Sperm activation for fertilization requires robust activity of the TAT-5 lipid flippase

Abstract

During fertilization, sperm and egg membranes signal and fuse to form a zygote and begin embryonic development. As lipids participate in signaling and membrane fusion, we investigated the role of lipid asymmetry in gametogenesis, fertilization, and embryogenesis. We show that the lipid flippase TAT-5, an essential P4-ATPase that maintains phosphatidylethanolamine asymmetry, is required for both oocyte formation and sperm activation, albeit at different levels of flippase activity. Loss of TAT-5 significantly decreases fertility in both males and hermaphrodites and decreases sperm activation. TAT-5 localizes to the plasma membrane of primary spermatocytes but is sorted away from maturing spermatids after meiosis. We also find that phosphatidylethanolamine asymmetry is lost in residual bodies prior to phosphatidylserine exposure. Our findings demonstrate that phosphatidylethanolamine asymmetry plays key roles during gametogenesis and sperm activation, expanding the roles of lipid dynamics in developmental cell fusion.

Introduction

Fertilization initiates embryonic development and depends on the formation of functional gametes. Oocytes and sperm must recognize each other and fuse their membranes to form the zygote (Deneke and Pauli, 2021; Krauchunas et al., 2016). The regulated asymmetry and symmetry of lipids across membrane bilayers is necessary for many processes, including cell fusion, signaling, vesicle release, and cell corpse clearance (Andersen et al., 2016; Folmer et al., 2009). In mammals, phosphatidylserine (PS) lipids found in the cytofacial leaflet of the plasma membrane are exposed to the surface of sperm and PS-binding receptors are required in the egg plasma membrane for fertilization (Rival et al., 2019), indicating that PS exposure and recognition promote sperm-egg fusion. However, PS lipids are only one of several classes of lipids normally enriched in the cytofacial leaflet and exposed during cell fusion (Lorent et al., 2020). Phosphatidylethanolamine (PE) lipids have also been implicated in cell fusion, and PE is exposed on capacitated sperm (Irie et al., 2017; Vries et al., 2003), but a function for PE exposure on sperm has not been studied.

The asymmetric distribution of lipids is facilitated by flippase proteins that enrich specific lipids in the cytoplasmic leaflet of membranes. Flippases act in opposition to scramblase proteins, which destroy lipid asymmetry by acting as channels to randomize lipid distribution across membrane bilayers (Figure 1A). Indeed, the regulated exposure of PS lipids on the surface of cells typically requires the inactivation of a flippase pump and the opening of a scramblase channel (Sakuragi and Nagata, 2023). The P4-ATPase family of flippase proteins are energy-dependent pumps that hydrolyze ATP to flip the hydrophilic headgroup of phospholipids across the hydrophobic barrier of the membrane, which establishes and maintains lipid asymmetry (Norris et al., 2024). P4-ATPases differ in their lipid specificity and only subsets of lipids are exposed on the cell surface in flippase mutant strains, allowing the examination of the role of distinct lipids in an otherwise asymmetric membrane.

Figure 1. Hypomorphic and null alleles of tat-5 have different effects on oogenesis and fertilization.

A. Schematic of the TAT-5 protein adapted from (Mark et al., 2013). A-actuator domain, N-nucleotide binding domain, P-phosphorylation domain. Orange lipids indicate phosphatidylethanolamine (PE) lipids. Arrow denotes the lipid flippase activity. Arrowheads indicate point mutations in DGET motif. The red line indicates the region of the protein deleted from the DNA in tm1741 mutants and the deletion (Δ) leads to a frame shift predicted to remove the remainder of the protein. B-G. Expression of pie-1p::mCherry::PH(PLC1∂1)::CTPD(OMA-1) in control (B), tat-5(D244T) (C), tat-5(E246Q) (D-E), and tat-5(Δ) (F-G) young adult hermaphrodites. The length of the uterus containing embryos is underlined with a dashed line to the vulva (V), while the proximal germ line containing oocytes is underlined with a dotted line. Images are oriented anterior to the left, dorsal up. Scale bar: 50 μm.

P4-ATPase flippases are subdivided into the non-essential P4A-ATPases and the essential P4B-ATPases (Bai et al., 2021), and some mammalian P4A-ATPases are thought to regulate lipid asymmetry in sperm and play a role in male fertility. Both P4A-ATPases ATP8B3 and ATP8B5 show testis-specific expression and localize to the acrosomal region of sperm (Gong et al., 2009; Wang et al., 2004; Xu et al., 2009), suggesting a potential role for lipid asymmetry in the acrosome reaction. The acrosome is a vesicle in the sperm head that fuses with the plasma membrane to release proteins that help the sperm penetrate the zona pellucida and reach the egg (Bianchi and Wright, 2020). ATP8B3 mutant sperm fail to bind to or penetrate the egg and prematurely expose PS on their surface (Wang et al., 2004). ATP8B5, originally named FetA, is thought to flip phosphatidylcholine (PC) and PE lipids (Xu et al., 2009), but male ATP8B5 knockout mice were fertile and ATP8B5 is a pseudogene in humans (Suzuki et al., 2024), suggesting that other P-type ATPases play redundant roles in sperm. The essential P4B-ATPases have conserved roles as lipid flippases (Bai et al., 2021), but are less well-studied than P4-ATPases. Mouse knockouts for the P4B-ATPase ATP9A are unable to give rise to progeny when knockouts are incrossed (Meng et al., 2023) suggesting that P4B-ATPases may have uncharacterized but conserved roles in animal fertility.

