Construction of the

The adult female reproductive system of Drosophila consists of a pair of ovaries, the genital ducts (common and lateral oviducts, uterus, vagina) and their accessory structures (two spermathecae, two accessory glands, and seminal receptacle), and the external genitalia (see Fig. 1). The ovaries are direct descendents of the embryonic gonads, whereas all other genital structures derive from a single genital imaginal disc. The formation of embryonic gonads implies the specification of pole plasm (germ plasm) by localized germ-line determinants early in embryogenesis (7). The pole plasm, in turn, is both necessary and sufficient for the formation of pole cells, the primordial germ cells (9). Pole cells migrate during gastrulation to reach the gonadal region, where they become surrounded by somatic mesodermal cells and form the embryonic gonads (10,11). The gonads remain in this undifferentiated state and have no direct connections with the genital disc until the larval-pupal transition (1). The first connections between the ovaries and lateral oviducts are established 54-60 h after pupariation (12). The undifferentiated larval ovaries

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

Ovarv

Ovarv

Drosophila Gastrulation
Fig. 1. The internal reproductive organ of the Drosophila female. (Adapted from ref. 2.)

are relatively small and consist of two cell types: large germ cells and small mesodermal cells; the latter will form the follicle cells, ovarian sheaths (epithelial and peritoneal), and other structures. Differentiation of adult ovaries begins during the mid third larval instar with the appearance of the terminal filaments as a consequence of tunica propria formation around the forming ovarioles (1). After pupariation, a set of rapid developmental events occurs, which will lead to formation of the mature ovary by the end of pupal life. Differentiation of the genital disc starts after puparium formation, and all of the genital ducts and their accessory structures will be present as primordia after 48 h.

The mature Drosophila ovary is composed of 12-16 ovarioles, joined at their tips by terminal filaments. The structural and functional unit of the ovary is the ovariole (see Fig. 2). Each ovariole is contained within a tube, protected by an ovariolar wall, which consists of tunica propria (or basement membrane), epithelial sheath (bilayered epithelial tissue), and peritoneal sheath (protective tissue). Both the epithelial and peritoneal sheaths contain nervous tissue. The musculature, set between the two layers of epithelial sheath, undergoes rhythmic contractions, pushing the eggs/egg chambers posteriorly. The peritoneal sheath contains tracheoles and holds the ovary together. The formation and differentiation of oocytes takes place in an assembly-line fashion inside the ovarioles. The apical region of the ovariole is called the germarium, and the following part is the vitellarium.

Drosophila Melanogaster Meiosis Diagram
Fig. 2. The formation and development of the egg chambers in ovarioles. Stages 1-14 of oogenesis and the corresponding stages of meiosis are indicated. tf-terminal filament; ovariole wall components: tunica propria, epithelial sheath, and peritoneal sheath. (Adapted from ref. 13.)

Oogenesis begins in the germarium with the asymmetrical division of a germ-line stem cell into a daughter stem cell and a cystoblast, as inferred from the partition of a spherical membranous organelle called the spectrosome (see Fig. 3). Four rounds of mitotic divisions of the cystoblast and its daughters (cystocytes) produce a cluster of 16 cells. Because each of these mitotic divisions is followed by incomplete cytokinesis, the cystocytes remain interconnected through arrested cleavage furrows, thus forming a 16-cell cyst. The arrested cleavage furrows develop into ring canals, and the 16-cell cyst contains 2 cells with 4 ring canals (also named pro-oocytes), 2 cells with 3 ring canals, 4 cells with 2 ring canals, and 8 cells with 1 ring canal each (see Fig. 3). The ring canals play important roles in the cystocyte divisions and they facilitate the growth and differentiation of the oocyte (14). The fusome, a continuous vesicular organelle descended from the spectrosome, connects the cystocytes inside the 16-cell cyst via the ring canals (see Fig. 4A).

Cystocyte divisions are confined to the anterior third of the germarium, known as region 1. As the 16-cell cyst moves posteriorly through regions 2a and 2b of the germarium, it undergoes a change of shape, and one of the two cystocytes with four ring canals differentiates as an oocyte, maintaining a mei-otic fate. The other pro-oocyte and 14 cystocytes will become nurse cells through endoreduplication cycles (see Figs. 2 and 4B-E; see also Chapter 6). Each 16-cell cyst acquires a monolayer of follicle cells derived from 2 somatic stem cells located near the junction with region 2b. In region 2b, the 16-cell cysts are initially lens shaped, then become round, and, finally, bud off in

Phalloidine Drosophila Egg Stage
Fig. 3. Schematic representation of the germ-line-specific divisions leading to formation of the 16-cell cyst.

germarium region 3 to form a new egg chamber. This stage 1 egg chamber subsequently enters the vitellarium where it continues to grow and moves further posteriorly.

