Gamete interactions require transmembranous immunoglobulin-like proteins with conserved roles during evolution

ABSTRACT

C. elegans spe-9 class genes are male germline-enriched in their expression and indispensable during sperm-oocyte fusion. Identification of mammalian orthologs that exhibit similar functions to these C. elegans genes has been a challenge. The mouse Izumo1 gene encodes a sperm-specific, immunoglobulin (Ig)-like transmembrane (TM) protein that is required for gamete fusion. We recently identified the C. elegans spe-45 gene, which shows male germline-enriched expression and encodes an Ig-like TM protein. spe-45 mutant worms produced otherwise normal spermatozoa that cannot fuse with oocytes, causing essentially the same phenotype as that seen in the Izumo1-knockout mice. By counting the number of self-sperm in the spermatheca of spe-45 hermaphrodites, it was found that this gene might be involved in sperm guidance from the uterus into the spermatheca, as well as gamete fusion. Moreover, we discovered that SPE-45 and IZUMO1 share certain functions for gamete fusion, which are presumably related to binding with cis- and/or trans-partners. Intriguingly, various organisms have Ig-like TM proteins that act during gamete interactions, indicating the wide-spread utility of Ig-like domains during fertilization.

Introduction

Fertilization is a key feature of sexual reproduction in eukaryotes. In mammals, such as the mouse, fertilization consists of multiple sperm interactions with the cumulus cell layer, zona pellucida and oocyte plasma membrane (Fig. 1, left).¹ The proteins thought to be important for these interactions evolve rapidly, likely in order to prevent the production of cross-species hybrids.^2-4 Thus, even if there is a conserved mechanism(s) among evolutionary distant species that are required for sperm-oocyte interactions during fertilization, it might be difficult to identify it by homology-based searches.

Figure 1.

Schematic comparison of fertilization in the mouse and C. elegans. (Left) In the mouse, a sperm must pass through the cumulus cell layer (CL, cells are yellow with a black nucleus) and the zona pellucida (ZP, pale blue) for entry into the perivilelline space (PV, light yellow), where the oocyte plasma membrane is exposed. The sperm becomes acrosome-reacted prior to contacting with the ZP,³⁴ and gamete fusion occurs on the equatorial segment of the acrosome-reacted sperm¹ (shown by a red square). (Right) Since a C. elegans oocyte lacks any types of accessory cells and egg coats such as the CL and ZP, respectively, a sperm directly binds to and fuses with the oocyte plasma membrane through the pseudopod^5,6 (red square).

Caenorhabditis elegans has several advantages as a model organism for studying sperm-oocyte fusion.^5,6 For example, a C. elegans oocyte lacks accessory cells and a thick egg coat, unlike mammalian oocytes (Fig. 1, right), so that the fusogenic reaction in C. elegans can be examined more simply than those in mammals. Also, four spe-9 class genes (spe-9,^7-9 spe-38,^10,11 spe-41/trp-3^11,12 and spe-42^13,14) are expressed specifically or predominantly in the C. elegans male germline and required during fertilization (in other words, “during gamete fusion”). Human and/or mouse homologs of these C. elegans genes can be predicted according to the domain architectures of each SPE-9 class protein (Table 1), but it is unknown whether those mammalian homologs are functionally related to the C. elegans spe-9 class genes.

Table 1.

C. elegans spe-9 class genes.

Gene	TM1	Domain2/Feature	Localization	Function	Ortholog3	Reference
spe-9	1	EGF (10)	PM of spermatid	Involvement in later step	DLL1 (h)	7-9
			Pseudopod of sperm	post gamete binding	DLL3 (h)
					DLL4 (h)
spe-38	4	No clear domain	MOs of spermatid	Regulation of SPE-41/TRP-3 relocation during spermiogenesis	Not found in mammals	10,11
			MOs and PM of sperm
spe-41/trp-3	6	TRPC family	MOs of spermatid	Regulation of Ca2+-influx during fertilization	TRPC6 (h)	11,12
			PM of sperm		Trpc6 (m)
spe-42	6	DC-STAMP (1)	Not determined	Involvement of gamete binding	DCTS2 (h)	13,14
		RING finger (1)			Dcts1 (m)
spe-45	1	Ig (1)	Not determined	Involvement in later step post gamete binding	Izumo1 (m)	15.17

Notes. Abbreviations used are as follows: TM, transmembrane; EGF, epidermal growth factor; TRPC, transient receptor potential-canonical; DC-STAMP, dendritic cell-specific transmembrane protein; Ig, immunoglobulin; PM, plasma membrane; MO, membranous organelles that are analogous to the acrosome;³⁵ DLL, Delta-like; DCST, DC-STAMP domain containing.

