Drosophila is a powerful model to study how lipids affect spermatogenesis. Yet, the contribution of neutral lipids, a major lipid group which resides in organelles called lipid droplets (LD), to normal sperm development is largely unknown. Emerging evidence suggests that LD are present in the testis and that loss of neutral lipid-and LD-associated genes causes subfertility; however, key regulators of testis neutral lipids and LD remain unclear. Here, we show that LD are present in early-stage somatic and germline cells within the Drosophila testis. We identified a role for triglyceride lipase brummer (bmm) in regulating testis LD, and found that whole-body loss of bmm leads to defects in sperm development. Importantly, these represent cell-autonomous roles for bmm in regulating testis LD and spermatogenesis. Because lipidomic analysis of bmm mutants revealed excess triglyceride accumulation, and spermatogenic defects in bmm mutants were rescued by genetically blocking triglyceride synthesis, our data suggest that bmm-mediated regulation of triglyceride influences sperm development. This identifies triglyceride as an important neutral lipid that contributes to Drosophila sperm development, and reveals a key role for bmm in regulating testis triglyceride levels during spermatogenesis.
This important study shows that lipid degradation is critical for spermatogenesis, with data supporting relevance of this finding across phyla. The authors contribute to a growing realization that lipid droplets have critical roles during differentiation and can influence cell fate, and use convincing methods to analyze the effects of loss of the lipase Brummer on germ line differentiation. This paper will be of interest to developmental and cell biologists working on gametogenesis.
Lipids play an essential role in regulating spermatogenesis across animals [(1)–(4)]. Studies in Drosophila have illuminated key roles for multiple lipid species in regulating sperm development [(5)–(7)]. For example, phosphatidylinositol and its phosphorylated derivatives participate in diverse aspects of Drosophila spermatogenesis including meiotic cytokinesis (111), somatic cell differentiation (12), germline and somatic cell polarity maintenance [(13)–(16)], and germline stem cell (GSC) maintenance and proliferation (17). Membrane lipids also influence sperm development (1819), whereas fatty acids play a role in processes such as meiotic cytokinesis (20) and sperm individualization (2122). While these studies suggest key roles for membrane lipids and fatty acids during Drosophila spermatogenesis, some of which are conserved in mammals [(23)–(25)], much less is known about how neutral lipids contribute to spermatogenesis.
Neutral lipids are a major lipid group that includes triglyceride and cholesterol ester, and reside within specialized organelles called lipid droplets (LD) (26). LD are found in diverse cell types (e.g. adipocytes, muscle, liver, glia, neurons) (272826), and play key roles in maintaining cellular lipid homeostasis. In nongonadal cell types, correct regulation of LD contributes to cellular energy production [(29)–(31)], sequestration and redistribution of lipid precursors [(32)–(36)], and regulation of lipid toxicity [(37)–(39)]. The importance of LD to normal cellular function in nongonadal cell types is shown by the fact that dysregulation of LD causes defects in cell differentiation, survival, and energy production (26374041). In the testis, much less is known about the regulation and function of neutral lipids and LD, and how this regulation affects sperm development.
Multiple lines of evidence suggest a potential role for neutral lipids and LD during spermatogenesis. First, genes that encode proteins associated with neutral lipid metabolism and LD are expressed in the testis across multiple species [(42)–(44)]. Second, testis LD have been identified in mammals and flies under both normal physiological conditions (2748) and after mitochondrial stress (49). Third, loss of genes associated with neutral lipid metabolism and LD cause subfertility phenotypes in both flies and mammals (2752). While studies suggest that mammalian testis LD contribute to steroidogenesis (5354), the spatial, temporal, and cell-type specific requirements for neutral lipids and LD in the testis have not been explored in detail in any animal. It remains similarly unclear which genes are responsible for regulating neutral lipids and LD during spermatogenesis.
To address these knowledge gaps, we used Drosophila to investigate the regulation and function of neutral lipids and LD during sperm development. Our detailed analysis of spermatogenesis under normal physiological conditions revealed the presence of LD in early-stage somatic and germline cells in the testis. We identified triglyceride lipase brummer (bmm) as a regulator of testis LD, and showed that this represents a cell-autonomous role for bmm. Importantly, we found that the bmm-mediated regulation of testis LD was significant for spermatogenesis, as both whole-body and cell-autonomous loss of bmm caused defects in sperm development. Given that our lipidomic analysis revealed an excess accumulation of triglyceride in animals lacking bmm, and that genetically blocking triglyceride synthesis rescued many spermatogenic defects associated with bmm loss, our data suggests that bmm-mediated regulation of triglyceride is important for normal Drosophila sperm development. This reveals previously unrecognized roles for neutral lipids such as triglyceride in regulating spermatogenesis, and for bmm in regulating sperm development under normal physiological conditions. Together, these findings advance knowledge of the regulation and function of neutral lipids during spermatogenesis.
Lipid droplets are present in early-stage somatic and germline cells
We previously reported the presence of small circular punctae (<1 μm) corresponding to LD near the apical tip of the testis (27). We confirm these results in w1118 males using neutral lipid stain BODIPY (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (Figure 1A). Importantly, we reproduced this spatial distribution of LD in two independent genetic backgrounds and at two additional ages (Figure 1B,1C). In all cases, LD were in a testis region that contains stem cells and early-stage somatic and germline cells (Figure 1A-A’, arrows), and in the hub, an organizing center and stem cell niche in the Drosophila testis (Figure 1A’’-A’’’, arrows) (55). LD were largely absent from the testis region occupied by spermatocytes (Figure 1A and A’, arrowheads). While LD may contain multiple neutral lipid species(56), cholesterol-binding fluorescent polyene antibiotic filipin III did not detect cholesterol within testis LD (Figure S1A), suggesting triglyceride is the main neutral lipid in Drosophila testis LD.
