Oogenesis and Development of Nurse Cells

Each of the two paired ovaries of the adult Drosophila female consists of a cluster of parallel ovarioles, where egg chambers are lying in a single-file arrangement (see Fig. 1A,B). Each egg chamber contains a branching chain of 16 interconnected cystocytes formed by mitotic division of a germarial cystoblast (2). Fifteen of the cystocytes differentiate into endopolyploid nurse cells (NCs) (see Fig. 1C). The function of these germ-line-derived cells is to synthesize RNA and protein molecules, which they transport through a system of ring canals into the oocyte (3) (see also Chapter 4).

The normal development of an egg chamber has been divided into 14 stages (4). Stages 1-6 are "previtellogenic," with intensive growth of all the cells that form each chamber. At stage 7, the process of transfer of yolk precursors from NCs into the oocyte begins. Vitellogenesis, accelerated growth by the oocyte

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

Diagram Oogenesis Drosophila

resulting from yolk accumulation, starts at stage 8. By stage 9, NCs also develop unusual nucleoli composed of a shell of interconnected fibers around the periphery of the nucleus. NCs retain more rRNA genes than other polyploid cells and synthesize rRNA at proportionately higher rates (5,6). By stage 10, NCs reach their maximum size and DNA content (1024-2048C) (7). Stage 11 is the shortest and marks the beginning of the postvitellogenic period, with apoptosis of NCs, and the completion of the vitelline membrane. During stages 12-14, beta yolk forms, and the egg shell and its appendages are synthesized.

The staging characteristics for Drosophila oogenesis were formulated after observations of Feulgen-stained, ovarian whole mounts (8) (see Subheading 3.1.). The NC nuclei in chambers belonging to stages 6-10 possessed a dispersed mass of Feulgen-positive threads (see Fig. 1B, S6). Nuclei in stages 3 and 4 contained densely staining bulbous structures (see Fig. 1B, S3 and S4). The NCs in stage 5 were at an obviously intermediate state. The nuclei nearest the oocyte contained dispersed chromosomes like those in stage 6, whereas the rest resembled stage 4.

We know that the chromosomes in NCs undergo a cycle of endomitotic DNA replications (9). In NCs at stages 1-4, the paired homologs go through four endocycles, and the DNA is completely replicated each time with a total DNA content of 64C. At stage 5, the bivalents fall apart, starting with the NCs closest to the oocyte, and the polytene univalents subdivide further to form 32 separate chromatids, each containing the 2C amount of DNA and held together by unreplicated regions. There are three to four replication cycles, depending on the distance of the NC from the oocyte. During these cycles, which take place during stages 6-10, only about 90% of the DNA is replicated (9).

Fig. 1. (A) A dorsal view of the internal reproductive system of an adult egg laying female Drosophila melanogaster. Two ovarioles have been pulled loose from the left ovary (From ref. 2, Copyright ©1967, used with permission, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.). (B) A diagram of a single ovariole and its investing membranes. S1-S6 = previtellogenic stages of egg chambers; 1, 2, and 3 = regions of the germarium. The distribution of the nucleolar material is drawn in the starred NC nucleus, whereas the other NC nuclei show the appearance of the chromosomal material (From ref. 2, Copyright ©1967, used with permission, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.). (C) A diagram of the steps in the production of a clone of 16 cystocytes. S = stem cell, Cb = cystoblast, Mj-M4 = four consecutive mitoses. The area in each cell is proportional to the volume of the cell. The canals connecting cells are labeled according to the division at which they were produced (see ref. 3) (From A Dictionary of Genetics, 6th ed, by Robert C. King and William D. Stansfield, ©1985, 1990, 1997, 2002 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc.)

Table 1

General Characteristics of Ovarian Phenotypes, Terminal Stage of Oogenesis, and Chromosome Morphology for Different otu Allelic Compositions

Table 1

General Characteristics of Ovarian Phenotypes, Terminal Stage of Oogenesis, and Chromosome Morphology for Different otu Allelic Compositions

OAC

Qa

Ta

pa

Oa

AS

ApC

ARC

pi

Hpt

Cb

Nb

7/7

2

0

26

72

p12

p12

12

284

151

76

0

11/11

0

0

53

47

p14

10B

10

89

82

113

266

7/11

1

4

48

47

14

10B

10

23

51

132

331

11/14

0

1

2

97

14

10B

10

_c

_c

_c

_c

Abbreviations: OAC = otu allelic composition; Q = quiescent germaria; T = ovarian tumors; P = PNC chambers; O = oocyte-NC chambers and mature eggs; AS = most advanced stage of oogenesis; APC = most advanced stage of polytene chromosomes; ARC = most advanced stage in replication cycle; P = pompons; HP = half-pompons; C = condensed; N = normal.

a Number per 100 ovarioles; QTPO values for 7/7 and 11/11 were from flies reared at 18°C; values for 7/11 and 11/14 are for flies reared at 23°C.

b Number of nuclei of each type observed on 20 slides prepared from 20 ovaries of flies of each genotype c No data available.

Source: Data from refs. 13,17,18.

Abbreviations: OAC = otu allelic composition; Q = quiescent germaria; T = ovarian tumors; P = PNC chambers; O = oocyte-NC chambers and mature eggs; AS = most advanced stage of oogenesis; APC = most advanced stage of polytene chromosomes; ARC = most advanced stage in replication cycle; P = pompons; HP = half-pompons; C = condensed; N = normal.

a Number per 100 ovarioles; QTPO values for 7/7 and 11/11 were from flies reared at 18°C; values for 7/11 and 11/14 are for flies reared at 23°C.

b Number of nuclei of each type observed on 20 slides prepared from 20 ovaries of flies of each genotype c No data available.

Source: Data from refs. 13,17,18.

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