Figures and data in An important role for triglyceride in regulating spermatogenesis

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Tables
Additional files

6 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement

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Lipid droplets (LD) are present in early-stage somatic and germ cells.

(A) Testis LD in *w¹¹¹⁸* animals visualized with neutral lipid dye BODIPY. (**A, A'**) Scale bar = 50 μm; (**A'', A'''**) scale bar = 15 μm. Asterisk indicates hub in all images. Arrows point to LD; arrowheads point to spermatocytes in A, B. Spermatocytes were identified as described in methods section. (B) Testis LD visualized with BODIPY in newly eclosed males from two wild-type genotypes. Scale bars: main image = 50 μm; inset image = 10 μm. (C) Testis LD from *w¹¹¹⁸* animals at different times post-eclosion. Scale bars = 50 μm. (D) Testis LD visualized with LipidTox Red in animals with somatic cell overexpression of GFP-LD (*Tj-GAL4*>*UAS-GFP-LD*). Green fluorescent protein (GFP)- and LipidTox Red-positive punctae are somatic LD (D–D'' arrows); LipidTox punctae without GFP indicate germline LD (D–D'' arrowheads). Scale bars = 10 μm. (E) Histogram showing the spatial distribution of somatic cell LD; error bars represent standard error of the mean (SEM). (F) Cumulative frequency distributions of somatic LD (blue line, data reproduced from E), zfh-1-positive somatic cells (zfh-1⁺ cells, orange line), and Eya-positive somatic cells (Eya⁺ cells, gray line). (G) Testis LD visualized with LipidTox Red in males with germline overexpression of GFP-LD (*nos-GAL4*>*UAS-GFP-LD*). GFP- and LipidTox Red-positive punctae indicate germline LD (arrows); LipidTox punctae without GFP indicate non-germline LD (arrowheads). Scale bars = 10 μm. (H) Histogram representing the spatial distribution of LD within the germline; error bars represent SEM. (I) Histogram representing the spatial distribution of LD and GFP fluorescence (green line) (arbitrary units, a.u.) in a representative testis of a *bam-GFP* animal (panel J). (J) Testis LD in a *bam-GFP* animal; arrows point to LD and arrowheads point to spermatocytes. Scale bar = 50 μm. See also Figure 1—figure supplement 1.

Figure 1—figure supplement 1

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Cholesterol is absent from testis lipid droplets.

(A) Testes stained with BODIPY (A) to detect neutral lipids and Filipin III (A') to detect free cholesterol. Scale bars = 50 μm.

Figure 2 with 2 supplements

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*brummer* regulates lipid droplets (LD) in both germline and somatic cells of the testis.

(**A–A''''**) Testis LD indicated by LipidTox Red in *bmm-GFP* animals. Arrows point to LD in all images. Arrowheads point to spermatocytes. Scale bars = 50 μm. Asterisks indicate the hub in all images. (B) Quantification of nuclear GFP intensity in testes isolated from *bmm-GFP* animals (n = 3). Germline stem cell (GSC), spermatogonia (SG), spermatocyte (SC). (C) Spatial distribution of LD (gray histogram) and GFP expression (green line) in testes from *bmm-GFP* animals as a function of distance from the hub (n = 3). LD near the apical region of the testis in *bmm^rev* (D) or *bmm¹* (E) animals. (F) LD further away from the apical tip in *bmm¹*animals. (**D–F**) Scale bars = 50 μm. (G) Histogram representing testis LD size distribution in *bmm^rev* (gray) and *bmm¹* (orange). (B,C,G) Error bars represent standard error of the mean (SEM). (H) Apical tip of the testes is at the left of the graph; individual dots represent a single LD and its relative position to the hub marked by an asterisk. Cumulative frequency distribution of the distance between LD and the apical tip of the testes are drawn as solid lines. See also Figure 2—figure supplement 1 and Figure 2—figure supplement 2.

Figure 2—figure supplement 1

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Expression of *brummer* and selected lipid metabolic genes during spermatogenesis in germline and somatic lineages.

(A) Pseudotime trajectory of germline (black line) based on single-cell RNA sequencing data (Li et al., 2021). Individual cells are labeled according to the annotation within the dataset. (B) Pseudotime trajectory of the somatic cells (black line) based on publicly available single-cell RNA sequencing data (Li et al., 2021). Individual cells are labeled according to the annotation within the dataset. Only one trajectory (branch b) is marked and used for panel D. (C) Rolling average of normalized transcript counts in the germline along the trajectory shown in panel A are plotted as a black line on the upper panel. Composition of cell types mapped on to the trajectory at each time point is shown at the bottom of panel B. (D) Rolling average of normalized transcript counts in somatic cells plotted as a black line along the trajectory shown in C (upper panel). Composition of cell types mapped on to the trajectory at each time point is shown at the bottom of panel D.

