交尾依存的な生殖幹細胞増殖を制御する神経内分泌機構の追究

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

交尾依存的な生殖幹細胞増殖を制御する神経内分泌機構の追究

筑波大学 (University of Tsukuba)201

Endocrine regulation of female germline stem cells in the fruit fly Drosophila melanogaster

Germline stem cells (GSCs) are critical for the generation of sperms and eggs in most animals including the fruit fly Drosophila melanogaster. It is well known that self-renewal and differentiation of female D. melanogaster GSCs are regulated by local niche signals. However, little is known about whether and how the GSC number is regulated by paracrine signals. In the last decade, however, multiple humoral factors, including insulin and ecdysteroids, have been recognized as key regulators of female D. melanogaster GSCs. This review paper summarizes the role of humoral factors in female D. melanogaster GSC proliferation and maintenance in response to internal and external conditions, such as nutrients, mating stimuli, and aging

Ovarian ecdysteroid biosynthesis and female germline stem cells

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

Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner

Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. Here, we show that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. Our study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication

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

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

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

<p>(A, D) Ecdysteroid levels in virgin (v) and mated (m) females in the ovarian somatic cell-specific (escort cells and follicle cells) <i>nvd</i> RNAi female flies. (A) <i>c587-GAL4</i> driver was crossed with control or <i>UAS</i> transgene strains as indicated. <i>UAS-nvd-Bm [wt]</i> and <i>UAS-nvd-Bm [H190A]</i> overexpressed the wild-type form and enzymatic inactive form of <i>Bombyx mori nvd</i> transgenes, respectively. (D) <i>nvd</i> 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 <i>nvd</i> RNAi animals with or without the <i>B</i>. <i>mori nvd</i> transgene (B), the ovarian somatic cell-specific <i>EcR</i> RNAi female flies and transheterozygous mutants for <i>EcR</i> (<i>EcR</i>^<i>A483T</i> and <i>EcR</i>^{<i>M554fs</i>}, mutants in the predicted ligand- binding domain) (E), ovarian somatic cell-specific <i>nvd</i> and <i>sad</i> 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 <i>nvd</i> 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 <i>U</i> test was used for B, E and F. Chi-square analysis was used for C. **<i>P</i> ≤ 0.01, *<i>P</i> ≤ 0.05, n.s., non-significant (<i>P</i> > 0.05).</p

Mating stimulates GSC proliferation.

<p>(A) <i>Drosophila</i> 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-<i>D</i>E-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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006123#pgen.1006123.ref004" target="_blank">4</a>], 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006123#pgen.1006123.s003" target="_blank">S1 Fig</a>. The number of germaria analyzed for H and I are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006123#pgen.1006123.s002" target="_blank">S2 Table</a>. Values are presented as the mean with standard error of the mean in G, H, I. For statistical analysis, a Mann-Whitney <i>U</i> test was used for D, H, I. Chi-square analysis was performed for F. A Student’s t-test was used for G. ***<i>P</i> ≤ 0.001, **<i>P</i> ≤ 0.01, n.s., non-significant (<i>P</i> > 0.05).</p

Neuronal <i>SPR</i> function is required and sufficient for a mating-induced increase in GSC numbers.

<p>(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 <i>SP</i> trans-heterozygous mutant adult male flies (<i>SP</i>^<i>0</i>/<i>SP</i>^<i>Δ</i>41). (B) Wild-type or <i>SPR</i> null homozygous female flies (<i>SPR</i>^{<i>Df(1)Exel6234</i>}) were mated with wild-type male flies. (C, E) Adult female flies overexpressing the membrane bound form of <i>SP</i> (<i>mSP</i>) or transgenic <i>SPR</i> RNAi were mated with wild-type male flies under the control of several neuronal GAL drivers (<i>elav-GAL4</i>, <i>ppk-GAL4</i> and <i>fru (NP21)-GAL4</i>) (C) or ovarian somatic cell-specific GAL4 driver (<i>c587-GAL4</i>) (E). Transgenes were driven by indicated GAL4 drivers. <i>NP21-GAL4</i> was used for driving transgenes in <i>fru</i>-positive neurons. (D) Frequency of mitotic GSCs was counted by staining with anti-phospho-histone H3 in <i>SPR</i> RNAi female flies. The numbers of germaria analyzed are shown inside the bars. For statistical analysis, a Mann-Whitney <i>U</i> test was used for (A, B, C and E). Chi-square analysis was performed for (D). **<i>P</i> ≤ 0.01, *<i>P</i> ≤ 0.05, n.s., non-significant (<i>P</i> > 0.05).</p