Sperm Incorporation Research Articles

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.

Sperm-oocyte signaling: the role of IZUMO1R and CD9 in PTK2B activation and actin remodeling at the sperm binding site†.

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

Sperm-oocyte contact induces outside-in signaling via PYK2 activation

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm – oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline – rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm – oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm – binding sites under conditions where sperm – oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm – oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

PTK2b function during fertilization of the mouse oocyte

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm–egg interactions and anaphase resumption

Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

PYK2: A calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte

Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm–oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone.

Essential role of maternal UCHL1 and UCHL3 in fertilization and preimplantation embryo development

Post-translational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1(gad-/-) mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1(gad-/-) mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex.

Analysis of the dynamic process of sperm penetration of the human oocyte using time-lapse cinematography

OBJECTIVE: Using time-lapse cinematography (TLC), we analyzed the dynamic process of human sperm penetration to investigate the mechanisms blocking polyspermy after sperm attachment. DESIGN: Retrospective visual analysis. MATERIALS AND METHODS: Between July 2004 and April 2008, TLC was performed on single, randomly selected oocytes after insemination, as previously described (Mio, 2008). The study focused on oocytes where the movements of the leading sperm (the one that penetrated the zona pellucida (ZP) most deeply) and the following sperm were captured in the same field. Sperm movements were then analyzed from the time of entry into the ZP to the incorporation of the leading sperm. RESULTS: We observed and recorded the process of sperm penetration and the sperm entry point in 22 embryos. In 3 of these embryos (target embryos), we were able to capture the movements of the leading and following sperm. Sperm attached to the oocyte membrane approx 96 minutes after insemination and 37 minutes later, the sperm head decondensed and disappeared. There was no difference in the time-course of the fertilization process of the target embryos and the other embryos observed. In the 3 target embryos, both leading and following sperm proceeded forward until the leading sperm attached on the ooplasmic membrane. The following sperm penetrated 12.9 μm (68.9%) of the ZP on average and, despite their active tail movements, stopped their progression within 10 seconds of the leading sperm attaching on the membrane. CONCLUSIONS: To date, the primary block to polyspermy in the mammalian oocyte is described as a zona reaction induced approximately 5 to 8 minutes after oocyte activation by sperm incorporation. In this study, 10 seconds after sperm-oocyte attachment, the following sperm were unable to continue further despite their sustained tail motility. This suggests that an unknown process has prevented the progression of the following sperm. Further investigations using TLC are underway to elucidate this mechanism of polyspermy prevention.

Effects of Ultraviolet Radiation on Gametic Function During Fertilization in Zebra Mussels(Dreissena polymorpha)

The gamete-specific effects of UV radiation on fertilization in the invasive freshwater zebra mussel (Dreissena polymorpha) are addressed. Both eggs and sperm of zebra mussels were highly sensitive to UVB. Sperm irradiated with 118 mJ/cm2 UVB showed decreased fertilization rates when inseminating nonirradiated eggs. Sperm binding was unaffected; however, acrosomal integrity and sperm incorporation into the egg cytoplasm were reduced by UVB irradiation. There was a decrease in first zygotic cleavage. The level of UVB needed to inhibit fertilization was lower than that required for inhibition of sperm motility. UVB-irradiated eggs also showed reduced function when inseminated by nonirradiated sperm. UVB-induced damage in eggs most commonly resulted in a stoppage or delay of meiosis. The levels effective in killing zebra mussel larvae should be sufficient to inhibit fertilization.

The role of centrosomes in mammalian fertilization and its significance for ICSI

Centrosome integrity is critically important for successful fertilization and embryo development. In humans, the sperm contributes the dominant centrosomal material containing centrioles and centrosomal components onto which oocyte centrosomal proteins assemble after sperm incorporation to form the sperm aster that is essential for uniting sperm and oocyte pronuclei. Increasingly, dysfunctional sperm centrosomes have been identified as a factor for sperm-derived infertility and heterologous Intracytoplasmic Sperm Injection (ICSI) has been used to assess centrosome and sperm aster formation and clearly established a relationship between infertility and sperm centrosomal dysfunction. ICSI has been used successfully to provide novel treatment to overcome male factor infertility and it may open up new possibilities to correct specific sperm-related centrosome dysfunctions at molecular levels. New data indicate that it is now possible to replace dysfunctional centrosomes with functional donor sperm centrosomes which may provide new treatment for couples in which infertility is a result of centrosome-related sperm dysfunctions.

Interactions between mitogen‐activated protein kinase and protein kinase C signaling during oocyte maturation and fertilization in a marine worm