Figure 2—figure supplement 2

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*brummer* regulates lipid droplets (LD) in both germline and somatic cells of the testis.

Representative images of *bmm^rev* (A and B) and *bmm¹* (C and D) testes with somatic overexpression of *GFP-LD* (*Tj-GAL4*>*UAS-GFP-LD*). Panels B and D contain magnified images of the area indicated by the boxes in panels A and C, respectively. In *bmm^rev* testes, LD were restricted to a region near the apical tip (A) of the testis in both somatic (**B–B'''**, arrows) and germline cells (**B–B'''**, arrowheads). In *bmm¹* testes, LD were present in both somatic (**C, D**, arrows) and germline cells (**C, D**, arrowheads), near the apical tip of the testis in a region corresponding to early-stage germ cells and in the region corresponding to spermatocytes. (**A, C**) Scale bars = 50 μm; (**B, D**) scale bars = 20 μm.

Figure 3

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*bmm* regulates germline lipid droplets (LD) in a cell-autonomous manner.

(A, B) Single confocal slices through a representative testis isolated from an individual carrying clones induced using the FLP-FRT system at 3 days post-clone induction. Clones are homozygous for an allele that encodes a functional *bmm* protein product (*bmm^rev*; **A–A'''**) or a loss-of-function *bmm* allele (*bmm¹,* **B–B'''**). GFP-negative areas mark homozygous clones in panels A and B; the boxed areas in A, A'' and B, B'' are shown in A', A''' and B', B''', respectively. In homozygous *bmm^rev* spermatocyte clones we detected no LD using neutral lipid dye LipidTox (**A''**, **A'''**). In contrast, spermatocyte clones homozygous for *bmm¹* have detectable LD (**B''**, **B'''**, arrows). Scale bars = 50 μm in A, A'' and B, B''; scale bars = 10 μm in A', A''' and B', B'''. (C) Number of testis LD in *bmm^rev* (gray) or *bmm¹* (orange) in FLP-FRT clones 3 days post-clone induction; dots represent measurements from a single clone. The number of cells in each cyst (CC) counted is indicated. There were significantly more LD in *bmm¹* spermatocyte (SC) clones (p = 0.026; Welch two-sample t-test) but not at other stages of development. Error bars represent standard error of the mean (SEM).

Figure 4 with 1 supplement

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A cell-autonomous role for *bmm* in regulating spermatogenesis.

Testes isolated from *bmm^rev* (A) and *bmm¹* (A') animals raised at 25°C stained with phalloidin. Scale bars = 100 μm. (B) Testis size in *bmm¹* and *bmm^rev* animals raised at 25°C. (C) Spermatid bundle number in *bmm¹* and *bmm^rev* testes from animals reared at 25°C. Representative images of *bmm^rev* (D) or *bmm¹* (E) testes stained with 4′,6-diamidino-2-phenylindole (DAPI) and anti-Vasa antibody. Arrows indicate germline stem cells (GSCs). Scale bar = 50 μm. The hub is marked by an asterisk in all images. (F) GSC number in *bmm¹* and *bmm^rev* testes. (G) Proportion of GSCs that were either *bmm¹* or *bmm^rev* clones at 3 and 14 days post-clone induction. (H) Representative images of *bmm^rev* (H) and *bmm¹* (H') testes carrying *bam-GFP*; data quantified in Figure 4—figure supplement 1J. Arrows indicate regions with high Bam-GFP. Scale bars = 50 μm. (I) Representative images of *bmm^rev* (I) or *bmm¹* (**I', I''**) testes stained with anti-Vasa antibody. Arrows indicate Vasa-positive cysts in *bmm¹* testis. Panel I'' is magnified from the boxed region in I'. (**I, I'**) Scale bars = 100 μm; (**I''**) scale bar = 50 μm. Maximum projection of *bmm^rev* (J) or *bmm¹* (J') testes stained with anti-Boule antibody (green) and DAPI (blue). Scale bars = 100 μm. Number of *bmm¹* and *bmm^rev* spermatocyte clones (K) or post-meiotic clones (L) at 3 and 14 days post-clone induction. (B,C,F,G,K,L) Error bars indicate standard error of the mean (SEM). See also Figure 4—figure supplement 1.

Figure 4—figure supplement 1

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Additional characterization of testis development and spermatogenesis in animals lacking *bmm*.