Drosophila spermatogenesis requires the codevelopment and differentiation of two cell lineages, the germline and the somatic cells (57). To identify LD in each lineage, we used the GAL4/UAS system to overexpress a transgene in which GFP is fused to the LD-targeting motif of motor protein Klarsicht (58) (UAS-GFP-LD). We targeted UAS-GFP-LD to somatic cells with Traffic jam (Tj)-GAL4 and to the germline using nanos (nos)-GAL4; LD were visualized using neutral lipid dye LipidTox. We found LD in the somatic cells of 0-day-old males (Figure 1D), and showed that the majority of somatic LD were <30 μm from the hub (Figure 1E). Because the somatic LD distribution coincided with a marker for somatic stem cells and their immediate daughter cells (Zinc finger homeodomain 1, Zfh-1) (Figure 1F; two-sample Kolmogorov-Smirnov test) (59), but not with a marker for late somatic cells (Eyes absent, Eya) (1260), our data suggests LD are present in early somatic cells. In the germline, GFP punctae corresponding to LD were found near the apical tip of the testis in 0-day-old males (Figure 1G,H). We found that the disappearance of germline LD coincided with peak expression of a GFP reporter that reflects the expression of Bag-of-marbles (Bam) protein in the testis (Bam-GFP) (61) (Figure 1I,1J). Because peak Bam expression signals the last round of transient amplifying mitotic cell cycle prior to the germline’s transition into the meiotic cell cycle [(62)–(64)], our data suggests that germline LD, like somatic LD, are present at early stages of germline development.
brummer plays a cell-autonomous role in regulating testis lipid droplets
Adipose triglyceride lipase (ATGL) is a critical regulator of neutral lipid metabolism and LD [(65)–(74)]. Loss of ATGL in many cell types triggers LD accumulation, and ATGL overexpression decreases LD number (3067687173737576). Given that the Drosophila ATGL homolog brummer (bmm) regulates testis LD induced by mitochondrial stress (49), we explored whether bmm regulates testis LD under normal physiological conditions. We first examined bmm expression in the testis by isolating this organ from flies in which a bmm promoter fragment drives GFP expression (bmm-GFP). Indeed, bmm-GFP accurately reproduces changes to bmm mRNA levels (77). GFP expression was present in the germline of bmm-GFP testes, and we found germline GFP levels were higher in spermatocytes than at earlier stages of sperm development (Figure 2A,2B; one-way ANOVA with Tukey multiple comparison test). Supporting this, our analysis of a publicly available single-cell RNA sequencing data set of the male reproductive organ (78) suggested a similar trend in bmm mRNA levels between different stages of germline (Figure S2A,S2B) and somatic cell (Figure S2C,S2D) development. Importantly, germline GFP levels were negatively correlated with testis LD in bmm-GFP flies (Figure 2A,2C), suggesting regions with higher bmm expression had fewer LD.
To test whether bmm regulates testis LD, we compared LD in testes from 0-day-old males carrying a loss-of-function mutation in bmm (bmm1) to control male testes (bmmrev)(67). bmm1 testes had significantly more LD across all LD sizes compared with control males (Figure 2D–2G; Welch two-sample t-test with Bonferroni correction), and showed a significantly expanded LD distribution (Figure 2D–2F,2H; two-sample Kolmogorov-Smirnov test). This suggests bmm normally restricts LD to the region near the apical tip of the testis, a role we confirm in both somatic and germline lineages (Figure S2E–S2H). Importantly, after inducing homozygous bmm1 or bmmrev clones in the testes using FLP-FRT system(79), we found bmm1 spermatocyte clones had significantly more LD at 3 days post-clone induction (Figure 2I; Welch two-sample t-test), a stage at which LD were absent from bmmrev clones. This indicates a previously unrecognized cell-autonomous role for bmm in regulating testis LD, a role we were unable to assess in somatic cells as we recovered no bmm1 somatic cell clones.
brummer plays a cell-autonomous role in regulating germline development
To determine the physiological significance of bmm-mediated regulation of testis LD, we investigated testis and sperm development in males without bmm function. In 0-day-old bmm1 males reared at 25°C, testis size was significantly smaller than in age-matched bmmrev controls (Figure S3A; Welch two-sample t-test), and the number of spermatid bundles was significantly lower (Figure S3B; Kruskal-Wallis rank sum test). Defects in testis size and sperm development were also observed in 14-day-old bmm1 males (Figure S3C,S3D Welch two-sample t-test). When the animals were reared at 29°C, a temperature that exacerbates spermatogenesis defects associated with changes in lipid metabolism (21), bmm1 phenotypes were more pronounced (Figure 3A-3C). This suggests loss of bmm affects testis development and spermatogenesis. Because similar phenotypes are observed in male mice without ATGL (52), and supplementing the diet of bmm1 males with medium-chain triglycerides (MCT) partially rescues the testis and spermatogenic defects we observed in flies (Figure S3E,S3F; one-way ANOVA with Tukey multiple comparison test), as it does in mice (5280), our data suggests flies are a good model to study how bmm/ATGL influences sperm development.
To explore spermatogenesis in bmm1 animals, we used germline-specific marker Vasa to visualize the germline in the testes of bmm1 and bmmrev males (Figure 3D,3E) (81). We observed a significant increase in the number of germline stem cells (GSC) (Figure 3F; Kruskal-Wallis rank sum test) and higher variability in GSC number in bmm1 males (p=5.7×10-12 by F-test). Given that GSC number is affected by hub size and GSC proliferation (8283), we monitored both parameters in bmm1 and bmmrev controls. While hub size in bmm1 testes was significantly larger than in testes from bmmrev controls (Figure S3G,S3H; Welch two-sample t-test), the number of phosphohistone H3-positive GSC, which indicates proliferating GSC, was unchanged in bmm1 animals (Figure S3I; Kruskal-Wallis rank sum test). While this indicates a larger hub may partly explain bmm’s effect on GSC number, bmm also plays a cell-autonomous role in regulating GSC, as we recovered a higher proportion of bmm1 clones in the GSC pool compared with bmmrev clones at 14 days after clone induction (Figure 3G; Welch two-sample t-test).
Beyond GSC, we uncovered additional spermatogenesis defects in bmm1 testes. Peak Bam-GFP expression in testes from 0-day-old bmm1 and bmmrev males showed that GFP-positive cysts with were significantly further away from the hub in bmm1 testes (Figure 3H,S3J; Welch two-sample t-test). Indeed, 15/18 bmm1 testes contained Vasa-positive cysts with large nuclei in the distal half of the testis (Figure 3I, arrowheads), a phenotype not present in bmmrev testes (0/8) (p=0.0005 by Pearson’s Chi-square test). Because these phenotypes are also seen in testes with differentiation defects (1384), we recorded the stage of sperm development reached by the germline in bmm1 testes. Most bmm1 testes contained post-meiotic cells in males raised at 25°C (Figure S3K); however, germline development did not progress past the spermatocyte stage in most bmm1 testes from animals raised at 29°C (Figure S3K). Testes from bmm1 males reared at 25°C also had a smaller Boule-positive area (Figure 3J,S3L; Welch two-sample t-test), and fewer individualization complexes and waste bags (Figure S3M,S3N; Kruskal-Wallis rank sum test). Together, these data indicate loss of bmm delays germline development. Because we recovered fewer bmm1 spermatocyte and spermatid clones 14 days after clone induction (Figure 3K,3L; Kruskal-Wallis rank sum test), this effect on germline development represents a cell-autonomous role for bmm.
