Gamete Interaction Research Articles

Environmental epigenetic interaction of gametes and early embryos†.

In recent years, the developmental origins of diseases have been increasingly recognized and accepted. As such, it has been suggested that most adulthood chronic diseases such as diabetes, obesity, cardiovascular disease, and even tumors may develop at a very early stage. In addition to intrauterine environmental exposure, germ cells carry an important inheritance role as the primary link between the two generations. Adverse external influences during differentiation and development can cause damage to germ cells, which may then increase the risk of chronic disease development later in life. Here, we further elucidate and clarify the concept of gamete and embryo origins of adult diseases by focusing on the environmental insults on germ cells, from differentiation to maturation and fertilization.

174 The impact of human pappiloma virus (HPV) on intra uterine insemination (IUI) outcome

Introduction and aims of the study: Human Pappiloma Virus is a nonenveloped, circular, double stranded DNA virus. HPVs are generally classified as low and high risk, according to their potential to cause genital cancer. The virus pathogenesis process includes either an infectious virion-producing pathway or a non infectious cell-transforming, cancer-inducing pathway. HPV virus presence in spermatozoa is associated with detrimental effects on sperm parameters and gametes interaction, causing also temporal subfertility.

Density-dependent patterns of multivariate selection on sperm motility and morphology in a broadcast spawning mussel.

Sperm cells exhibit extraordinary phenotypic variation, both among taxa and within individual species, yet our understanding of the adaptive value of sperm trait variation across multiple contexts is incomplete. For species without the opportunity to choose mating partners, such as sessile broadcast spawning invertebrates, fertilization depends on gamete interactions, which in turn can be strongly influenced by local environmental conditions that alter the concentration of sperm and eggs. However, the way in which such environmental factors impact phenotypic selection on functional gamete traits remains unclear in most systems. Here, we analyze patterns of linear and nonlinear multivariate selection under experimentally altered local sperm densities (densities within the capture zone of eggs) on a range of functionally important sperm traits in the broadcast spawning marine mussel, Mytilus galloprovincialis. Specifically, we assay components of sperm motility and morphology across two fertilization environments that simulate either sperm limitation (when there are too few sperm to fertilize all available eggs), or sperm saturation (when there are many more sperm than required for fertilization, and the risk of polyspermy and embryonic failure is heightened). Our findings reveal that the strength, form, and targets of selection on sperm depend on the prevailing fertilization environment. In particular, our analyses revealed multiple significant axes of nonlinear selection on sperm motility traits under sperm limitation, but only significant negative directional selection on flagellum length under sperm saturation. These findings highlight the importance of local sperm densities in driving the adaptation of sperm phenotypes, particularly those related to sperm motility, in broadcast spawning invertebrates.

Fertilization of Ascidians: Gamete Interaction, Self/Nonself Recognition and Sperm Penetration of Egg Coat.

Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm and eggs must undergo complex processes in a sequential order. Fertilization of marine invertebrate Ciona intestinalis type A (Ciona robusta) has been studied for more than a hundred years. Ascidian sperm are attracted by chemoattractants from eggs and bind to the vitelline coat. Subsequently, sperm penetrate through the vitelline coat proteolytically and finally fuse with the egg plasma membrane. Here, we summarize the fertilization mechanisms of ascidians, particularly from sperm-egg interactions to sperm penetration of the egg coat. Since ascidians are hermaphrodites, inbreeding depression is a serious problem. To avoid self-fertilization, ascidians possess a self-incompatibility system. In this review, we also describe the molecular mechanisms of the self-incompatibility system in C. intestinalis type A governed by three allelic gene pairs of s-Themis and v-Themis.

Cysteine-Rich Secretory Proteins (CRISP) are Key Players in Mammalian Fertilization and Fertility.