In the marine nemertean worm Cerebratulus, follicle-free oocytes re-initiate meiosis and undergo nuclear disassembly (=germinal vesicle breakdown, GVBD) after being stimulated to mature by seawater (SW) or cAMP-elevating drugs. Previously, it has been shown that inhibitors of mitogen-activated protein kinase (MAPK) or protein kinase C (PKC) signaling can reduce SW-induced GVBD in nemertean oocytes without affecting cAMP-induced GVBD. Thus, SW and cAMP elevators may trigger alternative pathways that vary in their dependence on MAPK and PKC. To further characterize such signaling cascades, immunoblotting analyses of MAPK and PKC activities were conducted on oocytes treated with U0126, an inhibitor of the MAPK kinase (MAPKK) that is responsible for activating MAPK. Based on these analyses and comparisons with the MAPKK inhibitor CI1040 that inactivates MAPK without preventing GVBD, U0126 seems to block GVBD via a non-MAPK-mediated effect that involves PKC. Moreover, evidence is presented for post-GVBD oocytes establishing positive feedback between MAPK and PKC signaling. Such feedback apparently allows the activities of both kinases to be maintained before insemination and to undergo concomitant downregulation after fertilization. Furthermore, in oocytes treated with MAPKK and PKC inhibitors during fertilization, sperm incorporation and polar body formation still occur, but normal cleavage is prevented. This suggests that although GVBD and aspects of post-fertilization activation may proceed in the absence of MAPK or PKC, such kinases are apparently required for proper embryogenesis. Collectively, these results are discussed relative to previous analyses of the interactions and functions of MAPK and PKC signaling during oocyte maturation and fertilization.

Fyn kinase activity is required for normal organization and functional polarity of the mouse oocyte cortex

The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally.

Human Sperm Centrosomal Function during Fertilization, a Novel Assessment for Male Sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.

Sperm incorporation and pronuclear development during fertilization in the freshwater bivalve Dreissena polymorpha

The invasive zebra mussel, Dreissena polymorpha (D. polymorpha), is proving to be a valuable model for understanding general mechanisms of fertilization, particularly regarding sperm incorporation. In the present study, we tracked the various components of the fertilizing sperm of D. polymorpha during sperm incorporation. During fertilization the sperm membrane remains associated with the egg surface as a distinct patch that disperses over time. This patch marked the site of sperm entry that occurs predominately on the CD blastomere. Taking advantage of the relatively unpigmented cytoplasm, real-time observations were made of the incorporated sperm nucleus as it decondensed and reformed as a developing pronucleus. Pronuclear enlargement occurred progressively and at rates comparable with previously reported fixed-time point observations. Sperm mitochondria were incorporated and separated from the sperm along the leading edge of the decondensing nucleus. Sperm mitochondria labeled with Mitotracker Green remained predominately associated with the CD blastomere following first cleavage and could be tracked to the 16-cell stage before the fluorescence was too faint to detect. Additionally, the demembranated sperm axoneme was incorporated, separated during nuclear decondensation, and remained visible in the egg cytoplasm up to 30 min postinsemination (PI). The present study provides one of the most complete descriptions of incorporation on multiple sperm components into the egg and coordinates fixed-time point observations with real-time observations of sperm within the remarkably transparent egg cytoplasm of zebra mussels.

Post-ICSI Cytoskeletal Dynamics during Fertilization

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.

Regulation of dynamic events by microfilaments during oocyte maturation and fertilization

Actin filaments (microfilaments) regulate various dynamic events during oocyte meiotic maturation and fertilization. In most species, microfilaments are not required for germinal vesicle breakdown and meiotic spindle formation, but they mediate peripheral nucleus (chromosome) migration, cortical spindle anchorage, homologous chromosome separation, cortex development/maintenance, polarity establishment, and first polar body emission during oocyte maturation. Peripheral cortical granule migration is controlled by microfilaments, while mitochondria movement is mediated by microtubules. During fertilization, microfilaments are involved in sperm incorporation, spindle rotation (mouse), cortical granule exocytosis, second polar body emission and cleavage ring formation, but are not required for pronuclear apposition (except for the mouse). Many of the events are driven by the dynamic interactions between myosin and actin filaments whose polymerization is regulated by RhoA, Cdc42, Arp2/3 and other signaling molecules. Studies have also shown that oocyte cortex organization and polarity formation mediated by actin filaments are regulated by mitogen-activated protein kinase, myosin light-chain kinase, protein kinase C and its substrate p-MARKS as well as PAR proteins. The completion of several dynamic events, including homologous chromosome separation, spindle anchorage, spindle rotation, vesicle organelle transport and pronuclear apposition (mouse), requires interactions between microfilaments and microtubules, but determination of how the two systems of the cytoskeleton precisely cross-link, and which proteins link microfilaments to microtubules to perform functions in eggs, requires further studies. Finally, the meaning of microfilament-mediated oocyte polarity versus embryo polarity and embryo development in different species (Drosophila, Xenopus and mouse) is discussed.

Cytoskeletal dynamics during mammalian gametegenesis and fertilization: Implications for human reproduction.

From gamete to neonate, human fertilization is a series of cell motilities (motion and morphological changes). Cytoskeletons play a role in cell motility as they work as a field worker in the cell. The present study is a review of dynamic motility of cytoskeletons (microfilaments and microtubules) during mammalian gamategenesis and fertilization. Dynamic and proper organization of cytoskeletons is crucial for the completion of oocyte maturation and spermatogenesis. By intracytoplasmic sperm injection, some difficulties in fertilization by sperm entry into the egg cytoplasm are overcome. However, the goal of fertilization is the union of the male and female genome, and sperm incorporation into an oocyte is nothing but the beginning of fertilization. Sperm centrosomal function, which introduces microtubule organization and promotes pronuclear apposition and first mitotic spindle formation, plays the leading role in the 'motility' of post-intracytoplasmic sperm injection events in fertilization. The present review introduces novel challenges in functional assessment of the human sperm centrosome. Furthermore, microtubule organization during development without the sperm centrosome (e.g. parthenogenesis) is mentioned. (Reprod Med Biol 2005; 4: 179-187).