brummer-dependent regulation of testis triglyceride levels affects spermatogenesis
ATGL catalyzes the first and rate-limiting step of triglyceride hydrolysis (738586). Loss of this enzyme or its homologs leads to excess triglyceride accumulation (2730677375) and shifts in multiple lipid classes (6689). To determine how loss of bmm affects spermatogenesis, we carried out mass spectrometry (MS)-based untargeted lipidomic profiling of bmm1 and bmmrev males. Hierarchical clustering of lipid species suggests that bmm1 and bmmrev males show distinct lipidomic profiles (Figure 4A). Overall, we detected 2464 and 1144 lipid features with high quantitative confidence in positive and negative ion modes, respectively. By matching experimental m/z, isotopic ratio, and tandem MS spectra to lipid libraries, we confirmed 293 unique lipid species (Supplemental table 1). We found 107 lipids had a significant change in abundance between bmm1 and bmmrev males (padj<0.05): 85 species were upregulated in bmm1 males and 22 lipid species were downregulated. Among differentially regulated species from different lipid classes, triglyceride had the largest residual above expected proportion (p=5.00×10-4 by Pearson’s Chi-squared test). This suggests triglyceride is the lipid class most affected by loss of bmm (Figure 4B,4C).
In bmm1 males, most triglyceride species (55/97) were significantly higher. Because we observed a positive correlation between the fold increase in triglyceride abundance with both the number of double bonds (p=7.52×10-8 by Kendall’s rank correlation test; Figure S4A) and the number of carbons (p=2.77×10-10 by Kendall’s rank correlation test; Figure S4B), our data align well with bmm/ATGL’s known role in regulating triglyceride levels(676873) and its substrate preference of long-chain polyunsaturated fatty acids(85). While we also detected changes in species such as fatty acids, acylcarnitine, and membrane lipids (Figure S4C–S4H), in line with recent Drosophila lipidomic data(9091), the striking accumulation of triglyceride in bmm1 males suggested that excess testis triglyceride in bmm1 males may contribute to their spermatogenic defects. To test this, we examined spermatogenesis in bmm1 males carrying loss-of-function mutations in midway (mdy). mdy is the Drosophila homolog of diacylglycerol O-acyltransferase 1 (DGAT1), and whole-body loss of mdy reduces whole-body triglyceride levels[(92)–(94)]. Importantly, testes isolated from males lacking both bmm and mdy (genotype mdyQX25/k03902;bmm1) had fewer LD than testes dissected from bmm1 males (Figures 4D,S4I; one-way ANOVA with Tukey multiple comparison test).
We found that testes isolated from mdyQX25/k03902;bmm1 males were significantly larger and had more spermatid bundles than testes from bmm1 males (Figure 4E–G; one-way ANOVA with Tukey multiple comparison test). The elevated number of GSC in bmm1 male testes was similarly rescued in mdyQX25/k03902;bmm1 males (Figure 4H; one-way ANOVA with Tukey multiple comparison test). These data suggest that defective spermatogenesis in bmm1 males can be partly attributed to excess triglyceride accumulation. Notably, at least some of these defects are cell-autonomous: RNAi-mediated knockdown of mdy in the germline of bmm1 males partially rescued the defects in testis size (Figure 4I; Kruskal-Wallis rank sum test with Dunn’s multiple comparison test) and GSC variance (Figure S4J; p=4.5 x 10-5 and 8.2 x 10-3 by F-test from the GAL4-and UAS-only crosses, respectively). bmm-mediated regulation of testis triglyceride therefore plays a previously unrecognized role in regulating sperm development.
In this study, we used Drosophila to gain insight into how the neutral lipids, a major lipid class, contribute to sperm development. We describe the distribution of LD under normal physiological conditions in the Drosophila testis, and show that LD are present at the early stages of development in both somatic and germline cells. While many factors are known to regulate LD in nongonadal cell types, we reveal a cell-autonomous role for triglyceride lipase bmm in regulating testis LD during spermatogenesis. Indeed, our data indicates loss of bmm delays germline differentiation leading to an accumulation of early-stage germ cells. These defects in germline differentiation can be partially explained by the excess accumulation of triglyceride in flies lacking bmm, as genetically blocking triglyceride synthesis rescues multiple spermatogenic defects in bmm mutants. Together, our data reveals previously unrecognized roles for LD and triglycerides during spermatogenesis, and for bmm as an important regulator of testis LD and germline development under normal physiological conditions.
One key outcome of our study was increased knowledge of LD regulation and function in the testis. Despite rapidly expanding knowledge of LD in cell types such as adipocytes or skeletal muscle, less is known about how LD influence spermatogenesis under normal physiological conditions. In mammals, testis LD contain cholesterol and play a role in promoting steroidogenesis (9596). In flies, we show that LD are present in the testis, and that excess accumulation of these LD affects sperm development. In nongonadal cell types, triglycerides provide a rich source of fatty acids for cellular ATP production, lipid building blocks to support membrane homeostasis and growth, and metabolites that can act as signaling molecules (26). Because ATP production, lipid precursors, and lipid signaling all play roles in supporting normal sperm development (9798), future studies will need to determine how each of these processes is affected when excess triglyceride accumulates in testis LD. This will provide critical insight into how triglyceride stored within testis LD contributes to overall cellular lipid metabolism during spermatogenesis. Because of the parallel spermatogenic defects we observed in bmm mutants and ATGL-deficient mice, we expect that these mechanisms will also operate in other species.
A more comprehensive understanding of neutral lipid metabolism during sperm development will also emerge from studies on the upstream signaling networks that regulate testis LD and triglyceride. Given that we show an important and cell-autonomous role for bmm in regulating testis LD and triglyceride, future studies will need to identify factors that regulate bmm in the testis. Based on public single-cell RNAseq data and the bmm-GFP reporter strain, our data suggest bmm mRNA levels are differentially regulated between early and later stages of sperm development. Candidates for mediating this regulation include the insulin/insulin-like growth factor signaling pathway (IIS), Target of rapamycin (TOR) pathway, and nuclear factor κB/Relish pathway (NFκB), as all of these pathways influence bmm mRNA levels in nongonadal cell types [(99)–(105)]. Beyond mRNA levels, Bmm protein levels and post-translational modifications may also be differentially regulating during spermatogenesis. For example, studies show that the proteins encoded by bmm homologs in other animals are regulated by phosphorylation (106), mediated by kinases such as adenosine monophosphate-activated protein kinase (AMPK) and protein kinase A (PKA) [(107)–(109)]. Importantly, many of these pathways, including IIS, TOR, AMPK, NFκB and possibly PKA influence Drosophila sperm development [(110)–(115)]. Identifying the signaling networks that influence bmm regulation during sperm development will therefore lead to a deeper understanding of how testis LD and triglyceride are coordinated with physiological factors to promote normal spermatogenesis. Because pathways such as IIS and AMPK, and others, regulate sperm development in other species [(116)–(118)], these insights may reveal conserved mechanisms that govern the regulation of cellular neutral lipid metabolism during sperm development.