To examine the role of PE lipid asymmetry during spermatogenesis and fertilization, we focused on the function of the essential P4B-ATPase, TAT-5, which is a C. elegans homolog of ATP9A. TAT-5 is ubiquitously expressed (Lyssenko et al., 2008), and the ATPase activity of TAT-5 is required to maintain the asymmetry of PE on cell surfaces, including the hermaphrodite gonad and embryos (Wehman et al., 2011). TAT-5 is required for fertility and embryogenesis, as loss of tat-5 results in sterility in most tat-5 deletion mutants and embryonic lethality after tat-5 RNAi treatment in C. elegans hermaphrodites (Wehman et al., 2011). Furthermore, the ATP-dependent flippase activity of TAT-5 is required for fertility, as a point mutation predicted to disrupt ATPase activity also results in sterility in most hermaphrodites, with oocytes or embryos rarely being laid (Pitts et al., 2023). The point mutation causing sterility (E246Q) is in one of the three globular cytosolic domains of P4-ATPases that change their conformation during the ATPase cycle of phosphorylation and dephosphorylation (Bai et al., 2021), specifically the conserved DGET motif of the actuator domain required for dephosphorylation and lipid flipping (Figure 1A). Given the conserved sterility phenotypes in tat-5 and ATP9A mutants, it is important to better characterize what roles TAT-5 and PE asymmetry have in female germ cells and determine whether TAT-5 and PE asymmetry also contribute to male germ cell function.

During spermatogenesis in animals including C. elegans, mouse, and humans, sperm shed organelles and remodel their cytoplasm to become efficient for fertilization (Breucker et al., 1985). This process of removal of the residual body, cytoplasmic waste bag, or cyst occurs across vertebrate and invertebrate organisms in order to generate individual sperm (Chu and Shakes, 2012). In vertebrates, this cellular remodeling occurs at the end of a post-meiotic differentiation program, but in C. elegans it occurs immediately after anaphase II (Winter et al., 2017). C. elegans secondary spermatocytes undergo incomplete cytokinesis and use myosin motors to enrich specific organelles and cytosolic proteins, including ribosomes, actin, and tubulin, in a growing structure called the residual body (Hu et al., 2019). Following meiosis II, spermatids bud off from the residual body to become individual haploid cells, with the spermatids inheriting a nucleus, mitochondria, and Major Sperm Protein (MSP)-containing Fibrous Body-Membranous Organelles (Ellis and Stanfield, 2014). The detached spermatids then undergo post-meiotic sperm differentiation, also known as spermiogenesis or sperm activation. This is where C. elegans sperm go from immotile spermatids to active spermatozoa with a pseudopod (Nelson et al., 1982; Nelson and Ward, 1980). After spermatid detachment, PS is exposed on the surface of the residual body, which signals for residual body removal by phagocytic pathways that mediate apoptotic cell removal (Huang et al., 2012). However, PE exposure during spermatogenesis has not yet been examined in C. elegans.

In this work, we find that TAT-5 is required for both male and female fertility in C. elegans. We show that TAT-5 localizes to the plasma membrane of spermatocytes and becomes enriched on the membrane of the residual body. We also find that PE is exposed on the surface of the residual body during partitioning of spermatid components, while PS exposure on residual bodies occurs after spermatids detach. Both tat-5 null and partial loss of function mutant males show reduced fertility and reduced sperm activation. Even a partial loss of TAT-5 activity caused improper localization of PE on the sperm surface. This work describes a new role of a P4B-ATPase during spermiogenesis.

Materials and Methods

C. elegans strains and culture conditions

Strains were cultured on C. elegans growth media MYOB plates with OP50 bacteria as modified from (Brenner, 1974). Analysis was conducted at 20°C for all fertility experiments, except fem-1(hc17ts) which are temperature sensitive, and the culture conditions are described in the sperm migration section. Fluorescent reporter strains were cultured on NGM plates with OP50 bacteria at 23˚C for imaging. Strains containing tat-5 mutations were balanced with the tmC18[dpy-5(tmIs1200)] balancer chromosome (Dejima et al., 2018). Homozygous mutant animals were selected by picking animals without myo-2::gfp expression in the pharynx. For the sperm migration experiment, adult fem-1(hc17ts) animals producing embryos were transferred to a new plate and shifted from 16°C to 25°C where they laid embryos. Once the resulting progeny from the upshifted adults reached the young adult stage, they were used in the experiment. A list of all strains used can be found in Supplemental Table 1.

Genotyping

All C. elegans sequences were from Wormbase (Sternberg et al., 2024). Genotyping was done as previously described (Maniates et al., 2023) using the primer sequences listed in Supplemental Table 2.

Phenotypic analysis

Brood size:

One homozygous L4 hermaphrodite animal was placed on an individual plate to lay progeny. This animal was transferred to a new plate every 24 hours for its reproductive lifetime. All live larval progeny, unhatched embryos, and unfertilized oocytes were counted.

Hermaphrodite fertility analysis after mating with male sperm:

To determine if reduced hermaphrodite fertility was due to defects solely in the sperm or also due to oocyte or embryonic lethality defects, one L4 hermaphrodite was crossed with four cylc-2::mNeonGreen(mon2); him-5(e1490) males. After 48 hours, it was assessed if sperm was transferred by the presence of mNeonGreen fluorescence in the hermaphrodite. For any plates where the male successfully transferred sperm, the presence of cross progeny was scored four days after setting up the cross. To quantify the extent that hermaphrodite fertility contributed to the observed phenotypes, we then quantified the number of fluorescent larval progeny one animal was able to generate after mating with four cylc-2::mNeonGreen(mon2); him-5(e1490) males.

Male fertility:

One control or mutant male and one dpy-11(e224) hermaphrodite were placed together on a plate and allowed to mate for 24 hours. After 24 hours, the hermaphrodite was transferred to a new plate every 24 hours until it stopped making any progeny. The number of Dpy and non-Dpy progeny was scored to determine male fertility rates.

