Species-specific Fertilization Research Articles

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

Molecular mechanisms and evolution of fertilization proteins.

Sexual reproduction involves a cascade of molecular interactions between the sperm and the egg culminating in cell-cell fusion. Vital steps mediating fertilization include chemoattraction of the sperm to the egg, induction of the sperm acrosome reaction, dissolution of the egg coat, and sperm-egg plasma membrane binding and fusion. Despite decades of research, only a handful of interacting gamete recognition proteins (GRPs) have been identified across taxa mediating each of these steps, most notably in abalone, sea urchins, and mammals. This review outlines and compares notable GRP pairs mediating sperm-egg recognition in these three significant model systems and discusses the molecular basis of species-specific fertilization driven by GRP function. In addition, we explore the evolutionary theory behind the rapid diversification of GRPs between species. In particular, we focus on how the coevolution between interacting sperm and egg proteins may contribute to the formation of boundaries to hybridization. Finally, we discuss how pairing structural information with evolutionary insights can improve our understanding of mechanisms of fertilization and their origins.

Reproductive strategies in the intercrossing corals Acropora donei and A. tenuis to prevent hybridization

Although many sympatric species of the coral Acropora spawns on the same day, hybridization is partly prevented by their species-specific fertilization manner. On the other hand, several species show bidirectional intercrossing, but their hybridization is prevented by slight time differences in their spawning and fertilization preference with conspecific sperm. However, it is scarcely described how the intercrossing species avoid hybridization. Here we demonstrate that hybridization of the intercrossing species Acropora tenuis and A. donei is likely prevented by differences in the timing of spawning, gametogenesis (differences in production numbers of matured sperm and eggs), population density, and fertilization preferences. We investigated three locations in Okinawa, Japan: Aka Island, Sesoko Island, and Oku. The timing of gamete release in the two species overlapped at Aka Island and Oku, but A. donei did not spawn vigorously at Sesoko Island. Histological observations indicated a decrease in the number of mature sperm and eggs in the weakly spawning colonies of A. donei at Sesoko Island. From the population density, as assessed by field surveys, we predicted that the encounter rate with heterospecific gametes for A. donei around Sesoko Island was higher than at the other locations. Thus, one predictable reason for the suppression of their gonadal development may be the avoidance of hybridization. Although synchronous spawning of A. tenuis and A. donei was observed at Aka Island and Oku, hybridization is likely infrequent, because sperm choice experiments demonstrated that the eggs preferred fertilization by conspecific sperm. Moreover, microsatellite analyses suggested that hybridization rarely occurred even at synchronous spawning sites, as the population structures of the two species at both Aka Island and Oku were not mixed. Collectively, the findings of this study highlight several strategies, tactics, and mechanisms of avoiding hybridization that are likely to be active in other species that hybridize in vitro.

The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization.

Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. We found that an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, is a crucial fertilization factor in zebrafish. Membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer not only is required for sperm-egg interaction but is also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged more than 200 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer's closest homolog in tetrapods, SPACA4, is restricted to the male germline in internally fertilizing vertebrates, which suggests that our findings in fish have relevance to human biology.

Bouncer keeps fertilization specific

Fertilization Fertilization needs to be highly efficient while remaining species-specific. However, despite decades of research, it is still unclear how these two requirements are met. Herberg et al. report the discovery of the Ly6/uPAR-type protein Bouncer as a species-specific fertilization factor in zebrafish (see the Perspective by Lehmann). Bouncer localizes to the egg membrane and is required for sperm entry. Remarkably, expression of Bouncer from another fish species (medaka) in zebrafish allowed for cross-species fertilization. Science , this issue p. [1029][1]; see also p. [974][2] [1]: /lookup/doi/10.1126/science.aat7113 [2]: /lookup/doi/10.1126/science.aau8356

Molecular interaction of zp3 to zp3r reveals a cross-species fertilization mechanism

