Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

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.

Has the concept of polyspermy prevention been invented in the laboratory?

There is no evidence, nor need, for a fast block to polyspermy in animal oocytes. The idea that oocytes have evolved a mechanism to allow the entry of one spermatozoon and repel all others has, however, gained consensus over the last century. The main culprit is the sea urchin, which has been used for over a century in in vitro studies of the fertilization process. Images of sea urchin oocytes with thousands of sperm attached to the surface are commonplace in textbooks and appeal to the nature of the reader implying an intriguing surface mechanism of sperm selection despite these oocytes being fixed for photography (Figure ). The abundance of gametes in this marine invertebrate and the ease of experimentation have given us the possibility to elucidate many aspects of the mechanism of fertilization, but has also led to ongoing controversies in reproductive biology, one being polyspermy prevention. Kinetic experiments by Rothschild and colleagues in the 1950s led to the hypothesis of a fast partial block to polyspermy in sea urchin oocytes that reduced the probability of a second spermatozoon from entering the oocyte by 1/20th. In the 1970s, Jaffe and colleagues suggested, with circumstantial evidence, that this partial block was due to the sperm-induced depolarization of the oocyte plasma membrane. However, the fate of supernumerary spermatozoa is determined well before the plasma membrane of the oocyte depolarizes. Transmembrane voltage does not serve to regulate sperm entry. Scholastic texts have inadvertently promulgated this concept across the animal kingdom with no logical correlation or experimentation and, as of today, a molecular mechanism to regulate sperm entry in oocytes has not been identified.

MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans.

In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.

Exposure to Cumulus Cell Secretome Improves Sperm Function: New Perspectives for Sperm Selection In Vitro.

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.

Species-Specific Gamete Interaction during Sea Urchin Fertilization: Roles of the Egg Jelly and Vitelline Layer.

In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications (the acrosome reaction, AR) upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs, Paracentrotus lividus eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied P. lividus eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca2+ signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.

Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Assisted Reproduction.

Remotely controllable microrobots are appealing for various biomedical in vivo applications. In particular, in recent years, our group has focused on developing sperm-microcarriers to assist sperm cells with motion deficiencies or low sperm count (two of the most prominent male infertility problems) to reach the oocyte toward in-vivo-assisted fertilization. Different sperm carriers, considering their motion in realistic media and confined environments, have been optimized. However, the already-reported sperm carriers have been mainly designed to transport single sperm cell, with limited functionality. Thus, to take a step forward, here, the development of a 4D-printed multifunctional microcarrier containing soft and smart materials is reported. These microcarriers can not only transport and deliver multiple motile sperm cells, but also release heparin and mediate local enzymatic reactions by hyaluronidase-loaded polymersomes (HYAL-Psomes). These multifunctional facets enable in situ sperm capacitation/hyperactivation, and the local degradation of the cumulus complex that surrounds the oocyte, both to facilitate the sperm-oocyte interaction for the ultimate goal of in vivo assisted fertilization.

Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum (ER) specializations (cortical ER clusters), which are thought to be essential for egg activation. In examination of cortical ER clusters, we observed that they were tethered to previously unreported fenestrae within the cortical actin layer. Furthermore, studies demonstrated that sperm preferentially bind to the plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global change in volume and shape that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males. In addition to these global changes, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, and then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

Involvement of cellular protrusions in gamete interactions.

Gamete fusion is of considerable importance in reproductive events, as it determines the gamete pairs or chromosomes that the next generation will inherit. To preserve species specificity with an appropriate karyotype, the fusion between gametes requires regulatory mechanisms to ensure limited fusion competency. In many organisms, gamete surfaces are not smooth, but present constitutive or transient cellular protrusions suggested to be involved in gamete fusion. However, the molecular mechanisms and the factors essential for the membrane-membrane fusion process and cellular protrusion involvement have remained unclear. Recent advances in the identification and functional analysis of the essential factors for gamete interaction have revealed the molecular mechanisms underlying their activity regulation and dynamics. In homogametic fertilization, dynamic regulation of the fusion core machinery on cellular protrusions was precisely uncovered. In heterogametic fertilization, oocyte fusion competency was suggested to correlate with the compartmentalization of the fusion essential factor and protrusion formation. These findings shed light on the significance of cellular protrusions in gamete fusion as a physically and functionally specialized site for cellular fusion. In this review, we consider the developments in gamete interaction research in various species with different fertilization modes, highlighting the commonalities in the relationship between gamete fusion and cellular protrusions.

