Gamete Recognition Research Articles

O-284 Evidence for gamete recognition mechanisms in humans

Abstract Egg-sperm binding and fusion are the central events of fertilization and are essential for the formation of a new individual. While decades of research have provided a thorough understanding of the cellular processes, the identity of molecules orchestrating these events, along with their mechanism of action, remain elusive. Often the interactions established by cell surface proteins are weak and transient, making them challenging to deal with using standard biochemical methods, like purifications that involve stringent washing and denaturing conditions. Therefore, our knowledge of membrane proteins required for the sperm-egg interaction remains scarce and the research is further complicated by the limited amount of biological material, especially the eggs. Thanks to experimental approaches specifically developed to investigate cell surface interactions, we identified the first binding pair essential for fertilization in mammals 1. The pair is made of the egg protein JUNO and the sperm protein IZUMO1. Recently another membrane protein, the Fc receptor-like 3 (MAIA), has been indicated as a binding partner of IZUMO1 and suggested to be required for human fertilization2, but the data needs to be further investigated. Indeed, the same biochemical methods used to identify the JUNO-IZUMO1 binding pair, found no direct evidence of binding between MAIA and IZUMO13. In the last decade more sperm proteins have been shown to be essential for sperm-egg interaction other than IZUMO1, and all findings have been validated using transgenic mouse models. Among these sperm proteins, Tmem95 was firstly discovered in bulls, it was later confirmed to be required for fertilization in mice, and new evidence suggests that its role is conserved in humans4,5. Notably, the role played by each of the novel sperm candidates remains elusive and little is known on whether and how these cell surface molecules interact with one another. Insights into the architecture of the mammalian egg-sperm fusion synapse are now emerging with the use of AlphaFold2, an artificial intelligence-based program capable of predicting proteins’ 3D structure. Remarkably, AlphaFold2 not only predicts the structure of extracellular proteins essential for fertilization, also suggests the formation of a pentameric complex composed of three sperm (IZUMO1, SPACA6 and TMEM81) and two egg (JUNO and CD9) proteins which is likely to be important for gamete recognition and fusion6,7. In a high throughput screening we have recently identified a novel binding partner of IZUMO1, expressed in human leukocytes, potentially linking the reproductive and immune system (unpublished).

Introgression of the gamete recognition molecule, bindin, in the sea urchin Diadema.

Hybridization is important in evolution, because it is a necessary (though not sufficient) step in the introgression of potentially adaptive variation between species. Bindin is a gamete recognition protein in echinoids and asteroids, capable of blocking cross-fertilization between species to varying degrees. Four species of the sea urchin genus Diadema are broadly sympatric in the Indo-Pacific: D. paucispinum, D. savignyi, D. clarki, and D. setosum. Data from three published studies, one of identification of hybrids through allozymes, one of the phylogeography of mitochondrial DNA, and one of the phylogeny of bindin, were combined to assess the degree of bindin introgression between these four species. I analyzed sequences of the ATPase 8 and ATPase 6 mitochondrial genes and of bindin, sampled throughout the species ranges, with an Isolation-Migration algorithm, IMa3. IMa3 uses a coalescent approach to produce Bayesian estimates of effective population sizes and gene flow between populations. The results showed that bindin alleles coalesce completely within the species bounds of D. clarki, and of D. setosum. The sister species D. paucispinum and D. savignyi, however, were estimated as having exchanged a bindin allele at an average of every one to two-and-a-half generations since they speciated from each other. As the allozyme study detected nine hybrids between three of these species in Okinawa (most of them between D. setosum and D. savignyi) in a single sample, hybrids between these species are produced, but bindin does not introgress. Therefore, bindin must not be efficient in blocking heterospecific fertilizations. Complete, or almost complete, reproductive isolation between species of Diadema must result from low hybrid fitness.