Objective: To evaluate the role of ZP3R in the species-specific fertilization mechanism. Methods: ZP3/ZP3R protein sequences of Mus musculus, Rattus norvegicus, and Cavia porcellus were downloaded from UNIPROT. Percentage of amino acids that was calculated by using the SIAS program. Protein sequences modeled was established by using the Modeller 9.14 program and glycosylation of the ZP3 using GlyProt program. Docking simulation of the ZP3R-ZP3 was performed between the same species and different species with PatchDock program. Results: Comparison of the ZP3R and ZP3 structure between species showed that ZP3 in these three species was more similar than ZP3R. Docking simulations of protein showed that changes in the pattern of the ZP3-ZP3R domain for interaction on cross-species compared to the same species. Changes in the pattern of binding ZP3R-ZP3 made sperm- egg binding was not functional and could inhibit cross-fertilization. Conclusions: ZP3R-ZP3 interaction is species-specific, and the role of ZP3R is greater than ZP3 in determining the species-specific recognition stage and sperm-egg binding.

Species-specific fertilization can benefit container nursery crop production.

Clark, M. J. and Zheng, Y. 2015. Species-specific fertilization can benefit container nursery crop production. Can. J. Plant Sci. 95: 251-262. To determine the responses of six container-grown shrub species to different controlled-release fertilizer (CRF) application rates, plant growth and root-zone traits were evaluated following fertilization with Polyon® 16-6-13, 5-6 month CRF incorporated at 0.60, 0.89, 1.19, 1.49 and 1.79 kg m-3 N. The six species tested at a southwestern Ontario, Canada, nursery were Cornus stolonifera ‘Flaviramea’ (yellow-twig dogwood), Euonymus alatus ‘Compactus’ (dwarf winged euonymus), Hydrangea paniculata ‘Grandiflora’ (Pee Gee hydrangea), Physocarpus opulifolius ‘Nugget’ (Nugget ninebark), Spiraea japonica ‘Magic Carpet’ (Magic Carpet spirea), Weigela florida ‘Alexandra’ (Wine and Roses weigela). Different species responded differently to the CRF rates applied. For the majority of species at the final harvest, growth index, plant height, canopy area, leaf area and above-ground dry weight were greater in high vs. low CRF rates; however, different species had different optimal CRF application rates or ranges: 1.49 kg m-3 N for Hydrangea and Spiraea, 1.19 kg m-3 N for Weigela, 1.19 to 1.49 kg m-3 N for Cornus and Physocarpus, and =0.60 kg m-3 N for Euonymus. Based on these species-specific optimal fertilizer rates or ranges, growers can group plant species with similar fertilizer demands, thereby reducing fertilizer waste and maximizing plant production.

Evolution of gamete attraction molecules: evidence for purifying selection in speract and its receptor, in the pantropical sea urchin Diadema.

Many free-spawning marine invertebrates, such as sea urchins, lack any courtship or assortative mating behavior. Mate recognition in such cases occur at the gametic level, and molecules present on the sperm and egg are major determinants of species-specific fertilization. These molecules must also coevolve in relation to each other in order to preserve functional integrity. When sea urchins release their gametes in seawater, diffusible molecules from the egg, termed sperm-activating peptides, activate and attract the sperm to swim toward the egg, initiating a series of interactions between the gametes. Although the compositions and diversity of such sperm-activating peptides have been characterized in a variety of sea urchins, little is known about the evolution of their genes. Here we characterize the genes encoding the sperm-activating peptide of the egg (speract) and its receptor on the sperm, and examine their evolutionary dynamics in the sea urchin genus Diadema, in the interest of determining whether they are involved in reproductive isolation between the species. We found evidence of purifying selection on several codon sites in both molecules and of selectively neutral evolution in others. The diffusible speract peptide that activates sperm is invariant across species, indicating that Diadema egg peptides do not discriminate between con- and hetero-specific sperm at this stage of the process. Speract and its receptor do not contribute to reproductive isolation in Diadema.

