Dynamic changes of the Golgi apparatus during bovine in vitro oocyte maturation.

For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.

Neural cadherin is expressed in human gametes and participates in sperm-oocyte interaction events.

Neural cadherin (N-cadherin) is a transmembrane glycoprotein involved in calcium-dependent cell-cell adhesion and signalling events. The previous evidence shows N-cadherin expression in the human gonads and gametes; however, N-cadherin subcellular localization in human spermatozoa and oocytes, and its involvement in fertilization remain to be characterized. In this study, expression of N-cadherin in human spermatozoa and testis was confirmed by RT-PCR and protein forms were identified using Western immunoblotting. N-cadherin localization in testicular and ejaculated spermatozoa, in cells that had undergone capacitation and acrosomal exocytosis, as well as in oocytes was assessed using immunocytochemistry. Participation of the adhesion protein in fertilization was evaluated using the HemiZona Assay (HZA) and the zona pellucida (ZP)-free hamster oocyte sperm penetration assay (SPA). Both the N-cadherin transcript and the mature protein form (135 kDa) were found in spermatozoa and testis. The protein was mainly immunolocalized in the acrosomal region of testicular, non-capacitated and capacitated spermatozoa, and was found in the equatorial segment after acrosomal exocytosis. N-cadherin was also detected in oocytes, in conjunction with beta-catenin, a member of the adhesion complex. Sperm incubation with anti N-cadherin antibodies did not affect their ability to interact with homologous ZP in the HZA; by contrast, presence of the antibodies in the SPA led to a significant (p < 0.01) reduction in the percentage of penetrated oocytes. In conjunction, results indicate that N-cadherin is a sperm protein of testicular origin localized in cellular regions involved in gamete interaction. N-cadherin would not participate in sperm-ZP interaction, but it would have a role in sperm-oolemma adhesion/fusion events.

Dynamics of microtubules, motor proteins and 20S proteasomes during bovine oocyte IVM.

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2-8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis.

Abnormal assembly of annulate lamellae and nuclear pore complexes coincides with fertilization arrest at the pronuclear stage of human zygotic development.

BACKGROUND: The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. METHODS AND RESULTS: We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. CONCLUSIONS: We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.

A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage.

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patient with severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phase contrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formation using antibodies against acetylated alpha- and beta-tubulins. RESULTS: Acephalic sperm, headless tails and abnormal alignments of the head-tail junction were observed. Flagella evidenced the features of dysplasia of the fibrous sheath. Bovine oocytes injected with patient's sperm showed male and female pronuclei but a faulty development of microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selection methods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts were performed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavage took place in all cycles, and in two of them positive betahCG plasma levels were detected but preclinical abortions ensued. CONCLUSIONS: We propose that the alterations in the head-tail junction and attachment, responsible for the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation, lack of syngamy and cleavage or defective embryos leading to early abortions.

Sperm ubiquitination in patients with dysplasia of the fibrous sheath.

BACKGROUND: Human sperm with structural abnormalities display an increased content of the cellular proteolytic marker peptide, ubiquitin. We investigated whether dysplasia of the fibrous sheath (DFS), a severe structural anomaly found in the sperm of some asthenozoospermic patients, is accompanied by (i) increased ubiquitination of the sperm surface and (ii) by increased ubiquitination of the sperm mitochondria. METHODS AND RESULTS: Five DFS patients and eight fertile donors were studied by immunocytochemistry with anti-ubiquitin antibodies. Increased cross-reactivity of the ubiquitinated mitochondrial epitopes was seen in 32-50% of DFS sperm, but only 2-4.1% of sperm from fertile donors. Sperm surface ubiquitination assessed by sperm-ubiquitin tag immunoassay (SUTI) and immunofluorescence demonstrated an increased sperm ubiquitination in all DFS patients. The average median value of ubiquitin-induced fluorescence in DFS patients was 25.8 counts (range 19.8-37.9), as opposed to 13.4 counts range (9.3-16.6) in fertile men. Sperm with 'stump tails', coiled tails, twin and triplet sperm, and clusters of immature spermatogenic cells were common. CONCLUSIONS: DFS sperm have increased cross-reactivity to anti-ubiquitin antibodies, a finding consistent with the ubiquitination of defective sperm shown in animal models. These results justify the use of ubiquitin-based assays for objective semen analysis in infertile men with heritable defects.

Pronuclear abnormalities and cytoskeletal organization during assisted fertilization in a patient with multifollicular ovarian response.

PURPOSE: To analyze the distribution of a tubulins and acetylated alpha tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. RESULTS: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed > or = 3 pronuclei (PN) and karyomeres after IVF-ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of > or =1 PN within the cytoplasm were the main events present in most oocytes after IVF-ICSI. CONCLUSIONS: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF-ICSI showed similar etiologies in this special group of zygotes.

Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa.

Dysplasia of the fibrous sheath (DFS) is an anomaly found in spermatozoa of severe asthenozoospermic patients. Marked hypertrophy and hyperplasia of the fibrous sheath is the common characteristic. Immunocytochemistry allowed us to visualize the distortions and incidence of tail structure abnormalities associated with this phenotype in six patients; four with a complete form and two with an incomplete form of this pathology previously diagnosed and studied by electron microscopy. Microtubules and fibrous sheaths were studied using monoclonal antibodies against alpha-acetylated tubulin and anti-FSC1 (the major protein component of the fibrous sheath). Mitochondrial sheaths were visualized using the mitochondrion-specific vital dye MitoTracker green FM(TM). Phase contrast and fluorescent microscopy of semen samples showed large numbers of spermatozoa with short, rigid, thick and irregular tails. As expected, anomalous and completely distorted fibrous sheaths, severe alterations of the axonemal microtubules and different patterns of mitochondrial sheath configurations were found. While ultrastructural studies of thin sections allow an in-depth knowledge of the internal organization of the sperm tail, fluorescence labelling of selected sperm components affords a unique view of the whole flagellum including topographical relationships of various organelles. The combination of these different approaches is essential for a comprehensive understanding of this particular pathology.

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure.

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.

Pregnancies established through intracytoplasmic sperm injection (ICSI) using spermatozoa with dysplasia of fibrous sheath.

AIM: Dysplasia of the fibrous sheath (DFS) is an anomaly found in asthenozoospermic patients with extremely low or absent motility. In order to determine the efficacy of ICSI in these patients, a retrospective analysis of ICSI results in DFS patients has been done. METHODS: Ten ICSI attempts were performed in 6 patients with diagnosis of Dysplasia of the Fibrous Sheath studied by transmission and scanning electron microscopy. RESULTS: In the cases studied, sperm concentration was (29.62 +/- 18.05) x 10(6)/mL, total motility was 1.14 +/- 1.31%. Progressive motility was 0% except for one case with 0.1% . One hundred and three preovulatory oocytes were obtained and 94 metaphase II oocytes were injected. Sixty-nine of them showed two pronuclei (fertilization rate: 73.4%). Forty-nine embryos were obtained and 34 were transferred (mean: 3.4 embryos per transfer). Five pregnancies were diagnosed by beta-hCG plasma level determinations that resulted to be one preclinical abortion, one clinical abortion and three deliveries. Another pregnancy (ongoing) was achieved from a cryopreserved embryo transfer. CONCLUSION: These results showed that ICSI provides a suitable solution for patients suffering from irreversible sperm defects such as DFS. Nevertheless, it is mandatory to inform couples of possible transmission risks to offspring, which are unknown at present. Only when the etiology of this problem is disclosed, it will be possible to assess the real genetic risk.