We thank Dr. Ronald Kühnlein for bmm1 and bmmrev lines (67), Dr. Michael Welte for UAS-GFP-LD(58), and Dr. Kaeko Kamei for bmm-GFP (77). We used stocks from the Bloomington Drosophila Stock Center (NIH P40OD018537) and Vienna Drosophila Resource Center (VDRC). We acknowledge critical resources and information provided by FlyBase (119) (supported by the National Human Genome Research Institute at the U.S. National Institutes of Health (U41 HG000739) and the British Medical Research Council (MR/N030117/1)). This work was supported by the Life Sciences Institutes Imaging Core, supported by the UBC GREx Biological Resilience Initiative. Funding for this study was provided by grants to EJR from the Canadian Institutes for Health Research (PJT-153072), Michael Smith Foundation for Health Research (16876), and the Canadian Foundation for Innovation (JELF-34879). GT was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC; 2018-04648), TH/HY/CW were supported by NSERC (2020-04895), MA was supported by the Jacob’s foundation. We would like to acknowledge that our research takes place on the traditional, ancestral, and unceded territory of the Musqueam people; a privilege for which we are grateful.
Conceptualization, C.C. and E.J.R.; Methodology, C.C. and Y.Y.P.; Software, C.C.; Investigation, C.C., H.Y., and Y.Y.P.; Lipidomics, M.A., H.Y., C.W., T.H.; Writing – Original Draft, C.C. and E.J.R.; Writing – Review and Editing, C.C., E.J.R., and Y.Y.P.; Supervision, E.J.R., G.T., and T.H.; Project administration, E.J.R.; Funding Acquisition, E.J.R., G.T., and T.H.
Declaration of interests
The authors declare no competing interests.
Materials and methods
Materials and Resource availability.
Drosophila strains and their source are listed in a Key Resources table. Further information and requests for resources and reagents should be directed to, and will be fulfilled by, lead contact Dr. Elizabeth J. Rideout (firstname.lastname@example.org).
Data and Code availability.
All raw data and results of statistical tests reported in this paper are located in Supplementary files 1-4. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Fly stocks were maintained at room temperature in 12:12 hour light:dark cycle. Unless otherwise indicated, all flies were raised at 25°C with a density of 50 larvae per 10 mL fly media. Because this project examines sperm development, we used male flies in all experiments. Fly media contained 20.5 g sucrose (SU10, Snow Cap), 70.9 g Dextrose (SUG8, Snow Cap), 48.5 g cornmeal (AO18006, Snow Cap), 30.3 g baker’s yeast (NB10, Snow Cap), 4.55 g agar (DR-820-25F, SciMart), 0.5 g calcium chloride dihydrate (CCL302.1, BioShop Canada), 0.5 g magnesium sulfate heptahydrate (MAG511.1, BioShop Canada), 4.9 mL propionic acids (P1386, Sigma-Aldrich), and 488 μL phosphoric acid (P5811, Sigma-Aldrich) per 1L of media. For diets with medium-or long-chain triglyceride, 4 g of coconut oil (medium chain triglyceride) or olive oil (long chain triglyceride) was added per 100 mL of media described above prior to cooling. Males were collected and dissected within 24 hours of eclosion unless otherwise indicated. Fixations were performed at room temperature with 4% paraformaldehyde (CA11021-168, VWR) in PBS for 20 minutes on a rotating platform followed by washing in PBS twice before staining. Fly strains used in our study are listed in a Key Resources table.
Testis cell stage classification and measurements.
Cells at an early stage of development (stem cells and early-stage somatic and germline cells) were located in the apical region of the testis, and were identified by their small and dense nuclei(120). GSC were defined as Vasa-positive cells in direct contact with the hub; proliferating GSC were identified as Vasa-positive cells in direct contact with the hub that were also phospho-H3 positive. Cells in the testis region occupied by primary spermatocytes were identified by their large cell size and decondensed chromosome staining occupying three nuclear domains(120). Spermatid bundles were identified by their condensed and needle-shaped nuclei, which roughly corresponds to nuclei with protamine-based chromatin(121). Testis size was measured by quantifying the length of a line drawn down the middle of a testis image; starting from the apical tip of the testis and ending where the testis meets the seminal vesicle.
FLP-FRT clone induction.
Adult males were collected at 3-5 days post-eclosion and heat-shocked three times at 37°C with a 10 min rest period at room temperature between heat shocks. After heat-shock, the flies were incubated at room temperature until dissection.
Fixed samples were rinsed three times with blocking solution containing 0.2% bovine serum albumin (A4503, Sigma-Aldrich), 0.3% Triton-X in PBS, then blocked for 1 hr on a rotating platform at room temperature. During the incubation, the blocking solution was changed every 15 minutes. After blocking, the sample were resuspended in blocking solution with the appropriate concentration of primary antibody (see Key Resources table), and incubated overnight at 4°C. Samples were rinsed three times with blocking solution after removing primary antibody, and blocked for one hour on a rotating platform in blocking solution. Secondary antibody was applied in blocking solution and left on the rotating platform at room temperature for 40 min. The sample was rinsed with blocking solution three more times, and washed four times for 15 min per wash in blocking solution. Testis samples were resuspended in Vectashield mounting media with DAPI (H-1200-10, Vector Laboratory) or SlowFade Diamond mounting media (S36972, Thermo Fisher Scientific) prior to mounting.
Lipid droplet staining.
Fixed testes were briefly permeabilized with 0.1% Triton-X in PBS for 5 min prior to applying phalloidin. For BODIPY staining, samples were suspended in PBS containing 10 μg/mL DAPI (2879083-5mg, PeproTech), 1:500 BODIPY 495/503 (Thermo Fisher Scientific D3922), and 1:1000 phalloidin iFluor647 (ab176759, Abcam) or 1:40 phalloidin TexasRed (T7471, Thermo Fisher Scientific). For staining with LipidTox Red, samples were suspended in PBS containing 10 μg/mL DAPI (2879083-5mg, PeproTech), 1:200 LipidTox Red (H34476, Thermo Fisher Scientific), and 1:1000 phalloidin iFluor647 (ab176759, Abcam). For staining free sterols, samples were prepared as for BODIPY staining with 50 μg/mL filipin in place of BODIPY for 30 min. Samples were incubated on a rotating platform for 40 minutes at room temperature. After incubation, samples were washed twice with PBS, then resuspended in SlowFade Diamond mounting media (Thermo Fisher Scientific S36972) prior to mounting.
