Roles of Nup133, Nup153 and membrane fenestrations in assembly of the nuclear pore complex at the end of mitosis

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by “Live CLEM” imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti‐GFP antibody, which captures GFP‐fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133‐coated beads or Nup153‐coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC‐like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133‐coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153‐coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations.     

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro.

It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.     

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium

In-house prepared medium was used routinely in our in-vitrofertilization (IVF) facility prior to the introduction of thecommercial ‘Medi-Cult’ products. A comparative studyof the in-vitro development of embryos cultured in two [T6 andEarle's balanced salt solution (EBSS)] humaninactivated serum(HlS)-supplemented media from days 0 to 5 showed that 44.7%(46/103) of the embryos developed to the blastocyst stage inthe T6 medium compared with 22.3% (23/103) in EBSS. Followingthe introduction of the commercial Medi-Cult IVF M2 medium,which is used routinely to culture fertilized eggs from days0 to 2, new baseline data were required for the in-vitro developmentof ‘spare’ embryos from days 2 to 5. When Medi-CultM3 medium was used, 35.6% (37/104) of the ‘spare’day 2 embryos achieved the blastocyst stage. However, if morphologicallysimilar (four normal nucleated blastomeres with no fragmentation)day 2 embryos were selected, an increase in the blastocyst rateto 50.0% (33/66) was achieved. This compared favourably withthe 45.0% blastocyst rate (published in the Medi-Cult literature)for M2/M3 medium cultured human embryos. A small series of experimentswith T6 $ HIS medium and human serum albumin (HSA)- supplementedHam's F-10, MCDB 302 and M3 media was undertaken to identifya suitable medium which could be used for the culture of M2medium day 2 embryos. Results show that M2 medium cultured embryosplaced in Ham's F-10 medium supplemented with 10 mg/ml HSA gavean acceptable 37.8% (14/45) blastocyst rate. Therefore, thismedium could be substituted for M3 medium in an emergency. Atotal of 483 IVF embryos donated by patients, which were surplusto the therapeutic IVF programme, were used for these studiesover a period of 30 months. Late day 2 IVF spare embryos wereassigned an embryo score based on a high-power phase-contrastmicroscopic examination prior to being placed in culture. Theembryo score provides an effective in-vitro parameter with whichembryos from different patients can be compared. The cleavageand development of individual embryos were monitored on days2 to 5. In some cases, the continuing normal development andviability of the day 5 cultured embryo were assessed by monitoringthe hatching, attachment and outgrowth of the cavitated blastocyst.