Identification and characterization of an L-fucose specific lectin GVSL involved in gamete recognition of Gracilaria vermiculophylla

The lectin-carbohydrate recognition system is especially important for red algae which female and male gametes are both non-motile. A sex-related lectin gene GvSL from Gracilaria vermiculophylla was cloned and expressed heterologously in Escherichia coli. ORF of GvSL was 684 bp in length and encoded a polypeptide of 228 amino acids with a deduced molecular weight of 25.8 kDa. Real-time PCR analysis showed that it up-regulated most in mature male gametophytes which was 15.4-fold and 21.94-fold higher than mature female gametophytes and immature male gametophytes respectively. The immunohistochemical staining also demonstrated that GvSL was mainly expressed in the spermatangium crypts of mature male gametophytes. Purified rGVSL showed significant agglutination of rabbit blood erythrocytes and only L-fucose showed the glycoinhibitory effect among the eight monosaccharides tested. The inhibitory tests further proved that GVSL and its complementary carbohydrate L-fucose composed a lectin-carbohydrate recognition system affecting the fertilization efficiency of G. vermiculophylla.

No evidence for a direct extracellular interaction between human Fc receptor-like 3 (MAIA) and the sperm ligand IZUMO1.

Fertilization involves the recognition and fusion of sperm and egg to form a previously unidentified organism. In mammals, surface molecules on the sperm and egg have central roles, and while adhesion is mediated by the IZUMO1-JUNO sperm-egg ligand-receptor pair, the molecule/s responsible for membrane fusion remain mysterious. Recently, MAIA/FCRL3 was identified as a mammalian egg receptor, which bound IZUMO1 and JUNO and might therefore have a bridging role in gamete recognition and fusion. Here, we use sensitive assays designed to detect extracellular protein binding to investigate the interactions between MAIA and both IZUMO1 and JUNO. Despite using reagents with demonstrable biochemical activity, we did not identify any direct binding between MAIA/FCRL3 and either IZUMO1 or JUNO. We also observed no fusogenic activity of MAIA/FCRL3 in a cell-based membrane fusion assay. Our findings encourage caution in further investigations on the role played by MAIA/FCRL3 in fertilization.

EGG CELL 1 contributes to egg-cell-dependent preferential fertilization in Arabidopsis.

During double fertilization in angiosperms, the pollen tube delivers two sperm cells into an embryo sac; one sperm cell fuses with an egg cell, and the other sperm cell fuses with the central cell. It has long been proposed that the preference for fusion with one or another female gamete cell depends on the sperm cells and occurs during gamete recognition. However, up to now, sperm-dependent preferential fertilization has not been demonstrated, and results on preferred fusion with either female gamete have remained conflicting. To investigate this topic, we generated Arabidopsis thaliana mutants that produce single sperm-like cells or whose egg cells are eliminated; we found that although the three different types of sperm-like cell are functionally equivalent in their ability to fertilize the egg and the central cell, each type of sperm-like cell fuses predominantly with the egg cell. This indicates that it is the egg cell that controls its preferential fertilization. We also found that sperm-activating small secreted EGG CELL 1 proteins are involved in the regulation of egg-cell-dependent preferential fertilization, revealing another important role for this protein family during double fertilization.

Climate Adaptation and Drift Shape the Genomes of Two Eel-Goby Sister Species Endemic to Contrasting Latitude.

Deciphering the role of climate adaptation in generating genetic divergence and hence speciation is a central question in evolution. Comparisons of genomes of closely related species spanning selective climate gradients are particularly informative in discerning the signatures of selection and thereby providing valuable information concerning the role of climate adaptation in speciation. Here we re-sequenced 99 genomes of the two sister eel-goby species Odontamblyopus lacepedii and O. rebecca, which are endemic to tidal mudflats spanning contrasting latitude gradients, to estimate the influence of divergent climate selection on shaping genome-wide patterns of divergence. The results indicated that genome-wide differentiation between the two species was evident (genome-wide FST = 0.313). Against a background of high baseline genomic divergence, 588 and 1202 elevated divergent loci were detected to be widespread throughout their genomes, as opposed to focused within small islands of genomic regions. These patterns of divergence may arise from divergent climate selection in addition to genetic drift acting through past glacial segregation (1.46 million years ago). We identified several candidate genes that exhibited elevated divergence between the two species, including genes associated with substance metabolism, energy production, and response to environmental cues, all putative candidates closely linked to thermal adaptation expected from the latitude gradient. Interestingly, several candidates related to gamete recognition and time of puberty, and also exhibited elevated divergence, indicating their possible role in pre-zygote isolation and speciation of the two species. Our results would expand our knowledge on the roles of latitude climate adaptation and genetic drift in generating and maintaining biodiversity in marine teleosts.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3′-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 μg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3′-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(β1-4)GlcNAc further supports the abundance of 3′-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3′-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Effect of MHC and inbreeding on disassortative reproduction: A data revisit, extension and inclusion of fertilization in sand lizards

