In mammals, eggs exhibit a series of transient increases in intracellular calcium ion (Ca²⁺) concentrations (Ca²⁺ oscillations) at the time of fertilization, which are prerequisite and sufficient for egg activation. Recent studies have been revealing the molecular mechanism of how sperm induce Ca²⁺ oscillations in fertilized eggs, that has remained unclear for a longtime. Especially in mammals, the sperm factor theory is being confirmed, which postulates that a cytoplasmic factor in the spermatozoon (the "sperm factor") is introduced into the egg through the sperm-egg cytoplasmic connection and activates the egg. In 2002, a new isoform of phospholipase C (PLC), PLC zeta, was identified as a strong candidate for the mammalian sperm factor. In this mini-review, the history of the mammalian sperm factor theory and the search for the mammalian sperm factor is reviewed. In addition, whether PLC zeta is truly the mammalian sperm factor or not is discussed.

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Polyspermy is penetration of the egg cytoplasm by more than a single spermatozoon, and in humans, polyspermy usually results in spontaneous abortion. Following sperm penetration in mammals, cortical granules (CGs), special organelles in eggs, release their contents into the perivitelline space. The CG exudates act on the zona pellucida, causing biochemical and structural changes that result in zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Significant advances have been made in elucidating signal molecules and signal transduction cascades that play important roles in the CG exocytosis and subsequent polyspermy block. Ca²⁺ oscillation is necessary and sufficient for CG exocytosis as well as for other events of egg activation. The Ca²⁺-dependent pathways and the proteins involved in membrane fusion may play pivotal roles in the regulation of CG exocytosis. Mammalian oocytes develop their ability to undergo CG exocytosis during maturation. This article reviews the signal molecules and signal transduction cascades involved in CG exocytosis.

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Although intracytoplasmic sperm injection (ICSI) is an innovative treatment for male infertility, a significant number of clinical cases of fertilization failure remain. ICSI overcomes the difficulty in fertilization of sperm entry into the egg cytoplasm. The goal of fertilization, however, is the union of the male and female genomes; sperm incorporation into the oocyte is only the start of fertilization. Human sperm must perform three vital functions after entering the egg: it contributes to the male genome, awakens the quiescent egg, and crystallizes the motility apparatus that unites the sperm and egg nuclei, consummating the fertilization process. During fertilization in most mammalian species, including humans, the sperm introduces the centrosome, which acts as a microtubule organizing center (MTOC). By promoting pronuclear apposition and mitotic spindle formation, the sperm plays the leading part in the induction of "motility", post-ICSI, in fertilization. The present review introduces the challenges remaining in functional assessment of the human sperm centrosome and discusses the biparental (e.g. rabbit) and maternal (e.g. parthenogenesis) centrosomal contributions to microtubule organization during development.

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The dynamic observation of human gametes and the early stage of embryos is of paramount interest for clarifying the physiological events during the fertilization process. We present our morphological analysis of human embryonic development after both intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) using time-lapse cinematography. The results of this study indicate that non-invasive imaging, time-lapse cinematography, is useful not only for elucidating the morphological events of fertilization in humans, but also for evaluating the physiological importance of these events during the early stages of human embryonic development. In addition, analysis of cellular activity and quality during fertilization and embryogenesis using this system will contribute to the future improvement of the clinical results of assisted reproductive technology (ART).

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In most mammals, the growth and development of the oocyte and its surrounding somatic cell compartment in the follicle occur in a highly coordinated and mutually dependent manner. Oocytes acquire developmental competence sequentially during follicle growth, finally gaining the ability to undergo complete meiotic and cytoplasmic maturation at the final stage of the preovulatory follicle. Fully-grown immature oocytes are tightly surrounded by compact layers of specialized granulosa cells called cumulus cells that form the cumulus-oocyte complex (COC). After a preovulatory surge of gonadotrophin, the cumulus cells organize a special muco-elastic extracellular matrix (ECM) that requires synthesis and deposition of a large amount of hyaluronan (HA) and HA-binding matrix glycoproteins. Many studies have reported that the formation of the COC matrix mass plays important roles in a variety of reproductive phenomenons: oocyte meiotic maturation with changes of junctional communication and cytoskeletal modification in COC, ovulation, fertilization and early embryo development. Recently, we identified the expressions of HA synthases and the HA receptor, CD44, in the porcine COC matrix. The interaction of HA and CD44 appears to be closely related to gap-junctional communications and meiotic resumption during oocyte maturation. This review describes the recent findings on the regulation and the presumptive mechanism of COC matrix molecules, and physiological features in COC expansion.