Image acquisition and processing.
All images were acquired on a Leica SP5 confocal microscope system with 20X or 40X objectives and quantified with Fiji image analysis software(122).
Drosophila extracts were prepared following the previously reported protocol(123). Briefly, 10 Drosophila males (∼10 mg) were weighed, 300 µL of ice-cold methanol/water mixture (9:1, v:v) was added to these males, and the samples were homogenized with glass beads using a bead beater (mini-beadbeater-16, BioSpec, Bartlesville, Ok, USA). Sample weight was used for sample normalization. Fly lysate was kept at-20°C for 4 hours for protein precipitation. Then, 900 µL of methyl tert-butyl ether was added and the solution was shaken for 5 min to extract lipids. To induce phase separation 285 µL of water was added, followed by centrifugation. The upper layer was separated, dried, and reconstituted in isopropanol/acetonitrile (1:1, v:v) for liquid chromatography-mass spectrometry (LC-MS) analysis. The volume of reconstitution solution was proportional to sample weight for normalization. Quality control (QC) samples were prepared by pooling 20 μL aliquot from each sample. The method blank sample was prepared using an identical workflow but without adding Drosophila.
Drosophila extracts were analyzed on an UHR-QqTOF (Ultra-High Resolution Qq-Time-Of-Flight) mass spectrometry Impact II (Bruker Daltonics, Bremen, Germany) interfaced with an Agilent 1290 Infinity II LC Systems (Agilent Technologies, Santa Clara, CA, USA). LC separation was performed using a Waters reversed-phase (RP) UPLC Acquity BEH C18 Column (1.7 µm, 1.0 mm ×100 mm, 130 Å) (Milford, MA, USA) maintained at 30°C. For positive ion mode, the mobile phase A was 60% acetonitrile in water and the mobile phase B was 90% isopropanol in acetonitrile, both containing 5 mM ammonium formate (pH = 4.8, adjusted by formic acid). For negative ion mode, the mobile phase A was 60% acetonitrile in water and the mobile phase B was 90% isopropanol in acetonitrile, both containing 5 mM NH4FA (pH = 9.8, adjusted by ammonium hydroxide). The LC gradient for positive and negative ion modes was set as follows: 0 min, 5% B; 8 min, 40% B; 14 min, 70% B; 20 min, 95% B; 23 min, 95% B; 24 min, 5% B; 33 min, 5% B. The flow rate was 0.1 mL/min. The injection volume was optimized to 2 µL in positive mode and 5 µL in negative mode using QC sample. The ESI source conditions were set as follows: dry gas temperature, 220 °C; dry gas flow, 7 L/min; nebulizer gas pressure, 1.6 bar; capillary voltage, 4500 V for positive mode and 3000 V for negative mode. The MS1 analysis was conducted using following parameters: mass range, 70-1000 m/z; spectrum type: centroid, calculated using maximum intensity; absolute intensity threshold: 250. Data-dependent MS/MS analysis parameters: collision energy: 16-30 eV; cycle time, 3 s; spectra rate: 4 Hz when intensity < 104 and 12 Hz when intensity > 105, linearly increased from 104 to 105. External calibration was applied using sodium formate to ensure the m/z accuracy before sample analysis.
The raw LC-MS data were processed using MS-DIAL (ver. 4.38)(124). The detailed MS-DIAL parameters are: MS1 tolerance, 0.01 Da; MS/MS tolerance, 0.05; mass slice width, 0.05 Da; smoothing method, linear weighted moving average; smoothing level, 3 scans; minimum peak width, 5 scans. Lipid features with high quantitative confidence were selected by the following criteria: retention time was within the gradient elution time (< 23 min); average intensity in QC samples is larger than 5-fold of the intensity in method blank sample. Lipid identification was performed by matching experimental precursor m/z, isotopic ratio and MS/MS spectrum against the LipidBlast libraries embedded in MS-DIAL. To improve the quantification accuracy, the measured MS signal intensities were corrected using serial diluted QC samples following the reported workflow(125).
Quantification and statistical analysis.
All microscopy images were quantified using Fiji software(122). For lipid droplet counts, a single optical slice through the middle of the testis containing the hub was used with the exception of FLP-FRT experiment where all lipid droplets within a GFP-negative cyst were counted (Figure 2I). All statistical analyses were done using R (obtained from https://cran.r-project.org). With exception of data concerning spatial distribution, and lipidomic data, Shapiro-Wilk test (via shapiro.test in base R) was used to assess normality of distribution prior to testing for significance. Kruskal-Wallis rank sum test (from the R package coin) and Dunn’s test (from the R package dunn.test) were used in place of Welch two-sample t-test and Tukey’s multiple comparison test when the assumption of normality was not met. For testing differences in variance between two populations, F-test (via var.test in base R) was used. For testing differences in spatial distribution, two-sample Kolmogorov-Smirnov test (via ks.test in base R) was used. All p-values are indicated in figures; extremely small p-values are listed as p<2.2 x 10-16.
Supplemental table and files
Supplemental table 1 – Table showing identified lipid species from untargeted lipidomic analysis.
Supplementary file 1 – Raw data and statistical outputs from Figure 1.
Supplementary file 2 – Raw data and statistical outputs from Figure 2 and Supplemental figure 2.
Supplementary file 3 – Raw data and statistical outputs from Figure 3 and Supplemental figure 3.
Supplementary file 4 – Raw data and statistical outputs from Figure 4 and Supplemental figure 4.