The harmful effects of close inbreeding have been recognized for centuries and, with the rise of Mendelian genetics, was realized to be an effect of homozygosis. This historical background led to great interest in ways to quantify inbreeding, its depression effects on the phenotype and flow-on effects on mate choice and other aspects of behavioral ecology. The mechanisms and cues used to avoid inbreeding are varied and include major histocompatibility complex (MHC) molecules and the peptides they transport as predictors of the degree of genetic relatedness. Here, we revisit and complement data from a Swedish population of sand lizards (Lacerta agilis) showing signs of inbreeding depression to assess the effects of genetic relatedness on pair formation in the wild. Parental pairs were less similar at the MHC than expected under random mating but mated at random with respect to microsatellite relatedness. MHC clustered in groups of RFLP bands but no partner preference was observed with respect to partner MHC cluster genotype. Male MHC band patterns were unrelated to their fertilization success in clutches selected for analysis on the basis of showing mixed paternity. Thus, our data suggest that MHC plays a role in pre-copulatory, but not post-copulatory partner association, suggesting that MHC is not the driver of fertilization bias and gamete recognition in sand lizards.

Genomic patterns of divergence in the early and late steps of speciation of the deep-sea vent thermophilic worms of the genus Alvinella

BackgroundThe transient and fragmented nature of the deep-sea hydrothermal environment made of ridge subduction, plate collision and the emergence of new rifts is currently acting to separate of vent populations, promoting local adaptation and contributing to bursts of speciation and species specialization. The tube-dwelling worms Alvinella pompejana called the Pompeii worm and its sister species A. caudata live syntopically on the hottest part of deep-sea hydrothermal chimneys along the East Pacific Rise. They are exposed to extreme thermal and chemical gradients, which vary greatly in space and time, and thus represent ideal candidates for understanding the evolutionary mechanisms at play in the vent fauna evolution.ResultsWe explored genomic patterns of divergence in the early and late stages of speciation of these emblematic worms using transcriptome assemblies and the first draft genome to better understand the relative role of geographic isolation and habitat preference in their genome evolution. Analyses were conducted on allopatric populations of Alvinella pompejana (early stage of separation) and between A. pompejana and its syntopic species Alvinella caudata (late stage of speciation). We first identified divergent genomic regions and targets of selection as well as their position in the genome over collections of orthologous genes and, then, described the speciation dynamics by documenting the annotation of the most divergent and/or positively selected genes involved in the isolation process. Gene mapping clearly indicated that divergent genes associated with the early stage of speciation, although accounting for nearly 30% of genes, are highly scattered in the genome without any island of divergence and not involved in gamete recognition or mito-nuclear incompatibilities. By contrast, genomes of A. pompejana and A. caudata are clearly separated with nearly all genes (96%) exhibiting high divergence. This congealing effect however seems to be linked to habitat specialization and still allows positive selection on genes involved in gamete recognition, as a possible long-duration process of species reinforcement.ConclusionOur analyses highlight the non-negligible role of natural selection on both the early and late stages of speciation in the iconic thermophilic worms living on the walls of deep-sea hydrothermal chimneys. They shed light on the evolution of gene divergence during the process of speciation and species specialization over a very long period of time.

Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria

BackgroundSpeciation genes contribute disproportionately to species divergence, but few examples exist, especially in vertebrates. Here we test whether Zan, which encodes the sperm acrosomal protein zonadhesin that mediates species-specific adhesion to the egg’s zona pellucida, is a speciation gene in placental mammals.ResultsGenomic ontogeny reveals that Zan arose by repurposing of a stem vertebrate gene that was lost in multiple lineages but retained in Eutheria on acquiring a function in egg recognition. A 112-species Zan sequence phylogeny, representing 17 of 19 placental Orders, resolves all species into monophyletic groups corresponding to recognized Orders and Suborders, with <5% unsupported nodes. Three other rapidly evolving germ cell genes (Adam2, Zp2, and Prm1), a paralogous somatic cell gene (TectA), and a mitochondrial gene commonly used for phylogenetic analyses (Cytb) all yield trees with poorer resolution than the Zan tree and inferior topologies relative to a widely accepted mammalian supertree. Zan divergence by intense positive selection produces dramatic species differences in the protein’s properties, with ordinal divergence rates generally reflecting species richness of placental Orders consistent with expectations for a speciation gene that acts across a wide range of taxa. Furthermore, Zan’s combined phylogenetic utility and divergence exceeds those of all other genes known to have evolved in Eutheria by positive selection, including the only other mammalian speciation gene, Prdm9.ConclusionsSpecies-specific egg recognition conferred by Zan’s functional divergence served as a mode of prezygotic reproductive isolation that promoted the extraordinary adaptive radiation and success of Eutheria.

Molecular Chaperone HSPA2 Distribution During Hyaluronic Acid Selection in Human Sperm

During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique.

Mechanisms of Sperm-Egg Interactions: What Ascidian Fertilization Research Has Taught Us.

Fertilization is an essential process in terrestrial organisms for creating a new organism with genetic diversity. Before gamete fusion, several steps are required to achieve successful fertilization. Animal spermatozoa are first activated and attracted to the eggs by egg-derived chemoattractants. During the sperm passage of the egg’s extracellular matrix or upon the sperm binding to the proteinaceous egg coat, the sperm undergoes an acrosome reaction, an exocytosis of acrosome. In hermaphrodites such as ascidians, the self/nonself recognition process occurs when the sperm binds to the egg coat. The activated or acrosome-reacted spermatozoa penetrate through the proteinaceous egg coat. The extracellular ubiquitin–proteasome system, the astacin-like metalloproteases, and the trypsin-like proteases play key roles in this process in ascidians. In the present review, we summarize our current understanding and perspectives on gamete recognition and egg coat lysins in ascidians and consider the general mechanisms of fertilization in animals and plants.

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 through functional divergence. Following duplication, paralogs may partition 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, suggestive of functional divergence 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.

Plasmodium falciparum Cysteine Rich Secretory Protein uniquely localizes to one end of male gametes

Fertilization is a central event during the life cycle of most eukaryotic organisms and involves gamete recognition and fusion, ultimately resulting in zygote formation. Gamete fertilization in the malaria-causing Plasmodium parasites occurs inside the mosquito midgut and represents a major bottleneck in the life cycle. Cysteine Rich Secretory Proteins (CRISPs) are key molecules involved in fertilization in vertebrates and the presence of a CRISP ortholog in human malaria infective Plasmodium falciparum suggested a possible role in fertilization. Strikingly, P. falciparum CRISP exhibited a unique terminal localization in the male microgamete. Parasites with a CRISP gene deletion (P. falciparum crisp−) proliferated asexually similar to wildtype NF54 parasites and differentiated into gametocytes. Further analysis showed that Plasmodium falciparum crisp− gametocytes underwent exflagellation to form male gametes and no apparent defect in transmission to the mosquito vector was observed. These data show that P. falciparum CRISP is a marker for the apical end of the microgamete and that it might only have an ancillary or redundant function in the male sexual stages.

Selection on genes associated with the evolution of divergent life histories: Gamete recognition or something else?

Gamete compatibility, and fertilization success, is mediated by gamete-recognition genes (GRGs) that are expected to show genetic evidence of a response to sexual selection associated with mating system traits. Changes in the strength of sexual selection can arise from the resolution of sperm competition among males, sexual conflicts of interest between males and females, or other mechanisms of sexual selection. To assess these expectations, we compared patterns of episodic diversifying selection among genes expressed in the gonads of Cryptasterina pentagona and C. hystera, which recently speciated and have evolved different mating systems (gonochoric or hermaphroditic), modes of fertilization (outcrossing or selfing), and dispersal (planktonic larvae or internal brooding). Cryptasterina spp. inhabit the upper intertidal of the coast of Queensland and coral islands of the Great Barrier Reef. We found some evidence for positive selection on a GRG in the outcrossing C. pentagona, and we found evidence of loss of gene function in a GRG of the self-fertilizing C. hystera. The modification or loss of gene functionality may be evidence of relaxed selection on some aspects of gamete interaction in C. hystera. In addition to these genes involved in gamete interactions, we also found genes under selection linked to abiotic stress, chromosomal regulation, polyspermy, and egg-laying. We interpret those results as possible evidence that Cryptasterina spp. with different mating systems may have been adapting in divergent ways to oxidative stress or other factors associated with reproduction in the physiologically challenging environment of the high intertidal. RESEARCH HIGHLIGHTS: Recent speciation between two sea stars was unlikely the result of selection on gamete-recognition genes annotated in this study. Instead, our results point to selection on genes linked to the intertidal environment and reproduction.

