Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

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.

Fluoxetine Mimics the Anorectic Action of Estrogen and Its Regulation of Circadian Feeding in Ovariectomized Female Rats.

Our previous study demonstrated that chronic estrogen replacement in ovariectomized rats reduces food intake and augments c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. Here, we hypothesized that serotonergic neurons in the central nervous system (CNS), which have anorectic action and play a role in regulating circadian rhythm, mediate the light phase-specific anorectic action of estrogen, and that selective serotonin reuptake inhibitors (SSRIs) mimic the hypophagic action of estrogen. Female Wistar rats were ovariectomized and treated with estradiol (E2) or cholesterol by subcutaneously implanting a silicon capsule containing E2 or cholesterol. Then, half of the cholesterol-treated rats were injected with the SSRI fluoxetine (5 mg/kg) (FLX group), while the remaining rats in the cholesterol-treated group (CON group) and all those in the E2 group were injected with saline subcutaneously twice daily at the onsets of the light and dark phases. Both E2 and FLX reduced food intake during the light phase but not the dark phase, and reduced body weight gain. In addition, both E2 and FLX augmented the c-Fos expression in the SCN, specifically during the light phase. These data indicate that FLX exerts estrogen-like antiobesity and hypophagic actions by modifying circadian feeding patterns, and suggest that estrogen regulates circadian feeding rhythm via serotonergic neurons in the CNS.

[Case of leptomeningeal carcinomatosis effectively treated with intrathecal chemotherapy using ventriculoperitoneal shunt].

A 70-year-old man presented with dizziness, headache and hearing loss. He was admitted to our hospital because of increasing unsteadiness of gait. Magnetic resonance imaging of the brain revealed meningeal thickening with enhancement. The lumbar puncture revealed high opening pressure. The cerebrospinal fluid showed pleocytosis, high carcinoembryonic antigen (CEA) concentration, and presence of neoplastic cells, leading to the diagnosis of leptomeningeal carcinomatosis. Systemic investigation for primary neoplasm identified a Bormman type 3 gastric cancer (papillary adenocarcinoma with micropapillary pattern). Except for the meninges, no metastatic lesions could be detected. A ventriculoperitoneal shunt (Codman Hakim Programmable Valve) was placed for management of intracranial hypertension and intrathecal chemotheray. He was started on oral S-1 (TS-1) combined with intrathecal methotrexate injection using the VP shunt reservoir. In two weeks, headache and hearing loss completely disappeared and gait disturbances started to improve. CSF findings also improved remarkably with disappearance of neoplastic cells and almost normalization of CEA. For the next five months, he was well on oral S-1 and monthly intrathecal chemotherapy, being able to walk using a walker and to stay at home. He subsequently developed posterior cortical symptoms such as prosopagnosia and cortical blindness and gradually lapsed into coma. He died from pneumonia one year after the onset of neurological symptoms. At autopsy, primary gastric cancer was found but much reduced in size. No peritoneal metastasis could be found. In the brain, leptomeningeal carcinomatosis involved the occipital lobes, the base of the temporal lobe, and the cerebellum. We suggest that intrathecal chemotherapy using ventriculoperitoneal shunt with programmable valve system could be an effective method for the treatment of meningeal carcinomatosis.

Involvement of syntaxin 18, an endoplasmic reticulum (ER)-localized SNARE protein, in ER-mediated phagocytosis.

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 gamma receptor, in which phagocytosis occurs in an IgG-mediated 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.

A new synthetic method for alpha-alkoxycarbonyl iminium salt and its reaction with nucleophiles.

An iminium salt was easily prepared using the oxidation of amino ketene silyl acetal with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and the subsequent nucleophilic addition to this iminium species proceeded efficiently to afford alpha-amino esters in good yields.