Mammalian fertilization is a complex process involving a series of successive sperm-egg interaction steps mediated by different molecules and mechanisms. Studies carried out during the past 30 years, using a group of proteins named CRISP (Cysteine-RIch Secretory Proteins), have significantly contributed to elucidating the molecular mechanisms underlying mammalian gamete interaction. The CRISP family is composed of four members (i.e., CRISP1-4) in mammals, mainly expressed in the male tract, present in spermatozoa and exhibiting Ca2+ channel regulatory abilities. Biochemical, molecular and genetic approaches show that each CRISP protein participates in more than one stage of gamete interaction (i.e., cumulus penetration, sperm-ZP binding, ZP penetration, gamete fusion) by either ligand-receptor interactions or the regulation of several capacitation-associated events (i.e., protein tyrosine phosphorylation, acrosome reaction, hyperactivation, etc.) likely through their ability to regulate different sperm ion channels. Moreover, deletion of different numbers and combination of Crisp genes leading to the generation of single, double, triple and quadruple knockout mice showed that CRISP proteins are essential for male fertility and are involved not only in gamete interaction but also in previous and subsequent steps such as sperm transport within the female tract and early embryo development. Collectively, these observations reveal that CRISP have evolved to perform redundant as well as specialized functions and are organized in functional modules within the family that work through independent pathways and contribute distinctly to fertility success. Redundancy and compensation mechanisms within protein families are particularly important for spermatozoa which are transcriptionally and translationally inactive cells carrying numerous protein families, emphasizing the importance of generating multiple knockout models to unmask the true functional relevance of family proteins. Considering the high sequence and functional homology between rodent and human CRISP proteins, these observations will contribute to a better understanding and diagnosis of human infertility as well as the development of new contraceptive options.

Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm

Cell–cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete–specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species–specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species–specific sperm–egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism.

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor’s sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

The social shape of sperm: using an integrative machine-learning approach to examine sperm ultrastructure and collective motility.

Sperm is one of the most morphologically diverse cell types in nature, yet they also exhibit remarkable behavioural variation, including the formation of collective groups of cells that swim together for motility or transport through the female reproductive tract. Here, we take advantage of natural variation in sperm traits observed across Peromyscus mice to test the hypothesis that the morphology of the sperm head influences their sperm aggregation behaviour. Using both manual and automated morphometric approaches to quantify their complex shapes, and then statistical modelling and machine learning to analyse their features, we show that the aspect ratio of the sperm head is the most distinguishing morphological trait and statistically associates with collective sperm movements obtained from in vitro observations. We then successfully use neural network analysis to predict the size of sperm aggregates from sperm head morphology and show that species with relatively wider sperm heads form larger aggregates, which is consistent with the theoretical prediction that an adhesive region around the equatorial region of the sperm head mediates these unique gametic interactions. Together these findings advance our understanding of how even subtle variation in sperm design can drive differences in sperm function and performance.

Knockin' on Egg's Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species.

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

αV Integrin Expression and Localization in Male Germ Cells.

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

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.

Oolemma Receptors in Mammalian Molecular Fertilization: Function and New Methods of Study.

Fertilization is a key process in biology to the extent that a new individual will be born from the fusion of two cells, one of which leaves the organism in which it was produced to exert its function within a different organism. The structure and function of gametes, and main aspects of fertilization are well known. However, we have limited knowledge about the specific molecules participating in each of the steps of the fertilization process due to the transient nature of gamete interaction. Moreover, if we specifically focus in the fusion of both gametes’ membrane, we might say our molecular knowledge is practically null, despite that molecular mechanisms of cell-to-cell adhesion are well studied in somatic cells. Moreover, between both gametes, the molecular knowledge in the egg is even scarcer than in the spermatozoon for different reasons addressed in this review. Sperm-specific protein IZUMO1 and its oocyte partner, JUNO, are the first cell surface receptor pair essential for sperm–egg plasma membrane binding. Recently, thanks to gene editing tools and the development and validation of in vitro models, new oocyte molecules are being suggested in gamete fusion such as phosphatidylserine recognition receptors. Undoubtedly, we are in a new era for widening our comprehension on molecular fertilization. In this work, we comprehensively address the proposed molecules involved in gamete binding and fusion, from the oocyte perspective, and the new methods that are providing a better understanding of these crucial molecules.

