The endoplasmic reticulum (ER) is thought to play an important structural and functional role in phagocytosis. According to this model, direct membrane fusion between the ER and the plasma or phagosomal membrane must precede further invagination, but the exact mechanisms remain elusive. Here, we investigated whether various ER-localized SNARE proteins are involved in this fusion process. When phagosomes were isolated from murine J774 macrophages, we found that ER-localized SNARE proteins (syntaxin 18, D12, and Sec22b) were significantly enriched in the phagosomes. Fluorescence and immuno-EM analyses confirmed the localization of syntaxin 18 in the phagosomal membranes of J774 cells stably expressing this protein tagged to a GFP variant. To examine whether these SNARE proteins are required for phagocytosis, we generated 293T cells stably expressing the Fc␥ receptor, in which phagocytosis occurs in an IgGmediated manner. Expression in these cells of dominant-negative mutants of syntaxin 18 or D12 lacking the transmembrane domain, but not a Sec22b mutant, impaired phagocytosis. Syntaxin 18 small interfering RNA (siRNA) selectively decreased the efficiency of phagocytosis, and the rate of phagocytosis was markedly enhanced by stable overexpression of syntaxin 18 in J774 cells. Therefore, we conclude that syntaxin 18 is involved in ER-mediated phagocytosis, presumably by regulating the specific and direct fusion of the ER and plasma or phagosomal membranes. INTRODUCTIONPhagocytosis is the coordinated process by which large foreign particles are internalized into a newly formed organelle, the phagosome. In professional phagocytes of the immune system, including macrophages, neutrophils, and dendritic cells, the internalization is triggered by activation of various types of cell surface receptors in the course of innate and adaptive immune responses. For example, in the case of immunoglobulin (Ig)-mediated phagocytosis, Fc receptors (Fc␥Rs) cluster where they contact an Ig-opsonized solid surface; this induces actin polymerization, resulting in the formation of pseudopods that engulf the particle . Subsequently, the phagosomes mature by fusing with other organelles of the endocytic pathway, leading to the formation of phagolysosomes (Downey et al., 1999;Vieira et al., 2002).In macrophages, every membrane fusion event during phagocytosis is thought to be mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Coppolino et al., 2001). It is currently believed that the assembly of SNARE proteins leads to a tight connection between the vesicle and the target membrane, which initiates the opening of the fusion pore . The SNARE complex forms an extended parallel four-helix bundle in order to fuse the two membranes Antonin et al., 2002). Three helices are extended from one membrane by proteins of the syntaxin and SNAP-25 families (Q-SNAREs) that contain a conserved glutamine residue at a central position called the "0" layer, and the remaining helix, extended from the opposite membr...

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

OBJECTIVE: We sought to evaluate specific sperm DNA damage, doublestranded DNA breaks (dsDNA), and its effect on embryo development and implantation.DESIGN: Over a year-long period, a prospective pilot study was carried out on sperm samples to evaluate the proportion of dsDNA on samples screened by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) to assess total DNA fragmentation. Once a correlation was established, we extrapolated dsDNA values retrospectively onto patients who had their ejaculates screened by TUNEL to evaluate clinical outcome.MATERIALS AND METHODS: Samples from consenting couples were screened for dsDNA rates by neutral Comet assay using an in-house protocol; 200 spermatozoa were assessed per patient. These samples were also assessed by TUNEL using a commercially available kit, analyzing at least 500 spermatozoa per patient. ICSI was performed in the standard fashion.RESULTS: The pilot study reported an average total DNA fragmentation of 11.3AE6% by TUNEL and an average dsDNA of 2.2AE3% by neutral Comet. The results showed a linear relationship between the overall SCF and dsDNA rates (R 2 ¼0.96). This equation was applied to extrapolate the dsDNA levels from 573 normozoospermic men (volume of 2.6AE1 mL, concentration of 42.3AE33x10 6 /mL, 43.1AE10% motility, and 4.2AE1% normal morphology) with an average SCF of 14.2AE8%. Therefore, on the basis of this preliminary test, we established a dsDNA threshold of 3%.A total of 417 couples (maternal age, 37.0AE4 yrs; paternal age, 38.6AE5 yrs) underwent 777 ICSI cycles and presented with dsDNA levels of 1.8AE0.6%. These cycles had a 73.0% fertilization rate, a 12.7% (123/966) implantation rate, and a 23.2% (102/440) clinical pregnancy rate (CPR), of which 15 were lost (14.7%), leaving a 19.7% (87/440) ongoing/delivery rate.There were 155 couples with dsDNA levels of 4.2AE1%. These couples underwent 268 ICSI cycles with a comparable maternal age of 37.3AE5 years, but with older male partners at 41.3AE8 years of age (P<0.001). These cycles had a comparable fertilization rate of 71.0%, implantation rate of 11.4% (53/ 464), and clinical pregnancy rate of 24.3% (44/181). However, these couples were much more likely to lose their pregnancy at 29.5% (13/44; P ¼ 0.03), leaving an ongoing/delivery rate of 17.1% (31/181).CONCLUSIONS: These findings provide further evidence that dsDNA has an important role in the success of a pregnancy generated by ICSI. dsDNA damage has been linked to aneuploidy and consequent pregnancy loss. The use of an assay in a laboratory setting to screen exclusively for dsDNA would help to identify paternally linked aneuploidy that traditional screening would only report as total SCF.

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