Mating-induced Ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis

Abstract

Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, we find that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. We find that GSC abscission failure is caused by increased Ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females, but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population.

Keywords: Drosophila; Cytokinesis; Ecdysone; Encystment; Mating; Stem cells.

Fig. 1.

Mating causes a significant increase in GSC abscission failure. (A) Diagram of testis niche and germline development. (B,C) Time-lapse of nanos-ABD-moe::GFP (green) and Myo-mCherry (magenta) during GSC divisions in non-mated (B) and mated (C) animals. Each image is 1-4 z-slices. Asterisks indicate niche. Dotted lines indicate a GSC and daughter(s) as they go through two rounds of mitotic division. (D) Percent GSC abscission failure under non-mating (n=12 testes; failed abscission: 1/135 divisions) and mating conditions (n=15 testes; failed abscission: 40/167 divisions). (E) GSC cell cycle length in non-mated (n=42 GSCs in 10 testes) and mated animals (n=43 GSCs in 14 testes). ****P<0.0001 (non-parametric Mann–Whitney U-test). ns, not significant. Error bars: s.d. All experiments n≥2 trials. Scale bar: 5 µm (for B,C).

Fig. 2.

Mating leads to decreased soma-germline adhesion and EGFR signaling. (A) Apical tip of the testis showing two-cell cyst (yellow box) and diagram of normal versus disrupted somatic encystment. (B-E) Magnified view of two-cell cysts from a control and non-mated testis showing typical E-cad accumulation at soma-germline junctions (white dotted line) (B,D) and a testis with somatic expression of DN-Rac1 and mated testis showing diminished E-cad levels at soma-germline contacts (C,E). (F) Quantification of E-cad fluorescence intensity represented as fold change relative to the respective controls (n≥40 two-cell cysts, minimum of 10 testes). (G,H) Immunofluorescent staining of somatic nuclei (green, Tj), niche cell junctions (magenta, FasciclinIII/FasIII) and dpErk (gray) in a non-mated testis (G) and a mated testis with diminished dpErk (H). (I) Quantification of dpErk fluorescence intensity in somatic nuclei represented as fold change (n≥345 somatic nuclei in at least six testes). ****P<0.0001 (non-parametric Mann–Whitney U-test). Error bars: s.d. All experiments n≥2 trials. Scale bars: 10 µm (A,G,H); 5 µm (B-E).

Fig. 3.

Ecdysone signaling but not germ cell depletion causes GSC abscission failure. (A,B,F-H) Time-lapse of nanos-ABD-moe::GFP (green) and Myo-mCherry (magenta) during GSC divisions in testes of males with optogenetic activation of Crz neurons (A″), expressing Bam-Hid (B″), fed 20E (F) and mated with somatic expression of DN-EcRB2 (H). Dotted lines indicate a GSC and daughter as they progress through successful (A″,B″,F) or failed (H) cytokinesis. (A′,B′) Diagrams of methods for sperm/germ cell depletion in the absence of mating. (A‴,B‴,G) Quantification of GSC abscission failure (n≥11 testes) (non-mated/mated data repeated from Fig. 1). (C,D) Immunostaining of Tj, E-cad and Broad in non-mated and mated males. (E) Quantification of broad fluorescence intensity represented as fold change (n≥345 somatic nuclei in at least six testes). (I) Quantification of E-cad fluorescence intensity at soma-germline contacts of two-cell cysts represented as fold change relative to the vehicle fed controls (n=35 two-cell cysts in 10 testes). **P<0.01, ****P<0.0001 (non-parametric Mann–Whitney U-test). ns, not significant. Error bars: s.d. All experiments n≥2 trials. Scale bars: 5 µm (A″,B″,F,H); 10 µm (C,D).

Fig. 4.

Mating-induced GSC abscission failure is transient but reoccurs with repeated bouts of mating. (A-C) Time-lapse of nanos-ABD-moe::GFP (green) and Myo-mCherry (magenta) during GSC divisions in testes of 2 days mated (A) 2 days mated+1 day of rest (B) and 2 days mated+1 day of rest+2 days mated (C) males. (D) De-differentiation of a two-cell cyst in 2 days mated+1 day of rest+2 days mated testes. Dotted lines indicate a GSC and daughter as they undergo failed (A,C) or successful (B) abscission, and a two-cell cyst that de-differentiates and reenters the niche (D). (E) Quantification of GSC abscission failure and de-differentiation (n≥11 testes). (F-I) Model for Ecdysone-mediated control of somatic encystment in non-mating (F,G) and mating (H,I) conditions. (J) Model of the potential adaptive function for decreased encystment. *P<0.05, ****P<0.0001 (non-parametric Mann–Whitney U-test). ns, not significant. Error bars: s.d. All experiments n≥2 trials. Scale bar: 5 µm.