Sperm activation:

L4 males were placed on plates without any hermaphrodites for 24 hours. The males were dissected on a Histobond slide (VWR, 16005–108) in either Sperm Media (Singaravelu et al., 2011) or Sperm Media plus 200 μg/ml pronase. A coverslip was placed, and the slide was incubated for 5–10 minutes. Sperm were then observed using DIC.

Pseudopod measurements:

Using data collected from sperm activation, the length of the pseudopod was measured in Fiji (Schindelin et al., 2012) as a ratio of the length of the pseudopod and cell body divided by the cell body to account for any differences in the cell body (Figure 6N). Only sperm with visible pseudopods were measured.

Figure 6. TAT-5 is required for spermiogenesis.

A-E. Schematic (A) and representative images of DAPI sperm transfer to fem-1(hc17ts) hermaphrodite animals (B) after mating with him-5(e1490) (C), tat-5(Δ); him-5(e1490) (D), or tat-5(D244T); him-5(e1490) (E) males. A few representative spermatids in the spermatheca (yellow arrowheads) and uterus (white arrowheads) are indicated. F. Prevalence of sperm transferred to fem-1(hc17ts) hermaphrodites. n=10 for all groups. G-I. Representative images of spermatids with insets of sperm to show nuclear placement. J-L. Representative images of sperm after activation with Pronase treatment. M. Quantification of sperm activation based on morphology. Each dot represents the sperm resulting from two dissected males (n=10 males dissected for each genotype). Statistical significance was calculated using the Mann-Whitney U test. N. Schematic of pseudopod measurement. O. Quantification of pseudopod length as a ratio of cell body and pseudopod measurement/cell body measurement for him-5(e1490) n=348, tat-5(Δ); him-5(e1490) n=45 and tat-5(D244T); him-5(e1490) n=146.

Sperm migration:

To assess the ability of male sperm to locate and crawl towards the spermatheca, one L4 male and one young adult fem-1(hc17ts) hermaphrodite were placed together on a mating plate and allowed to mate overnight at 20°C. The next day, the hermaphrodite animal was stained with DAPI (described below), and the location of the sperm was assessed.

Off center nuclei:

To assess the off centered nuclei phenotype, male sperm dissected in sperm media were examined as described in (Shakes and Ward, 1989a).

DAPI staining:

DAPI staining was completed as previously described (Maniates et al., 2023) where animals were fixed with methanol and then stained with Vectashield Plus DAPI (H-1200–10, Vector Laboratories).

Light and fluorescence microscopy

Images of live animals mounted in M9 buffer on 4% agarose pads were collected on a Zeiss Axio Observer 7 microscope with a Plan-Apo 20X 0.5 NA oil objective and Excelitas Technologies X-Cite 120LED Boost illumination using a Hamamatsu ORCA-Fusion sCMOS camera controlled by 3i SlideBook6 software. Images for DAPI stained animals were collected on a Zeiss Universal microscope using a 20x objective with 0.75 NA with a ProgRes camera (Jenoptik) using ProgRes CapturePro software. Sperm activation images were collected on the Universal microscope using a 40x objective with 0.75 NA.

Localization of GFP::TAT-5 during spermatogenesis

Fluorescence microscopy of dissected sperm from the gfp::tat-5(wur36); him-5(e1490) strain and corresponding him-5(e1490) control spermatids were completed using a Zeiss Elyra7 Lattice Structured Illumination Microscope (SIM2) using a 488nm laser and a 63x water objective with a 1.2 NA. For time-lapse imaging of GFP::TAT-5b, unmated adult males were rinsed in M9 buffer before mounting in 0.1 μm Polybead microspheres (Polysciences) on a 10% agarose pad. A z-stack with 2 μm steps was collected every 2 minutes using a Nikon TI2E microscope with an AX-R confocal using a 488 nm laser, GaAsP detectors, and a 60X oil objective with 1.42 NA controlled by NIS Elements.

Lipid staining and quantification

Unmated adult day 1 or 2 males were dissected in 4-well slides (ibidi) in sperm media. Live sperm were stained for 30 minutes in 1:200 Alexa488-Annexin V (AnxV) (Invitrogen), 0.1 μM Duramycin-LC-Biotin (DLB) (MTTI), 1.5 μg/mL Alexa594-Streptavidin (Invitrogen) in sperm media. Stained sperm were imaged in sperm media on a Zeiss Axio Observer 7 microscope with a Plan-Apo 40X 1.4 NA oil objective and Excelitas Technologies X-Cite 120LED Boost illumination. Images were collected with a Hamamatsu ORCA-Fusion sCMOS camera controlled by 3i SlideBook6 software. Mean fluorescence intensity of a 3-pixel line was measured around the surface of spermatids using FIJI (NIH) and a neighboring region not containing cells. In addition, a circle smaller than the spermatid (typically ~5 μm radius) was used to measure the mean fluorescence intensity of the cytoplasm. The neighboring background fluorescence was subtracted from the spermatid surface fluorescence or the cytoplasm fluorescence.

