Description
We report the identification of a new allele of
catp-4
, which we refer to as
catp-4
(
as46
)
.
CATP-4
is a subunit of a Na+/K+-ATPase required for sperm activation, sperm motility, and fertility in
C. elegans
(Wang et al., 2021). The
as46
allele was isolated from a forward genetics EMS (ethyl methanesulfonate) screen for sterile mutants, similar to the one described in Singaravelu et al., 2015. Brood sizing assays show that both
catp-4
(
as46
)
hermaphrodites and
catp-4
(
as46
)
males have significantly reduced fertility (p<0.0001) compared to wild-type
N2
(
Figure 1A
and 1B).
To determine the reason for reduced fertility of
catp-4
(
as46
)
hermaphrodites we examined whether sperm were produced and correctly localized within the spermatheca. Sperm nuclei appear as small bright punctae by DAPI staining. It was observed that at 6 hours after the L4 larval stage, there was no visual difference between the complement of sperm in unmated
catp-4
(
as46
)
hermaphrodites and unmated
N2
hermaphrodites (
Figure 1C
). At this time point spermatogenesis should be complete but the first ovulation has not yet taken place. However, at 24 hours after the L4 stage, when multiple ovulations have occurred, the number of sperm in the spermathecae for
catp-4
(
as46
)
hermaphrodites appeared noticeably reduced relative to wild type. We also observed sperm in the uterus which was not observed for wild-type
N2
hermaphrodites (
Figure 1C
). This loss of sperm is indicative of sperm migration and motility defects. As an egg moves from the spermatheca to the uterus sperm are pushed out into the uterus and must crawl back into the spermatheca for the opportunity to fertilize a subsequent egg (L'Hernault, 2006; Ward & Carrel, 1979). If they are unable to migrate back to the spermatheca, the sperm are rapidly lost as ovulations continue. Consistent with our observation of rapid sperm loss from the spermathecae, spermatids dissected from
catp-4
(
as46
)
mutant males suffer from severe morphological defects (
Figure 1D
). Instead of having a normal spherical morphology, the spermatids are angular and distorted. In addition, when these spermatids are exposed to Pronase to trigger sperm activation
in vitro
, they form pseudopods that are shorter than wild-type controls (
Figure 1D
). To quantify the relative length of the pseudopods we measured the aspect ratio for the sperm, by dividing the sperm length (cell body and pseudopod) by the sperm width (cell body). A ratio closer to 1 indicates shorter pseudopods. The aspect ratio is significantly smaller (p < 0.0001) for
catp-4
(
as46
);
him-5
sperm (1.336 ± 0.01188, n = 140 ) compared to
him-5
sperm (1.448 ± 0.01381, n = 140 ) (
Figure 1E
). Taken together our data indicate that the
as46
mutation causes defects in sperm activation and motility.
Whole genome sequencing identified that the
as46
allele is a missense point mutation in exon 4 of
C01G12.8
,
aka
catp-4
. The missense mutation causes an amino acid change from glycine (G) to arginine (R) at the 532nd amino acid position (
Figure 1E
). The mutation was confirmed by Sanger sequencing. The phenotype we observe for
as46
matches the phenotype of
catp-4
null alleles already reported (Wang et al., 2021).
To verify that the observed phenotype is due to the missense mutation in the
catp-4
gene, we performed a complementation test at 25°C where the
as46
phenotype is the most severe. If two strains carry a mutation in the same gene, then upon crossing a heterozygous worm from one strain to a homozygous mutant worm from the other strain, we expect 50% of the F1 progeny to show the mutant phenotype. When heterozygous
catp-4
(
ok2056
)
/+ males were crossed with homozygous
as46
hermaphrodites, 45.8% of F1 hermaphrodites showed the
catp-4
phenotype of reduced progeny production (11 out of 24). Furthermore when
heterozygous
as46
/+
males were crossed with homozygous
catp-4
(
ok2056
)
hermaphrodites, 54.2% (13 out of 24) of F1 hermaphrodites showed the
catp-4
phenotype. Therefore, we conclude that the two alleles fail to complement and
as46
is an allele of
catp-4
.
EMBL-EBI MUSCLE was used to align the
C. elegans
CATP-4
protein with Na+/K+-ATPases from other organisms. We found that the
as46
mutation changes a highly conserved glycine (
Figure 1F
). This conserved glycine residue must be critical for
CATP-4
protein function as substitution of an arginine at this site recapitulated the phenotype of a complete loss of function of
catp-4
. The glycine lies between two ATP binding sites, one of which is only four amino acids away. We hypothesize that since the substitution of glycine with arginine is in close proximity to an ATP binding site, it potentially affects the structure of the ATP binding site, and ultimately the protein's function. However, further experiments will be needed to confirm this hypothesis.