¹The number of TM domains in each predicted protein.

²Protein domains that are present in each predicted protein besides the TM domain. The number of those domains is shown in parentheses.

³Human (h) and/or mouse (m) orthologous genes were predicted by WormBase (www.wormbase.org), except for Izumo1, on the basis of their nucleotide and/or predicted protein sequences.

By a reverse genetic approach, we identified C. elegans spe-45, which has expression confined to the male germline and encodes a transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain (Fig. 2).¹⁵ Our study showed that spe-45 is a functional ortholog of Izumo1, a mouse gene encoding a sperm Ig-like TM protein required during gamete fusion.¹⁶ Singaravelu et al. also identified the spe-45 gene in a forward genetic screen for spe-9 class mutants.¹⁷

Figure 2.

Domain architectures of immunoglobulin-like transmembrane proteins that are involved during gamete interactions in diverged organisms.Transmembrane (TM, blue box) proteins with the immunoglobulin (IG)-like (green) or IG-like folded filamin (IG_FLMN, purple) domains from a variety of species, which play important roles in gamete interactions. A black bar represents a 100-amino acid (AA) stretch. IZUMO, IZUMO domain.

In this commentary, we review these data and discuss the conserved roles of TM proteins containing Ig-like domains during gamete interactions in diverse organisms.

spe-45 is a novel spe-9 class gene

Our work is guided by the hypothesis that, despite obvious differences in sperm and egg appearance (compare the left and right halves of Figure 1), gamete fusion during fertilization in mammals will have conserved features shared with C. elegans. If any mammalian orthologs of the C. elegans spe-9 class genes act during gamete fusion, we can predict that an ortholog of the mouse Izumo1–like gene will play a key role during C. elegans fertilization. This hypothesis triggered our recent work.¹⁵ Mouse IZUMO1 contains one Ig-like and one TM domain (Fig. 2)¹⁶ and, guided by these structural features, we identified C. elegans F28D1.8 as a candidate gene that showed male germline-enriched expression.¹⁵

The tm3715 deletion mutation of F28D1.8 caused hermaphrodites to be self-sterile because of a defect(s) in either or some of male germline functions (therefore, F28D1.8 was renamed spe-45).^15,17 However, spe-45(tm3715) mutant males produced spermatids with wild-type-like cytology, number and activation rate into spermatozoa.^15,17 A wild-type (N2) adult hermaphrodite has ∼300 self-sperm right after the L4 stage and nearly all are sequentially consumed by fertilization, resulting in disappearance of sperm from the spermatheca by 72 h post L4 stage. In contrast, spe-9 class mutant hermaphrodites that were at 72 h post L4 stage retained ∼52% (spe-9), ∼100% (spe-42) or ∼63% (spe-45) of the spermatozoa observed right after the L4 stage.¹⁵ Like the other spe-9 class mutants spe-9^7,9 and spe-42,^13,14 spe-45(tm3715) laid very few fertilized eggs,^15,17 consistent with the failure of mutant self-sperm in the spermatheca to participate in fertilization.

These data suggest that the following distinctive defects characterize spe-9 class mutants: spe-9 and spe-45 sperm can bind to oocytes, but fail to undergo later steps during gamete fusion in the spermatheca. These unfertilized oocytes, with sperm bound to their surface, are squeezed through the spermathecal valve and ejected into the uterus. In some cases, the mutant sperm remain bound to oocytes when they enter the uterus. However, ejection into the uterus involves the spermathecal valve being swept over the oocyte surface and this frequently dislodges bound sperm. These now-free sperm are attracted to and subsequently crawl back into the spermatheca where they await the next oocyte. In contrast, spe-42 sperm seem to be incapable of binding to the oocyte plasma membrane. Izumo1-deficient mouse spermatozoa cannot fuse with oocytes despite successful sperm binding to the oocyte plasma membrane,¹⁶ which is essentially the same phenotype as C. elegans spermatozoa that lack SPE-45.

Another possibility is that spe-9 and/or spe-45 might be required for sperm guidance, in addition to their roles in gamete fusion. F-series prostaglandins that are synthesized from yolk lipoprotein complex-derived unsaturated fatty acids act as sperm guidance factors. Therefore, when genes involved in the synthesis are mutated, spermatozoa become partly defective in their relocation from the uterus into the spermatheca.^18-20 These phenotypes seem similar to those of spe-9 and spe-45 mutants, resulting in the incomplete sperm recruitment into the spermatheca. Currently the sperm prostaglandin-receptor(s) is not known, but SPE-9 and/or SPE-45 could function in the prostaglandin binding to spermatozoa and/or subsequent sperm reaction(s).