Results

TAT-5 is required for fertility in hermaphrodites

As the ATPase activity of TAT-5 is required to produce embryos (Wehman et al., 2011), we wanted to determine how TAT-5 ATPase activity impacts fertility. A large-scale screen found that tat-5 knockdown resulted in visible defects in hermaphrodite gonads (Green et al., 2011), so we examined the localization of a germ line plasma membrane reporter, pie-1p::mCherry::PH(PLC1∂1)::CTPD(OMA-1). The plekstrin homology (PH) domain of phospholipase C (PLC) binds to PI4,5P₂ lipids in the plasma membrane (Kim et al., 1996), and the C-terminal phosphodegrons (CTPD) of OMA-1 lead to protein degradation in embryos after the first mitotic division (Beer et al., 2019; Nishi and Lin, 2005). The mCh::PH::CTPD reporter was crossed into two strains with point mutations in the conserved DGET motif of the Actuator domain of P4-ATPases: a partial loss-of-function mutant changing Aspartate 244 to Threonine, tat-5(D244T), and an ATPase-dead mutant changing Glutamate 246 to Glutamine, tat-5(E246Q) (Figure 1A) (Pitts et al., 2023). We also crossed mCh::PH::CTPD into a null allele tat-5(tm1741), hereafter tat-5(Δ), which has a deletion of the DNA encoding most of the Actuator domain that causes a frame shift predicted to remove the remainder of the protein (Wehman et al., 2011). In control and tat-5(D244T) mutant hermaphrodites, embryos are visible in the uterus by DIC (Figure 1B-C), while most tat-5(E246Q) and tat-5(Δ) mutants lacked embryos (Figure 1D, 1F). In all strains, mCh::PH::CTPD localized to the plasma membrane in both the distal and proximal germ cells, including the large cylindrical oocytes. The oocytes in tat-5(D244T) mutants appeared similar to control worms (Figure 1B–C). However, the regular sizing and spacing of oocytes was disrupted in tat-5(E246Q) and tat-5(Δ) mutants (Figure 1D–G). To characterize the cells in the uterus of rare tat-5(E246Q) mutants with potential embryos, we examined whether the cells continued to express mCh::PH::CTPD. The CTPD degron causes degradation of the mCh::PH::CTPD membrane reporter in fertilized embryos after the 1-cell stage (Beer et al., 2019). Unfertilized oocytes in the uterus of tat-5(E246Q) and tat-5(Δ) mutants are labeled with mCh::PH::CTPD (Figure 1E–G), while the fertilized embryos appear as dark ovals from the 2-cell stage onward (Figure 1E, 1G). The presence of unfertilized oocytes in tat-5(E246Q) and tat-5(Δ) mutants suggested that TAT-5 and PE asymmetry may play a role during fertilization in addition to oogenesis.

Differential requirements of TAT-5 activity for oocyte and embryonic development

To ascertain the full effect that TAT-5 has on fertility, we counted the brood size of a strain with a deletion that acts as a null mutation, tat-5(Δ) (Wehman et al., 2011). The tat-5 null mutants produced no live larval progeny (Figure 2A), in contrast to wild type animals (N2), which produced on average 273 larval progeny (Figure 2A). Instead, the average tat-5 null mutant produced 7 unhatched embryos and 29 unfertilized oocytes (Figure 2B–C), confirming that there are defects in germ cell development in addition to embryogenesis. The number of unhatched embryos and unfertilized oocytes was not significantly different than wild type (Figure 2B–C). However, combined with the absence of larval progeny (Figure 2A), these data show that the germ line in tat-5 null mutants produces significantly fewer ovulation events (Figure 2D), which are the sum of larval progeny, unhatched embryos, and unfertilized oocytes.

Figure 2. TAT-5 is required for female fertility and embryonic development.

A-E. Progeny analysis of 10 N2, 11 tat-5(Δ), and 11 tat-5(D244T) hermaphrodites. Each point represents one animal analyzed. A. Number of live larval progeny. B. Number of unhatched embryos. C. Number of unfertilized oocytes. D. Total number of ovulation events corresponds to the sum of larval progeny, unhatched embryos, and unfertilized oocytes (A-C). E. Percentage of successful fertilization events (larval progeny and unhatched embryos compared to total number of ovulation events). F. Number of fluorescent larval progeny produced after mating with cylc-2::mNG; him-5(e1490) males. n=12 for N2 and n=14 for tat-5(Δ), and tat-5(D244T). Error bars ± SEM. One way ANOVA, **** p<0.0001, ns p>0.05.

We then analyzed the brood size of the tat-5(D244T) partial loss of function allele, which is predicted to lead to a 3-fold loss in ATPase activity and lipid flipping based on similar mutations in a P4A-ATPase (Coleman et al., 2012). The tat-5(D244T) mutant hermaphrodites did not produce live larval progeny, similar to the null allele (Figure 2A). However, tat-5(D244T) mutants retained significant fertility, averaging 125 unhatched embryos and 114 unfertilized oocytes, significantly more than wild type or the null allele (Figure 2B–C). Comparing the total number of oocytes that passed through the spermatheca, tat-5(D244T) mutants did not have significantly reduced ovulation events compared to N2 wild type controls, unlike the tat-5(Δ) null allele (Figure 2D). These data indicate that a low level of TAT-5 activity is sufficient for oocyte development but not for embryogenesis.

We next asked whether TAT-5 plays a role in fertilization. We reanalyzed the brood counts for evidence of self-fertilization by comparing the number of embryonic and larval progeny with the total number of ovulation events. Both tat-5(D244T) partial loss of function and tat-5(Δ) null mutants showed a significant decrease in the percentage of successful fertilization events compared to N2 wild type controls (Figure 2E). This data suggests that TAT-5 activity is important for fertilization.

To assess whether sperm from males with wild type tat-5 could recover fertility and embryogenesis in tat-5 mutants, we mated hermaphrodites with control cylc-2::mNG; him-5 males for two days. We performed this series of crosses twice, once scoring for the presence of larval cross progeny, and once counting the number of fluorescent larval progeny. Wild type N2 hermaphrodites were able to produce fluorescent cross progeny that was sired by control male sperm 100% of the time (n=22), as expected since C. elegans preferentially use male sperm when available (LaMunyon and Ward, 1995; Singson et al., 1999). Mated N2 hermaphrodites produced hundreds of fluorescent larval cross progeny (Figure 2F, n=12). In contrast, only one of the 24 tat-5(Δ) null mutant hermaphrodites (4%) was able to generate any larval cross progeny after mating. However, the larvae were not counted from the single tat-5(Δ) null with progeny in the first set of 14 crosses and we did not observe any larval cross progeny from 10 mated tat-5 null mutants in the second set of crosses (Figure 2F). This decrease in cross progeny is consistent with the observed defects in oogenesis, and these data suggest that the sterility in tat-5 null mutant hermaphrodites is primarily due to oocyte defects.