Supplemental figure legends
- 1.Phosphoinositide signaling in sperm developmentSemin Cell Dev Biol 59:2–9https://doi.org/10.1016/j.semcdb.2016.06.010
- 2.Sterols in spermatogenesis and sperm maturationJ Lipid Res 54:20–33https://doi.org/10.1194/jlr.R032326
- 3.Metabolic regulation is important for spermatogenesisNat Rev Urol 9:330–338https://doi.org/10.1038/nrurol.2012.77
- 4.Lipid metabolism and Drosophila sperm developmentSci China Life Sci 55:35–40https://doi.org/10.1007/s11427-012-4274-2
- 5.The stem cell niche: lessons from the Drosophila testisDev Camb Engl 138:2861–2869https://doi.org/10.1242/dev.056242
- 6.Investigating Spermatogenesis in Drosophila melanogasterMethods San Diego Calif 68:218–227https://doi.org/10.1016/j.ymeth.2014.04.020
- 7.Drosophila spermiogenesis: Big things come from little packagesSpermatogenesis 2:197–212https://doi.org/10.4161/spmg.21798
- 8.A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesisDevelopment 127
- 9.The Class I PITP Giotto Is Required for Drosophila CytokinesisCurr Biol 16:195–201https://doi.org/10.1016/j.cub.2005.12.011
- 10.PIP2 Hydrolysis and Calcium Release Are Required for Cytokinesis in Drosophila SpermatocytesCurr Biol 15:1401–1406https://doi.org/10.1016/j.cub.2005.06.060
- 11.Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesisBMC Cell Biol 8https://doi.org/10.1186/1471-2121-8-15
- 12.Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cuesDev Camb Engl 143https://doi.org/10.1242/dev.139782
- 13.Identification of genetic networks that act in the somatic cells of the testis to mediate the developmental program of spermatogenesisPLOS Genet 13https://doi.org/10.1371/journal.pgen.1007026
- 14.The polarity protein Baz forms a platform for the centrosome orientation during asymmetric stem cell division in the Drosophila male germlineeLife 4https://doi.org/10.7554/eLife.04960
- 15.Membrane Targeting of Bazooka/PAR-3 Is Mediated by Direct Binding to Phosphoinositide LipidsCurr Biol 20:636–642https://doi.org/10.1016/j.cub.2010.01.065
- 16.The Dlg Module and Clathrin-Mediated Endocytosis Regulate EGFR Signaling and Cyst Cell-Germline Coordination in the Drosophila TestisStem Cell Rep 12:1024–1040https://doi.org/10.1016/j.stemcr.2019.03.008
- 17.Male Germline Stem Cell Division and Spermatocyte Growth Require Insulin Signaling in DrosophilaCell Struct Funct 34:61–69https://doi.org/10.1247/csf.08042
- 18.Disruption of Sphingolipid Metabolism Elicits Apoptosis-Associated Reproductive Defects in DrosophilaDev Biol 309:329–341https://doi.org/10.1016/j.ydbio.2007.07.021
- 19.Drosophila Lysophospholipid Acyltransferases Are Specifically Required for Germ Cell DevelopmentMol Biol Cell 20:5224–5235https://doi.org/10.1091/mbc.e09-05-0382
- 20.A Role for Very-Long-Chain Fatty Acids in Furrow Ingression during Cytokinesis in Drosophila SpermatocytesCurr Biol 18:1426–1431https://doi.org/10.1016/j.cub.2008.08.061
- 21.Drosophila spermatid individualization is sensitive to temperature and fatty acid metabolismSpermatogenesis 5https://doi.org/10.1080/21565562.2015.1006089
- 22.The fatty acid elongase NOA is necessary for viability and has a somatic role in Drosophila sperm developmentJ Cell Sci 120:2924–2934https://doi.org/10.1242/jcs.006551
- 23.Male meiotic cytokinesis requires ceramide synthase 3-dependent sphingolipids with unique membrane anchorsHum Mol Genet 24:4792–4808https://doi.org/10.1093/hmg/ddv204
- 24.Elovl4 and Fa2h expression during rat spermatogenesis: a link to the very-long-chain PUFAs typical of germ cell sphingolipidsJ Lipid Res 59:1175–1189https://doi.org/10.1194/jlr.M081885
- 25.ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in miceJ Lipid Res 52:245–255https://doi.org/10.1194/jlr.M011346
- 26.Lipid Droplets and Cellular Lipid MetabolismAnnu Rev Biochem. 81:687–714https://doi.org/10.1146/annurev-biochem-061009-102430
- 27.A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown. Hassan BA, editorPLOS Biol 18https://doi.org/10.1371/journal.pbio.3000595
- 28.Cell biology of lipid dropletsCurr Opin Cell Biol 20:378–385https://doi.org/10.1016/j.ceb.2008.05.009
- 29.Lipid droplet dynamics in skeletal muscleExp Cell Res 340:180–186https://doi.org/10.1016/j.yexcr.2015.10.023
- 30.Dual Lipolytic Control of Body Fat Storage and Mobilization in DrosophilaPLOS Biol 5https://doi.org/10.1371/journal.pbio.0050137
- 31.Fatty Acid Trafficking in Starved Cells: Regulation by Lipid Droplet Lipolysis, Autophagy, and Mitochondrial Fusion DynamicsDev Cell 32:678–692https://doi.org/10.1016/j.devcel.2015.01.029
- 32.Adipose triglyceride lipase regulates eicosanoid production in activated human mast cellsJ Lipid Res 55:2471–2478https://doi.org/10.1194/jlr.M048553
- 33.Patatin-like phospholipase domain–containing protein 3 promotes transfers of essential fatty acids from triglycerides to phospholipids in hepatic lipid dropletsJ Biol Chem 293:6958–6968https://doi.org/10.1074/jbc.RA118.002333
- 34.Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae⋆Biochim Biophys Acta BBA-Mol Cell Biol Lipids 1801:1314–1322https://doi.org/10.1016/j.bbalip.2010.08.004
- 35.Adipose triglyceride lipase acts on neutrophil lipid droplets to regulate substrate availability for lipid mediator synthesisJ Leukoc Biol 98:837–850https://doi.org/10.1189/jlb.3A0515-206R
- 36.Quantitative modeling of triacylglycerol homeostasis in yeast-metabolic requirement for lipolysis to promote membrane lipid synthesis and cellular growth: Triacylglycerol mobilization in yeastFEBS J 275:5552–5563https://doi.org/10.1111/j.1742-4658.2008.06681.x
- 37.Antioxidant Role for Lipid Droplets in a Stem Cell Niche of DrosophilaCell 163:340–353https://doi.org/10.1016/j.cell.2015.09.020
- 38.The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/DCell Metab 26:719–737https://doi.org/10.1016/j.cmet.2017.08.024
- 39.DGAT1-Dependent Lipid Droplet Biogenesis Protects Mitochondrial Function during Starvation-Induced AutophagyDev Cell 42:9–21https://doi.org/10.1016/j.devcel.2017.06.003
- 40.three’s a party: lysosomes, lipid droplets, and the ER in lipid trafficking and cell homeostasisCurr Opin Cell Biol 59:40–49https://doi.org/10.1016/j.ceb.2019.02.011