Bovine And Human Zona Pellucida 3 Gene Glycans Site Prediction Using In Silico Analysis

Zona pellucida is one of the protective layer of the egg cell and has a function as an intermediary species-specific fertilization. Glycoproteins of human and bovine zona pellucida is composed of three types, namely ZP1, ZP2 and ZP3. ZP3 gene has amino acid sequence homology with other mammals. Oligosaccharides components of the zona pellucida glycans are composed from units of asparagine residues (N -linked) and serine/threonine (O -linked). The aims of this study was to analyze the DNA sequences of human and bovine and further predicts glycans site on amino acid sequence of human and bovine ZP3. In this study, ZP3 gene fragments have been isolated from bovine and humans were analyzed in Silico. This work were conducted by comparing the data of DNA sequence from human and bovine PCR product using NCBI BLAST. The results showed that there were similarities at amino acid positions number 23-38. bZP3 sequence had three glycans site (Asn-X-Thr/Ser) and one site on hZP3 glycans. One of the sites was conserved between the two species.

Spermatozoa and seminal plasma enzyme activity in Persian sturgeon,Acipenser persicus, in relation to short-term storage

An important component of semen examination is the comparison between the characteristics of fresh and stored semen (Lahnsteiner, Berger, Weismann & Patzner 1996). The relationships among the parameters of fresh and cryopreserved semen may be determined to identify markers for prediction of the effectiveness of the cryopreservation procedure for fish semen (Lahnsteiner et al. 1996). As the quality of cryopreserved semen is significantly lower than that of fresh semen, such markers are important for successful cryobanking of fish semen (Piros, Glogowski, Kolman, Rzemieniecki, Domagala, Horvath, Urbanyi & Ciereszko 2002). The handling and storage of gametes is central to many assisted reproductive procedures and may be broadly considered as either short-term storage, at temperatures above 0°C, or long-term storage at temperatures below 0°C. Short-term storage of fish gametes may be necessary when the availability of male and female gametes for in vitro fertilization is asynchronous (Lahnsteiner, Weismann & Patzner 1997) or if the sources of gametes are spatially separated. Preservation may also be necessary during transport to a facility with the capacity to cryopreserve gametes for long-term storage, in certain instances when spermatozoa and oocytes have been removed from moribund or recentlydead animals in the field. Several factors can, however, affect the quality and viability of the stored sperm. In this regard, the assessment of the biochemical characteristics of sperm, particularly in terms of enzyme characteristics, can provide new insights into spermatozoa motility and fertilization ability, thus creating opportunities for improving artificial reproduction and germplasm resource conservation procedures (Li, Hulak & Linhart 2008). The enzymology of fish gametes is not well known, when compared with that associated with mammalian semen. Detailed results of research on the enzymes of fish spermatozoa are available with respect to aspartate aminotransferase (AST), lactate dehydrogenase (LDH), acid phosphatase (AcP), b-N-acetylglucosaminidase (b-N AGase) and arylsulfatase (AS) (Schmehl, Graham & Erdhal 1987; McNiven, Gallant & Richardson 1992; Ciereszko & Dabrowski 1994; Piros et al. 2002;). AS and b-N AGase are among the key enzymes, localized within the mammalian acrosome, that play a pivotal role during penetration of the oocyte (Nikolajczyk & O’Rand 1992; Brandon, Srivastava, Heusner & Fayer-Hosken 1997). The activity of AS and b-N AGase during fertilization in sturgeon can provide useful information regarding speciesspecific fertilization and prevention of polyspermy (when a single egg is fertilized by more than one sperm) as postulated by Cherr and Clark (1985). AcP, one of the enzymes found in sperm, has been described in mammalian spermatozoa and seminal plasma (Yousef, Diamandis, Jung & Diamandis 2001) and has also been observed in spermatozoa

Cross species fertilization and development investigated by cat sperm injection into mouse oocytes

The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts.

In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis

It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented. Human ZP1 (amino acid residues 26-551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues. The MAbs generated against ZP1 recognized specifically the zona matrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of approximately 75 and approximately 85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the G(i)-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction. These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis.

