When sperm from sibling and non-sibling male house mice compete for the same eggs in vitro, fertilization is biased toward the unrelated males' sperm even though the competitors do not differ in sperm number or swimming performance — evidence that the egg side of the interaction selects against genetically related sperm (Firman & Simmons 2015, Ecology Letters). What mediates this egg-driven sperm selection is unknown: the responsible gametic recognition molecules have not been identified, it is unresolved whether dissimilarity at the major histocompatibility complex (MHC) or another genotype-specific cue drives the bias, and it is unsettled at which stage the selection acts (chemoattractant secretions, sperm-egg binding, or post-contact signalling). No molecular identification of this egg-driven bias in house mice has been reported; adjacent progress includes a proteomic shortlist of cumulus-matrix candidates in mice, human follicular-fluid chemoattraction and MHC-dissimilarity sperm-performance studies, and mixed evidence on the phenomenon's generality in other taxa. The problem remains open in its original generality.

Background

In species with internal fertilization, sexual selection continues after mating. Sperm competition — the race between the sperm of rival males within a female's reproductive tract — is the best-studied postmating process, but females are not passive arenas: through cryptic female choice, female-driven mechanisms at or after mating can bias which male's sperm is used for fertilization. One hypothesized benefit of cryptic female choice is inbreeding avoidance: mating among close relatives (inbreeding) increases homozygosity and the expression of deleterious recessive alleles, reducing offspring fitness (inbreeding depression), so a polyandrous female that cannot avoid mating with a relative could still avoid fertilizing her eggs with his sperm. Demonstrating sperm selection at the gamete level is difficult, because a non-random paternity share can also arise from subtle differences in sperm competitiveness between males, or from differential mortality of embryos sired by different males.

The house mouse (Mus musculus domesticus) is the key model for this question. The major histocompatibility complex (MHC) — the extraordinarily polymorphic vertebrate gene region that encodes immune-recognition proteins and also shapes body odour and mate choice — has long been suspected to act at fertilization itself: in vitro fertilization (IVF, i.e. fertilization of eggs in a culture dish rather than inside the female) with MHC-congenic mouse strains produced blastocysts with non-random MHC combinations, an effect that shifted with an ongoing viral epidemic and that was interpreted as eggs selecting sperm by MHC haplotype (Wedekind et al. 1996; Rulicke et al. 1998). In red junglefowl, paternity biases against related males' sperm are better predicted by MHC similarity than by genome-wide relatedness, although that effect disappeared under artificial insemination, implicating the female tract and male phenotypic cues as well as gametes (Lovlie et al. 2013). The central mouse evidence is Firman and Simmons (2015): in IVF assays, cumulus-oocyte complexes (each unfertilized egg surrounded by several thousand cumulus cells embedded in a hyaluronic-acid-rich extracellular matrix) were incubated with mixed sperm from a sibling and a non-sibling of the female. Embryo genotyping revealed a consistent fertilization bias toward the unrelated male's sperm, while the number of motile sperm and sperm swimming performance did not differ between competitors — leaving an egg-driven interaction as the proposed mechanism of cryptic female choice, with the expression or secretion of gametic proteins suggested as its possible molecular basis.

None of the molecules involved is known. The classic mammalian gamete-recognition components — for example the sperm membrane protein IZUMO1 and its egg-surface receptor JUNO, an essential pair for sperm-egg fusion whose gene pair carries population-level genetic diversity (Allingham & Floriano 2021) — are conserved, species-level requirements for fertilization rather than kin-discriminating cues. Human follicular fluid (the fluid surrounding the oocyte, a natural source of sperm chemoattractants, i.e. diffusible chemicals that guide sperm swimming) differentially attracts the sperm of different males, so chemoattraction can in principle support gamete-mediated mate choice in an internal fertilizer (Fitzpatrick et al. 2020) — yet the attractant molecules are unidentified, and mammalian sperm chemotaxis lacks even species specificity, which makes a kin-specific chemoattractant mechanism non-trivial. On the mouse egg side, proteomic work has implicated the cumulus extracellular matrix as a mediator of fertilization outcomes and identified a set of abundant, rapidly evolving matrix proteins (Keeble et al. 2021), but without any test of kin discrimination. Whether MHC dissimilarity or another genotype-specific cue drives the mouse fertilization bias, and whether the bias arises before gamete contact (via secreted chemoattractants), at binding to the cumulus or zona pellucida (the glycoprotein coat of the egg), or after sperm-egg contact, remains undetermined.

Problem Statement

Determine the molecular basis of the egg-driven fertilization bias toward unrelated males' sperm observed in vitro in house mice (Firman & Simmons 2015). Concretely: (1) identify the gametic recognition molecule(s) or cue(s) — egg-side (oocyte surface, zona pellucida, cumulus cells, or their secretions) and/or sperm-side — whose interaction generates the bias against sperm of related males; (2) determine whether the relevant cue is dissimilarity at the major histocompatibility complex (MHC) or some other genotype-specific cue; and (3) establish at which stage of the gamete interaction the selection acts — chemoattractant-mediated sperm guidance before contact, sperm binding to the cumulus or zona pellucida, or post-contact sperm-egg signalling. The responsible molecule or cue — egg-side or sperm-side — must be one through which eggs respond differentially to the sperm of related versus unrelated males, and one that causally produces the kin-biased fertilization pattern, so that experimentally manipulating it (blocking, removing, or otherwise perturbing the molecule or cue) in controlled in vitro fertilization kin-competition assays removes or restores the bias. A rigorous demonstration that the egg-driven bias itself does not occur under the original experimental conditions, with the confounds of sperm competitiveness and differential embryo mortality excluded, would equally close the question by removing the phenomenon to be explained.

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