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Polyploidy in the nuclei of ovarian nurse and follicle cells of the silk moth Hyalophora cecropia.

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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. [2]. 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 [3]. 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 [4]. 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
[7]. 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 [3]; 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 [9]. 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 [12]. 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 [13]. 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 [12] (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 [3]. 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. [14]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. Hough-Evans BR, Jacobs-Lorena M, Cummings MR, Britten RJ, Davidson, EH: Complexity
of RNA in eggs of Drosophila rnelanogasfer and Musca dornestica. Genetics 95,81 (1980).
3. Telfer WH: Development and physiology of the oocyte-nurse cell synctium. Adv Insect Physiol
22,223 (1975).
4. Oishi M, Locke J, Wyatt G: The ribosomal RNA genes of Locusta rnigratoria: Copy number
and evidence for underreplication in a polyploid tissue. Can J Biochem Cell Biol 63, 1064
(1985).
5. Cave MD: Absence of amplification of ribosomal DNA in the polytrophic meroistic ovary of
the giant silkworm, Antheraea pernyi. Wilhelm Roux’s Arch Dev Bioll84,135 (1978).
6. Cave MD, Sixby J: Absence of ribosomal DNA amplification in a meroistic polytrophic ovary.
Exp Cell Res 102,23 (1976).
7. Renkawitz R, Kunz W: Independent replication of the ribosomal RNA genes in the polytrophicmeroistic ovaries of Calliphora erythrocephala, Lkosophila hydei, and Sarcophage barbata. Chromosoma
53, 131 (1975).
8. King RC, Buning J: The origin and functioning of insect oocytes and nurse cells. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology. Kerkut GA, Gilbert LI, eds.
Pergamon Press, Oxford, vol 1, pp 37-82 (1985).
9. Paglia LM, Berry SJ, Kastern WH: Messenger RNA synthesis, transport, and storage in silkworm ovarian follicles. Dev Biol52, 173 (1976).
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Cardoen et al.
10. Suzuki Y, Gage P, Brown D: The genes for silk fibroin in Bombyx mori. J Mol Biol 70, 637
(1972).
11. Mulligan PK, Rasch EM: Determination of DNA content in the nurse and follicle cells from
wild-type and mutant Drosophilu melunoguster by DNA-Feulgen cytophotometry. Histochemistry 82,233 (1985).
12. Cardoen J, Schoofs L, Broeckaert D, Van Mellaert H, Verachtert B, De Loof A: Polyploidisation
and localisation of poly(A + )RNA in the different cell types of the vitellogenic meroistic ovary
of the fleshfly, Surcophugu bullutu. Histochemistry 85,305 (1986).
13. Berry SJ: Insect nucleic acids. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, vol10, pp. 219-253 (1985).
14. Mahowald AP, Caulton JH, Edward MK and Floyd AD: Loss of centrioles and polyploidization
in follicle cells of Drosophilu melunoguster. Exp Cell Res 178,404 (1979).
15. Thireos G, Kafatos F: Cell-free translation of silk moth chorion mRNAs: identification of
protein precursors and characterization of cloned DNAs by hybrid-selected translation.
Dev Biol78,36 (1980).
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