Gamete Fusion Research Articles

Zygospore formation in Zygnematophyceae predates several land plant traits.

Recent research on a special type of sexual reproduction and zygospore formation in Zygnematophyceae, the sister group of land plants, is summarized. Within this group, gamete fusion occurs by conjugation. Zygospore development in Mougeotia, Spirogyra and Zygnema is highlighted, which has recently been studied using Raman spectroscopy, allowing chemical imaging and detection of changes in starch and lipid accumulation. Three-dimensional reconstructions after serial block-face scanning electron microscopy (SBF-SEM) or focused ion beam SEM (FIB-SEM) made it possible to visualize and quantify cell wall and organelle changes during zygospore development. The zygospore walls undergo strong modifications starting from uniform thin cell walls to a multilayered structure. The mature cell wall is composed of a cellulosic endospore and exospore and a central mesospore built up by aromatic compounds. In Spirogyra, the exospore and endospore consist of thick layers of helicoidally arranged cellulose fibrils, which are otherwise only known from stone cells of land plants. While starch is degraded during maturation, providing building blocks for cell wall formation, lipid droplets accumulate and fill large parts of the ripe zygospores, similar to spores and seeds of land plants. Overall, data show similarities between streptophyte algae and embryophytes, suggesting that the genetic toolkit for many land plant traits already existed in their shared algal ancestor. This article is part of the theme issue 'The evolution of plant metabolism'.

The signal that stimulates mammalian embryo development.

Embryo development is stimulated by calcium (Ca2+) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a "toolkit", a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca2+ from the egg's intracellular stores which in mammals is followed by repetitive Ca2+ spikes known as Ca2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca2+ signal and describe the steps of the signal transduction mechanism that activates the egg's developmental program and turns it into an embryo.

Single-cell transcriptomics reveal transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis

The Plasmodium falciparum life cycle includes obligate transition between a human and mosquito host. Gametocytes are responsible for transmission from the human to the mosquito vector where gamete fusion followed by meiosis occurs. To elucidate how male and female gametocytes differentiate in the absence of sex chromosomes, we perform FACS-based cell enrichment of a P. falciparum gametocyte reporter line followed by single-cell RNA-seq. In our analyses we define the transcriptional programs and predict candidate driver genes underlying male and female development, including genes from the ApiAP2 family of transcription factors. A motif-driven, gene regulatory network analysis indicates that AP2-G5 specifically modulates male development. Additionally, genes linked to the inner membrane complex, involved in morphological changes, are uniquely expressed in the female lineage. The transcriptional programs of male and female development detailed herein allow for further exploration of the evolution of sex in eukaryotes and provide targets for future development of transmission blocking therapies.

The emerging role of cysteine-rich peptides in pollen-pistil interactions.

Unlike early land plants, flowering plants have evolved a pollen tube that transports a pair of non-motile sperm cells to the female gametophyte. This process, known as siphonogamy, was first observed in gymnosperms and later became prevalent in angiosperms. However, the precise molecular mechanisms underlying the male-female interactions remain enigmatic. From the landing of the pollen grain on the stigma to gamete fusion, the male part needs to pass various tests: how does the stigma distinguish between compatible and incompatible pollen? what mechanisms guide the pollen tube towards the ovule? what factors trigger pollen tube rupture? how is polyspermy prevented? and how does the sperm cell ultimately reach the egg? Successful male-female communication is essential for surmounting these challenges, with cysteine-rich peptides (CRPs) playing a pivotal role in this dialogue. In this review, we summarize the characteristics of four distinct classes of CRPs, systematically review recent progress in the role of CRPs in four crucial stages of pollination and fertilization, consider potential applications of this knowledge in crop breeding, and conclude by suggesting avenues for future research.

Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse.

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

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.

What determines mate choices? Heterospecific mating in Sympetrum dragonflies

Abstract Heterospecific interactions are an important phenomenon of animal epigamic behaviour; however, they remain understudied within important groups, such as insects. Dragonflies are an ideal group for research on interspecific mating and possible hybridisation as they are easily identifiable, large insects, with a conspicuous mating process, often striking territorial behaviour and several mating systems. Using the genus Sympetrum as the model group, we examined heterospecific mating at three different levels. In the field, we identified whether species identity, time and weather affected heterospecific mating frequency. One important part of heterospecific mating is whether the process is completed. For dragonflies, this means that flying in tandem is followed by successful copulation, gamete fusion and oviposition (which comprise mating completeness). In a mesocosm experiment, we determined mating completeness (tandems, copulation, oviposition) of hetero‐ and homospecific pairs and the possible role of species density in heterospecific mating. In the laboratory, we compared the viability of the offspring from heterospecific pairs with different epigamous behaviour. We found heterospecific mating to be a relatively common phenomenon unaffected by environmental variables, that was primarily influenced by species identity, temporal distribution and abundance of dragonfly species. Consequently, the presence of counterparts of other species is the main predictor of the frequency of heterospecific mating. The probability of completed epigamic behaviour (copulation and subsequent oviposition) connected with gamete fusion is lower in heterospecific mating. Generally, based on our results we can assume that successful heterospecific mating (leading to gamete fusion) occurs in closely related species (e.g., Sympetrum striolatum and Sympetrum vulgatum). However, as pre‐copulatory barriers are not strongly developed in some dragonfly groups, less closely related species (e.g., Sympetrum sanguineum and S. striolatum) also may mate. This phenomenon requires further study as it may present a threat to the survival of some species in the context of changing environmental conditions, including climate change.