SPE-45 and IZUMO1 share a common function through their Ig-like domains

To examine if SPE-45 and IZUMO1 are functionally related to each other, we constructed a transgene encoding chimeric SPE-45, where the native Ig-like domain was removed and replaced by the mouse IZUMO1 Ig-like domain. When the transgene was expressed in spe-45(tm3715) hermaphrodites, the self-sterility of the mutant was rescued to ∼77% of the level observed for the wild-type transgene.¹⁵ To our knowledge, this finding was the first to show that two genes involved in fertilization are functionally orthologous to each other, even though they are evolutionally distant (∼900 million years)²¹ and show a low level of amino acid identity (8.7%).¹⁵

The precise roles played by these Ig-like domains in IZUMO1 and SPE-45, however, are currently unknown. Sperm IZUMO1 is known to bind to egg JUNO, a glycosylphosphatidylinositol (GPI)-anchored folate receptor-like protein.^22-24 This binding is indispensable for sperm-oocyte fusion in mice, and the IZUMO domain of IZUMO1 (Fig. 2) is required for the gamete interaction.^22-24 Moreover, Inoue et al. suggested the presence of a secondary IZUMO1 receptor on the oocyte surface that possibly acts after IZUMO1 binding to JUNO.²⁴ Therefore, SPE-45's Ig-like domain might indirectly act in binding to an oocyte SPE-45 receptor or participate in a later step(s) after the primary ligand-receptor binding during gamete fusion.

It seems probable that the SPE-45, like IZUMO1, interacts with a receptor on the oocyte surface and, perhaps, the Ig-like domain plays a role. Since C. elegans seems to have no genes encoding mouse JUNO-like proteins,²⁵ GPI-anchored proteins or folate-related proteins such as FOLT (folate transporter family) proteins, which contain multiple TM domains, might be alternative candidates of the oocyte-resident receptor.

Ig-like domains play key roles during gamete interactions in various organisms

Besides C. elegans SPE-45 and mouse IZUMO1, other Ig-like TM proteins from a variety of organisms have been found to participate in gamete interactions (Fig. 2). The mutant mouse line BART97b, where the Spaca6 gene encoding an Ig-like TM protein is deleted, produces spermatozoa that appear to be incapable of fusing with the oocyte plasma membrane.²⁶ Another mouse example is the Bsg gene that encodes a single-pass TM protein containing two Ig-like domains. Both male and female Bsg-knockout mice are sterile and, in males, spermatogenesis is arrested.²⁷ Later work revealed that the BSG protein also acts during sperm interactions with the cumulus cells and the zona pellucida.²⁸ Arabidopsis and Chlamydomonas have GEX2^29,30 and FUS1,^31,32 respectively, both of which are single-pass TM proteins with Ig-like filamin repeat domains. These two proteins are involved in gamete fusion or attachment.

As in the case of SPE-45 and IZUMO1, it remains unclear how Ig-like domains found in the mouse, plant and algal proteins are involved in gamete interactions. However, the Hydractinia (a cnidarian) Alr1 and Alr2 genes might provide clues to the functions of Ig-like domains during fertilization. These two genes encode TM proteins with multiple Ig-like domains that play critical roles in recognition of self- versus non-self (allo-recognition) through trans-homotypic interactions of Alr proteins.³³ Perhaps the other Ig-like TM proteins shown in Figure 2, like the Alr proteins, associate with the same and/or other TM proteins containing Ig-like domains to form cis- and/or trans-protein complexes that are important for fertilization. For example, on the mouse sperm surface, IZUMO1 and SPACA6 might interact with each other, although any functional relationship between them is presently unclear. Since the C. elegans genome has both male and female germline-specifically or predominantly expressed genes encoding Ig-like TM proteins besides spe-45,¹⁵ there might be a SPE-45 partner(s) with such the domain architecture on the surface of spermatozoa and/or oocytes.

Abbreviations

TM

transmembrane

Ig

immunoglobulin

GPI

glycosylphosphatidylinositol.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by MEXT KAKENHI Grant Number 24112716 and JSPS KAKENHI Grant Numbers 23870029 and 24570241 to H.N., Japan. We were also supported by grants from the NSF IOB-0544180, NIH GM082932 and NIH HD066577 and by funds from Emory College to S.W.L., USA.