Ovarian ecdysteroid biosynthesis and female germline stem cells

Abstract

The germline stem cells (GSCs) are critical for gametogenesis throughout the adult life. Stem cell identity is maintained by local signals from a specialized microenvironment called the niche. However, it is unclear how systemic signals regulate stem cell activity in response to environmental cues. In our previous article, we reported that mating stimulates GSC proliferation in female Drosophila. The mating-induced GSC proliferation is mediated by ovarian ecdysteroids, whose biosynthesis is positively controlled by Sex peptide signaling. Here, we characterized the post-eclosion and post-mating expression pattern of the genes encoding the ecdysteroidogenic enzymes in the ovary. We further investigated the biosynthetic functions of the ovarian ecdysteroid in GSC maintenance in the mated females. We also briefly discuss the regulation of the ecdysteroidogenic enzyme-encoding genes and the subsequent ecdysteroid biosynthesis in the ovary of the adult Drosophila.

Keywords: Drosophila; Halloween gene; ecdysone; mating; sex peptide; steroid hormone.

Figure 1.

Transcriptional regulation of ecdysteroidogenic enzyme genes in the ovary. (A) The ecdysteroid biosynthesis pathway. Cholesterol is converted into 20-hydroxyecdysone (active form of ecdysone) by several ecdysteroidogenic enzymes (Shown in bold). (B) Temporal changes in ecdysteroidogenic enzyme genes in virgin female flies in post-eclosion period (n = 4). Most of the genes showed higher expression levels at 6 hours or 15–21 hours post-eclosion (nobo, nvd, sro, spo, phm, and shd). (C) Relative changes in ecdysteroidogenic enzyme gene expression in ovary. Ovaries were dissected from age-matched virgin and mated females at 16 hours post-mating. Some genes showed significant increase in mated female flies compared to virgin female flies (sro, spo, phm, sad, and shd). These increased expressions after mating were suppressed when female flies mated with SP null male flies (except for spo). Values are presented as the mean with standard error of the mean in B. For statistical analysis, t-test with Holm's correction was used for B, Student's t-test was used for C. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, NS, non-significant (P > 0.05).

Figure 2.

The role of ecdysteroid biosynthesis on the regulation of GSC maintenance. (A) Protocol for all experiments in this figure. One-week-old females were mated with males and used for the assay 1 week after mating. (B) Drosophila germarium. Germline stem cell (GSC) resides in a niche, comprising somatic cells called cap cells, terminal filament, and escort stem cells. GSCs are identifiable by their typical spectrosome morphology and their location (adjacent to the niche cells). GSC produces one self-renewing daughter and one cystoblast (CB) that differentiates into a germline cyst. The cystoblast divides four times with incomplete cytokinesis (2 cc: 2-cell cyst, 4 cc: 4-cell cyst, 8cc: 8-cell cyst and 16 cc: 16-cell cyst). (C) Drosophila ovary is composed of 15–20 ovarioles. The continuous developing egg chamber is divided into 14 stages. (D) Left: Frequencies of germaria containing zero, one, two and three GSCs (left y-axis), and average number of GSCs per germarium (right y-axis) in mated females. Ovarian neverland (nvd) knockdown in ovarian somatic cells (escort cells and follicle cells, using c587-GAL4) reduced average GSC number as compared to the control (P = 0.006145). Right: Temporal change in GSC number in virgin females (1-day-old and 1-week-old) and mated females (2-week-old), (n ≥ 94). (E and F) The average number of germline cyst (E) and egg chamber in each stage (F) was not changed in ovarian nvd RNAi female flies (c587>nvd RNAi). (G) UAS-nvd-Bm [wt] and UAS-nvd-Bm [H190A] were used for overexpressing the wild-type form and enzymatic inactive form of Bombyx mori nvd transgenes, respectively. Ovarian ecdysteroid decreased in c587>nvd RNAi female flies as compared to the control flies (P = 0.0233). This reduction was restored by overexpressing UAS-nvd-Bm [wt] but not UAS-nvd-Bm [H190A]. (H) GSC phenotype in c587>nvd RNAi animals was restored by overexpressing UAS-nvd-Bm [wt] but not UAS-nvd-Bm [H190A]. (I) GSC phenotype in c587>nvd RNAi flies was rescued by oral administration of 20E or 7dC. Values are presented as the mean with standard error of the mean in G. The numbers of samples examined are indicated in parentheses in D, E, F, H and I. For statistical analysis, Wilcoxon rank sum test was used for D, E and F. t-test with Holm's correction was used for G. Steel-Dwass test was used for H and I. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, NS, non-significant (P > 0.05).

Figure 3.

Model for this study. Ecdysteroid biosynthesis in the ovary is differentially regulated in different adult life stages, including the post-eclosion and pre-mated stage (upper column), the post-mating early stage, and the post-mating late stage (lower column). In post-eclosion stage, ecdysteroidogenic enzyme gene expression is regulated by unknown tropic stimuli and may be involved in controlling the ovarian ecdysteroid biosynthesis to initiate oogenesis. In post-mated early stage, SP stimulates ecdysteroid biosynthesis via upregulation of biosynthesis enzyme gene expression, which control GSC proliferation. Ovarian ecdysteroid biosynthesis is also required for GSC maintenance in post-mated late stages.