Sperm induction of somatic cell-cell fusion as a novel functional test.

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

Genome dosage alteration caused by chromosome pyramiding and shuffling effects on karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids.

We developed an array of Zea-Tripsacum tri-hybrid allopolyploids with multiple ploidies. We unveiled that changes in genome dosage due to the chromosomes pyramiding and shuffling of three species effects karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids. Polyploidy, or whole genome duplication, has played a major role in evolution and speciation. The genomic consequences of polyploidy have been extensively studied in many plants; however, the extent of chromosomal variation, genome dosage, phenotypic diversity, and heterosis in allopolyploids derived from multiple species remains largely unknown. To address this question, we synthesized an allohexaploid involving Zea mays, Tripsacum dactyloides, and Z. perennis by chromosomal pyramiding. Subsequently, an allooctoploid and an allopentaploid were obtained by hybridization of the allohexaploid with Z. perennis. Moreover, we constructed three populations with different ploidy by chromosomal shuffling (allopentaploid × Z. perennis, allohexaploid × Z. perennis, and allooctoploid × Z. perennis). We have observed 3 types of sexual reproductive modes and 2 types of asexual reproduction modes in the tri-species hybrids, including 2n gamete fusion (2n + n), haploid gamete fusion (n + n), polyspermy fertilization (n + n + n) or 2n gamete fusion (n + 2n), haploid gametophyte apomixis, and asexual reproduction. The tri-hybrids library presents extremely rich karyotype heterogeneity. Chromosomal compensation appears to exist between maize and Z. perennis. A rise in the ploidy of the trihybrids was linked to a higher frequency of chromosomal translocation. Variation in the degree of phenotypic diversity observed in different segregating populations suggested that genome dosage effects phenotypic manifestation. These findings not only broaden our understanding of the mechanisms of polyploid formation and reproductive diversity but also provide a novel insight into genome pyramiding and shuffling driven genome dosage effects and phenotypic diversity.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

Phospholipase C Zeta 1 (PLCZ1): The Function and Potential for Fertility Assessment and In Vitro Embryo Production in Cattle and Horses.

Phospholipase C Zeta 1 (PLCZ1) is considered a major sperm-borne oocyte activation factor. After gamete fusion, PLCZ1 triggers calcium oscillations in the oocyte, resulting in oocyte activation. In assisted fertilization, oocyte activation failure is a major cause of low fertility. Most cases of oocyte activation failures in humans related to male infertility are associated with gene mutations and/or altered PLCZ1. Consequently, PLCZ1 evaluation could be an effective diagnostic marker and predictor of sperm fertilizing potential for in vivo and in vitro embryo production. The characterization of PLCZ1 has been principally investigated in men and mice, with less known about the PLCZ1 impact on assisted reproduction in other species, such as cattle and horses. In horses, sperm PLCZ1 varies among stallions, and sperm populations with high PLCZ1 are associated with cleavage after intracytoplasmic sperm injection (ICSI). In contrast, bull sperm is less able to initiate calcium oscillations and undergo nuclear remodeling, resulting in poor cleavage after ICSI. Advantageously, injections of PLCZ1 are able to rescue oocyte failure in mouse oocytes after ICSI, promoting full development and birth. However, further research is needed to optimize PLCZ1 diagnostic tests for consistent association with fertility and to determine whether PLCZ1 as an oocyte-activating treatment is a physiological, efficient, and safe method for improving assisted fertilization in cattle and horses.

Sperm-Induced Ca2+ Release in Mammalian Eggs: The Roles of PLCζ, InsP3, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca2+ concentration. These Ca2+ oscillations are due to the production of InsP3 within the egg and the subsequent release of Ca2+ from the endoplasmic reticulum into the cytosol. The generation of InsP3 is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP3 production and Ca2+ release which then stimulates further InsP3 production. Most cytosolic Ca2+ increases in eggs at fertilization involve a fast Ca2+ wave; however, due to the limited diffusion of InsP3, this means that InsP3 must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca2+ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr2+ medium stimulates Ca2+ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP3 receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca2+ oscillations.

