1. Cell Biology
Download icon

Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male reproductive advantage in C. elegans

  1. Chia-An Yen
  2. Dana L Ruter
  3. Christian D Turner
  4. Shanshan Pang
  5. Sean P Curran  Is a corresponding author
  1. Leonard Davis School of Gerontology, University of Southern California, United States
  2. Department of Molecular and Computation Biology, Dornsife College of Letters, Arts, and Sciences, University of Southern California, United States
  3. School of Life Sciences, Chongqing University, China
  4. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, United States
Research Article
Cite this article as: eLife 2020;9:e52899 doi: 10.7554/eLife.52899
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
alh-6 fertility defects are sperm-specific.

(A) Proline catabolism pathway. (B–C) alh-6 hermaphrodites have reduced brood size when fed OP50 (B) or HT115 (C) diets. (D) alh-6 hermaphrodites lay increased number of unfertilized oocytes, but few dead embryos. (E) Mated reproductive assay scheme utilizes males to maximize reproductive output (as in F) and can exploit males harboring GFP to differentiate progeny resulting from self- versus male-sperm (as in G). (F) Wild type (WT) and alh-6 hermaphrodites mated with WT males yield similar number of total progeny. (G) WT hermaphrodites mated with alh-6;gst-4p::gfp males yield more non-GFP progeny (indicating self-fertilization) than hermaphrodites mated with WT males harboring gst-4p::gfp. Statistical comparisons by unpaired t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 1—figure supplement 1
Mitochondrial localization of ALH-6 in the germline.

UV integrated alh-6::gfp strain under its endogenous promoter reveals expression of ALH-6 in hermaphrodite (A–B) and male (C–D) germline. a and c are DIC images while b and d are GFP images. Scale bar for all images is 10µm.

Figure 1—figure supplement 2
alh-6 hermaphrodite reproductive span is similar to wild type (WT) on different diets.

(A–B) Progeny output time-courses are plotted as % total progeny for each time point. WT and alh-6 mutant have similar output on OP50 (A) and HT115 (B). Significance indicate differences in progeny output at a particular time point done by multiple t-tests. *, p<0.05; *F*, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 1—figure supplement 3
alh-6 fertility defects are sperm-specific.

(A) Day four adult WT hermaphrodites mated to either gst-4p::gfp or alh-6;gst-4p::gfp males yield similar total brood size. (B) Day four adult alh-6 hermaphrodites mated to either gst-4p::gfp or alh-6;gst-4p::gfp males yield similar total brood size. Comparisons made with unpaired t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 2 with 1 supplement
alh-6 males have sperm defects on both OP50 and HT115 diets.

(A–C) sperm phenotypes on OP50 diet. (A) Sperm quantity is similar between wild type (WT) and alh-6 mutant day one adult males. (B) Spermatid size is reduced in alh-6 mutant day one adult males as compared to age matched WT males. (C) Sperm activation is impaired in alh-6 mutant day one adult males relative to age-matched WT males. (D–F) sperm phenotypes on HT115 diet. (D) Sperm quantity is reduced in alh-6 mutant day one adult males compared to age-matched WT males. (E) Spermatid size is reduced in alh-6 mutant day one adult males as compared to age matched WT males fed HT115. (F) Sperm activation is impaired in alh-6 mutant day one adult males relative to age-matched WT males fed HT115. Statistical comparisons of sperm number and size by unpaired t-test and sperm activation by Fisher’s exact test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 2—figure supplement 1
Activation defects of alh-6 spermatids.

(A) alh-6 hermaphrodites have reduced sperm number as day one adults. (B) Spermiogenesis stages (round, spike, protrusion, pseudopod). Spermatozoa with fully formed pseudopods are considered activated in Pronase and Monensin experiments. (C) alh-6 male spermatids treated with Pronase are stalled at the ‘spikes’ stage compared to WT male spermatids. (D) alh-6 male spermatids treated with Monensin have reduced activation compared to WT male spermatids. (E) alh-6 male spermatids treated with Monensin are stalled at the ‘protrusion stage’ compared to age-matched WT spermatids. Statistical comparisons of sperm number by unpaired t-test and sperm activation by Fisher’s exact test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 3 with 1 supplement
Transcriptional patterns define developmental- and adult-specific consequences to loss of alh-6 activity.

Gene Ontology (GO) term enrichment analysis of RNA-Seq data. (A) Transcriptional changes at L4 stage are enriched for metabolism and sperm-specific genes. (B) Transcriptional changes at day three adulthood are enriched for changes in glutathione activity, oxidoreductase activity, and muscle-specific genes. All studies performed in at least biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 3—figure supplement 1
RNA-Sequencing data of WT and alh-6 hermaphrodites at L4 and day three adulthood.

