Journal of Insect Physiology 46 2000 1239–1248 www.elsevier.comlocatejinsphys
Vitellogenic ovarian follicles of Drosophila exhibit a charge- dependent distribution of endogenous soluble proteins
Russell W. Cole, Richard I. Woodruff
Department of Biology, West Chester University, West Chester, PA 19383-8102, USA Received 17 September 1999; accepted 19 January 2000
Abstract
In ovarian follicles of Drosophila, soluble endogenous charged proteins are asymmetrically distributed dependent upon their ionic charge. Reversal of the normal ionic difference across the intercellular bridges which connect nurse cells to their oocyte results in
a redistribution of these proteins. Twelve soluble endogenous acidic proteins were identified by 2-D gel electrophoresis as being present in both oocytes and nurse cells in samples run on four or more gels. Of these, following osmotically induced reversal of
the electrical transbridge gradient the concentration of seven proteins decreased in the oocyte while nurse cell concentrations of all twelve proteins increased. Of seven basic proteins analyzed, following reversal of the electrical gradient the concentration of all
seven increased in oocytes. Four of these decreased in nurse cells, while nurse cell concentrations of the remaining three basic proteins also appeared to decrease, but yielded spots too faint for measurement. Data presented here demonstrate that, as in the
Saturniidae, the ionic gradient across the nurse cell-oocyte intercellular bridges of the dipteran, Drosophila, can influence the distribution of soluble endogenous charged molecules.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Ionic gradient; Cytosolic proteins; Membrane potential
1. Introduction
In insects, as throughout most of the animal kingdom, developing oocytes are most commonly supported by
sibling cells called nurse cells, to which they are joined by open channels of cytoplasm called intercellular
bridges Telfer, 1975. During pre-ovulation develop- ment the oocyte nucleus becomes a dormant germinal
vesicle GV and the oocyte acquires yolk via endo- cytosis Telfer, 1965. Meanwhile, the nurse cell nuclei
become highly endopolyploid and active, with most of the RNA produced destined to pass through the inter-
cellular bridges and become sequestered in the oocyte Bier, 1963a,b; Pollack and Telfer, 1969. Intercellular
bridges, the products of incomplete cytokinesis, most often function to maintain synchrony among syncytial
cells Fawcett et al., 1959, yet in ovarian follicles this function must be circumvented as oocyte and nurse cells
Corresponding author. Tel.: +
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1-610-436- 2183.
E-mail address: rwoodruffmail.wcupa.edu R.I. Woodruff.
0022-191000 - see front matter
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become both morphologically and physiologically differ- ent Fig. 1. As the two cell types differentiate along
separate lines some mechanism must exist which allows them to retain autonomy while still maintaining the open
bridges needed to transport copious amounts of RNA
Fig. 1. Micrograph of a stage 10 Drosophila ovarian follicle.
Ooc =
oocyte, NC =
nurse cells. Accumulated yolk renders the large oocyte opaque. Also visible are individual epithelial cells and the
nuclei of several of the 15 nurse cells. Scale bar =
100 µ
m. SSEE optics.
1240 R.W. Cole, R.I. Woodruff Journal of Insect Physiology 46 2000 1239–1248
from their sites of synthesis in the nurse cells to the sites where these molecules will be sequestered in the oocyte.
Particularly in Drosophila, the oocyte is precisely structured, eventually containing organization which
influences post-fertilization development Nusslein-Vol- hard et al., 1987. This organized “pre-programming” is
achieved within a multicellular complex which must itself be highly and actively regulated. Indeed, the devel-
opmental sequence of follicular activities that generate egg structure implies an exacting set of cellular controls.
Morphological polarity, manifested as an anterior-pos- terior orientation with respect to both the ovariole and
the whole organism of the oocyte-nurse cell syncytium, is apparent when the follicle is first formed. In Hyalo-
phora cecropia
, an electrically-based physiological polarity initiates close to the onset of vitellogenesis
Woodruff and Telfer 1973, 1980, and continues as a steady-state phenomenon for several days, until the nurse
cells disintegrate about 24 h before the end of vitellogen- esis. This physiological polarity involves a metabolically
driven difference in [Ca
2 +
]
i
between the oocyte and the nurse cells Woodruff et al., 1991; Woodruff and Telfer,
1994, which establishes an electrical gradient focused across the bridges connecting the two cell types.
Recently, this transbridge ionic gradient has been shown to influence the distribution of charged endogenous cyto-
solic proteins Cole and Woodruff, 1997. This current, seemingly through action of the cytosolic proteins whose
distribution it regulates, also enforces changes in the transcriptional activity of the oocyte nucleus Woodruff
et al., 1998.
The ovarian follicles of Drosophila resemble those of Hyalophora
, but possess more nurse cells n =
15 and reside in a more conventional blood ion environment.
Reports from different labs on the existence of a trans- bridge gradient in Drosophila have varied. Bohrmann et
al. 1986 and Sun and Wyman 1987, 1993, found no significant difference, while Woodruff et al. 1988,
Woodruff 1989, Verachtert et al. 1989, Verachtert and De Loof 1989 and Singleton and Woodruff 1994
all found significant differences between the steady-state potentials of nurse cells and the oocyte to which they
are attached. An experimentally supported answer explaining the differing results has now been put forth,
and centers around the composition of the media in which the measurements were performed Singleton and
Woodruff, 1994. Experimental evidence revealed that the steady-state membrane potential E
m
of nurse cells was more affected by osmolarity than E
m
of oocytes. The osmolarity of adult female hemolymph was measured to
be 250 mOsmol, at which osmolarity nurse cells were shown to be more electronegative than the oocyte to
which they were attached. At increasingly higher osmol- arity the difference between cell types first decreased to
0 and then reversed.
Microinjection of fluorescently labeled lysozyme, in either the positive or the negative form, has provided
evidence that a charge-dependent asymmetric distri- bution of proteins can occur in Drosophila Woodruff et
al., 1988, but lysozyme is an exogenous protein in this system, and soluble endogenous proteins might be regu-
lated by other means. Thus in the present study we have utilized 2-D gel electrophoresis to analyze the distri-
bution of charged endogenous cytosolic proteins from the ovarian follicles of Drosophila. Soluble proteins
could be susceptible to iontophoretic effects, while pro- teins bound to cytoskeletal elements, membranes and
other cytoplasmic structures would not be. Bound pro- teins are present in such perfusion that, if not removed,
they obscure the influence of the electrical gradient upon the soluble proteins. As in a previous study Cole and
Woodruff, 1997, a necessary step was to separate sol- uble proteins from those which were bound. To achieve
this, we harvested soluble proteins from nurse cell or from oocyte extracts by centrifugation and ultrafiltration.
We furthermore took advantage of the effect on the transbridge electrical gradient wrought by changes in
osmolarity Singleton and Woodruff, 1994. This pro- vided a non-invasive non-pharmacological means to
reverse the direction of the gradient. If the transbridge gradient actually does influence the distribution of
charged soluble molecules, the distributions of both acidic and basic proteins should be affected in opposite
manners. Relative to controls incubated in a 255 mOs- mol. medium, in follicles incubated at 400 mOsmol. the
concentrations of soluble acidic proteins should diminish in the oocytes, and increase in the nurse cells. Similarly,
the relative concentrations of soluble basic proteins should decrease in the nurse cells and increase in the
oocytes of follicles incubated at high osmolarity.
The experiments reported here show that the distri- butions of most of the soluble proteins responded to
changes in the osmolarity of the incubation medium exactly as if they were responding to the transbridge
electrical gradient.
2. Materials and methods