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.

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

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. Ecological and behavioral factors have been suggested to underlie stickleback reproductive isolation and incipient speciation, but reproductive proteins mediating gamete recognition during fertilization have so far remained unexplored. To begin to investigate the contribution of reproductive proteins to stickleback reproductive isolation, we have characterized the stickleback egg coat proteome. We find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3, as in other teleost fish. Our molecular evolutionary analyses indicate that across teleosts, ZP3 but not ZP1 has experienced positive Darwinian selection. Mammalian ZP3 is also rapidly evolving, and surprisingly some residues under selection in stickleback and mammalian ZP3 directly align. Despite broad homology, however, we find differences between mammalian and stickleback ZP proteins with respect to glycosylation, disulfide bonding, and sites of synthesis. Taken together, the changes we observe in 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.

Reproductive isolation between two cryptic sponge species in New Zealand: high levels of connectivity and clonality shape Tethya species boundaries

Although commonly reported among widespread marine taxa, hybridization has only been recorded for one species of marine sponge to date. Sponges have evolved an array of sexual and asexual reproductive strategies, but it remains unclear if the lack of reported hybrids in this phylum is due to their reproductive diversity (which promotes reproductive isolation) or instead due to a lack of data. In this study, we aimed to determine whether hybridization occurs between two cryptic species of Tethya burtoni (T. burtoni sp. A and T. burtoni sp. B) that live sympatrically in central New Zealand. We also examined how both small-scale population structure (for five locations) and asexual reproduction contributed to instances (or lack thereof) of hybridization for these sponges. Using 11 microsatellite markers, we found no evidence of hybridization between species. Both species exhibited differences in distribution, where one species was present at all five locations, but the other was only detected at three locations. For these three locations (all within 20 km of each other), both species exhibited high levels of gene flow. Asexual buds did not appear to disperse far, and groups of clonal individuals were found within areas of < 900 cm2. Asexual reproduction, therefore, did not play an obvious role in connectivity between populations, but instead appeared to be important for population maintenance. While specific mechanisms for reproductive isolation, such as gamete recognition, between both T. burtoni species remain unknown, we suggest that such mechanisms likely exist due to the strong differentiation found between both species (FST = 0.191, P < 0.0001). Further, the observed high levels of both gene flow and asexual reproduction may also act to reduce the potential for hybridization by maintaining genetic diversity and increasing the chance of mating with a conspecific individual.

Deleterious variants in genes regulating mammalian reproduction in Neanderthals, Denisovans and extant humans.

