The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract

Unique patterns of inheritance and selection on Y chromosomes lead to the evolution of specialized gene functions. Yet characterizing the function of genes on Y chromosomes is notoriously difficult. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. WDY mutants produce mature sperm with beating tails that can be transferred to females but fail to enter the female sperm storage organs. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as wild-type sperm’s and that the mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely cause the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggest that sperm tail beating and propulsion do not always correlate. Furthermore, we find significant differences in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs of WDY that are Y-linked and those that are autosomal. Given that WDY appears to be evolving under positive selection, our results suggest that WDY’s functional evolution coincides with its transition from autosomal to Y-linked in Drosophila melanogaster and its most closely related species. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. In contrast to WDY, PRY mutants do swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.02.526876 doi: bioRxiv preprint Figure S1: CRISPR target site on exon 2 of WDY had no identifiable duplicates Exon    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Drosophila Stocks and Husbandry
Flies were reared on a cornmeal-agar-sucrose medium (recipe available at https://cornellfly.wordpress.com/s-food/) at 25°, with a 12 hr light-dark cycle. The stocks used in this study are described in Table S1.

Generation of a WDY Mutant with CRISPR
Three 20 base pair guide RNAs were designed to target exon 2 of WDY, a region of the gene with no known duplications (Figure S1) 1 . We also targeted ebony, a visible Co-CRISPR marker 2 . Guide sequences were incorporated into pAC-U63-tgRNA-Rev (Addgene, Plasmid #112811) which is analogous to the "tgFE" construct from [3]. This was done by appending guide RNA sequences to tracrRNA core and tRNA sequences from pMGC (Addgene, Plasmid #112812) through tailed primers (Table S3) to create inserts that were then inserted by Gibson Assembly into a SapI-digested pAC-U63-tgRNA-Rev (Addgene, Plasmid #112811). The plasmid backbone contained attB, and we used Phi-C31 to integrate it into an attP-9A site on chromosome 3R. The construct was injected into yw nanos-phiC31; PBac{y+-attP-9A}VK00027 by Rainbow Transgenic Flies Inc. Transformants were identified by eye color from a mini-white+ marker. Transformants express the four guides ubiquitously under the U6:3 promoter as a single polycistronic transcript that is processed by the endogenous cellular tRNA processing machinery (RNase P and Z) to release the individual mature gRNAs and interspersed tRNAs. The transformants were balanced and inserts were confirmed by PCR and sequencing. A few of the transformants had light-red eyes, but we only used those with dark-red eyes.
Different combinations of transformants and germline Cas-9 drivers were tested for editing efficiency (data not shown). Males containing vasa-Cas9 and our guide RNAs were sterile, while females showed a 6.2% CRISPR efficiency, based on the generation of ebony mutants. In contrast, F1 males from crosses with nanos-Cas9 drivers on chromosomes 2 and 3 produced progeny. F2 progeny (from male and female crosses combined) showed that these two lines had editing efficiencies of 2.2% and 2.6%, respectively. Our observation of higher efficiency and sterility from vasa-Cas9 is consistent with the earlier 4 and higher somatic 5,6 protein expression of Vasa versus Nanos, as well as the RNAi phenotype of WDY 7 . We proceeded to make stable mutants by crossing transformant #1 to nanos-Cas9 in a compound chromosome background.
Our crossing scheme for creating WDY alleles is shown in Figure S3. We combined the nanos-Cas9 driver on chromosome 3 with a Y chromosome marked with 3xP3-tdTomato 8 . We also combined the guideexpressing insert on 3R with a compound X ( ̂ ; C(1)M4, y [1] ). CRISPR editing occurred in F1 females (̂Y 3XP3-tdTomato ) that carried the marked Y chromosome. By crossing to a compound X-Y (̂, C(1;Y)1, y [1]) we were able to establish balanced lines from 55 ebony and 5 non-ebony F2 flies. Males were of genotype ̂ Y 3XP3-tdTomato and were fertile regardless of CRISPR-mediated edits of the free Y. We screened these lines for visible deletions in the WDY target site -first by gel, then by sequencing. Alleles derived from our crossing scheme are listed in Table S4 and described in Figure S4. They are maintained as stable lines with ̂ females and ̂ males; the free Y chromosome is edited.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made In several of our lines, we saw varying, intermediate degrees of position effect variegation (PEV) ( Figure  S2). This corresponded with either failed amplification at the target site in WDY or the presence of several bands of unexpected size. Based on our previous results when editing FDY with CRISPR 7 , we hypothesized that these mutations were large deletions in the Y chromosome, and thus did not phenotype these mutants for sperm or fertility characteristics. C(1)M4 contains white [mottled-4], a PEV marker that is highly sensitive to Y-chromosome dosage. ̂ females with C(1)M4 have mostly white eyes, while ̂ females have an almost entirely red eye. We previously showed that lines with visibly altered PEV lacked large sections of the Y chromosome 7 . Such deletions may be caused by the presence of uncharacterized copies of the target region present in unassembled regions of the Y chromosome.

