Polyploidy in the nuclei of ovarian nurse and follicle cells of the silk moth Hyalophora cecropia.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 15:93-100 (1 990) Polyploidy in the Nuclei of Ovarian Nurse and Follicle Cells of the Silk Moth, Hyalophora cecropia Johan Cardoen, Cornelius Watson, Arnold DeLoof, and Spencer J. Berry Department of Biology, Wesleyan University, Middletown, Connecticut (C.W., S.J.B.); Laboratory for Developmental Physiology, Zoological Institute, Catholic University of Leuven, Belgium (J.C., A . D . ) The ploidy of ovarian nurse cells of Hyalophora cecropia was determined at three arbitrarily chosen stages of follicle development. C-values ranged from 8,192 to 65,536 for the nurse cells, and increased i n orderly geometric fashion with the age of the follicle. The ploidyof accompanying follicle cells increased from 8 to 512C during the same development sequence. Key words: insect development, oogenesis, DNA content INTRODUCTION In the ovaries of several groups of more advanced insects such as Diptera and Lepidoptera, the follicles are classified as polytrophic because the developing oocytes are connected to trophic ”nurse cells.” Nqrse cells are of particular interest to biologists because of certain structural features, and because they are the major, if not the sole, source of the ooplasmic RNAs stored in the egg prior to fertilization. The interesting structural features include: the large size of the nuclei (-0.05 mm), and the cytoplasmic continuity which they maintain with each other and with the developing oocyte they service. The large size of nurse cell nuclei appears to be necessary to accommodate the highly reiterated genome. The management and regulation of a nucleus of the size and complexity of the ovarian nurse cell should provide a model for nuclear structure and organization on an exaggerated macro scale. The specific activities associated with mitosis would be eliminated, but all other aspects of nuclear metabolism, including replication and transcription should be present. In many highly polyploid cells, such as the silk and salivary glands, a single, specific, Acknowledgments: We are very grateful to the Monsanto Co. and Dr. Howard Scheiderman for a generous research grant, and to NATO for a travel grant to support this research. Dr. Frederick M. Cohan helped in the analysis and presentation of the data. Received April 3,1990; accepted June25,1990. Address reprint requeststo Spencer J. Berry, Department of Biology, Wesleyan University, Middletown, CN 06457. 0 1990 Wiley-Liss; Inc. 94 Cardoen et al. product of the unique sequences predominates, and thus the variety of gene activities which must be regulated is presumably reduced. Nurse cells, by contrast, can be expected to transcribe a variety of genes in addition to ribosomes and other “generic” translational machinery. In giant silkmoths related to Hyalophora cecropia, Pollack and Telfer [l],estimate that 3 pg of RNA is stored in each egg. The sequence complexity for the oocyte mRNA of the housefly, Musca domesfica, is estimated at 2.4 x lo7, and the number of copies of each unique sequence is set at 760,000 by HoughEvans et al. . This prodigious amount of very diversified RNA molecules is apparently synthesized using the nurse cell chromosomes as template, since the oocyte nucleus is synthetically quiescent during oogenesis . The template for ribosomal RNA in panoistic ovaries, such as those of the locust and cockroach, is provided by temporary amplification of specific chromosomal regions . Assays for specific amplification of the ribosomal locus in the meroistic, polytrophic ovaries of lepidopterans have proven negative [5,6]. In dipterans, which also have nurse cells, only slight (135%)overreplication, or even under-replication of the rDNA has been detected by Renkawitz and Kunz . These observations suggest that total, rather than specific amplification of the genome may be utilized by the nurse cells to provide sufficient template. The ovarian follicles of H. cecropia consist of seven nurse cells and one oocyte, all interconnected by cytoplasmic connectives (ring canals, or fusomes; see reviews by Telfer ; King and Buning [S]). Once this cluster of presumptive nurse cells and oocyte (cystocytes)is formed from the germ line, it is surrounded by a palisade of mesodermally-derived follicle cells. Newly synthesized RNAs, including mRNAs as well as