To determine the extent that sperm with wild type tat-5 could recover embryogenesis in tat-5 mutants, we also examined the larval progeny resulting from mating cylc-2::mNG; him-5 males with tat-5 partial loss-of-function mutant hermaphrodites. 95% of tat-5(D244T) hermaphrodites (n=20) were able to generate fluorescent cross progeny after mating, confirming that tat-5(D244T) mutant hermaphrodites produce more functional oocytes than tat-5 null mutants. However, the number of fluorescent larval progeny from 10 mated tat-5(D244T) mutant hermaphrodites was significantly decreased compared to wild type (Figure 2F), averaging 23 fluorescent larval progeny (Figure 2F). As hundreds of ovulation events were observed in most tat-5(D244T) hermaphrodites (Figure 2D), this disparity suggests that zygotic tat-5(wt) is only rarely able to rescue embryos from maternal tat-5(D244T) mutants. The limited rescue is consistent with the rounded cell morphology and gastrulation defects that begin early in tat-5 mutant embryos at stages before zygotic transcription has started (Wehman et al., 2011). However, mating tat-5(D244T) hermaphrodites did increase the number of larval progeny from zero (Figure 2A, n=11) to a range from two to 123 (Figure 2F, n=10). Therefore, zygotic tat-5(wt) can sometimes be sufficient to rescue embryogenesis in a partial loss-of-function background. Together, these results suggest that TAT-5 ATPase activity has differential requirements in oocyte development and embryogenesis, in addition to TAT-5 playing an important role in fertilization.

TAT-5 localization is dynamic in the male germ line

To better understand the roles of TAT-5 in germ cell development and fertilization, we examined where a GFP-tagged TAT-5 localizes in hermaphrodite and male germ lines. The tat-5 gene has four isoforms (a-d) with two transcriptional start sites, one start site encoding tat-5b and tat-5d near the neighboring gene ent-6 and one start site ~4 kb away encoding tat-5a and tat-5c (Sternberg et al., 2024). As co-expression of neighboring genes from operons is common in the hermaphrodite germ line (Reinke and Cutter, 2009), we examined TAT-5 localization using a strain with GFP knocked into the N-terminus of the tat-5b and tat-5d isoforms (Park et al., 2024), hereafter GFP::TAT-5b. Consistent with the previous study of this knock-in (Park et al., 2024), GFP::TAT-5b labeled the plasma membrane of oocytes and embryos as well as cortical granules in the oocyte cytosol. We observed that GFP::TAT-5b appeared brighter in the hermaphrodite spermatheca region than in neighboring oocytes (Figure 3A), suggesting that TAT-5 could be enriched in sperm or spermathecal cells. To investigate TAT-5 localization to sperm cells, we imaged younger hermaphrodites whose sperm have not yet migrated into the spermatheca. At L4 and early adult stages, GFP::TAT-5b fluoresced brightly in the plasma membrane of spermatocytes and the proximal arm of the gonad (Figure 3B–C), demonstrating that TAT-5 is expressed during spermatogenesis. We also crossed the GFP knock-in to him-5 mutants to generate males expressing GFP::TAT-5b. In the adult male germ line, GFP::TAT-5b fluorescence was first observed on the plasma membrane during meiotic prophase (Figure 3D) and appeared to disappear after gamete differentiation (arrowhead in Figure 3D). These dynamic patterns suggest that TAT-5b/d protein expression and localization are regulated during spermatogenesis.

Figure 3. TAT-5 is expressed in both male and hermaphrodite germlines.

A-C. GFP::TAT-5b expression in membranes of adult (A), early adult (B) and L4 (C) hermaphrodites. The uterus containing embryos is underlined with a dashed line to the vulva, the spermatheca region is indicated with an S, and the proximal germ line containing oocytes is underlined with a dotted line. Scale bar represents 50 μm for left and center panels, 25 μm for right panels. D. GFP::TAT-5b expression in adult him-5 males. The arrowhead indicates the division zone where spermatocytes complete meiosis and form residual bodies. E-I. Expression of GFP::TAT-5b in spermatogenic cells at the indicated stages. Scale bar represents 5 μm.

To determine where TAT-5 localizes through different stages of spermatogenesis, we dissected GFP::TAT-5b-expressing males and imaged their sperm. We found that GFP::TAT-5b localized to the plasma membrane in primary and secondary spermatocytes through anaphase II (Figure 3E–G), consistent with the bright fluorescence in the prophase and meiotic regions of the male germ line (Figure 3D). However, GFP::TAT-5b was later enriched in the residual body membrane in comparison to the plasma membrane of budding spermatids (Figure 3H), suggesting that the most proximal structures labeled in the male germ line correspond to forming residual bodies (arrowhead in Figure 3D). After separation from the residual body, mature spermatids did not show GFP::TAT-5b on the plasma membrane (Figure 3I). Instead, the remaining green fluorescence appeared similar to mature spermatids that do not express any GFP (Supplemental Figure 1). These data are consistent with the loss of GFP::TAT-5b fluorescence in the proximal male gonad where mature spermatids reside (right of the arrowhead in Figure 3D). To observe GFP::TAT-5b dynamics, we imaged intact adult males using time-lapse microscopy. GFP::TAT-5b on the plasma membrane of a spermatocyte appears to enrich towards the center of the cell and is asymmetrically inherited by the forming residual body (Video). Consequently, GFP::TAT-5b fluorescence progressively disappeared from budding spermatids. The absence of GFP::TAT-5b in mature spermatids suggests that TAT-5 activity is likely required earlier during spermatogenesis.