Sterility, Mating, and Sperm Storage
Crosses and experiments were done with flies 2-5 days after eclosion (dAE). To test for sterility, we crossed individual XY males to 4 Canton S virgin females in a food containing vial with wet yeast. Adults were transferred to a new vial after one week. Crosses were scored for the presence of progeny. 15-20 crosses were tested per line. For experiments that required timing from the start of mating, one Canton S virgin female was mated to three males of a given genotype and flies were observed. Once mating began the time was noted. Females were analyzed or flash frozen in liquid nitrogen for 30 minutes, 2 hours, or 24 hours after the start of mating (30 mASM, 2 hASM, 24 hASM). Reproductive tracts were dissected from frozen females in PBS, fixed in 4% paraformaldehyde, and mounted in Vectashield with DAPI. Samples were imaged on an Echo Revolve microscope or a Leica DMRE confocal microscope.

Sperm Counting with Imaris software
To quantify sperm transferred, female reproductive tracts 30mASM were imaged on a Leica DMRE confocal using standardized settings. Two μm Z-stacks through each sample were collected. Using Imaris 9.8.0 software (RRID:SCR_007370), first the female reproductive tract was extracted in each image by manually drawing a contour surface. The mating plug and cuticle were specifically excluded due to their high autofluorescence 9 Second, protamine-labelled sperm heads were automatically detected using the "Surfaces" function (smoothing and background elimination enabled, 2.0 µm surface grain size, 1 µm diameter of largest sphere, 2.747-13.048 manual threshold, >15 quality, <0.8 sphericity). Counts of transferred sperm from control and WDY males were statistically compared using a Student's t-test in R software.

Sperm-tail Beat Frequency Analysis
Tail-beat frequency was measured for sperm dissected from the reproductive tracts of males 2-5dAE or females 2-5 dAE and 30 mASM into PBS. Sperm were released into a 15 ul drop of PBS on a glass slide by tearing the male seminal vesicle or female uterus. Sperm were observed under brightfield optics with an Olympus BX51WDI microscope and a 50x LMPLFLN objective. Eight second raw movie clips at 1280x720 resolution and 60 frames per second were captured from 4-6 different regions around the sperm mass using a Canon EOS Rebel T6 camera. Dissected sperm masses all contain sperm tails beating at a range . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.02.526876 doi: bioRxiv preprint of frequencies -we specifically quantified the beat frequency of the 1-2 fastest-beating sperm tails from each clip.
To measure the sperm tail-beating frequency, video clips were imported to FIJI (RRID:SCR_002285) using the ffmpeg plug-in. From each clip, we measured beat frequencies of the 1-2 fastest-beating sperm, limited to tails that were not overlapping or entangled with other sperm tails. A selection line was drawn across an isolated section of sperm tail. A 1-pixel "Multi Kymograph" was generated which shows pixel intensities across the selection line on the X-axis for each frame along the Y-axis. The beating of the sperm tail appears as a traveling wave form. The number of beats and the number of frames were counted for the region where the sperm tail remained in focus and isolated from other tails. Beat frequency was then calculated as: Hz = (# beats X 60 fps) / # frames. Ten measurements were made per individual fly. Approximately one-third of samples were scored blind, and statistical analysis indicated consistent results whether samples were scored blind or not.
Sperm tail-beat frequencies were measured from a minimum of three individuals of each allele. Using the lme4 package in R, linear mixed models were fitted to the data, incorporating the individual as a random effect and experimental batch and the experimenter who measured beat frequency as fixed effects. We then ran a Likelihood-Ratio test to compare the model with and without "Genotype" as a fixed effect.

Sperm Swimming Analysis
Videos of sperm swimming were acquired from either male reproductive tracts or female reproductive tracts 1 hASM. Tracts were dissected and mounted in 15 µL PBS. Spacers (2 layers of double-stick tape) were used to avoid compression of the tissue by the coverslip. Fluorescent sperm heads were recorded through screen recording of the preview window on an Echo Revolve. We used ffmpeg (RRID:SCR_016075) to convert videos to constant frame rate of 60 fps and .mov format. Videos were then imported into FIJI (RRID:SCR_002285) using the ffmpeg plugin. We manually tracked sperm heads across 60 frames using the "Manual Tracking" plugin in FIJI. The tracking shown in Figure 3 represent movement across 30 frames.

WDY Annotation and Sequence Comparisons
EF hand motifs were identified by searching (using Geneious software, RRID:SCR_010519) for the canonical and pseudo PROSITE motif consensus sequences defined in [ 10 ] and allowing for a maximum 1 base pair mismatch. Because the pseudo-EF hand motif contains a variable size region, there were two potential start locations in the sequence -residue 44 or 47. However, the Alphafold prediction showed residues that should form the loop region would instead form part of the alpha helix in the motif beginning at residue 44. We therefore favored the motif beginning at residue 47. Locations of the calcium binding residues were determined based on the consensus sequence logograms in [ 10 ].
Three WD40 domains 11 were originally identified in the protein sequence based on homology. Flybase reported a handful of WD40 repeats (2 for Pfam and 8 for SMART) were identified. 4 to 16 of these repeat domains may together form a circular beta propeller structure called a WD40 domain [12][13][14] ; . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.02.526876 doi: bioRxiv preprint however, insufficient WD40 repeats were identified in WDY to predict the presence of a WD40 domain. We used the a structural prediction of D. melanogaster WDY by Alphafold (PDB B4F7L9) 15 to identify the locations of the characteristic β-propeller, consisting of 4 antiparallel sheets 13 . WDY is predicted to form two WD40 domains -one with 6 WD40 repeats and one with 7 WD40 repeats.
WDY ortholog sequences were obtained as described in Table S6. The proteins were aligned in Geneious using a BLOSUM cost matrix with a gap open cost of 10 and a gap extend cost of 0.1.