Multifaceted roles of the egg perivitelline layer in avian reproduction: Functional insights from the proteomes of chicken egg inner and outer sublayers.

The avian egg perivitelline layer (PL) is a proteinaceous structure that encloses the egg yolk. It consists of the inner and the outer perivitelline layers (IPL and OPL, respectively) that are assumed to play distinct roles in bird reproduction. To gain insight into their respective function, we analyzed the proteome of IPL and OPL in chicken unfertilized eggs after mechanical separation, using a GeLC-MS/MS strategy. Of the 412 proteins identified, 173 proteins were uniquely recovered in IPL and 98 proteins in OPL, while 141 proteins were identified in both sublayers. Genes coding the most abundant proteins were shown to be expressed either in the liver/ovary (IPL formation) or in the oviduct (OPL formation), but rarely in both. The presence of oviduct-specific proteins (including LYZ, VMO1, AvBD11, PTN, OVAL and LOC10175704) in IPL strongly suggests that they participate in the physical association of IPL to OPL, whose tight attachment was further evidenced by analyses of IPL/OPL interfaces (by scanning electron microscopy). Functional annotation of identified proteins revealed functions associated with fertilization and early development for IPL, while OPL would rather participate in egg defense and embryogenesis. Collectively, our data highlight the complementary functions of IPL and OPL that are major determinants of bird reproductive success. SIGNIFICANCE: The present study unveils for the first time the individual proteomes of the two sublayers composing the chicken egg perivitelline layer (PL), which allowed to assign their respective putative biological roles in avian reproduction. The combination of proteomics with gene expression and ultrastructural analyses provides insightful data on the structure and biochemistry of the avian PL. The functional annotation of PL proteins highlights the multifaceted biological functions of this structure in reproduction including fertilization, embryonic development, and antimicrobial protection. This work will stimulate further research to validate predicted functions and to compare the physiology and the functional specificities of PL in egg-laying species.

A stallion spermatozoon's journey through the mare's genital tract: In vivo and in vitro aspects of sperm capacitation.

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these 'fertilization supporting factors' would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes.

Microenvironment of the male and female reproductive tracts regulate sperm fertility: Impact of viscosity, pH, and osmolality.

BACKGROUND: Terminally differentiated mammalian sperm are exposed to gradients of viscosity, pH, and osmolality both in the male and female reproductive tract during their perilous journey to quest the ovum. The complex physicochemical factors play an integral role in preparing sperm for the fertilization process. OBJECTIVES: To elucidate the influence of the reproductive tract microenvironment especially viscosity, pH, and osmolality in regulating sperm functional and fertilization competence. MATERIALS AND METHODS: The data used in this review were collected from the research papers and online databases focusing on the influence of viscosity, pH, and osmolality on sperm function. DISCUSSION: The gradients of viscosity, pH, and osmolality exist across various segments of the male and female reproductive tract. The changes in the viscosity create a physical barrier, pH aid in capacitation and hyperactivation, and the osmotic stress selects a progressive sperm subpopulation for accomplishing fertilization. The sperm function tests are developed based on the concept that the male genotype is the major contributor to the reproductive outcome. However, recent studies demonstrate the significance of sperm genotype-environment interactions that are essentially contributing to reproductive success. Hence, it is imperative to assess the impact of physicochemical stresses and the adaptive ability of the terminally differentiated sperm, which in turn would improve the outcome of the assisted reproductive technologies and male fertility assessment. CONCLUSION: Elucidating the influence of the reproductive tract microenvironment on sperm function provides newer insights into the procedures that need to be adopted for selecting fertile males for breeding, and ejaculates for the assisted reproductive technologies.

Sperm bauplan and function and underlying processes of sperm formation and selection.

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.

Role of Integrins in Sperm Activation and Fertilization.

In mammals, integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrin receptors are an important part of signalization pathways and have an ability to transmit signals into and out of cells and participate in cell activation. In addition to somatic cells, integrins have also been detected on germ cells and are known to play a crucial role in complex gamete-specific physiological events, resulting in sperm-oocyte fusion. The main aim of this review is to summarize the current knowledge on integrins in reproduction and deliver novel perspectives and graphical interpretations presenting integrin subunits localization and their dynamic relocation during sperm maturation in comparison to the oocyte. A significant part of this review is devoted to discussing the existing view of the role of integrins during sperm migration through the female reproductive tract; oviductal reservoir formation; sperm maturation processes ensuing capacitation and the acrosome reaction, and their direct and indirect involvement in gamete membrane adhesion and fusion leading to fertilization.