TAT-5 activity prevents PE exposure on the surface of spermatids

Given the role of TAT-5 in maintaining phosphatidylethanolamine (PE) asymmetry on the plasma membrane of embryonic blastomeres and the distal germ line of hermaphrodites (Beer et al., 2018; Wehman et al., 2011), we wanted to determine whether a decrease in TAT-5 ATPase activity also causes PE exposure on sperm. To generate mutant males, we crossed the null and partial loss-of-function tat-5 alleles to him-5(e1490) mutants. To examine PE exposure, we stained dissected sperm with duramycin, a non-permeable PE-binding lantibiotic (Navarro et al., 1985). Duramycin staining appeared as an inconsistent number of dim puncta on the surface of him-5 control spermatids (Figure 4A, 4G), consistent with mature spermatids maintaining PE asymmetry on most of their plasma membrane. In contrast, we observed a >5-fold increase in duramycin staining on spermatids dissected from tat-5(D244T); him-5 males (Figure 4B–C), with the staining appearing along the entire cell surface of the partial loss-of-function mutants (Figure 4B). Duramycin staining was also more variable in tat-5(D244T); him-5 spermatids, requiring the data to be plotted on a log scale (Figure 4C). Similar to the hermaphrodite gonad after tat-5 RNAi treatment (Beer et al., 2018), the male tat-5(D244T); him-5 gonad also stained brightly with duramycin (Supplemental Figure 2), suggesting that PE is exposed throughout germ cell development when TAT-5 flippase activity is reduced. These results imply that high levels of TAT-5 ATPase activity are required to maintain lipid asymmetry in mature spermatids. As GFP::TAT-5b was not observed on the plasma membrane of mature spermatids (Figure 3I and S1, Video), mature spermatids are likely to inherit PE asymmetry that was established by TAT-5 earlier during development.

Figure 4. TAT-5 is required for phosphatidylethanolamine asymmetry in mature spermatids.

A-B. Representative images of him-5(e1490) (A) or tat-5(D244T); him-5(e1490) (B) mature spermatids after duramycin and annexin V staining. Scale bar is 5 μm. C. Surface intensity of Annexin V (AnxV) and duramycin (DLB) fluorescence over background. Each dot represents a single mature spermatid. D-G. Representative images of him-5(e1490) spermatocytes, budding spermatids, and residual bodies after duramycin and annexin V staining.

To confirm whether tat-5 mutant spermatids specifically lose PE asymmetry, we co-stained dissected sperm with Annexin V, which binds preferentially to exposed phosphatidylserine (PS) (Thiagarajan and Tait, 1990). We did not observe plasma membrane labeling on either him-5 control or tat-5(D244T); him-5 spermatids (Figure 4A–B). However, we could measure a two-fold increase in dim green fluorescence on tat-5(D244T); him-5 spermatids (Figure 4C), but this is likely due to green autofluorescence in mature spermatids (Supplemental Figure 1). We also did not observe a clear correlation between Duramycin and Annexin V staining intensity (Figure 4C), confirming that PE exposure does not always correlate with PS exposure. Therefore, TAT-5 maintains PE asymmetry in the male germ line, not PS asymmetry.

PE is exposed on residual bodies after meiosis

Given the enrichment of GFP::TAT-5b on the residual body membrane during spermatid budding (Figure 3H, Video), we analyzed the timing of PE and PS exposure during residual body formation. We dissected him-5 males and stained with duramycin and Annexin V before imaging developing sperm cells. Primary spermatocytes stained weakly with duramycin (Figure 4D). We observed intermediate labeling of the forming residual body with duramycin during partitioning stages (Figure 4E–F), and duramycin stained the residual body brighter after the spermatids separated from the residual body (Figure 4G, n=14). These data suggest that PE asymmetry is lost gradually and specifically on the residual body membrane. Furthermore, these lipid domains with exposed PE appear to be kept separate from the asymmetric domain with cytofacial PE within the shared plasma membrane during spermatid budding.

We next compared the timing and localization of PE exposure to PS exposure. Consistent with PS-mediated phagocytic clearance of residual bodies (Huang et al., 2012), we observed Annexin V labeling the residual body membrane after its release from mature spermatids (Figure 4G). However, primary spermatocytes, budding spermatids, and forming residual bodies appeared unstained by Annexin V (Figure 4D–F), in contrast to duramycin. Together, these results indicate that the residual body membrane progressively loses PE asymmetry before losing PS asymmetry.

During our staining experiments, we also observed bright puncta near the residual body labeling with both duramycin (n=10/14) and Annexin V (n=8/14) (Figure 4E, 4G), consistent with a stronger loss of lipid asymmetry on discrete structures, such as small extracellular vesicles released during spermatogenesis or dying cell fragments resulting from dissections. Thus, lipid asymmetry appears to be regulated differentially in budding spermatids, the residual body, and dying cells.

TAT-5 is required for male fertility

Given the observed dynamics of TAT-5 localization and PE exposure in sperm, we next sought to determine the impact of TAT-5 and disrupted PE asymmetry on sperm maturation and function. Examination of him-5 male strains after DAPI staining showed that both the tat-5(Δ) null and tat-5(D244T) hypomorphic mutant germ lines appear normal (Figure 5A, n=10), producing many sperm with chromatin masses of uniform size (bracketed inset). Thus, sperm appear to have progressed through meiosis to produce numerous haploid spermatids in tat-5 mutant males.

Figure 5. TAT-5 is required for male fertility.