(A) Testis size was smaller in *bmm¹* mutant animals compared with *bmm^rev* controls at <24 hr post-eclosion when raised at 29°C (Welch two-sample t-test). (B) The number of spermatid bundles was significantly lower in *bmm¹* mutant animals compared with *bmm^rev* controls at <24 hr post-eclosion when raised at 29°C (Kruskal–Wallis rank sum test). (C) Testis size was significantly smaller in *bmm¹* mutant males compared with *bmm^rev* control males at 14 days post-eclosion (Welch two-sample t-test). (D) While the median number of spermatid bundles was not significantly different between *bmm¹* mutant males and *bmm^rev* control males at 14 days post-eclosion (Welch two-sample t-test), 8/27 *bmm¹* testis had no spermatid bundles, a phenotype absent in age-matched *bmm^rev* males (0/22) (p = 0.0163, Pearson’s Chi-squared test), suggesting a subtle defect is present. (E) Food supplemented with 4% medium-chain triglyceride (MCT), but not long-chain triglyceride (LCT), significantly increased testis length in *bmm¹* animals but had no effect on this phenotype in *bmm^rev* control animals (one-way analysis of variance [ANOVA] with Tukey multiple comparison test). (F) Food supplemented with 4% medium-chain triglyceride significantly increased the number of spermatid bundles in *bmm¹* testes but had no effect on this phenotype in *bmm^rev* control animals (one-way ANOVA with Tukey multiple comparison test). (G) Representative images of *bmm^rev* (G, G') or *bmm¹* (**G''**, **G'''**) testes stained for FasIII (G, **G''**) and Vas (G', **G'''**). Scale bars = 25 μm. (H) Quantification of hub area in *bmm^rev* or *bmm¹* testes showed a significantly larger hub size in *bmm¹* testes (Welch two-sample t-test). (I) The number of germline stem cells (GSCs) undergoing mitosis (phospho-histone H3⁺ GSC/total GSC) was not significantly different between *bmm¹* and *bmm^rev* testes (Kruskal–Wallis rank sum test). (J) The distance between the hub and the first Bam-GFP-positive cyst (Figure 3H) was significantly higher in *bmm¹* testes than in *bmm^rev* testes (Welch two-sample t-test). (K) All *bmm^rev* testes and most *bmm¹* testes contained spermatids when raised at 25°C; however, the most advanced stage of spermatogenesis observed in the majority of *bmm¹* testes isolated from animals reared at 29°C was the spermatocyte stage. (L) Testes isolated from *bmm¹* animals showed a significantly smaller Boule-positive area than control testes (Welch two-sample t-test). (M) Testes isolated from *bmm¹* animals contain fewer individualization complexes than *bmm^rev* control testes (Kruskal–Wallis rank sum test). (N) Fewer waste bags were present in testes isolated from *bmm¹* animals compared with *bmm^rev* control testes (Kruskal–Wallis rank sum test). (O) Proportion of Zfh-1-positive clones that were homozygous for either *bmm¹* or *bmm^rev* at 3 and 14 days post-clone induction (Kruskal–Wallis rank sum test). (P) The location of Zfh-1-positive clones homozygous for either *bmm¹* or *bmm^rev* at 3 and 14 days post-clone induction measured as the distance from the center of the hub. Clones homozygous for *bmm¹* locate significantly closer to the hub than the wild-type clones (Kruskal–Wallis rank sum test). Error bars indicate standard error of the mean (SEM).

Figure 5 with 1 supplement

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Loss of *bmm* disrupts triglyceride homeostasis and leads to spermatogenic defects.

(A) Hierarchical clustering of lipid species detected in *bmm^rev* and *bmm¹* animals. (B) Histograms showing the proportion of significant species in each lipid class with different levels between *bmm¹* and *bmm^rev*. Numbers on histograms indicate the number of species with differences in abundance. (C) Volcano plot showing fold change in abundance of triglyceride (green; 97 species) and non-triglyceride lipids (gray; 186 species) in our dataset. (D) Arrows indicate testis lipid droplets (LD) stained with LipidTox Red in *bmm^rev* (D), *bmm¹* (D'), or *mdy^QX25/k03902; bmm¹* (**D''**) animals. (E) Whole testes isolated from *bmm^rev* (E), *bmm¹* (E'), or *mdy^QX25/k03902;bmm¹* (**E''**) animals stained with anti-Vasa antibody (red) and DAPI (blue). Arrowheads indicate spermatid bundles. Scale bars = 100 μm. (F) Testis size in *bmm^rev*, *bmm¹*, and *mdy^QX25/k03902;bmm¹* animals. Spermatid bundles (G) and number of germline stem cells (H) in *bmm^rev*, *bmm¹*, and *mdy^QX25/k03902;bmm¹* animals. (I) Testis size in animals with germline-specific *mdy* knockdown (*nos-GAL4*>*mdy RNAi; bmm¹*) compared with controls (*nos-GAL4*>*+; bmm¹* and +>*mdy RNAi; bmm¹*). Error bars indicate standard error of the mean (SEM). See also Figure 5—figure supplement 1.