Identification of Cell Surface Proteins of the Sea Star Mature Egg using Complimentary Methods

Multiple events occur between the sperm and egg membranes at fertilization, including sperm-egg binding, species-specific discrimination, membrane fusion, and egg activation. To date, only a handful of egg membrane proteins have been identified, and none of these are known to be involved in the egg activation pathway at fertilization. An excellent model organism to address how these events take place is the Pacific bat star, Patiria miniata, due to the quantity of synchronized oocytes that are large in size, nearly transparent, and amenable to many different techniques. To address this lack of knowledge regarding the egg cell surface, three databases were developed to explore the membrane proteome of the mature sea star egg. These databases utilized the first sea star mature egg transcriptome for the sea star, which has 96,723 unigenes and is available on NCBI (BioProject ID: 389668). The first database is a prediction of all mature egg transcripts that have putative transmembrane alpha-helical sequences using two different software programs: Phobius and TMHMM, which use different methods for prediction. After combining results from both programs, 15,501 transcripts were predicted to encode a transmembrane domain. The majority possessed a single transmembrane domain (9744); however, many were predicted to have multiple transmembrane domains. The second database categorized proteins into five major signaling families, each of which have been hypothesized to play a role in gamete interaction in different species. This database identified unique representatives of 26 Tetraspannins, 28 Receptor tyrosine kinases, 8 A Disintegrin and Metalloproteases (ADAMS), 140 G-protein coupled receptors, and 9 Integrins. The final database represents the membrane proteins currently present in the mature egg as identified by mass spectrometry. In the mature egg membrane preparation, 951 proteins were identified by mass spectrometry. Of these, 167 matched with the transcripts identified using the topology prediction software (Database 1) and 272 were matched with transcripts belonging to the five families of interest (Database 2). Interestingly, overlap between all three databases identified five proteins that are present in the unfertilized egg and belong to one of the signaling families predicted to be involved at the surface. These proteins were an integrin alpha and beta-like isoform, and three different tetraspannins. A major goal of this work is to provide targets for future functional studies of receptors involved in activation of fertilization at the egg surface.

Function of Small Peptides During Male-Female Crosstalk in Plants.

Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and development. Successful sexual reproduction in plants requires extensive communication between male and female gametophytes, their gametes, and with the surrounding sporophytic tissues. In the past decade, it has been well-documented that small peptides participate in many important reproductive processes such as self-incompatibility, pollen tube growth, pollen tube guidance, and gamete interaction. Here, we provide a comprehensive overview of the peptides regulating the processes of male-female crosstalk in plant, aiming at systematizing the knowledge on the sexual reproduction, and signaling of plant peptides in future.

Evolutionary consequences of environmental effects on gamete performance.

Variation in pre- and post-release gamete environments can influence evolutionary processes by altering fertilization outcomes and offspring traits. It is now widely accepted that offspring inherit epigenetic information from both their mothers and fathers. Genetic and epigenetic alterations to eggs and sperm-acquired post-release may also persist post-fertilization with consequences for offspring developmental success and later-life fitness. In externally fertilizing species, gametes are directly exposed to anthropogenically induced environmental impacts including pollution, ocean acidification and climate change. When fertilization occurs within the female reproductive tract, although gametes are at least partially protected from external environmental variation, the selective environment is likely to vary among females. In both scenarios, gamete traits and selection on gametes can be influenced by environmental conditions such as temperature and pollution as well as intrinsic factors such as male and female reproductive fluids, which may be altered by changes in male and female health and physiology. Here, we highlight some of the pathways through which changes in gamete environments can affect fertilization dynamics, gamete interactions and ultimately offspring fitness. We hope that by drawing attention to this important yet often overlooked source of variation, we will inspire future research into the evolutionary implications of anthropogenic interference of gamete environments including the use of assisted reproductive technologies. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Human Zona Pellucida Glycoproteins: Binding Characteristics With Human Spermatozoa and Induction of Acrosome Reaction.

Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes encoding human ZP glycoproteins are one of the causative factors leading to abnormal ZP matrix and infertility in women. Relevance of the human ZP glycoproteins in ‘sperm–oocyte’ binding has been delineated by using either transgenic animal models expressing human zona proteins or purified native/recombinant human zona proteins. Studies based on the purified native/recombinant human zona proteins revealed that ZP1, ZP3, and ZP4 primarily bind to the capacitated acrosome-intact human spermatozoa whereas ZP2 binds to acrosome-reacted spermatozoa. On the contrary, human spermatozoa binds to the eggs obtained from transgenic mouse lines expressing human ZP2 but not to those expressing human ZP1, ZP3, and ZP4 suggesting that ZP2 has an important role in human ‘sperm–oocyte’ binding. Further studies using transgenic mouse lines showed that the N-terminus of human ZP2 mediate the taxon-specific human sperm–oocyte binding. Both glycans and protein-protein interactions have a role in human gamete interaction. Further studies have revealed that the purified native/recombinant human ZP1, ZP3, and ZP4 are competent to induce acrosome reaction. Human sperm binds to the mouse transgenic eggs expressing human ZP1-4 instead of mouse ZP1-3 proteins, penetrated the ZP matrix and accumulated in the perivitelline space, which were acrosome-reacted suggesting that human ZP2 in transgenic mouse model also induce acrosome reaction. In humans N-linked glycosylation of zona proteins have been shown to play an important role in induction of the acrosome reaction. Hence in humans, based on studies using transgenic mouse model as well as purified native/recombinant zona proteins, it is likely that more than one zona protein is involved in the ‘sperm–oocyte’ binding and induction of the acrosome reaction.

ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction.

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Mannosylated glycoconjugates on the surface of activated sperm in the giant freshwater prawn are crucial for sperm binding with the egg vitelline envelop.

The unusual morphology and poorlydefined acrosome-like structure in the mature sperm of the giant freshwater prawn Macrobrachium rosenbergii has led to difficulties in identifying the state of sperm activation. Mature distal vas deferens sperm (dVSp) can be activated by the calcium ionophore A23187 to show acrosome reaction-like enzymatic activities that increase their binding and penetration capabilities. However, these short-lived enzymatic activities limit their usefulness as a marker of sperm activation for further qualitative and quantitative analyses, leading to our examining the alterations in the exposureof sperm surface glycoconjugates both as markers of sperm activation and for their role in gamete interaction. Our results showed that after A23187 treatment, there was an increased exposure of mannosylated glycoconjugates on the sperm surface revealed by significant Concanavalin A (Con A) staining. Furthermore, sodium metaperiodate pre-treatment, Con A pre-incubation, or co-incubation with α-mannose monosaccharides all significantly reduced A23187-induced dVSp binding to the egg vitelline envelop, demonstrating the importance of sperm surface mannosylated glycoconjugates in the binding process. These same pre-treatments of sperm also resulted in the inhibition of thebinding of soluble vitelline envelop proteins (MrVE) to both the sperm surface and to mannosylated dVSp protein bands. Therefore, the present study demonstrated the importance of the exposure of mannosylated glycoconjugates on the surface of activated dVSp, both as a reliable marker of sperm activation and as a binding factor in the gamete interaction process. Furthermore, these findings allow for a better understanding of the surface glycoconjugate-mediated interaction process between gametes in this species of prawn.

Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection.

Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation; instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the utero-tubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37°C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time- and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating ( Ded et al., 2020 ). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm.

Indirect parental effects on offspring viability by egg-derived fluids in an external fertilizer.

The capacity for parents to influence offspring phenotypes via nongenetic inheritance is currently a major area of focus in evolutionary biology. Intriguing recent evidence suggests that sexual interactions among males and females, both before and during mating, are important mediators of such effects. Sexual interactions typically extend beyond gamete release, involving both sperm and eggs, and their associated fluids. However, the potential for gamete-level interactions to induce nongenetic parental effects remains under-investigated. Here, we test for such effects using an emerging model system for studying gamete interactions, the external fertilizer Mytilus galloprovincialis. We employed a split-ejaculate design to test whether exposing sperm to egg-derived chemicals (ECs) from a female would affect fertilization rate and offspring viability when those sperm were used to fertilize a different female's eggs. We found separate, significant effects of ECs from non-fertilizing females on both fertilization rate and offspring viability. The offspring viability effect indicates that EC-driven interactions can have nongenetic implications for offspring fitness independent of the genotypes inherited by those offspring. These findings provide a rare test of indirect parental effects driven exclusively by gamete-level interactions, and to our knowledge the first evidence that such effects occur via the gametic fluids of females.