Methods
C. elegans
maintenance
All
C. elegans
strains were maintained according to (Brenner, 1974). Worms were cultured on MYOB plates seeded with
OP50
E. coli
. Strains were maintained at 16°C. For experiments, adult worms were placed at 16°C for 2 days and then their eggs/L1 progeny were shifted to 25°C. All experiments were carried out at 25°C unless stated otherwise.
Hermaphrodite self-fertility and male fertility
Fertility was determined by measuring the brood size of individual animals. To determine hermaphrodite self-fertility, L4 hermaphrodites were selected and put on individual plates. These hermaphrodites were transferred to new plates every 24 hours until they stopped laying any embryos. To determine male fertility, males were put on individual plates with
fog-2
(
oz40
)
L4 hermaphrodites in a 4:1 ratio.
fog-2
(
oz40
)
hermaphrodites do not produce sperm and therefore all progeny are known to be sired by the males. After 24 hours, the males were removed and the hermaphrodites were transferred daily to new plates until no new embryos were seen on the plates. For both hermaphrodite self-fertility and male fertility assays, once the hermaphrodite was removed from the plate the progeny were grown at 20°C and counted 3 days later. Hermaphrodites that disappeared or died during the experiment were excluded from the analysis.
DAPI staining
Hermaphrodites were selected at the L4 stage and aged for either 6 hours or 24 hours. The hermaphrodites were fixed with ethanol and stained with DAPI (1 mg/ml) (Clarke et al., 2018). Images were taken using a Zeiss Observer D.1 with a 40X objective and captured with an AxioCAM 503 camera.
Spermatid morphology and sperm activation
Young adult males were separated from hermaphrodites for approximately 24 hours prior to the experiment. To determine spermatid morphology and sperm activation, males were dissected in Sperm Media (50 mM HEPES, 25 mM KCl, 45 mM NaCl, 1 mM MgSO
4
, 5 mM CaCl
2
, 10 mM Dextrose, pH 7.8) modified from (L'Hernault SW & Roberts TM, 1995) or Sperm Media with Pronase (200 µg/ml), respectively. DIC images were taken using a Zeiss Observer D.1 with a 40X objective and captured with an AxioCAM 503 camera. The aspect ratio was calculated by dividing the sperm's total length of the cell body and the pseudopod by the width of the cell body modified from (Hansen et al., 2015).
Whole genome sequencing
The one step whole genome sequencing and SNP mapping strategy for mutant identification was employed wherein
catp-4
(
as46
)
hermaphrodites were crossed with Hawaiian strain
CB4856
males (Doitsidou et al., 2010). The F2 progeny (approximately 90 worms) that exhibited the
as46
phenotype were lysed and prepped as in (Jaramillo-Lambert et al., 2015). Sequencing was done at the University of Delaware Sequencing and Genotyping Center using an Illumina sequencer. The data analysis was done using Mimodd v0.1.9 (
https://mimodd.readthedocs.io/en/latest/nacreousmap.html
).
Sanger sequencing
An approximately 5.5 kb genomic sequence was amplified from lysed
N2
and
catp-4
(
as46
) worms. For Sanger sequencing, we used a forward sequencing primer (5'-AGATACTGCGAGATGATTCG-3') 261 bp upstream of the site of the mutation. The Sanger sequencing results for
N2
and
catp-4
(
as46
)
were trimmed for low quality bases and aligned to the
catp-4
gene sequence from Wormbase (Sternberg et al., 2024).
Complementation test
In order to create trans-heterozygotes we needed males carrying an allele of
catp-4
. Therefore, we crossed
catp-4
(
ok2056
)
and
catp-4
(
as46
)
L4 hermaphrodites to young adult
N2
males at 20°C. The heterozygous
catp-4
(
ok2056
) male progeny and heterozygous
catp-4
(
as46
)
male progeny were crossed into
catp-4
(
as46
)
L4 hermaphrodites and
catp-4
(
ok2056
) L4 hermaphrodites respectively at 25°C to perform the complementation test. Their F1 progeny were singled out to determine whether they had reduced fertility.
Statistical analysis
t-tests and statistics were performed using GraphPad Prism version 9.1.2 for Windows, GraphPad Software, Boston, Massachusetts USA,
www.graphpad.com
Software
Figure 1C
and
Figure 1D
were assembled using Fiji (version 1.54p) (Schindelin et al., 2012) plugin Quickfigures (version 2023.2) (Mazo, 2021). The aspect ratio was measured using the line tool in Fiji. The MUSCLE multiple sequence alignment was executed using the job dispatcher on the EMBL EBI website (Madeira et al., 2024).