Developmental Biology

An important role for triglyceride in regulating spermatogenesis

Charlotte F. Chao
Yanina-Yasmin Pesch
Huaxu Yu
Chenjingyi Wang
Maria J. Aristizabal
Tao Huan
Guy Tanentzapf
Elizabeth J. Rideout author has email address

Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Department of Chemistry, The University of British Columbia, Vancouver, BC, Canada V6T 1Z1
Department of Biology, Queen’s University, Kingston, ON, Canada K7L 3N6

https://doi.org/10.7554/eLife.87523.3

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Figures and data

Lipid droplets are present in early-stage somatic and germ cells.
(A) Testis lipid droplets (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). 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, grey 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 Supplemental Figure 1.

brummer regulates lipid droplets in both germline and somatic cells of the testis.
(A-A’’’’) Testis lipid droplets (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 (grey histogram) and GFP expression (green line) in testes from bmm-GFP animals as a function of distance from the hub (n=3). (D,E) 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 (grey) and bmm¹ (orange). (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 LDs and the apical tip of the testes are drawn as solid lines. See also Supplemental Figure 2 and 3.

bmm regulates germline lipid droplets 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 panel 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’’ and A’’’). In contrast, spermatocyte clones homozygous for bmm¹ have detectable LD (B’’ and 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 (grey) 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.

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. (D,E) Representative images of bmm^rev (D) or bmm¹ (E) testes stained with DAPI and anti-Vasa antibody. Arrows indicate germline stem cells (GSC). 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^revclones 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 S4J. 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. (J) 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 bmm¹and bmm^rev spermatocyte clones (K) or post-meiotic clones (L) at 3 and 14 days post-clone induction. See also Supplemental Figure 4.

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 triglycerides (green; 97 species) and non-triglyceride lipids (grey; 186 species) in our dataset. (D) Arrows indicate testis 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). See also Supplemental Figure 5.

Model of bmm-mediated lipid droplet regulation in the Drosophila testis.
Schematic representation summarizing bmm-mediated lipid droplet regulation in the testis during development.

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.

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 [78]. Individual cells are labeled according to the annotation within the data set. (B) Pseudotime trajectory of the somatic cells (black line) based on publicly available single-cell RNA sequencing data [78]. Individual cells are labeled according to the annotation within the data set. 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.

brummer regulates lipid droplets in both germline and somatic cells of the testis.
(A-D) Representative images of bmm^rev (A and B) and bmm¹(C and D) testes with somatic over-expression of GFP-LD (Tj-GAL4>UAS-GFP-LD). Panel F and H contain magnified images of the area indicated by the boxes in panel 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 bmm¹testes, 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.

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 (A; Welch two-sample t-test). (B) The number of spermatid bundles was significantly lower in bmm¹mutant 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^revcontrol 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 bmm¹testis had no spermatid bundles, a phenotype absent in age-matched bmm^revmales (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 ANOVA with Tukey multiple comparison test). (F) Food supplemented with 4% medium chain triglyceride significantly increased the number of spermatid bundles in bmm¹testes 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 Fas3 (G and G’’) and Vas (G’ and G’’’). Scale bars=25 μm. (H) Quantification of hub area in bmm^revor bmm¹ testes showed a significantly larger hub size in bmm¹testes (Welch two-sample t-test). (I) The number of germline stem cells (GSC) undergoing mitosis (phospho-histone H3⁺ GSC/total GSC) was not significantly different between bmm¹ and bmm^revtestes (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^revtestes (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 bmm¹animals 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 wildtype clones (Kruskal-Wallis rank sum test).

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 panel 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 bmm¹males. (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 bmm¹males. (F) The number of carbon and the degree of saturation of fatty acids (FA) and acyl-carnitines (ACar) with significant changes in abundance between bmm¹and 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 panel 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 LD in bmm¹ testes to control levels (one-way ANOVA with Tukey multiple comparison test). (J) Germline-specific loss of mdy in bmm¹ animals did not reduce 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).

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