(A) Number of genes that are significantly upregulated in alh-6(lax105) compared to WT at L4 and Day three adult stages. (B) Number of genes that are significantly downregulated in alh-6(lax105) compared to WT at L4 and Day three adult stages. FDR = 0.05. (C) alh-6 mutants display increased expression of metabolic enzymes to reduce P5C levels. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 4 with 1 supplement
Loss of FAD homeostasis in alh-6 mutants leads to sperm dysfunction.

(A) Metabolic pathways utilize adenine dinucleotide cofactors to maintain redox balance in cells. (B–C) FAD+ levels are reduced in alh-6 mutant animals fed OP50 (B) or HT115 (C) at the L4 developmental stage. (D) FAD biosynthetic pathway. (E–G) Dietary supplement of riboflavin restores FAD level (E), sperm size (F), and sperm activation (G) in alh-6 mutants. (H–I) RNAi knockdown of R10H10.6 (H) or flad-1 (I) in WT males reduces their sperm size compared to L4440 vector control. (J–K) RNAi knockdown of R10H10.6 (J) or flad-1 (K) in WT males impairs sperm activation upon Pronase treatment. Statistical comparisons of sperm size by ANOVA. Statistical comparisons of activation by fisher’s exact test with p-value cut-off adjusted by number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 4—figure supplement 1
Adenine nucleotide cofactor homeostasis is disrupted in alh-6 mutants.

(A) FAD levels are unchanged between WT and alh-6 mutant animals fed OP50 at day three adulthood. (B) WT hermaphrodites mated to alh-6;gst-4p::gfp males fed OP50 supplemented with 2.5 mM riboflavin results in increase in total brood size compared to WT hermaphrodites mated to non-supplemented alh-6;gst-4p::gfp males. (C) Dietary riboflavin supplement increased spermatid size of WT males. (D) WT males fed OP50 diet supplemented with riboflavin have similar % spermatid activated upon Pronase treatment as those without riboflavin supplement. (E) R10H10.6 one expression is modestly reduced by whole animal RNAi via RT-PCR verification. (F) flad-1 expression is reduced by whole animal RNAi via RT-PCR verification. (G) NAD levels are unchanged between WT and alh-6 animals at L4 and day three adulthood (H) NADH level is unchanged between aged matched WT and alh-6 hermaphrodites at both L4 and Day three adulthood. (I) NAD+/NADH level is unchanged between aged matched WT and alh-6 hermaphrodites at both L4 and Day three adulthood. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Statistical comparisons done by unpaired t-test for all experiments except for sperm activation, which is done by Fischer’s exact test. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 5 with 1 supplement
Mitochondrial dynamics drive sperm quality.

(A–E) JC-1 dye stained mitochondria of WT (A–B), alh-6 mutant (C–D); (B and D) are ImageJ detection of JC-1 stained sperm mitochondria area which are quantified in (E). (F) Mitochondria in alh-6 mutant spermatids have reduced JC-1 red/green fluorescence ratio, indicating mitochondria depolarization. (G–H) RNAi knockdown of FAD biosynthetic pathway genes, R10H10.6 (G) or flad-1 (H) increases mitochondrial fusion in WT spermatids. (I) Dietary supplement of FAD precursor riboflavin restores mitochondrial fusion in alh-6 spermatids to WT level. (J–K) eat-3 or fzo-1 RNAi decreases mitochondrial fusion in both WT (J) and alh-6 (K) mutant spermatids. (L) drp-1 mutation increases mitochondrial fusion in both WT and alh-6 spermatids. (M) drp-1 mutation significantly impairs sperm activation in both WT and alh-6 mutant spermatids. (N) fzo-1 RNAi restores sperm activation in alh-6 mutant. (O) eat-3 RNAi reduces sperm activation in WT males but not alh-6 males. Statistical comparisons of JC-1 Red/Green FL ratio by unpaired t-test. Statistical comparisons of mitochondria fusion by ANOVA. Statistical comparisons of sperm activation by Fisher’s exact test with p-value cut-off adjusted by number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 5—figure supplement 1
fzo-1 is involved in mitochondrial dynamics aberration in alh-6 spermatids.