- 41.Expanding Roles for Lipid DropletsCurr Biol 25https://doi.org/10.1016/j.cub.2015.04.004
- 42.Hormone-sensitive lipase deficiency disturbs the fatty acid composition of mouse testisProstaglandins Leukot Essent Fatty Acids 88:227–233https://doi.org/10.1016/j.plefa.2012.12.005
- 43.Transcriptome Analysis of Testis from HFD-Induced Obese Rats (Rattus norvigicus) Indicated Predisposition for Male InfertilityInt J Mol Sci 21https://doi.org/10.3390/ijms21186493
- 44.Proteomic analysis of murine testes lipid dropletsSci Rep 5https://doi.org/10.1038/srep12070
- 45.Changes in carbohydrate metabolism of testicular germ cells during meiosis in the ratEur J Endocrinol 138:322–327
- 46.CYCLIC VARIATIONS IN SERTOLI CELL LIPID CONTENT THROUGHOUT THE SPERMATOGENIC CYCLE IN THE RATReproduction 43:1–8https://doi.org/10.1530/jrf.0.0430001
- 47.Morphometric analysis of Leydig cells in the normal rat testisJ Cell Biol 84:340–354https://doi.org/10.1083/jcb.84.2.340
- 48.Changes in the lipid inclusion/Sertoli cell cytoplasm area ratio during the cycle of the human seminiferous epitheliumReproduction 80:335–341https://doi.org/10.1530/jrf.0.0800335
- 49.Mitochondrial fusion regulates lipid homeostasis and stem cell maintenance in the Drosophila testisNat Cell Biol 21:710–720https://doi.org/10.1038/s41556-019-0332-3
- 50.PLIN1 deficiency affects testicular gene expression at the meiotic stage in the first wave of spermatogenesisGene 543:212–219https://doi.org/10.1016/j.gene.2014.04.021
- 51.Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertilityMol Reprod Dev 75:565–577https://doi.org/10.1002/mrd.20800
- 52.Long-chain fatty acid triglyceride (TG) metabolism disorder impairs male fertility: a study using adipose triglyceride lipase deficient miceMHR Basic Sci Reprod Med 23:452–460https://doi.org/10.1093/molehr/gax031
- 53.Studies on the source of cholesterol used for steroid biosynthesis in cultured Leydig tumor cellsJ Biol Chem 257:14231–14238
- 54.Hormone-sensitive lipase deficiency alters gene expression and cholesterol content of mouse testisReproduction 153:175–185https://doi.org/10.1530/REP-16-0484
- 55.The stem cell niche: lessons from the Drosophila testisDevelopment 138:2861–2869https://doi.org/10.1242/dev.056242
- 56.Lipid Droplet BiogenesisAnnu Rev Cell Dev Biol 33:491–510https://doi.org/10.1146/annurev-cellbio-100616-060608
- 57.Specification, migration and assembly of the somatic cells of the Drosophila gonadDevelopment 121:1815–1825https://doi.org/10.1242/dev.121.6.1815
- 58.Targeting the motor regulator Klar to lipid dropletsBMC Cell Biol 12https://doi.org/10.1186/1471-2121-12-9
- 59.Zfh-1 Controls Somatic Stem Cell Self-Renewal in the Drosophila Testis and Nonautonomously Influences Germline Stem Cell Self-RenewalCell Stem Cell 3:44–54https://doi.org/10.1016/j.stem.2008.05.001
- 60.A somatic role for eyes absent (eya) and sine oculis (so) in drosophila spermatocyte developmentDev Biol 258:117–128https://doi.org/10.1016/S0012-1606(03)00127-1
- 61.A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cellDevelopment 130:1159–1170https://doi.org/10.1242/dev.00325
- 62.Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineageProc Natl Acad Sci U S A 106:22311–22316https://doi.org/10.1073/pnas.0912454106
- 63.Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in DrosophilaPLoS Genet 10https://doi.org/10.1371/journal.pgen.1004797
- 64.DiNardo S. bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesisDevelopment 124:4361–4371
- 65.YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae*J Biol Chem 278:23317–23323https://doi.org/10.1074/jbc.M302577200
- 66.The impact of genetic stress by ATGL deficiency on the lipidome of lipid droplets from murine hepatocytesJ Lipid Res 54:2185–2194https://doi.org/10.1194/jlr.M037952
- 67.Brummer lipase is an evolutionary conserved fat storage regulator in DrosophilaCell Metab 1:323–330https://doi.org/10.1016/j.cmet.2005.04.003
- 68.Defective Lipolysis and Altered Energy Metabolism in Mice Lacking Adipose Triglyceride LipaseScience 312:734–737https://doi.org/10.1126/science.1123965
- 69.ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-α and PGC-1Nat Med 17:1076–1085https://doi.org/10.1038/nm.2439
- 70.Adipose triacylglycerol lipase deletion alters whole body energy metabolism and impairs exercise performance in miceAm J Physiol-Endocrinol Metab 297https://doi.org/10.1152/ajpendo.00190.2009
- 71.ATGL/CGI-58-Dependent Hydrolysis of a Lipid Storage Pool in Murine EnterocytesCell Rep 28:1923–1934https://doi.org/10.1016/j.celrep.2019.07.030
- 72.Obese Yeast: Triglyceride Lipolysis Is Functionally Conserved from Mammals to Yeast *J Biol Chem 281:491–500https://doi.org/10.1074/jbc.M508414200
- 73.Fat Mobilization in Adipose Tissue Is Promoted by Adipose Triglyceride LipaseScience 306:1383–1386https://doi.org/10.1126/science.1100747
- 74.A beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion in miceDiabetologia 59:2654–2663https://doi.org/10.1007/s00125-016-4105-2
- 75.Lipid Droplet Protein LID-1 Mediates ATGL-1-Dependent Lipolysis during Fasting in Caenorhabditis elegansMol Cell Biol 34:4165–4176https://doi.org/10.1128/MCB.00722-14
- 76.ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cellsJ Lipid Res 57:1162–1174https://doi.org/10.1194/jlr.M066415
- 77.A Drosophila Model for Screening Antiobesity AgentsBioMed Res Int 2016:1–10https://doi.org/10.1155/2016/6293163
- 78.Fly Cell Atlas: a single-cell transcriptomic atlas of the adult fruit flybioRxiv
- 79.Analysis of genetic mosaics in developing and adult Drosophila tissuesDevelopment 117:1223–1237
- 80.Effect of lipid metabolism on male fertilityBiochem Biophys Res Commun 485:686–692https://doi.org/10.1016/j.bbrc.2017.02.103
- 81.The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4ANature 335:611–617https://doi.org/10.1038/335611a0
- 82.Headcase Promotes Cell Survival and Niche Maintenance in the Drosophila Testis. Bökel C, editorPLoS ONE 8https://doi.org/10.1371/journal.pone.0068026
- 83.Somatic support cells restrict germline stem cell self-renewal and promote differentiationNature 407:750–754https://doi.org/10.1038/35037606