A. Representative images of DAPI staining of him-5(e1490), tat-5(Δ); him-5(e1490), and tat-5(D244T); him-5(e1490) males. Brackets indicate region with mature spermatids. Scale bar is 20 μm. B. Percentage of matings with dpy-11(e224) hermaphrodites generating cross progeny, n=10, 12, and 32 crosses. C. Number of cross progeny after successful mating with dpy-11(e224) hermaphrodites for him-5(e1490) n=10 or tat-5(D244T); him-5(e1490) n=11 males. D. Percentage of cross progeny generated after mating males with dpy-11(e224) hermaphrodites. B. Fisher’s exact test, p<0.0001. C-D. Error bars ± SEM. One way ANOVA, *** p<0.001.

To understand how sperm contribute to the fertility phenotypes observed for hermaphrodites (Figure 2), we mated tat-5; him-5 mutant males with a dpy-11(e224) hermaphrodite and observed their ability to sire non-Dumpy progeny. Control him-5 males generated cross progeny 10 out of 10 times (Figure 5B). In contrast, we found that tat-5(Δ) null mutant males were unable to generate cross progeny (n=11) (Figure 5B). Hypomorphic tat-5(D244T) males also failed to sire cross progeny two-thirds of the time (n=21/32), siring significantly less frequently than him-5 males, but more often than tat-5(Δ) null mutant males (Figure 5B). These defects could be due to decreased function of tat-5 mutant sperm or failure of tat-5 mutant males to mate.

As tat-5(D244T) males sire cross progeny one-third of the time (n=11/32), we could analyze the fitness of sperm after a successful mating with dpy-11(e224) hermaphrodites. Matings with hypomorphic tat-5(D244T) males produced significantly fewer cross progeny compared to him-5 control matings (Figure 5C), with most matings producing only one-eighth the cross progeny (n=9/11). When the mating data was analyzed daily, we found that sperm from wild type males primarily produced cross progeny for the first few days after mating, with self-progeny first dominating 4 days after mating (Figure 5C). In contrast, the tat-5 hypomorph consistently produced less than 50% cross progeny every day after a successful mating (Figure 5D). These data suggest that male sperm with reduced TAT-5 activity were not able to compete with hermaphrodite sperm.

TAT-5 is required for sperm activation and migration

For male sperm to outcompete hermaphrodite sperm after mating, male sperm first migrate inside the hermaphrodite gonad from the vulva to the spermatheca. To assess sperm migration, males were mated with feminized strains unable to produce sperm. When raised at the restrictive temperature, fem-1(hc17ts) mutants produce no sperm, allowing us to use DAPI staining to assess the number of mated fem-1(hc17ts) mutants that had sperm transferred and localized to the spermatheca (Figure 6A). As expected, unmated fem-1 mutants showed no sperm in the spermatheca at the restrictive temperature (n=10, Figure 6B, 6F), while mating with him-5(e1490) resulted in many sperm in the spermatheca in 93% of matings (n=14/15, Figure 6C, 6F). In contrast, after overnight mating with tat-5 mutant males, we never observed many sperm in the fem-1 spermatheca (Figure 6F). Two-thirds of tat-5 null mutant (n=7/11) or tat-5(D244T) mutant (n=7/10) matings resulted in no sperm detected in the spermatheca by DAPI staining (Figure 6D, 6F). However, one-third of tat-5 null mutant (n=3/11) or tat-5(D244T) mutant (n=3/10) matings without visible sperm did result in fertilized embryos (Figure 6F), which indicates that at least a few sperm were transferred. In cases where both sperm and embryos were present (Figure 6E–F), we sometimes observed sperm in the fem-1 uterus after mating with tat-5 mutant males. These results suggest that tat-5 mutant sperm rarely take up residence in the spermatheca, even in the absence of competing hermaphrodite sperm.

To understand why tat-5 sperm were unable to reach or stay in the spermatheca, we examined the morphology of mature spermatids dissected in sperm media. Although dissected tat-5 mutant spermatids appeared mostly normal, we noticed that there were changes to nuclear positioning in 10% of tat-5 mutant spermatids (Figure 6H–I), a six-fold increase in off-center nuclei over him-5 controls (Table 1). However, whether the off-center nuclei phenotype contributes to sperm migration defects is unclear as sperm which lack chromatin are still able to migrate (Sadler and Shakes, 2000).

Table 1.

Nuclear Position in Spermatids

Genotype	Centered Nuclei	On-side Nuclei	Percent Abnormal
him-5(e1490)	725	11	1.5%
tat-5(Δ); him-5(e1490)	806	91	10.1%
tat-5(D244T); him-5(e1490)	706	75	9.6%

Modeled after (Shakes and Ward, 1989a)

For sperm to migrate, they need to activate and transition from a round immotile spermatid to a motile spermatozoon with a pseudopod (Shakes and Ward, 1989a). Therefore, we wanted to know if tat-5 mutant sperm were able to change their morphology in the presence of an in vitro sperm activator, Pronase. After Pronase treatment, 80% of dissected him-5 control sperm activated and formed pseudopods (Figure 6J, 6M). In contrast, more than 80% of tat-5(Δ) and 69% of tat-5(D244T) mutant sperm maintained a round immotile morphology in the presence of Pronase (Figure 6K–M), suggesting that tat-5 mutant sperm have defects in changing their cell shape in the presence of an activation signal.

To determine whether the activated tat-5 mutant sperm were able to form normal pseudopods, we quantified pseudopod length in proportion to the cell body (Figure 6N). In the 20% of tat-5(Δ) sperm able to produce a pseudopod, pseudopods were slightly shorter compared to the control (Figure 6O). However, there was no significant difference in pseudopod length in the 31% of tat-5(D244T) mutant sperm able to form a pseudopod (Figure 6O). These results confirm that low levels of TAT-5 activity are required for changes in cell shape important for migration, which could explain why tat-5 mutant sperm are unable to establish or maintain their residence in the spermatheca after mating.