Figure 5—figure supplement 1

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Lipidomic analysis of animals lacking *bmm*.

(A) Higher fold-changes of triglycerides in *bmm¹* animals were associated with less saturation in the acyl-groups (Kendall’s rank correlation test). (B) Higher fold-changes of triglycerides in *bmm¹* animals were associated with higher number of carbons in the acyl-groups (Kendall’s rank correlation test). Each dot represents a single triglyceride species for panels B and C. (C) Volcano plot of identified lipids; monoglycerides shown in blue and diglycerides shown in orange. Many monoglycerides and diglycerides show increase in fold-change in *bmm¹* males. (D) The number of carbon and the degree of saturation of monoglycerides (MAG) and diglycerides (DAG) with significant changes in abundance between *bmm¹* and *bmm^rev* males. (E) Volcano plot of identified lipids; fatty acids shown in magenta and acyl-carnitine shown in green. Many fatty acids show an increase in fold-change while many acyl-carnitines show a decrease in fold-change in *bmm¹* males. (F) The number of carbon and the degree of saturation of fatty acids (FA) and acyl-carnitines (ACar) with significant changes in abundance between *bmm¹* and *bmm^rev* males. (G) Volcano plot of identified lipids; membrane lipids shown in yellow. (H) The number of carbon and the degree of saturation of membrane lipids with significant changes in abundance between *bmm¹* and *bmm^rev* males. For panels G and H, PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanolamine; SM: sphingomyelin; PG: phosphatidylglycerol. (I) Loss of *mdy* function rescued the elevated number of lipid droplets (LD) in *bmm¹* testes to control levels (one-way analysis of variance [ANOVA] with Tukey multiple comparison test). (J) Germline-specific loss of *mdy* in *bmm¹* animals did not reduce germline stem cell (GSC) numbers, but the variance in GSC numbers was significantly rescued (*nos-GAL4*>*+; bmm¹* vs *nos-GAL4*>*mdy RNAi; bmm¹*: p = 4.5 × 10⁻⁵;+*>mdy RNAi; bmm¹* vs *nos-GAL4* >*mdy RNAi; bmm¹:* p = 0.0082 by F-test). Error bars indicate standard error of the mean (SEM).

Figure 6

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Model of *bmm*-mediated lipid droplet regulation in the *Drosophila* testis.

Schematic representation summarizing *bmm*-mediated lipid droplet regulation in the testis during development.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Anti-Vasa (rabbit, polyclonal)	Gift from Dr. R. Lehman, MIT		IF (1:200)
Antibody	Anti-Eya (mouse monoclonal)	Developmental Studies Hybridoma Bank (DSHB)	eya10H6	IF (1:50)
Antibody	Anti-zfh1 (mouse polyclonal)	Gift from Dr. J. Skeath, WUSTL		IF (1:1000)
Antibody	Anti-boule (rabbit polyclonal)	Gift from Dr. S. Wasserman, UCSD		IF (1:1000)
Antibody	Anti-phospho-histone H3 (mouse monoclonal)	Millipore Sigma	05-1354	IF (1:1000)
Strain, strain background	w¹¹¹⁸	Bloomington Drosophila stock center	3605	3605
Strain, strain background	CantonS	Bloomington Drosophila stock center	64349	64349
Strain, strain background	OregonR	Bloomington Drosophila stock center	25211	25211
Strain, strain background	bmm¹	Gift from Dr. R. Kühnlein; Grönke et al., 2005
Strain, strain background	bmm^rev	Gift from Dr. R. Kühnlein; Grönke et al., 2005
Strain, strain background (Drosophila melanogaster)	mdy[Qx25], cn[1], bw[1]/CyO, I(2)DTS513[1]	Bloomington Drosophila stock center	5095	5095
Strain, strain background (Drosophila melanogaster)	y[1],w[67c23];P{lacW}Cse1[k03802],mdy[k03902]/CyO	Bloomington Drosophila stock center	10536	10536
Strain, strain background (Drosophila melanogaster)	w[1118];P{GD1749}v6367 (UAS-mdy-RNAi)	Vienna Drosophila resource center	6367	6367
Strain, strain background (Drosophila melanogaster)	nos-GAL4::VP16	Bloomington Drosophila stock center	7303	7303
Strain, strain background (Drosophila melanogaster)	Tj-GAL4	Gift from Dr. D. Godt, University of Toronto
Strain, strain background (Drosophila melanogaster)	c587-GAL4	Bloomington Drosophila stock center	67747	67747
Strain, strain background (Drosophila melanogaster)	Bam-GFP	Chen and McKearin, 2003
Strain, strain background (Drosophila melanogaster)	bmm-GFP	Gift from Dr. K. Kamei; Men et al., 2016
Strain, strain background (Drosophila melanogaster)	GFP-LD	Gift from Dr. M. Welte; Yu et al., 2011
Strain, strain background (Drosophila melanogaster)	P{neoFRT}82B, bmm[1]	This study		Flies available from E. Rideout, made as in ‘Fly Husbandry’
Strain, strain background (Drosophila melanogaster)	P{neoFRT}82B, bmm[rev]	This study		Flies available from E. Rideout, made as in ‘Fly Husbandry’
Strain, strain background (Drosophila melanogaster)	bam-GFP, bmm[1]	This study		Flies available from E. Rideout, made as in ‘Fly Husbandry’
Strain, strain background (Drosophila melanogaster)	bam-GFP, bmm[rev]	This study		Flies available from E. Rideout, made as in ‘Fly Husbandry’
Software, algorithm	Fiji	https://imagej.net/software/fiji/
Software, algorithm	R	https://cran.r-project.org