Mammalian sperm hyperactivation regulates navigation via physical boundaries and promotes pseudo-chemotaxis.

Mammalian sperm migration within the complex and dynamic environment of the female reproductive tract toward the fertilization site requires navigational mechanisms, through which sperm respond to the tract environment and maintain the appropriate swimming behavior. In the oviduct (fallopian tube), sperm undergo a process called "hyperactivation," which involves switching from a nearly symmetrical, low-amplitude, and flagellar beating pattern to an asymmetrical, high-amplitude beating pattern that is required for fertilization in vivo. Here, exploring bovine sperm motion in high-aspect ratio microfluidic reservoirs as well as theoretical and computational modeling, we demonstrate that sperm hyperactivation, in response to pharmacological agonists, modulates sperm-sidewall interactions and thus navigation via physical boundaries. Prior to hyperactivation, sperm remained swimming along the sidewalls of the reservoirs; however, once hyperactivation caused the intrinsic curvature of sperm to exceed a critical value, swimming along the sidewalls was reduced. We further studied the effect of noise in the intrinsic curvature near the critical value and found that these nonthermal fluctuations yielded an interesting "Run-Stop" motion on the sidewall. Finally, we observed that hyperactivation produced a "pseudo-chemotaxis" behavior, in that sperm stayed longer within microfluidic chambers containing higher concentrations of hyperactivation agonists.

The Fertilization Enigma: How Sperm and Egg Fuse.

Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm-egg fusion, have remained mysterious for many decades. Here, we summarize our current molecular understanding of sperm-egg interaction, focusing mainly on mammalian fertilization. Given the fundamental importance of sperm-egg fusion yet the lack of knowledge of this process in vertebrates, we discuss hallmarks and emerging themes of cell fusion by drawing from well-studied examples such as viral entry, placenta formation, and muscle development. We conclude by identifying open questions and exciting avenues for future studies in gamete fusion.

The potential role of assisted oocyte activation in the management of a POSEIDON group 3 patient with fertilization failure: A case report and literature review.

OBJECTIVE: Assisted oocyte activation combined with ICSI (ICSI-AOA) has been reported to improve fertilization outcomes of couples with oocyte activation deficiency (OAD). Although there's no sufficient evidence to support ICSI-AOA as routine use, it might be beneficial for POSEIDON group 3 patients with suspected oocyte-related OAD. CASE REPORT: A 29-year-old female presented with a history of primary infertility for two years. She was classified as a POSEIDON group 3 patient and had a total fertilization failure history. With the help of ICSI-AOA, six oocytes were successfully fertilized. Pregnancy was later confirmed after embryo transfer. A living infant was born after 34 weeks of pregnancy. CONCLUSION: OAD should be taken into consideration for POSEIDON group 3 patients since low Antimüllerian hormone is associated decreased quality. Further research needs to be done to understand the mechanism underlying oocyte-related OAD and the potential role of ICSI-AOA in young patients with suboptimal ovarian response.

Limited proteolysis by acrosin affects sperm-binding and mechanical resilience of the mouse zona pellucida.

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Methyl-beta cyclodextrin and creatine work synergistically under hypoxic conditions to improve the fertilization ability of boar ejaculated sperm.

Although successful fertilization is completed by only 150 sperm in the pig oviduct, more than 50,000 sperms are required to achieve a fertilization rate of more than 70% by pig in vitro fertilization (IVF). In this study, to improve the efficiency of pig IVF, the effects of hypoxic conditions and treatment with creatine and methyl-beta cyclodextrin (MßCD) on the glycolytic pathway were investigated. Under low O2 conditions, zig-zag motility was strongly induced within 30 min; however, the induction disappeared at 60 min. Although caffeine suppressed zig-zag motility under low O2 conditions, creatine induced and sustained zig-zag motility until 120 min. Additionally, pretreatment with MßCD for 15 min greatly enhanced zig-zag motility via ATP production in sperm incubated with creatine under low O2 conditions. Sperm pretreated with MßCD were used for IVF in medium containing creatine under low O2 conditions. A fertilization rate of approximately 70% was achieved with only 1.0 x 104 sperms/mL, and there were few polyspermic embryos. Therefore, our novel method was beneficial for efficient production of pig embryos in vitro. Moreover, the zig-zag motility may be a novel movement which boar capacitated sperm exhibit in the culture medium.