Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila

Abstract

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

Fig 1. Mating stimulates GSC proliferation.

(A) Drosophila germarium. GSCs (red) reside in a niche, comprising somatic cells such as cap cells (orange), terminal filament, and escort stem cells. GSCs are identifiable by their typical spectrosome morphology (yellow) and their location (adjacent to the cap cells). GSC division produces one self-renewing daughter and one cystoblast that differentiates into a germline cyst. (B) Representative examples of the germaria of wild-type flies containing one, two, or three GSCs in each germaria. Samples were stained with 1B1 (green) and anti-DE-cadherin (magenta) antibodies, which visualized GSCs (dotted circles) and overall cell membranes, respectively. (C) Protocol for GSC analysis for all experiments in this study. 3-day-old females were mated with males and used for assay 1 day after mating. (D) Frequencies of germaria containing one, two, and three GSCs (left y axis), and average number of GSCs per germarium (right y axis) in virgin and mated females in wild-type flies. Mated females showed increased GSC numbers as compared with virgin females. (E) Frequencies of GSC expressing phosphorylated Mad (pMad), which is a stem cell marker [4], were almost the same in mated female flies as compared with virgin female flies. (F) Frequency of mitotic GSCs was counted by staining with anti-phospho-histone H3, which is a marker for mitotic cell division. Mated females showed an increased rate of mitotic GSCs as compared with virgin females. (G) The number of cap cells was counted by staining with anti-Lamin-C antibody, which is a marker for the cap cells. The number of germaria analyzed is shown in parentheses in E, and inside bars in D, F, and G. (H, I) Temporal change in GSC numbers in virgin and mated females. Females were mated with males for the first time 3 days after eclosion (1^st mating) (H) and in the second time 7 days after 1^st mating (2^nd mating) (I). Also see S1 Fig. The number of germaria analyzed for H and I are shown in S2 Table. Values are presented as the mean with standard error of the mean in G, H, I. For statistical analysis, a Mann-Whitney U test was used for D, H, I. Chi-square analysis was performed for F. A Student’s t-test was used for G. ***P ≤ 0.001, **P ≤ 0.01, n.s., non-significant (P > 0.05).

Fig 2. Neuronal SPR function is required and sufficient for a mating-induced increase in GSC numbers.

(A, B, C and E) Frequencies of germaria containing one, two, and three GSCs (left y axis), and average number of GSCs per germarium (right y axis) in virgin (v) and mated (m) female flies. (A) Wild-type females were mated with wild-type and SP trans-heterozygous mutant adult male flies (SP⁰/SP^Δ41). (B) Wild-type or SPR null homozygous female flies (SPR^{Df(1)Exel6234}) were mated with wild-type male flies. (C, E) Adult female flies overexpressing the membrane bound form of SP (mSP) or transgenic SPR RNAi were mated with wild-type male flies under the control of several neuronal GAL drivers (elav-GAL4, ppk-GAL4 and fru (NP21)-GAL4) (C) or ovarian somatic cell-specific GAL4 driver (c587-GAL4) (E). Transgenes were driven by indicated GAL4 drivers. NP21-GAL4 was used for driving transgenes in fru-positive neurons. (D) Frequency of mitotic GSCs was counted by staining with anti-phospho-histone H3 in SPR RNAi female flies. The numbers of germaria analyzed are shown inside the bars. For statistical analysis, a Mann-Whitney U test was used for (A, B, C and E). Chi-square analysis was performed for (D). **P ≤ 0.01, *P ≤ 0.05, n.s., non-significant (P > 0.05).

Fig 3. Ovarian ecdysteroid biosynthesis controls a mating-induced increase in GSC numbers.