(A) JC-1 stained alh-6 spermatid mitochondria show sensitivity to mitochondrial uncoupler CCCP treatment. Intensity of red mitochondria species (J-aggregates) are dissipated while that of green mitochondria species (monomers) are intensified, indicating membrane depolarization (Note that CCCP treatment is known to cause cellular swelling). Scale bar = 1 uM. (B) alh-6 spermatid mitochondria stained with MitoTracker Red CMXRos show increased fusion compared to WT male spermatids. Scale bar = 0.5 uM. (C) Spermatids of alh-6 males fed HT115 diet still display increased mitochondrial fusion compared to spermatids of age-matched WT males. (D) Riboflavin supplementation did not alter mitochondria fusion in spermatids of WT males. (E) eat-3 and fzo-1 RNAi knockdown in WT and alh-6 mutants are verified using RT-PCR. (F) fzo-1 expression is increased in alh-6 mutants, while eat-3 expression is not significantly increased. Statistical comparisons done by unpaired t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 6 with 1 supplement
alh-6 and FAD function cell autonomously in the germline to regulate sperm function.

(A–C) Germline-specific RNAi of alh-6 does not change sperm size (A), but does impair sperm activation (B) and increases mitochondrial fusion in sperm (C). (D–F) Germline-specific rescue of WT alh-6 in alh-6 mutant male animals increases sperm size (D) and restores activation (E) and mitochondrial dynamics (F). Statistical comparisons of sperm size and mitochondrial fusion in spermatids by unpaired t-test. Similarly, (G–L) germline-specific RNAi of R10H10.6 and flad-1 do not change sperm size (G,J), impair sperm activation (H,K), and increase mitochondrial fusion in sperm (I,L). Statistical comparisons of sperm activation by Fisher’s exact test with p-value cut-off adjusted by number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Figure 6—figure supplement 1
Germline expression of WT alh-6 is sufficient to rescue sperm defect.

(A–B) Soma-restricted RNAi of alh-6 slightly reduces sperm size (A), but does not affect sperm activation (B). (C–F) Whole animal RNAi of alh-6 reduces sperm size (C), impairs activation (D), and increases mitofusion in spermatids (E). (F) alh-6 expression is reduced by whole animal RNAi as shown in RT-PCR verification. Statistical comparisons done by unpaired t-test for all experiments except for sperm activation, which is done by Fischer’s exact test. All studies performed in biological triplicate; refer to Supplementary file 1 for n for each comparison.

Model of alh-6 and FAD mediated male reproductive senescence.
Author response image 1

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain (C. elegans)N2Caenorhabditis Genetics Center (CGG)Laboratory reference strain (wild type)
Strain (C. elegans)SPC321PMID: 24440036Genotype: alh-6(lax105)
Strain (C. elegans)SPC326PMID: 24440036alh-6p::alh-6::gfp
Strain (C. elegans)SPC447This paperGenotype: alh-6(lax105);laxEx025(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)
Strain (C. elegans)SPC455This paperGenotype: alh-6(lax105);laxEx033(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)
Strain (C. elegans)SPC473This paperGenotype: alh-6(lax105);laxEx051(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)
Strain (C. elegans)CL2166Caenorhabditis Genetics Center (CGG)Genotype: gst4-p::gfp
Strain (C. elegans)SPC223PMID: 24440036Genotype: alh-6(lax105);gst-4p::gfp
Strain (C. elegans)DCL569Caenorhabditis Genetics Center (CGG)Genotype: [mkcSi13(sun-1p::rde-1::sun-1 3'UTR + unc-119(+)) II; rde-1(mkc36) V
Strain (C. elegans)CU6372Caenorhabditis Genetics Center (CGG)Genotype: drp-1(tm1108)
Strain (C. elegans)GR1948PMID: 24684932Genotype: mut-14(mg464);smut-1(tm1301) V.
Chemical compound, drugRiboflavinMillipore SigmaR9504Concentration used: 2.5 mM
Commercial Assay or kitFAD Colorimetric/Fluorometric Assay KitBioVisionK357
Commercial Assay or kitNAD/NADH Quantification Colorimetric KitBioVisionK337
Chemical compound, drugPronaseMillipore SigmaP8811Concentration used: 200 ug/mL
Chemical compound, drugeBioscience Monensin Solution (1000X)Thermo Fisher Scientific00-4505-51Concentration used: 100 nM
Chemical compound, drugMitoProbe JC-1 Assay KitThermo Fisher ScientificM34152Concentration used: JC-1 15 uM, CCCP 50 uM
Chemical compound, drugMitoTracker Red CMXRosThermo Fisher ScientificM7512Concentration used:
100 uM dried on plate
SoftwareGraphPad PrismGraphPad Prism (https://graphpad.com)RRID:SCR_015807Version 6
SoftwareImageJImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)