Inducible degradation of dosage compensation protein DPY-27 facilitates isolation of Caenorhabditis elegans males for molecular and biochemical analyses

Abstract

Biological sex affects numerous aspects of biology, yet how sex influences different biological processes have not been extensively studied at the molecular level. Caenorhabditis elegans, with both hermaphrodites (functionally females as adults) and males, is an excellent system to uncover how sex influences physiology. Here, we describe a method to isolate large quantities of C. elegans males by conditionally degrading DPY-27, a component of the dosage compensation complex essential for hermaphrodite, but not male, development. We show that germ cells from males isolated following DPY-27 degradation undergo meiosis and spermiogenesis like wild type and these males are competent to mate and sire viable offspring. We further demonstrate the efficacy of this system by analyzing gene expression and performing affinity pull-downs from male worm extracts.

Keywords: Caenorhabditis elegans; DPY-27; Genetics of Sex; dosage compensation; males; meiosis; spermiogenesis.

Fig. 1.

Enrichment of male worms by conditional degradation of DPY-27. a) Strategy for isolation of male cultures. b) Plate phenotype following auxin degradation of DPY-27, where greater than 98% of the worms are males. c) Quantification of male enrichment in WT, him-8(me4), sun-1p::TIR1; dpy-27::AID; him-8(me4) and mex-5p::TIR1; dpy-27::AID; him-8(me4) in the absence (−auxin) and presence (+auxin) of 1 mM auxin. A minimum of 5 plates were counted for males; mean and 95% confidence intervals are shown. Statistical comparisons between + and − auxin for sun-1p::TIR1; dpy-27::AID; him-8(me4): P = 0.001 and mex-5p::TIR1; dpy-27::AID; him-8(me4): P = 0.0005 by Mann–Whitney.

Fig. 2.

DPY-27 depletion does not affect male meiosis. a) Quantification of RAD-51 in indicated regions of the germline. Box whisker plots show number of RAD-51 foci per nucleus. Horizontal line of each box represents the median, top and bottom of each box represent medians of upper and lower quartiles, lines extending above and below boxes indicate SD, and individual data points are outliers from 5% to 95%. Comparisons by Mann–Whitney revealed no statistical differences between the strains. PZ, proliferative zone; TZ, transition zone; EP, early pachytene; MP, mid-pachytene; LP, late pachytene. Three germlines were scored for each strain/condition. Number of nuclei scored in each region: WT, PZ = 559; TZ =136; EP = 132; MP = 175; LP = 132; him-8(me4), PZ = 452; TZ = 115; EP = 134; MP = 141; LP = 135; sun-1p::TIR1; dpy-27::AID; him-8(me4) − auxin, PZ = 391; TZ = 126; EP = 117; MP = 155; LP = 168; sun-1p::TIR1; dpy-27::AID,; him-8(me4) + auxin, PZ = 671; TZ = 187; EP = 197; MP = 209; LP = 206. b) Number of COSA-1 foci in mid-late-pachytene in indicated strains/conditions; mean and 95% confidence intervals are shown. Comparisons by Mann–Whitney revealed no statistical differences between the strains. Number of nuclei analyzed: wild type = 189 (from 3 worms); him-8(me4) = 182 (from 3 worms); sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin = 549 (from 8 worms); sun-1p::TIR1; dpy-27::AID; him-8(me4) +auxin = 584 (from 8 worms). c) Images of the division zone of male gonads labeled with anti-tubulin (cyan) to visualize the spindle and DAPI (magenta) to visualize the DNA from WT (N2), him-8(me4) and sun-1p::TIR1; dpy-27::AID; him-8(me4) – and + auxin. Scale bar = 5 microns. There was no significant difference in the number of metaphase and anaphase meiosis I and II spindles/gonad in the different genotypes/conditions (N2 = 13.5 ± 3.7; him-8(me4)=12.83 ± 2.1; sun-1p::TIR1; dpy-27::AID; him-8(me4) – auxin = 11.83 ± 2.3 and + auxin = 13.5 ± 1.9/gonad) by Mann–Whitney.

Fig. 3.

Spermiogenesis, sperm quality, and competition are not perturbed following DPY-27 degradation in males. a) Micrographs of sperm isolated from N2 (WT), him-8(me4), and sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin and +auxin and in the presence of Pronase E (+Pronase E), which leads to sperm activation. Scale bar = 10 microns. b) Quantification of sperm activation. Comparisons by Mann–Whitney revealed no statistical differences between the strains/conditions. The number of sperm examined: N2 (WT) = 544; him-8(me4) = 1,104; sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin = 602; sun-1p::TIR1; dpy-27::AID; him-8(me4) +auxin = 647. c) Total progeny sired. Mean and 95% confidence intervals are shown. Comparisons by Mann–Whitney revealed no statistical differences between the strains/conditions. Number of crosses examined: N2 (WT) = 6; him-8(me4) = 7; sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin = 6; sun-1p::TIR1; dpy-27::AID; him-8(me4) +auxin = 7. d) Embryonic viability of fog-2(q71) progeny sired by indicated males. Mean and 95% confidence intervals are shown. Comparisons by Mann–Whitney revealed no statistical differences between the strains/conditions. Number of crosses examined: wild type = 10; him-8(me4) = 8; sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin = 10; sun-1p::TIR1; dpy-27::AID; him-8(me4) +auxin = 9. e) Sperm competition assays were performed with unc-119(ed3) hermaphrodites. Mean and 95% confidence intervals are shown. Comparisons by Mann–Whitney revealed no statistical differences between the strains/conditions. Number of crosses examined: wild type = 10; him-8(me4) = 7; sun-1p::TIR1; dpy-27::AID; him-8(me4) −auxin = 7; sun-1p::TIR1; dpy-27::AID; him-8(me4) +auxin = 8.

Fig. 4.

Molecular and biochemical analyses of males isolated following DPY-27 depletion. a) Fold change of mRNA expression for vit-2 (hermaphrodite enriched) and sncp-1.3 (male-enriched) in sun-1p::TIR1; dpy-27::AID; him-8(me4) in the absence and presence of auxin relative to wild type. Fold change was determined using the 2^−ΔΔCq method. ama-1 (housekeeping) mRNA levels were used as a reference. Statistical comparisons by Mann–Whitney between − and + auxin for both vit-2 and sncp-1.3, P = 0.0022. Raw Cq values and calculations are provided in Supplementary File 3. b) Mass spectrometry of peptides identified from male extracts (BRD-1 = 97.6% males; BRD-1::GFP::3xFLAG = 98.5% males). Venn diagram (generated by https://bioinfogp.cnb.csic.es/tools/venny/) shows unique peptides from BRD-1::GFP::3xFLAG and peptides from untagged control (BRD-1) with common peptides in middle. A small number of proteins with known interactions with BRC-1-BRD-1 (unique) as well as sperm proteins that are likely nonspecific interactions (common) are highlighted. The complete data set of nonspecific peptides and peptides unique to BRD-1::GFP::3xFLAG are available in Supplementary File 4.