Additional files

Supplementary file 1 Raw data and statistical outputs from Figure 1.: https://cdn.elifesciences.org/articles/87523/elife-87523-supp1-v1.xlsx
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Supplementary file 2 Raw data and statistical outputs from Figures 2 and 3.: https://cdn.elifesciences.org/articles/87523/elife-87523-supp2-v1.xlsx
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Supplementary file 3 Raw data and statistical outputs from Figure 4.: https://cdn.elifesciences.org/articles/87523/elife-87523-supp3-v1.xlsx
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Supplementary file 4 Raw data and statistical outputs from Figure 5.: https://cdn.elifesciences.org/articles/87523/elife-87523-supp4-v1.xlsx
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Supplementary file 5 Identified lipid species from untargeted lipidomic analysis.: https://cdn.elifesciences.org/articles/87523/elife-87523-supp5-v1.csv
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MDAR checklist: https://cdn.elifesciences.org/articles/87523/elife-87523-mdarchecklist1-v1.docx
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Charlotte F Chao
Yanina-Yasmin Pesch
Huaxu Yu
Chenjingyi Wang
Maria J Aristizabal
Tao Huan
Guy Tanentzapf
Elizabeth Rideout

(2024)

An important role for triglyceride in regulating spermatogenesis

eLife 12:RP87523.

https://doi.org/10.7554/eLife.87523.4

Figures

Lipid droplets (LD) are present in early-stage somatic and germ cells.

Cholesterol is absent from testis lipid droplets.

brummer regulates lipid droplets (LD) in both germline and somatic cells of the testis.

Expression of brummer and selected lipid metabolic genes during spermatogenesis in germline and somatic lineages.

brummer regulates lipid droplets (LD) in both germline and somatic cells of the testis.

bmm regulates germline lipid droplets (LD) in a cell-autonomous manner.

A cell-autonomous role for bmm in regulating spermatogenesis.

Additional characterization of testis development and spermatogenesis in animals lacking bmm.

Loss of bmm disrupts triglyceride homeostasis and leads to spermatogenic defects.

Lipidomic analysis of animals lacking bmm.

Model of bmm-mediated lipid droplet regulation in the Drosophila testis.

Tables

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

MDAR checklist

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Lipid droplets (LD) are present in early-stage somatic and germ cells.

Cholesterol is absent from testis lipid droplets.

brummer regulates lipid droplets (LD) in both germline and somatic cells of the testis.

Expression of brummer and selected lipid metabolic genes during spermatogenesis in germline and somatic lineages.

brummer regulates lipid droplets (LD) in both germline and somatic cells of the testis.

bmm regulates germline lipid droplets (LD) in a cell-autonomous manner.

A cell-autonomous role for bmm in regulating spermatogenesis.

Additional characterization of testis development and spermatogenesis in animals lacking bmm.

Loss of bmm disrupts triglyceride homeostasis and leads to spermatogenic defects.

Lipidomic analysis of animals lacking bmm.

Model of bmm-mediated lipid droplet regulation in the Drosophila testis.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

MDAR checklist

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