ribosomal, are transported to the ooplasm via the cytoplasmic connectives . At the end of the vitellogenic period, the nurse cell nuclei become pycnotic, the connectives are severed, and the degenerating nurse cell cluster is segregated from the oocyte by the developing chorion, secreted by the follicle cells. Extremely high levels of polyploidy are common in specialized insect cells. For example, Suzuki et al. [lo], report a ploidy value of 170,000 for the silk gland cells of Bornbyx mori. In most tissues, the level of ploidy is more modest, but the nurse cells of Diptera reach, 1,024C in Drosophila [ l l ] and in Sarcophagu . The haploid C values for lepidopteran nuclei have been determined by both cytospectrophotometry and by biochemical methods, and are a surprisingly consistent 1 pg/diploid genome for the species examined to date . The only data for H . cecropia nurse cells is the unpublished estimate the DNA content at 30,000 pghucleus. This value was arrived at by measuring the total extractable DNA from a known number of nuclei [13, Table 21. In this paper, we determine the distribution of ploidy values and examine the evolution of increased ploidy of individual nurse cell nuclei as the development of the follicle progresses. We also examine the change in ploidy of the mesodermally-derived follicle cells which surround the nurse cells. MATERIALS AND METHODS Pupae of H. cecropia were obtained from commercial dealers, and stored at 5°C until use, when they were transferred to 25°C and allowed to initiate adult Nurse Cell Polyploidy 95 development. Ovaries were carefully dissected under Ringer solution until strings of follicles were exposed. Clusters of nurse cells were carefully removed from follicles of specific sizes (Fig. lA), and were gently squashed between microscope slides coated with Silane (Sigma, St. Louis, MO). These pairs of slides were then immersed in various fixatives, including AFA (ethyl alcohollformaldehyde/acetic acid, 75/20/5), glutaraldehyde (4% in 0.1 M cacodylate buffer, pH 7.4), or 2% formaldehyde (0.1 M phosphate buffer, pH 7.4). Fixatives were allowed to penetrate between the slides by capillarity, and then the slides were separated and the tissue fixed for 2 h. AFA-fixed squashes for cytospectrophotometry were stained by the Fuelgen procedure as described by  (Fig. 1B). Hydrolysis was carried out for 50 min in 4N HCl at 28°C. Conditions for optimal hydrolysis were determined by a preliminary series of determinations. Cytospectrometric determinations were carried out on a Vickers M-86 scanning-integrating microdensitometer (Vickers Fig. 1. A: String of developing ovarian follicles of H. cecropia subdivided into small, medium, and large follicles to indicate the rough categories referred to in the text. B: Feulgen stained nuclear-squash preparation of a nurse cell from a "medium" size follicle. 96 Cardoen et al. Ltd., United Kingdom) at 560 nm. Spermatid nuclei of H . cecropia were used to determine the haploid DNA content and as reference for the microdensitometer. At least 50 spermatid reference values and 40 or more nurse cell or follicle cell determinations were made for each class of follicle examined. DNA absorption values of nurse cell and follicle cell nuclei were converted into arbitrary units by means of a calibration factor (164.53), which takes into account differences in measuring scan frame sizes and objective power and were expressed as log2 of total extinction. These were grouped as frequency of “C-values” and the trimodal distributions expressed as the mean. RESULTS Follicles were arbitrarily divided into small, medium, and large on the basis of the rough proportion between nurse cell and oocyte volumes. These distinctions are illustrated in Figure 1A. Nuclear DNA values for the nurse cells are illustrated in Figure 2. As the volume of the nurse cell “cap” increases (average volume for “small”, 1.17 mm3; for “medium”, 2.2. mm3; for ”large”, 3.60 mm3), a clear trend toward increased ploidy and increased follicle size is apparent. The proportion of the follicle occupied by the nurse cell cap diminishes however, because of the faster increase in the volume of the oocyte. The range of ploidy values in the “small” follicles is from 8,192C to 32,768C, but the predominant value is 8,192C (52%). In the “medium” follicles, the predominant values are 16,834C (48%) and 32,768C (49%). In the ”large” follicles, 32,768 (67%)is the predominant C-value, but five nuclei (9%), showed a further increase to 65,536C. One nucleus in a cap apparently about to undergo inversion, exhibited a C-value of 131,000 (data not included). A number of caps which had begun inversion were examined. In these cells, the C-values were reduced, and the Feulgen staining appeared faded. C-values for follicle cell nuclei were followed in the same preparations. The follicle cells examined were those stretched over the nurse cell cap, rather than surrounding the oocyte. These cells did not show any mitotic figures, and thus the increase in surface areas as the nurse cell cap volume increases must also be accompanied by endoreplication. The DNA content of each stage was distributed over several classes (Fig. 3). There is a shift of the predominant ploidy classes towards higher degrees of ploidy. The observed peaks on a log2 scale coincide perfectly with successive doublings of DNA. The DNA content of the nuclei of the follicle cells of the small follicles ranges from 8 to 32C with 32C (51%)as the predominant class. The medium-size follicle exhibit a range from 16 to 128C with 64 (60%) more prominent. In large follicles, the predominant class is 128C (49%), while the range is 64 to 512C. DISCUSSION The cytospectrophotometric data confirm and extend earlier unpublished estimates of the ploidy of nurse cell nuclei at 30,000 [12, Table 21. Variation of ploidy values may, in part, be attributable to errors in classifyingfollicles according to size, but is more likely attributable to non-synchronous cycles of replication by nurse cells in the same cluster . Inspection of histological sections Nurse Cell Polyploidy 100 97 S ma1I 70 60 loo 90 1 0 50 - 0 40 - c c a 3 0- 2 LL 30 20 10 Medium - '"3 70 60 50 Large - 20 40 30 10 - .-0 81926 16834C 32768C 65536C DNA content (polyploidy level) Fig. 2. Distribution of ploidy values for nurse cell nuclei grouped arbitrarily according to follicle development stage. 98 Cardoen et al. 100 Small 70 60 i Medium 100 80 70 60 : 50 - 40 : 30 : i Large 100 70 60 40 30 - 50 20 10 - 8C 16C 32C 64C 128C 256C 512C DNA Content (polyploidy level) Fig. 3. Distribution of ploidy values for follicle cell sheath which invests nurse cell caps illustrated in Figure 2. Nurse Cell Polyploidy 99 of follicles indicates that at least one nurse cell in each cluster is smaller than the others. While the nurse cells of Diptera appear to have a maximum DNA content which does not exceed 2,048C [11,12], the nurse cells of Lepidoptera attain much higher values. The fairly commonly observed value of 65,00OC, and the single value of 131,000, indicate that 30,000-ploid is not a predetermined end-point, but that endopolyploidy can continue beyond that level. The ultimate fate of the nurse cell nuclei is more problematical, because, rather than shrivel and become pycnotic, as we anticipated from histological observations, they seem to maintain their volume, but to fade in terms of Feulgen stainability. The ploidy of the surrounding follicle cells was monitored because they were included in the preparation, but more important because they served as an internal calibration standard for the larger changes in the nurse cells. The follicle cells are of somatic (mesodermal) rather than germ-line origin, and the relatively modest increases in ploidy most likely reflect an attempt to maintain the nuclearkytoplasm volume ratio as the cells are stretched to enclose the enlarging mass of nurse cells. The increase in ploidy is orderly and geometric as the predominant classes progress from 32 to 64C, and finally to 512C as the age of the follicle increases. In Drosophilu and Sarcophage the DNA content of the follicle cells ranges from 8 to 64C [14,12], and Mahowald et al. also observed a correlation between the loss of centrioles and polyploidization in the follicle cells of Drosophila. The greater DNA content of H . cecropia follicle cells presumably subserves the same functions. Both vitelline membrane and chorion as well as some vitellogenin synthesis is carried out by the follicle cells. Thireos and Kafatos 1151 reported that specific amplification of the genes for chorion proteins occurs in the follicle cell nuclei. LITERATURE CITED 1. Pollack SB, Telfer WH: RNA in cecropia moth ovaries: Sites of synthesis, transport, and storage. J Exp Zoo1 170, 1 (1969). 2. 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