tobias, C E, Arch biochem biophys 42 (1953) 2.
12. Lapham, L W, Excerpta med intern congr 100 (1965) 445.
Received December 19, 1968
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