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Fertilization is a lengthy process that culminates when the male and female pronuclei fuse in the oocyte cytoplasm. The final stage of fertilization is mediated by the sperm centrosome, which induces microtubule organization into the first mitotic spindle. Despite its critical role, only few functional analyses of the sperm centrosome have been performed until now. Here, we review the recent literature with regard to sperm centrosomal functions during human fertilization, as well as the development of functional assays for the human sperm centrosome. We then address various challenges for fertilization failure resulting from centrosomal dysfunction. Cytological analyses of oocytes that fail to complete fertilization following intracytoplasmic sperm injection (ICSI) have shown that some cases are associated with sperm centrosomal dysfunction. Human sperm can organize a sperm aster even within the oocytes of other mammals. This property has been utilized as a means of assessing the centrosome function. In some patients with teratozoospermia, the sperm does show evidence of impaired centrosomal function. Some clinical and basic challenges for overcoming the fertilization failure caused by sperm centrosomal dysfunction have been reported. The sperm centrosome plays an important role in the phase of the fertilization process after ICSI, i.e. within the oocyte's cytoplasm. The next generation of assisted reproductive technologies (ART) will likely incorporate analyses of sperm centrosomal function as well as techniques designed to counter sperm centrosome dysfunction.

During mammalian fertilization, a centrosome is introduced by the sperm during the first cell cycle to organize a radial array of microtubules known as the sperm aster. In nature, multiple human sperm centrosomes may exist in the same egg cytoplasm during polyspermy. However, critical information concerning individual sperm centrosomal function with regards to the latter case remains unknown. We subsequently examined the sperm aster formation after injection of multiple human sperm into a bovine egg. When 2 fertile human sperm were simultaneously microinjected into different regions of the same bovine egg cytoplasm, no difference in sperm aster formation rate was observed compared to cases in which a single sperm was injected. Two human sperm were also microinjected into bovine eggs 30-, 60- and 120-minute intervals apart from one another, and no difference in sperm aster formation rates were observed. Among eggs in which 1 sperm aster was organized, there was no observable bias towards the first or second injected sperm. These findings indicated that when multiple human sperm are present in a single egg cytoplasm, each centrosome can function independently from the other. This fact suggests the possibility of transplanting a normal sperm centrosome into an egg with a sperm known to have centrosomal dysfunction.

Objective: To report a successful pregnancy after intracytoplasmic sperm injection (ICSI) with artificial oocyte activation (AOA) on a patient whose fertilization rate after ICSI was extremely low; and to report on cytologic analyses of the fertilization failure (FF) eggs after ICSI and a biologic assessment of the sperm of this patient., Design: Case report with an assessment of gamete function., Setting: University hospital and an experimental laboratory., Patient(s): A couple with severe oligoasthenozoospermia, whose seventh attempt at ICSI ended in the failure., Intervention(s): Cytologic analyses of FF eggs after ICSI, AOA after ICSI, and analyses of human sperm oocyte activation ability and centrosomal function., Result(s): Fertilization arrest after ICSI was observed in FF eggs at various stages of fertilization. After artificial oocyte activation by exposure to ionomycin, clinical pregnancy was confirmed, and a healthy baby was born. As assessed by heterologous ICSI of human sperm into bovine oocytes, there was no significant difference in the oocyte activation rates between the patient's and control sperm, but the sperm centrosomal function was low in the patient's sperm (48.5% vs. 69.6%)., Conclusion(s): We report a successful pregnancy after ICSI with AOA using a calcium ionophore, after critical cytologic analyses of the FF eggs. Furthermore, sperm centrosomal function was low, indicating that sperm's ability to process the events of fertilization after the oocyte activation was poor in this patient.

Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo-micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (gamma-tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P = 0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected.

The human spermatozoal centrosome acts as a microtubule organizing center and is essential for male and female pronuclear migration and apposition. In this study, we assess centrosomal function of spermatozoa from infertile patients using heterologus intracytoplasmic sperm injection (ICSI) into bovine eggs. Spermatozoa from 15 infertile patients undergoing assisted reproductive technology (ART) treatment and 3 fertile donors were tested. Microtubules and DNA were imaged by immuocytochemistry and epifluorescence microscopy. Decondensed female chromosomes and sperm nuclei, pronuclear formation and sperm aster formation were examined. The average rate of sperm aster formation using spermatozoa from infertile individuals was lower (47.0%) than that with spermatozoa from fertile individuals (66.1%). We compared the sperm aster formation rates after ART with various clinical parameters, including semen characteristics, pronuclear formation rates, embryonic cleavage rates and pregnancy outcome. Clinical semen characteristics and the rate of pronuclear formation appeared independent of sperm centrosomal function. In contrast, the centrosomal function had a substantial effect on embryonic cleavage rate and pregnancy after ART. These results suggested that centrosomal function is essential for pregnancy and embryonic development. The method described using bovine eggs is suitable to assay human centrosome function and predict pregnancy after ART.