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This study investigated the activation of oocytes after microinjection of sperm extracts (SE) from miniature pig spermatozoa. SE was prepared from miniature pig sperm by sonication for 35 min and centrifugation 100,000 g for 1 h. Injection of SE 0.3 mg/ml into porcine oocytes produced the highest activation rate (41.8%). SE injection into bovine oocytes induced similar activation (43.3%). The porcine oocyte activation rate (63.4%) was significantly (p<0.05) improved when SE was purified with a 2-D clean-up kit. When SE was heat treated at 60°C for 30 min, it failed in activation. However, SE succeeded in activation when stored at 4°C for 48 h or was freeze-thawed 5 times using liquid nitrogen. The activation rates were 42.1% and 31.4%, respectively. The results indicate that SE from miniature pig sperm might have an oocyte activation factor, and that the factor might have stability at low temperatures, though it appears to be inactivated at high temperatures.

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In our previous study, we observed decreases in the rate of fertilization and increases in the rate of polyspermy when mouse ova were exposed to hypergravity during in vitro fertilization. Moreover, the structures in mouse ova were changed by hypergravity. However, it was not determined whether the change in the rate of fertilization was caused by gravitational force acting directly on a process during fertilization or by structural changes in the ovum caused by hypergravity. We investigated the effect of prefertilization hypergravity, which excluded the direct acts on a process during fertilization, on in vitro fertilization. The rates of monospermy, polyspermy, and non-fertilization were investigated under various gravity loading conditions: Pre-G group, 3G-load (centrifugal force) for 2 h before insemination; During-G group, 3G-load for 6 h during fertilization; Pre&During-G group, 3G-load for 2 h before insemination and 6 h during fertilization; Non-G group, 3G-load was not performed. The rates of polyspermy in all G-load groups were significantly higher than in the Non-G group. Furthermore, the rate of polyspermy in the Pre-G group was the highest. Two-hour exposure to hypergravity before insemination increased the rate of polyspermy, and this may be due to structural changes in the cytoskeleton of the secondary oocyte.

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The purpose of this study was to test the hypothesis that an extracellular vitrification solution might be unnecessary after equilibration of bovine blastocysts with a vitrification solution. We assessed hatching in culture and the development to normal calves of bovine blastocysts vitrified on a nitrocellulose membrane filter that absorbed extracellular vitrification solution. Twenty in vitro produced bovine blastocysts were vitrified. All embryos were collected after warming and classified as excellent at the start of culture and all hatched between 48-96 h of culture. Transfer of three vitrified blastocysts derived in vivo given to three recipients resulted in the birth of one healthy calf on December 3, 2004. These results indicate that an extracellular vitrification solution is unnecessary after equilibration of embryos and that this new method of vitrification of embryos on a membrane filter is useful for the cryopreservation of bovine blastocysts.

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Cycloheximide (CHX) is a reversible inhibitor of bovine meiotic resumption. This study examined the timing of nuclear maturation in bovine oocytes treated with CHX and determined the optimum maturation interval in cultures for their subsequent development. CHX prevented the nuclear maturation of nearly all oocytes for 24 h. In an inhibitor-free medium, the majority of the CHX-treated oocytes underwent germinal vesicle breakdown (GVBD) after 6 h of culture, while the control oocytes did so at 10 h. In the maturation culture, the majority of the CHX-treated oocytes had reached metaphase II by 16 h whereas the control oocytes took 20 h. However, the CHX-treated oocytes that matured for 16 h developed into blastocysts at low rates while those that were matured for 20 h had a development rate similar to that of the control oocytes. These results indicate that the nuclear maturation of CHX-treated oocytes did accelerate, but these oocytes needed the same maturation time as required by the control oocytes for their subsequent development to blastocysts.

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