(A, D) Ecdysteroid levels in virgin (v) and mated (m) females in the ovarian somatic cell-specific (escort cells and follicle cells) nvd RNAi female flies. (A) c587-GAL4 driver was crossed with control or UAS transgene strains as indicated. UAS-nvd-Bm [wt] and UAS-nvd-Bm [H190A] overexpressed the wild-type form and enzymatic inactive form of Bombyx mori nvd transgenes, respectively. (D) nvd RNAi female flies were fed food supplemented with ethanol (EtOH; for control) and 7-dehydrocholesterol (7dC). (B, E and F) Frequencies of germaria containing one, two, and three GSCs (left y axis), and average number of GSCs per germarium (right y axis) of follicle cell-specific nvd RNAi animals with or without the B. mori nvd transgene (B), the ovarian somatic cell-specific EcR RNAi female flies and transheterozygous mutants for EcR (EcR^A483T and EcR^M554fs, mutants in the predicted ligand- binding domain) (E), ovarian somatic cell-specific nvd and sad RNAi female flies that were fed food supplemented with EtOH (for control), 7dC and 20E (F). (C) Frequency of mitotic GSCs was counted by staining with anti-phospho-histone H3 in nvd RNAi female flies. Values are represented as the mean with standard error of the mean in A and B. The numbers of samples examined are indicated in parentheses in A and D. The numbers of germaria analyzed are shown inside bars in B, C, E and F. For statistical analysis, Dunnett’s test was used for A and D. A Mann-Whitney U test was used for B, E and F. Chi-square analysis was used for C. **P ≤ 0.01, *P ≤ 0.05, n.s., non-significant (P > 0.05).

Fig 4. Ovarian ecdysteroid biosynthesis is required for reproductive output.

(A–C) The number of laid eggs was measured in ovaries of follicle cell-specific nvd RNAi female flies. (A) Temporal changes in laid eggs were measured for 7 days. (B) Total number of laid eggs in 7 days after mating is shown. (C) Total number of laid eggs in 48 hours in nvd RNAi female flies that were fed food supplemented with EtOH (for control), 7dC and 20E. Feeding of 7dC or 20E rescued decreased egg number phenotype in nvd RNAi. Values are presented as the mean with standard error of the mean in A. Each value is plotted as a dot. Box plot shows 25–75% (box), median (band inside) and minima to maxima (whiskers) in B and C. For statistical analysis, a Student’s t-test was used for A and B. Dunnett’s test was used for C. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.

Fig 5. Neuronal SP signaling controls a mating-induced increase in GSC numbers dependent on ecdysteroid.

(A) Ecdysteroid levels in virgin (v) and mated (m) female ovaries isolated from adult female flies overexpressing the membrane bound form of SP (mSP) and transgenic SPR RNAi. Transgenes were driven by ppk-GAL4 or fru (NP21)-GAL4. (B) Mating-induced expression of ecdysteroid biosynthesis enzyme genes in the ovaries. The y-axis represents fold changes of transcript levels between the ovaries of mated and virgin females. Box plot shows 25–75% (box), median (band inside) and minima to maxima (whiskers). Most of the enzyme genes, except for nobo, transcriptionally increased by mating in control female ovaries (Gray: ppk/+). Increased transcript levels of nvd and phm by mating were suppressed in SPR RNAi females (Purple: ppk>SPR RNAi). (C and D) Frequencies of germaria containing one, two, and three GSCs (left y axis), and average number of GSCs per germarium (right y axis) in virgin (v) and mated (m) female flies of neuronal SPR RNAi adult female flies that were fed food supplemented with EtOH (for control) and 20E. (D) Females mated with SP null mutant males did not show mating-induced increase in GSCs and this phenotype was not rescued by oral administration of 20E. (E) Schematic of neuroendocrine control of a mating-induced increase in GSC numbers. Neuronal sex peptide signaling induced by mating increases GSC numbers via activated ovarian ecdysteroid biosynthesis. Since overexpression of SP, but not feeding of 20E into virgin females was sufficient to induce an increase in GSC numbers, there might be another pathway activated by SP to control GSC numbers (dotted arrow). Values are presented as the mean with standard error of the mean in A. The numbers of samples examined are indicated in parentheses in A, or inside bars in C, D. For statistical analysis, Dunnett’s test was used for A, Student’s t-test was used for B, a Mann-Whitney U test were used for C and D. ***P≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, n.s., non-significant (P > 0.05).