Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

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".     

Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes

The objective of this study was to determine microtubule assembly and chromatin configuration in porcine oocytes during the first cell cycle following round spermatid injection into matured porcine oocytes in the presence or absence of electrical stimulation. The oocytes with two large pronuclei and two polar bodies were classified as normal fertilization at 6 to 8 h following injection. The incidence of normal fertilization following round spermatid injection with electrical stimulation was significantly higher (21/45, 47%) than that following injection alone (6/39, 15%). Although a small microtubular aster was organized near the decondensed spermatid chromatin in some oocytes (2/6, 33%, spermatid injection alone; 9/21, 29%, spermatid injection and electrical stimulation), it did not enlarge nor fill the cytoplasm. Instead, a dense network of microtubules in the cytoplasm was organized from cortex. At 12 to 15 h after injection, we classified the oocytes with closely apposed pronuclei as normal fertilization. The electrical stimulation following spermatid injection enhanced (P < 0.05) the incidence of normal fertilization (18/54, 33%) compared with spermatid injection alone (7/52, 13%). During pronuclear movement, the maternally derived microtubules filled the whole cytoplasm, which appeared to move male and female chromatin. Mitosis and two-cell division were observed at 20 to 24 h after spermatid injection with electrical stimulation (12/41, 29%). At mitotic metaphase, the microtubular spindle had focused astral poles, and chromosomes were aligned on the spindle equator. During mitosis, asters were assembled at each spindle pole, and they filled the cytoplasm. These results suggested that round spermatid nuclei of the pig can develop into a morphologically normal pronucleus in matured porcine oocytes and are competent to participate in syngamy with the ootid chromatin. In addition, functional microtubules for complete fertilization with spermatid were not associated with male-derived centrosome but were organized solely from maternal stores. Mol. Reprod. Dev. 50:221–228, 1998. © 1998 Wiley-Liss, Inc.     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.     

Centrosomal function assessment in human sperm using heterologous ICSI with rabbit eggs: a new male factor infertility assay

Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as "unexplained infertility," and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy.     

Cat fertilization by mouse sperm injection

Summary Interspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

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, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.     

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration. © 1996 Wiley-Liss, Inc.     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization

Anti-tubulin immunofluorescence microscopy is used here to demonstrate the configurations of the microtubule-containing structures which participate in the pronuclear movements of sea urchin fertilization. This technique shows that the egg is devoid of microtubules until after the fertilizing sperm is fully incorporated. All the microtubules which appear during the course of fertilization are organized around the base of the sperm head and the sperm aster thus formed behaves in a way that could account for the characteristic motions of the male and female pronuclei as documented by time-lapse video microscopy. Extension of astral microtubules appears to be responsible for the slow (ca. 2.5 μm min^?1) movement of the sperm aster into the cytoplasm of the egg; the rapid (ca. 15 μm min^?1) migration of the female pronucleus to the sperm aster seems to depend on connection of the female pronucleus to microtubules of the sperm aster. Continued extension of astral microtubules after the pronuclei are brought into conjunction can account for the centripetal motion of the paired (or fused) pronuclei and for the positioning of the zygote nucleus in the center of the egg. The behavior of astral microtubules during these motions suggests that they are capable of transmitting both pushing and pulling forces. All the pronuclear movements, and the assembly of detectable microtubules, are sensitive to the microtubule inhibitors griseofulvin and colchicine. Because of this sensitivity, and since all the observable microtubules within the egg during fertilization arise at the sperm aster, it is concluded that the pronuclear movements of fertilization result from the actions of the sperm aster. The pronuclear movements of sea urchin fertilization represent a simple but striking example of microtubule-mediated motility.     

Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

Male pronuclear formation and sperm mitochondria in porcine oocytes following intracytoplasmic injection of pig or mouse sperm

In the present study we determined the chromatin organization and fate of introduced mitochondria in porcine embryos following intracytoplasmic injection of pig or mouse sperm cells. At 3, 6, 9 and 12 h following injection of pig or mouse spermatozoa or isolated sperm heads, the oocytes were fixed and stained with propidium iodide. Between 3 and 6 h following injection, both porcine and murine sperm chromatin developed into pronuclei. The male and female pronuclei were apposed within 12 h in porcine oocytes following sperm injection from either source. We also introduced foreign mitochondria from either mouse or pig sperm midpiece into porcine oocytes following sperm injection. While porcine sperm mitochondria rapidly disappeared from the actively developing porcine oocytes, mouse sperm mitochondria remained in the embryos until the 8-cell stage. These results suggest that pronuclear formation and movement occur between 6 and 12 h following sperm incorporation into the cytoplasm, and that foreign mitochondria are selectively removed in a species-specific manner.     

Chromatin and microtubule organisation in maturing and pre-activated porcine oocytes following intracytoplasmic sperm injection

Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.     

Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.