Inhomogeneous Morphology and Elasticity of Mouse Oocyte Zona Pellucida Pre- And Post-Fertilization

The mature mammalian oocyte is encapsulated within a thick membrane, the zona pellucida (ZP), which is composed of a small number of glycoproteins. The proteins are secreted by the oocyte during maturation. Sperm has to penetrate the ZP first before it reaches the oocyte. The ZP controls for species-specific fertilization and acts as the barrier to polyspermy, i.e. it prevents a multiplicity of sperms from reaching the oocyte. All ZP proteins contain a characteristic ZP domain but, otherwise, their 3-D structure is unknown as is the architecture of the zona membrane. Upon fertilization, one of the proteins (ZP2) is cleaved near its N-terminal. The cleavage is believed to alter the ZP structure which, henceforth, acts as a barrier to further sperm penetration.We used the atomic force microscope (AFM) to examine both the ZP structure and mechanical properties of the wildtype mouse ZP under physiological conditions. For that purpose, patches of isolated mouse ZP were immobilized on polylysine coated mica. Imaging revealed two predominant membrane surface morphologies consistent with previously reported electron microscopy images of the outer and inner membrane surfaces, respectively, from different species. One is a rough, ruffled surface and the other is a smoother surface with the appearance of a tighter construction. In addition to the surfaces, the structure across the wall thickness was visualized at high resolution revealing a layered, well organized architecture. Elastic modulus estimates from force-indentation data also showed systematic variability mirroring the morphological inhomogeneity. For example, significant differences in elasticity were measured between the rough regions, hypothesized to be outer surfaces, and the smoother regions. Interestingly, these properties also appear to undergo appreciable changes upon fertilization pointing to the structural change effected by the ZP2 cleavage.

A carbohydrate-based mechanism of species recognition in sea urchin fertilization

In the present review, we describe a systematic study of the sulfated polysaccharides from marine invertebrates, which led to the discovery of a carbohydrate-based mechanism of sperm-egg recognition during sea urchin fertilization. We have described unique polymers present in these organisms, especially sulfated fucose-rich compounds found in the egg jelly coat of sea urchins. The polysaccharides have simple, linear structures consisting of repeating units of oligosaccharides. They differ among the various species of sea urchins in specific patterns of sulfation and/or position of the glycosidic linkage within their repeating units. These polysaccharides show species specificity in inducing the acrosome reaction in sea urchin sperm, providing a clear-cut example of a signal transduction event regulated by sulfated polysaccharides. This distinct carbohydrate-mediated mechanism of sperm-egg recognition coexists with the bindin-protein system. Possibly, the genes involved in the biosynthesis of these sulfated fucans did not evolve in concordance with evolutionary distance but underwent a dramatic change near the tip of the Strongylocentrotid tree. Overall, we established a direct causal link between the molecular structure of a sulfated polysaccharide and a cellular physiological event - the induction of the sperm acrosome reaction in sea urchins. Small structural changes modulate an entire system of sperm-egg recognition and species-specific fertilization in sea urchins. We demonstrated that sulfated polysaccharides - in addition to their known function in cell proliferation, development, coagulation, and viral infection - mediate fertilization, and respond to evolutionary mechanisms that lead to species diversity.

In the urochordate Ciona intestinalis zona pellucida domain proteins vary among individuals

Fertilization in hermaphroditic urochordates requires efficient mechanisms to prevent self-mating. By comparing gonadal transcripts of three genetically unrelated Ciona intestinalis individuals by suppression subtractive hybridization (SSH), we have previously identified a highly polymorphic gene coding for a transmembrane receptor which is expressed in follicle cells surrounding the mature oocyte [Kürn U, Sommer F, Hemmrich G, Bosch TCG, Khalturin K. Allorecogmition in urochordates: identification of a highly variable complement receptor-like protein expressed in follicle cells of Ciona. Dev Comp Immunol 2007; 31(4): 360–71]. Here, we show that in addition to follicle cells, oocytes also express variable genes. We have identified four novel Ciona genes coding for zona pellucida (ZP) domain proteins which are expressed in developing oocytes and which significantly vary among individuals. ZP containing proteins are thought to play a crucial and conserved role in controlling fertilization. Our results provide strong evidence that in Ciona components of both follicle cells and oocytes show a high degree of interindividual variability and, therefore, may take part in the prevention of self-mating as well as in the maintenance of species-specific fertilization.