The of Effects of Light and a Combination of Growth Regulators on the Induction of Somatic Embryogenesis in Orchid Rhynchostylis gigantea (LindI.) Ridl.

The squirrel-tail orchid (Rhynchostylis gigantea) belongs to the Orchidaceae family. This orchid is indigenous to Southeast Asia and is scented and arranged in a bouquet of dangling stems. Its uniqueness makes it commercially desirable. In vitro culture has been utilized for orchid proliferation for a very long time to aid in the development of orchid seedlings. However, not all orchid species react the same way. Through somatic embryogenesis without gamete fusion, in vitro culture techniques can produce new plants during the entire embryonic phase. Endogenous and exogenous hormones, as well as light, influence the success of embryogenesis. Determining the optimal environmental parameters (light, combination and concentration of growth regulators, and their interaction) for inducing somatic embryogenesis from leaf explants in R. gigantea is the purpose of the present study. The leaves of R. gigantea clone 19 were utilized as explants in this investigation, utilizing a completely random factorial design. The first factor is a combination of growth regulator types and concentrations, and the second factor is light. Explants that are alive and growing at a rapid rate indicate that the in vitro culture is successful. Light and the combination of growth regulators significantly affected the percentage of viable explants, the beginning date of callus formation, and the number of embryogenic calluses produced. The interaction of two components (light and a combination of growth regulators) did not affect the three characteristics, with the exception of the proportion of somatic embryogenesis. Incubation in the dark is the optimal environment for initiating somatic embryogenesis in explants. The optimal combination of plant growth regulators for inducing somatic embryogenesis was 0.5 mgL-1 TDZ and 0.1 mgL-1 BAP. Bright light and a concentration of 1.0 mgL-1 TDZ + 0.1 mgL-1 BAP were the optimal interaction conditions for the induction of somatic embryogenesis in R. gigantea.

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca 2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1 , the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca 2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure.

Swimming zygote formation mediated by symmetrically arranged cell fusion sites in the gametes of Chlamydomonas eugametos (Chlorophyceae, Chlorophyta)

SUMMARYThe spatial arrangement of cell fusion sites and eyespots of the gametes in vis‐à‐vis mating pairs of Chlamydomonas eugametos, corresponding to the swimming zygote of Chlamydomonas reinhardtii, were studied using field emission scanning electron microscopy and light microscopy, respectively. After the adhesion of mt+ and mt− gametes, each of which has one eyespot and two flagella elongated from the cell anterior, gamete–gamete fusion occurred at the anterior papilla on the cell apex of each gamete, leading to the formation of a vis‐à‐vis mating pair, indicating that the cell fusion site is arranged symmetrically relative to the flagellar beat plane. In vis‐à‐vis mating pairs, two eyespots could be seen on the same or on opposite sides of the cell. As only the mt+ flagella and eyespot are functional for swimming and phototaxis in the vis‐à‐vis mating pair of C. eugametos, it is suggested that coordinated alignment of the parental gamete flagella and eyespots in the swimming zygote and asymmetric positioning of the gamete mating structure/cell fusion sites, which is prevalent in C. reinhardtii and other chlorophytes, is not necessary for C. eugametos.

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

The biphasic life cycle of the non-spinose planktonic foraminifera is characterised by an aberrant coiling signature

The complete lifecycle of the planktonic foraminifera has continued to remain obscure. Benthic foraminifera exhibit an obligate alternation of generations between haploid gamonts and diploid agamonts, yet despite years of observation, the fusion of gametes to form a zygote, gamont or agamont has never been observed in the planktonic groups. Recent evidence from Neogloboquadrina pachyderma culture confirms that they do have a biphasic reproductive cycle and the agamonts may exhibit dimorphism in coiling direction. In this study we examine the morphology of normal and aberrant coiling tests of both N. pachyderma and N. incompta from three Arctic and North Atlantic water masses, to explore whether aberrant coiling is an indicator of a biphasic life cycle in the non-spinose planktonic foraminifera group as a whole. Using a Nano-Computed Tomography scanning approach, we morphologically compared the proloculus and growth trajectories of aberrant and normal coiling tests. Aberrant coiling tests were found to have a slightly larger proloculus and test size, consistent with a left coiling N. pachyderma agamont in the literature. This degree of dimorphism is insufficient to discriminate agamonts in the wild population and coiling direction remains their only distinguishable feature without further morphological investigation. The constant low-level aberrant coiling signature in a range of non-spinose planktonic foraminiferal genera implies that a biphasic life cycle is the principal mode of reproduction in the non-spinose planktonic group.