Discussion

This work describes the first role for a P4B-ATPase in sperm and confirms the importance of TAT-5 for oogenesis and embryogenesis. We demonstrated that a loss of tat-5 decreases male and hermaphrodite fertility by disrupting both oocyte production and sperm activation, although only oogenesis can proceed with a low level of TAT-5 activity. Our results suggest that TAT-5 flippase activity is critical during spermatogenesis to establish PE asymmetry on the spermatid plasma membrane, in addition to the female germ line (Beer et al., 2018; Wehman et al., 2011). We demonstrated that even with low levels of TAT-5 activity in the tat-5(D244T) mutant, PE was exposed on the surface of mature spermatids that were unable to activate to form a motile pseudopod (Figure 7). Our work, combined with others, demonstrates the importance of regulating membrane asymmetry in sperm (Flesch and Gadella, 2000). Given the infertility of ATP9A knockout mice (Meng et al., 2023) it will be important to determine how ATP9A and ATP9B, the mammalian P4B-ATPases homologous to TAT-5, promote male fertility.

Figure 7. Model for the role of TAT-5 in sperm.

A. TAT-5 internalizes phosphatidylethanolamine (PE) on the surface of primary and secondary spermatocytes, establishing lipid asymmetry for the spermatid plasma membrane (blue outline) and allowing normal activation and migration. PE is normally exposed on the residual body membrane (red outline). B. Loss of TAT-5 activity results in PE exposure on the outside of the spermatid membrane (red outline), off-center nuclei, and defects in sperm activation to form pseudopods.

Cytosolic proteins that are not needed during sperm activation or fertilization are typically segregated into the residual body (Nelson and Ward, 1980), suggesting that the multipass transmembrane protein TAT-5 could be intentionally segregated away from the membrane of maturing spermatids to become enriched in the residual body membrane. TAT-5 is the first transmembrane protein in C. elegans to be specifically localized to the membrane of the forming residual body, suggesting that not only cytosolic components are segregated to the residual body after meiosis. This is consistent with the phenotype and localization of the single-pass transmembrane protein Germinal Angiotensin-I-converting enzyme (gACE) which is male sterile and becomes enriched in the residual body in mammals (Ramaraj et al., 1998; Takeuchi et al., 2009, 2007). Furthermore, our lipid staining experiments demonstrated that lipid asymmetry is specifically and progressively lost in the residual body and not in budding spermatids, suggesting the existence of lateral sorting mechanisms for lipids and proteins in the membrane during spermatogenesis. However, membrane trafficking can also occur through endocytosis, which could lead to preferential lysosomal degradation of TAT-5 in budding spermatids. Therefore, GFP::TAT-5b will be a useful marker to investigate the mechanisms of membrane sorting after male meiosis, which may be related to or distinct from the myosin-dependent mechanisms of cytosolic protein and organelle sorting (Hu et al., 2019).

Furthermore, TAT-5 may help localize other proteins or organelles to the residual body, based on the known roles of TAT-5 homologs in endolysosomal trafficking (Hua et al., 2002; Hua and Graham, 2003; McGough et al., 2018; Meng et al., 2023; Tanaka et al., 2016; Wicky et al., 2004). Consistently, we observed an increase in off-center nuclei (Figure 6H–I, Table 1), which could suggest that either organelle positioning or content are disrupted in tat-5 mutant sperm. Therefore, characterizing the positioning of other organelles and proteins in spermatids may give insights into how TAT-5 regulates sperm activation for fertilization.

We were surprised to discover that residual bodies began losing PE asymmetry during their formation, despite the localization of the PE flippase TAT-5 to residual bodies. This suggests that TAT-5 ATPase activity establishes the asymmetry of the spermatid membrane during the primary and secondary spermatocyte stages, but that TAT-5 is quickly inactivated in residual bodies during spermatogenesis. As mitochondria are sorted into the budding spermatids and away from the residual body (Huang et al., 2012), this could lead to regional differences in ATP availability for P4-ATPases like TAT-5. In addition, the rapid PE exposure suggests that lipid scramblases are quickly activated in forming residual bodies to disrupt lipid asymmetry. Notably, PE exposure preceded PS exposure, suggesting different regulation of PS flippases, PE flippases like TAT-5, and non-specific lipid scramblases. Determining how flippase and scramblase activities are controlled to disrupt lipid asymmetry in residual bodies will be important to understand how cells balance the structural and signaling roles of lipid asymmetry.

Although we discovered a fine choreography of lipid localizations during spermatogenesis, the role that PE asymmetry plays during sperm activation is unclear. One possible explanation for the loss of pseudopod formation in tat-5 mutant sperm is an inability to sense the activation signal with PE exposed, which could be due to the mislocalization of signaling proteins. However, given the unusually rounded morphology of cells in tat-5 mutant embryos (Wehman et al., 2011), the round morphology of mutant sperm could also be due to cytoskeletal defects or lipid-induced changes to the biophysical properties of the membrane itself. Therefore, it will be important to determine how lipid asymmetry regulates sperm for fertilization, as these lipid functions are likely to be conserved in other developmental and pathogenic cell fusion events.

Supplementary Material

Sup Figures

Supplemental Figure 1. Spermatid autofluorescence.

Mature spermatids from control him-5(e1490) males that do not express GFP imaged for green fluorescence with conditions identical to Figure 3I.

Supplemental Figure 2. Phosphatidylethanolamine is exposed on male tat-5 mutant gonads.

DIC and fluorescence images of a gonad dissected from an adult tat-5(D244T); him-5(e1490) male after duramycin staining. Scale bar is 10 μm.

Sup Video 1

Supplemental Video. GFP::TAT-5b localization after meiosis.

Adult WEH787 male expressing GFP::TAT-5b imaged every 2 minutes. GFP::TAT-5b in the indicated spermatocyte becomes enriched in the center of the cell and is inherited in the residual body. The budding spermatids progressively lose GFP::TAT-5b fluorescence. Projection of 2 images 2 μm apart.