Adaptive Evolution of Fertilization Proteins within a Genus: Variation in ZP2 and ZP3 in Deer Mice (Peromyscus)

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.

The specificity of interactions between proteins and sulfated polysaccharides

Sulfated polysaccharides are capable of binding with proteins at several levels of specificity. As highly acidic macromolecules, they can bind non-specifically to any basic patch on a protein surface at low ionic strength, and such interactions are not likely to be physiologically significant. On the other hand, several systems have been identified in which very specific substructures of sulfated polysaccharides confer high affinity for particular proteins; the best-known example of this is the pentasaccharide in heparin with high affinity for antithrombin, but other examples may be taken from the study of marine invertebrates: the importance of the fine structure of dermatan sulfate (DS) to its interaction with heparin cofactor II (HCII), and the involvement of sea urchin egg-jelly fucans in species specific fertilization. A third, intermediate, kind of specific interaction is described for the cell-surface glycosaminoglycan heparan sulfate (HS), in which patterns of sulfate substitution can show differential affinities for cytokines, growth factors, and morphogens at cell surfaces and in the intracellular matrix. This complex interplay of proteins and glycans is capable of influencing the diffusion of such proteins through tissue, as well as modulating cellular responses to them.

Full-length sequence of VERL, the egg vitelline envelope receptor for abalone sperm lysin

Abalone sperm use 16 kDa lysin to create a hole in the egg vitelline envelope (VE) by a species-specific, nonenzymatic mechanism. To create the hole, lysin binds tightly to VERL (the VE receptor for lysin), a giant, unbranched glycoprotein comprising 30% of the VE. Binding of lysin to VERL causes the VERL molecules to lose cohesion and splay apart creating the hole. Lysin and VERL represent a cognate pair of gamete recognition proteins, one male the other female, which mediate fertilization. The coevolution of such cognate pairs may underlie the establishment of species-specific fertilization which could be a component of the mechanism to achieve reproductive isolation and hence new species. Here we present the full-length cDNA sequence (11,166 bp) of VERL from the red abalone ( Haliotis rufescens). There are 42 amino acids from the start Met residue to the beginning of the first ‘VERL repeat’. Most of VERL (9981 bp; 89.4%) consists of 22 tandem repeats of a ∼153 amino acid sequence that is predicted to be β-sheet. The last VERL repeat is followed by 353 non-repeat amino acid residues containing a furin cleavage site (RTRR), a ZP domain and a hydrophobic COOH-terminus with a 3′ UTR of only 10 nucleotides. VERL repeats 3–22 have been subjected to concerted evolution and consequently have almost identical sequences. Curiously, comparisons of repeats from other species shows that repeats 1 and 2 of red abalone VERL have not been subjected to concerted evolution since the divergence of the red species from the other six California species.

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin.

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.

Abalone lysin: the dissolving and evolving sperm protein.

Abalone sperm lysin is a non-enzymatic protein that creates a hole for sperm passage in the envelope surrounding the egg. Lysin exhibits species-specificity in making the hole and it evolves rapidly by positive selection. Our studies have focused on combining structural, biochemical, and evolutionary data to understand the mechanism of action and evolution of this remarkable protein. Currently, more is known about lysin than about any other protein involved in animal fertilization. We present an hypothesis to explain lysin's rapid evolution and the evolution of species-specific fertilization in this order of mollusks. We also propose a two-step model for lysin's action in which a dimer of lysin binds species-specifically to its glycoprotein receptor, and then monomerizes and binds the receptor in a non-species-specific manner. This experimental system yields data relevant to the general problem of molecular recognition between cell surfaces, and is also important to our thinking about how new species arise in the sea. BioEssays 23:95-103, 2001.