Recently, the immunoglobulin superfamily protein IZUMO on the sperm surface was identified as being essential for sperm-egg fusion. Although we examined the expression of IZUMO on sperm of 13 male infertile patients with severe oligozoospermia and/or athenozoospermia and 12 infertile patients with fertilization failure in previous conventional IVF to determine whether it is varied in infertility patients, we could not identify any patients with IZUMO negative by immunocytochemistry.

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 during fertilization of spermatozoon entry into the egg cytoplasm. The goal of fertilization, however, is the union of the male and female genomes; spermatozoon incorporation into the oocyte is only the initiation of fertilization. During fertilization in most mammalian species, including humans, the spermatozoon introduces the centrosome, which acts as a microtubule organizing center (MTOC). By promoting pronuclear apposition and mitotic spindle formation, the spermatozoon plays the leading part in the induction of "motility" post-ICSI in fertilization. The present review introduces the remaining challenges in functional assessment of the human sperm centrosome and discusses the biparental (for example, rabbit) and maternal (for example, parthenogenesis) centrosomal contributions to microtubule organization during development.

Background: In human fertilization, sperm centrosome function is essential for male and female pronuclear movement and fusion. In this study, we investigated the possibility of restoring human sperm centrosomal function in sperm exhibiting abnormalities in microtubule organization., Methods: Semen was obtained from both a fertile donor and a patient with dysplasia of the fibrous sheath (DFS). Following heterologous ICSI using human sperm, we examined microtubules and chromatin configuration in bovine oocytes. Sperm were treated with dithiothreitol (DTT) prior to ICSI, while the oocytes were treated with the cytoskeletal stabilizer paclitaxel after ICSI., Results: The combination of DTT and paclitaxel treatment induced microtubule organization in dead sperm from the fertile donor following heterologous ICSI. This treatment, however, was not effective for DFS sperm. In addition, expression of centrin, a protein functioning within the sperm centrosome, was reduced in DFS sperm from that of the normal levels observed in fertile donor sperm., Conclusion: These results indicate that sperm centrosomal function could be induced by the treatment of sperm with DTT before ICSI and of oocytes with paclitaxel after ICSI. DFS sperm are likely to exhibit such severe dysfunction of sperm centrosome that cannot be compensated for by this treatment; therefore, this method may be a practical way to discern the degree of sperm centrosomal dysfunction.

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".

Problems: Intracytoplasmic sperm injection (ICSI ) has been described as the 'cure' for male sterility because a single sperm can now be directly introduced into an egg with some chance of pregnancy. While ICSI has revolutionized the practice of assisted reproductive techniques (ART), there are few molecular and cellular studies about its safety and efficacy. Even by using ICSI, fertilization in humans succeeds only if the sperm effectively accomplishes a number of tasks including 'post-ICSI events' in fertilization. To assess the function of human sperm after ICSI, we used heterologous ICSI with human sperm into animal eggs. Egg activation, sperm decondensation and sperm centorosomal function were examined in sperm from fertile men and infertile patients., Methods: Sperm from fertile men and infertile patients were injected into hamster, rabbit and bovine eggs by Piezo micromanipulator, and studied in decondensation of sperm nuclei, egg activation and microtubule organization., Results: Decondensation human sperm head following ICSI into hamster eggs occurred initially form basal lesion, and apical portion of sperm nuclei which is surrounded by acrosome and perinuclear theca, still condensed in early pronuclear stage. Radial array of microtubules from sperm centrosome 'sperm aster' which is essential for pronuclear movement was observed in 30% rabbit eggs following ICSI with human sperm. By heterologous ICSI system with fertile human sperm and bovine eggs, 83.3% of eggs was activated and 60% eggs had sperm aster, indicating that bovine Piezo ICSI system is appropriate for assessing human sperm oocyte activation ability and human sperm centrosomal function. Oocyte activation and sperm centrosomal function were significantly low in sperm from men with globozoospermia and men with dysplasia of fibrous sheath., Conclusion: These assays indicate differences of the process of fertilization between in vitro fertilization and ICSI, and reflect the human sperm function especially for the 'post-ICSI events' in fertilization. More molecular and cellular analyses in fertilization by ICSI are needed for improvement of ART.

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., (Copyright 2004 Wiley-Liss, Inc.)

Objective: To determine the possible use of the mammalian acrosomal marker vehicle-associated membrane protein (VAMP)/synaptobrevin to detect acrosome abnormalities in human sperm., Design: Analysis of human sperm after fixation and staining with an anti-VAMP antibody., Setting: An academic research institution., Patient(s): Semen samples from consenting patients who were participating in an infertility treatment program., Intervention(s): Human sperm samples were fixed, permeabilized with detergent, and examined by immunocytochemistry., Main Outcome Measure: Immunostaining., Result(s): Observation of sperm from patients with no obvious sperm morphological defects revealed normal looking acrosomes, as assessed by VAMP immunostaining. However, severe acrosome malformations were detected in other cases. The observations registered varied from the absence of a fully formed organelle in samples of patients with globozoospermia to abnormal VAMP staining in samples from patients with acrosomal defects., Conclusion: VAMP/synaptobrevin may be a useful marker for the functional assessment of acrosomal status in human sperm.

