Recurrent Duplication and Diversification of Acrosomal Fertilization Proteins in Abalone

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another important mechanism that facilitates the generation of molecular novelty. Following duplication, paralogs may parse ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, consistent with a subfunctionalization model where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.

Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.

Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

Transcriptome analysis provides a blueprint of coral egg and sperm functions

Background Reproductive biology and the evolutionary constraints acting on dispersal stages are poorly understood in many stony coral species. A key piece of missing information is egg and sperm gene expression. This is critical for broadcast spawning corals, such as our model, the Hawaiian species Montipora capitata, because eggs and sperm are exposed to environmental stressors during dispersal. Furthermore, parental effects such as transcriptome investment may provide a means for cross- or trans-generational plasticity and be apparent in egg and sperm transcriptome data. Methods Here, we analyzed M. capitata egg and sperm transcriptomic data to address three questions: (1) Which pathways and functions are actively transcribed in these gametes? (2) How does sperm and egg gene expression differ from adult tissues? (3) Does gene expression differ between these gametes? Results We show that egg and sperm display surprisingly similar levels of gene expression and overlapping functional enrichment patterns. These results may reflect similar environmental constraints faced by these motile gametes. We find significant differences in differential expression of egg vs. adult and sperm vs. adult RNA-seq data, in contrast to very few examples of differential expression when comparing egg vs. sperm transcriptomes. Lastly, using gene ontology and KEGG orthology data we show that both egg and sperm have markedly repressed transcription and translation machinery compared to the adult, suggesting a dependence on parental transcripts. We speculate that cell motility and calcium ion binding genes may be involved in gamete to gamete recognition in the water column and thus, fertilization.

Proteomics support the threespine stickleback egg coat as a protective oocyte envelope

After the end of the last ice age, ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Stickleback populations are reproductively isolated to varying degrees, despite the fact that they can be crossed in the lab to produce viable offspring. Ecological and behavioral factors have been suggested to underlie incipient stickleback speciation. However, reproductive proteins represent a previously unexplored driver of speciation. As mediators of gamete recognition during fertilization, reproductive proteins both create and maintain species boundaries. Gamete recognition proteins are also frequently found to be rapidly evolving, and their divergence may culminate in reproductive isolation and ultimately speciation. As an initial investigation into the contribution of reproductive proteins to stickleback reproductive isolation, we characterized the egg coat proteome of threespine stickleback eggs. In agreement with other teleosts, we find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3. We explore aspects of stickleback ZP protein biology, including glycosylation, disulfide bonding, and sites of synthesis, and find many substantial differences compared to their mammalian homologs. Furthermore, molecular evolutionary analyses indicate that ZP3, but not ZP1, has experienced positive Darwinian selection across teleost fish. Taken together, these changes to stickleback ZP protein architecture suggest that the egg coats of stickleback fish, and perhaps fish more generally, have evolved to fulfill a more protective functional role than their mammalian counterparts. Data are available via ProteomeXchange with identifiers PXD017488 and PXD017489.

Maternal control of gamete choice during fertilization

Sexually reproducing organisms generate male and female haploid gametes, which meet and fuse at fertilization to produce a diploid zygote. The evolutionary process of speciation is achieved and maintained by ensuring that gametes undergo productive fusion only within a species. In animals, hybrids from cross-species fertilization events may develop normally, but are usually sterile (Fitzpatrick, 2004). Metazoan sperm and eggs have several features to ensure that the gametes, which have evolved independently and also in conflict with each other, are competent to undergo fertilization (Firman, 2018). Fertilization is a specific process that is ideally supposed to result in randomized fusion of compatible egg and sperm. Here, I will discuss key processes driven by maternal factors in the egg that dictate earliest stages of gamete recognition, gamete choice and fusion in metazoans.

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.