The vitellarium contains egg chambers from stage 2 to stage 14, which is the mature egg (13-16) (see Fig. 2). (The most important morphological features of each stage of oogenesis are listed in Appendix A.) The oocyte gradually increases in size during stages 2-10a, acquiring cytoplasm from the nurse cells (see Subheading 1.5.) and yolk (vitellogenesis) beginning at stage 8. Three

Fig. 4. (opposite page) (A) Stem cell at metaphase stained for tubulin (green), a-spectrin (red), and DNA (TOTO-3, blue). Note the asymmetric nature of this division as inferred from the apical position of the fusome. Scale bar: 5 |im. (B) Stage 7 egg chamber stained for lamin (green) and DNA (blue). (C) Stage 6 egg chamber stained for tubulin (green), Orbit (red), and DNA (blue). The Orbit protein accumulates behind the oocyte nucleus, whereas the microtubule bundles extend across the cyst. (D) Stage 7 egg chamber, stained for actin (red), Filamin (green), and DNA (blue). Notice the subcortical actin network in the oocyte. Scale bar identical for B-D: 25 |im. (E) Stage 5 egg chamber stained for actin (red) and Filamin (green). Note the four ring canals of the oocyte and the colocalization of the two proteins in the ring canals. Scale bar: 5 |im. (F) Stage 4 egg chamber stained with anti-lamin (green) and DNA (red). The nurse cell nuclei contain similar amounts of DNA. The oocyte nucleus, situated at the posterior of the egg chamber, contains the karyosome. (G) Stage 10 egg chamber stained as in (F), showing the oocyte nucleus in the anterior dorsal corner. Note the lamin accumulation in the oocyte nucleus. Squamous follicle epithelium covers the nurse cells and columnar epithelium is seen on the oocyte. (H) Stage 8 egg chamber stained with anti-tubulin (green), anti-CP190 (red, and panel 2), and DNA (blue, and panel 3). CP190 protein is seen in the nurse cells and accumulates in the oocyte nucleus around stage 8. Note the small size of the karyosome as inferred from the DNA staining (panel 3). (I) Cystocyte divisions visualized through staining for tubulin (green), a-spectrin (red, and panel 2), and DNA (blue). The left cyst shows metaphase spindles

Pole Gene Antibody

Fig. 4. (continued) associated with the fusome at of one of their poles. The right cyst shows fusome "plugs" migrating towards the old fusome. (J,K) Stage 13 oocyte. The nuclear lamina of the oocyte nucleus was stained with anti-lamin antibody (green) and the DNA with propidium iodide (red). Note the anterior-dorsal position of the oocyte nucleus with the highly condensed karyosome. (L,M) Breakdown of the oocyte nuclear membrane during stage 13, stained as in (J) and (K). Lamin becomes dispersed in the cytoplasm during the disassembly of the nuclear envelope. (See color plate 3 in the insert following p. 242.)

Fig. 4. (continued) associated with the fusome at of one of their poles. The right cyst shows fusome "plugs" migrating towards the old fusome. (J,K) Stage 13 oocyte. The nuclear lamina of the oocyte nucleus was stained with anti-lamin antibody (green) and the DNA with propidium iodide (red). Note the anterior-dorsal position of the oocyte nucleus with the highly condensed karyosome. (L,M) Breakdown of the oocyte nuclear membrane during stage 13, stained as in (J) and (K). Lamin becomes dispersed in the cytoplasm during the disassembly of the nuclear envelope. (See color plate 3 in the insert following p. 242.)

major yolk constituents have been identified: protein-containing particles, gly-cogen-rich particles, and lipid droplets. Yolk proteins are synthesized in the fat body (17), released into the hemolymph, and subsequently taken up by the oocyte through endocytic activity (18). In addition, yolk proteins are produced by follicle cells (19). The lipid droplets appear in nurse cells at stage 8 and are transported to the oocyte, whereas the glycogen-rich particles do not appear until stage 13 and are probably synthesized in the oocyte. Maturation of the oocyte occurs in stages 12-14 with the complete resorbtion of the nurse cells, and the completion of eggshell formation, while in the oocyte, meiosis arrests at metaphase I.

The eggshell is a multilayered protein structure deposited by follicle cells (20,21). The inner layers include the vitelline membrane, the waxy layer, and the inner chorion layer. The outer layers include the endochorion and the exo-chorion. Once eggshell formation is completed, the follicle cells undergo apoptosis (22) and are resorbed as the mature egg is released from the ovary. The eggshell has regional specializations like the anterior dorsal appendages and posterior aeropyle (both involved in gas exchange), the micropyle, and the operculum, ringed on three sides by a collar. The collar exhibits ultrastuctural perforations in the chorion and vitelline membrane, and it is easily split, letting the larva escape through the operculum. The micropyle juts out near the ventral collar and surrounds a canal for sperm entry, playing an important role in fertilization.

Egg production depends on the availability of protein (1,23), sex peptide (24,25), juvenile hormone (26,27), ecdysone (28,29), and the insulin-signaling pathway (30,31). The newly eclosed Drosophila female has a vitellarium consisting of three to six egg chambers, all in previtellogenic stages. Egg chambers/ oocytes in vitellogenic stages appear during the first day of adult life, and inseminated females will be able to lay eggs the second day after eclosion. In nutrient-rich conditions, approximately two to three eggs per ovariole per day are produced. The germ-line and somatic stem cells, as well as their progeny, adjust their proliferation rates in response to nutrition without affecting the number of active stem cells (31). Under conditions of nutrient limitation, egg production can be regulated by programmed cell death at two precise developmental points: (1) in region 2a/2b within the germarium and (2) in stage 8 egg chambers at the onset of vitellogenesis (28,29,31).

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