Were Neanderthals and Denisovans (referred here also as extinct hominidae) carrying deleterious variants in genes regulating reproduction? The majority of extinct hominidae analyzed here, presented a considerable number of deleterious variants per individual in proteins regulating different aspects of reproduction, including gonad and uterine function, and gametogenesis. Neanderthals, Denisovans and extant humans were interfertile and hybridized while occupying geographically overlapping areas in Europe and Asia. This is evidenced by the small archaic genome component (average ∼2%) present in non-African extant humans. The genome of eight extinct hominidae, together with five human genome databases, plus 44 mothers and 48 fathers (fertile controls), were screened to look for deleterious variants in 1734 protein-coding genes regulating reproduction. Ancient DNA from six Neanderthals and two Denisovans dated between ∼82000 and 43000 calibrated years was retrieved from the public European Nucleotide Archive. The hominins analyzed include Altai, Vindija 33.15, 33.19, 33.25 and 33.26, El Sidron 1253, Denisova 3 and 11. Their DNA was analyzed using the CLC Genomics Workbench 12, by mapping overlapping paired-end reads (Illumina, FASTQ files) to the human genome assembly GRCh37 (hg19) (Vindija 33.19, 33.25, 33.26, Denisova 3 and Denisova 11) or by analyzing BAM files (Altai, El Sidron 1253 and Vindija 33.15) (human genome reference, GRCh37 (hg19)). Non-synonymous reproductive variants were classified as deleterious or tolerated (PolyPhen-2 and SIFT analyses) and were compared to deleterious variants obtained from extant human genome databases (Genome Aggregation Database (GnomAD), 1000 Genomes, the Haplotype Map (HapMap), Single Nucleotide Polymorphism Database (dbSNPs)) across different populations. A genetic intersection between extant or extinct DNA variants and other genetic disorders was evaluated by annotating the obtained variants with the Clinical Variant (ClinVar) database. Among the eight extinct hominidae analyzed, a total of 9650 non-synonymous variants (only coverage ≥20 reads included; frameshift mutations were excluded) in 1734 reproductive protein-coding genes were found, 24% of which were classified as deleterious. The majority (73%) of the deleterious alleles present in extant humans that are shared between extant humans and extinct hominidae were found to be rare (<1%) in extant human populations. A set of 8044 variants were found uniquely in extinct hominidae. At the single-gene level, no extinct individual was found to be homozygous for deleterious variants in genes necessary for gamete recognition and fusion, and no higher chance of embryo-lethality (calculated by Mendelian Genetics) was found upon simulated mating between extant human and extinct hominidae compared to extant human-extant human. However, three of the eight extinct hominidae were found to be homozygous for 48-69 deleterious variants in 55 genes controlling ovarian and uterine functions, or oogenesis (AKAP1, BUB1B, CCDC141, CDC73, DUSP6, ESR1, ESR2, PATL2, PSMC3IP, SEMA3A, WT1 and WNT4). Moreover, we report the distribution of nine Neanderthal variants in genes associated with a human fertility phenotype found in extant human populations, one of which has been associated with polycystic ovarian syndrome and primary congenital glaucoma. While analyzing archaic DNA, stringent filtering criteria were adopted to screen for deleterious variants in Neanderthals and Denisovans, which could result in missing a number of variants. Such restraints preserve the potential for detection of additional deleterious variants in reproductive proteins in extinct hominidae. This study provides a comprehensive overview of putatively deleterious variants in extant human populations and extinct individuals occurring in 1734 protein-coding genes controlling reproduction and provides the fundaments for future functional studies of extinct variants in human reproduction. This study was supported by the Department of Biological Science and by the Office of Research and Sponsored Programs at the University of Tulsa (Faculty Research Grant and Faculty Research Summer Fellowship) to M.A. and the University of Tulsa, Tulsa Undergraduate Research Challenge (TURC) program to E.L.; no conflict of interest to declare. N/A.

Localización de arilsulfatasa A durante la incubación in vitro de espermatozoides humanos en medio de capacitación

ObjectiveTo determine the localization and distribution of the ArylsulfataseA receptor (ARSA) in human spermatozoa before and after their incubation in capacitation medium for 1 and 4hours. Material and methodsSemen samples were obtained from five normozoospermic donors. Capacitation was by swim-up technique using capacitation medium for 1 and 4hours. Localization of the ARSA receptor was assessed by indirect immunofluorescence using confocal microscopy. A minimum of 200cells were observed in each physiological condition. ResultsBefore incubation, no representative pattern was observed among the cells positive for this biomarker (8.61%). This percentage increased significantly after incubation in the capacitation medium for 1 and 4hours (61.86% and 63.38% respectively). A majority pattern was observed among the capacitated cells, with intense labelling in the acrosomal region (27.11% and 28.20% after 1 and 4hours respectively). It should be noted that the pattern corresponding to fluorescence at the level of the periacrosomal region was not observed in the spermatozoa prior to incubation. Only after incubation in capacitation medium for 1 and 4hours, 9.13% and 12.78% of cells with such distribution were detected. ConclusionsIn vitro capacitation, regardless of time, favours the immunolocalization of ARSA in the cephalic region of the spermatozoa. The most representative subpopulation after this process was the one in which ARSA was intensely and homogeneously distributed in the acrosome region, involved in primary gamete recognition.