- 84.Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila malesDevelopment 122:1331–1341
- 85.Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferasesJ Biol Chem 287:41446–41457https://doi.org/10.1074/jbc.M112.400416
- 86.Adipose Triglyceride Lipase and Hormone-sensitive Lipase Are the Major Enzymes in Adipose Tissue Triacylglycerol Catabolism *J Biol Chem 281:40236–40241https://doi.org/10.1074/jbc.M608048200
- 87.Late onset of neutral lipid storage disease due to novel PNPLA2 mutations causing total loss of lipase activity in a patient with myopathy and slight cardiac involvementNeuromuscul Disord 27:481–486https://doi.org/10.1016/j.nmd.2017.01.011
- 88.Neutral lipid storage disease: a possible functional defect in phospholipid-linked triacylglycerol metabolismBiochim Biophys Acta BBA-Mol Basis Dis 1096:162–169https://doi.org/10.1016/0925-4439(91)90055-E
- 89.Neuronal lipolysis participates in PUFA-mediated neural function and neurodegenerationEMBO Rep 21https://doi.org/10.15252/embr.202050214
- 90.Physiological and metabolomic consequences of reduced expression of the Drosophila brummer triglyceride Lipase. Melkani GC, editorPLOS ONE 16https://doi.org/10.1371/journal.pone.0255198
- 91.Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid dropletsCell Biology https://doi.org/10.1101/2021.08.02.454724
- 92.PERILIPIN-Dependent Control of Lipid Droplet Structure and Fat Storage in DrosophilaCell Metab 12:521–532https://doi.org/10.1016/j.cmet.2010.10.001
- 93.Mutations in the midway Gene Disrupt a Drosophila Acyl Coenzyme A: Diacylglycerol AcyltransferaseGenetics 160:1511–1518
- 94.Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune functionPLOS Genet 16https://doi.org/10.1371/journal.pgen.1009192
- 95.Studies on the source of cholesterol used for steroid biosynthesis in cultured Leydig tumor cellsJ Biol Chem 257:14231–14238
- 96.Hormone-sensitive lipase deficiency alters gene expression and cholesterol content of mouse testisReproduction 153:175–185https://doi.org/10.1530/REP-16-0484
- 97.Lipid droplets and cellular lipid metabolismAnnu Rev Biochem 81:687–714https://doi.org/10.1146/annurev-biochem-061009-102430
- 98.Dynamics and functions of lipid dropletsNat Rev Mol Cell Biol 20:137–155https://doi.org/10.1038/s41580-018-0085-z
- 99.High-fat-diet-induced obesity and heart dysfunction are regulated by the TOR pathway in DrosophilaCell Metab 12:533–44https://doi.org/10.1016/j.cmet.2010.09.014
- 100.NF-κB Shapes Metabolic Adaptation by Attenuating Foxo-Mediated Lipolysis in DrosophilaDev Cell 49:802–810https://doi.org/10.1016/j.devcel.2019.04.009
- 101.Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signallingMol Syst Biol 7https://doi.org/10.1038/msb.2011.36
- 102.The Drosophila forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signalingJ Biol 2https://doi.org/10.1186/1475-4924-2-20
- 103.Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent responseEMBO J 21:6162–73
- 104.Transcriptional feedback control of insulin receptor by dFOXO/FOXO1Genes Dev 19:2435–2446https://doi.org/10.1101/gad.1340505
- 105.Drosophila Kruppel homolog 1 represses lipolysis through interaction with dFOXOSci Rep 7https://doi.org/10.1038/s41598-017-16638-1
- 106.Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocationJ Proteome Res 6:3256–3265https://doi.org/10.1021/pr070158j
- 107.Identification and functional characterization of protein kinase A phosphorylation sites in the major lipolytic protein, adipose triglyceride lipaseEndocrinology 153:4278–4289https://doi.org/10.1210/en.2012-1127
- 108.Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survivalNature 457:210–214https://doi.org/10.1038/nature07536
- 109.Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotypeCell Metab 13:739–748https://doi.org/10.1016/j.cmet.2011.05.002
- 110.Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cuesDev Camb Engl 143:3914–3925https://doi.org/10.1242/dev.139782
- 111.Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and HpoEMBO J 33:2295–2313https://doi.org/10.15252/embj.201387500
- 112.Male Germline Stem Cell Division and Spermatocyte Growth Require Insulin Signaling in DrosophilaCell Struct Funct 34:61–69https://doi.org/10.1247/csf.08042
- 113.Innate immune signalling drives loser cell elimination during stem cell competition in the Drosophila testisDevelopmental Biology https://doi.org/10.1101/2020.03.05.979161
- 114.Drosophila LKB1 is required for the assembly of the polarized actin structure that allows spermatid individualization. White-Cooper H, editorPLOS ONE 12https://doi.org/10.1371/journal.pone.0182279
- 115.Combover interacts with the axonemal component Rsp3 and is required for Drosophila sperm individualizationDev Camb Engl 146https://doi.org/10.1242/dev.179275
- 116.Inactivation of AMPKα1 induces asthenozoospermia and alters spermatozoa morphologyEndocrinology 153:3468–3481https://doi.org/10.1210/en.2011-1911
- 117.AMPK Function in Mammalian SpermatozoaInt J Mol Sci 19https://doi.org/10.3390/ijms19113293
- 118.An Essential Role for Insulin and IGF1 Receptors in Regulating Sertoli Cell Proliferation, Testis Size, and FSH Action in MiceMol Endocrinol 27:814–827https://doi.org/10.1210/me.2012-1258
- 119.FlyBase 2.0: the next generationNucleic Acids Res 47https://doi.org/10.1093/nar/gky1003
- 120.Drosophila Cytogenetics Protocols:45–75https://doi.org/10.1385/1-59259-665-7:45
- 121.Drosophila spermiogenesisSpermatogenesis :197–212https://doi.org/10.4161/spmg.21798
- 122.Fiji: an open-source platform for biological-image analysisNat Methods 9:676–682https://doi.org/10.1038/nmeth.2019
- 123.Parallel metabolomics and lipidomics enables the comprehensive study of mouse brain regional metabolite and lipid patternsAnal Chim Acta 1136:168–177https://doi.org/10.1016/j.aca.2020.09.051
- 124.MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysisNat Methods 12:523–526https://doi.org/10.1038/nmeth.3393
- 125.Patterned Signal Ratio Biases in Mass Spectrometry-Based Quantitative MetabolomicsAnal Chem 93:2254–2262https://doi.org/10.1021/acs.analchem.0c04113