To evaluate whether the morphology of the sperm midpiece observed by high magnification microscopy relates to sperm centrosomal function. Sperm selected by conventional microscopy were defined as controls. By high magnification microscopy, sperm with straight midpieces were defined as Group 1, while those with tapering midpieces were defined as Group 2. Heterologous ICSI of human sperm into bovine oocytes was used to assess human sperm centrosomal function and analysis of sperm aster formation. The total rate of sperm aster formation was 80.5% in Group 1, which was significantly higher ( P < 0.05) than the rate of 33.3% seen for Group 2. Furthermore, sperm aster formation rates tended to be higher for Group 1 than for the controls. This study demonstrates improvement of sperm aster formation rates by selecting sperm on the basis of midpiece morphology. The injection of selected sperm bearing morphologically straight midpieces may contribute to improved expression of sperm centrosomal function, providing a positive effect on fertilization after ICSI. [ABSTRACT FROM AUTHOR]

Objective: To investigate the structural changes in the cytoskeleton (microtubules, microfilaments) and examine the expression of centrosomal functional proteins during human spermiogenesis. Design: Immunofluorescent staining of human spermiogenic cells. Setting: University hospital and IVF clinic. Patient(s): Human testicular tissues were obtained by testicular sperm aspiration (TESA) under informed consent. Three cases of obstructive azoospermia, with confirmed normal spermatogenesis, were examined. Intervention(s): Spermatogenic cells were fixed with microtubule-stabilizing buffer. Immunocytochemical detection of microtubules, microfilaments, and centrosome was performed using monoclonal antibodies against α- and β-tubulin, phalloidin, and functional centrosomal proteins. Main Outcome Measure(s): Samples were examined using epifluorescence and laser scanning confocal microscopes. Result(s): During the Sb2 period, microtubules formed the manchette structure, which extended from the equator of the nucleus through the cytoplasm. Microfilaments were organized in the periacrosamal region during spermiogenesis (Sa to Sd). Although centrin was observed throughout the spermiogenic period, γ-tubulin was detected only in the Sb2 period. Conclusion(s): Dynamic cytoskeletal movement was observed during human spermiogenesis. Cytoskeletal rearrangements in the Sb2 period appear to play important roles in the morphologic changes that occur during human spermiogenesis. Studies of the cytoskeletal system during spermiogenesis may help identify some causes of male infertility (e.g., teratozoospermia, maturation arrest). [Copyright &y& Elsevier]

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) [ABSTRACT FROM AUTHOR]

In most mammalian fertilization, the sperm introduces the centrosome, which acts as a microtubule organizing center (MTOC) and is essential for pronuclear movement. In rabbit fertilization, biparental centrosomal contribution in microtubule organization has been suggested.To reveal the function and inheritance of the centrosome during rabbit fertilization, we compared microtubule organization and early embryonal development following intracytoplasmic sperm injection (ICSI) with and without sperm centrosome. Sperm centrosome was removed by sonication, and the isolated sperm head was injected by a Piezo-driven micromanipulator. Samples were studied by light microscope after immunocytological stain.The sperm aster formation was observed 2–3 h after ICSI with intact sperm. In contrast, microtubules were organized between the male and female pronucleus without a nucleation site in the eggs after ICSI with an isolated sperm head. In the late pronuclear stage following ICSI with an isolated sperm head, microtubule organization was the same as in late pronuclear stage eggs after intact sperm injection. The first mitotic spindle was organized in eggs following ICSI with an isolated sperm head, as observed in eggs following ICSI with an intact sperm.These results indicate that the MTOC is in oocyte cytoplasm during fertilization and fulfils the function when the sperm centrosome is absent. (Reprod Med Biol 2005;4: 169–178) [ABSTRACT FROM AUTHOR]

Sperm morphology is correlated with fertility in men, yet the existing, subjective sperm morphology assays provide only a limited insight into patients' infertility. Here, we provide the experimental background for a new, objective and automated semen assay, based on the cross-reactivity of defective human spermatozoa with antibodies against a proteolytic marker peptide, ubiquitin. Using immunofluorescence and flow cytometry, we screened the spermatozoa from 17 infertility patients and two fertile donors for their cross-reactivity with anti-ubiquitin antibodies. Thirteen out of 17 patients, but neither of two fertile donors, displayed an increased binding of anti-ubiquitin antibodies to sperm surface, that reflected the occurrence of abnormalities in these samples and was corroborated by available clinical data. Highest correlation coefficient (r = –0.432) was obtained with the cleavage rate after IVF. The contribution of male factor was revealed in several couples previously diagnosed with idiopathic infertility. Ubiquitin-cross-reactive sperm-surface proteins thus seem to be a universal marker of semen abnormalities, including sperm head and tail defects and semen contaminants such as spermatids, leukocytes and cellular debris. We propose that the sperm-ubiquitin tag immunoassay (SUTI) may be a valuable diagnostic tool in treatment of male factor and idiopathic infertility. [ABSTRACT FROM PUBLISHER]

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis. [Copyright &y& Elsevier]