Artificial blastocyst collapse prior to vitrification significantly improves Na+/K+-ATPase-dependent post-warming blastocoel re-expansion kinetics without inducing endoplasmic reticulum stress gene expression in the mouse.

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus).

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Serological HLA class I alleles in Senegalese blood donors detected HBs Ag positive.

We analysed the HLA class I alleles in 96 blood donors HBs Ag positive compared with 93 healthy control individuals (HBs negative). The most frequent HLA-A, -B, -C alleles found were, A23 (33.6%); A2 (25%); A30 (25%); B8 (31.5%); B7 (16.3%); B58 (11.9%); B35 (11.9%); B49 (11.9%); B53 (10.8%); Cw7 (39.1%); Cw3 (36.9%); Cw4 (36.9%). Significant differences (P<0.001) were found between the blood donors and the controls for the following HLA alleles, A1; A23; B8 and Cw3. The detection of HBe antigen was positive in 26/84 blood donors. It was observed a significant difference (P<0.01; odds ratios (OR)=6.25) between positive and negative HBe antigens blood donors for HLA-A1 allele.

[Association between Class I HLA alleles and HBs antigen carrier status among blood donors in Senegal]. / Association entre allèles HLA de classe I et portage de l'antigene HBs chez des donneurs de sang au Sénégal.

The course of hepatitis B virus (HBV) infection may be influenced by the host immune response. A prospective study was carried out in ninety-eight subjects (mean age = 23 years) HBs antigens carriers of hepatitis B and living in Dakar, Senegal. We analysed the HLA-A, -B, and C antigens distribution compared to that one of a control (HBs negative) healthy senegalese population (n = 96) living in Dielmo village where a longitudinal study was set-up since 1990. The HLA class I typing was performed by microlymphocytotoxicity assays. The most frequent HLA-A, -B, -C antigens found were: locus A: A23 (33.6%), A2 (25%), A30 (25%), locus B: B8 (31%), B7 (16.3%), B58 (11.9%), B35 (11%), B49 (11%), B53 (10.8%) and locus C: Cw7 (39.1%), Cw17 (39.1%), Cw3 (36.9%), Cw4 (36.6%). Significant differences (P < 0.001) were found between the donors and the control group for the following HLA antigens: A1, A23, B8 and Cw3. The detection of HBe antigen was positive in 26/84 blood donors. It was observed a significant difference (p < 0.01) between positive and negative HBe donors for HLA-A1 allele with an odds ratio of 6.25. All the donors carrying the HLA haplotype: A1-B8-Cw7 (11.5%) were positive in HBe antigen. HLA: B8-Cw7 haplotype (detected in 8.5% of positive donors) seems to be likely associated with a liver cancer according to many reports. An adequate follow-up should be set-up for positive HBe subjects carrying a susceptible HLA type.

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

Antigen-induced long-term potentiation of nicotinic synaptic transmission in the superior cervical ganglion of the guinea pig.

1. Recordings of evoked postganglionic compound action potentials (CAPs) evoked by preganglionic stimulation were obtained from guinea pig superior cervical ganglia (SCGs) in vitro to study the effects of specific antigen challenge on ganglionic synaptic transmission. SCGs were removed from guinea pigs actively sensitized to ovalbumin. 2. Exposing SCGs from sensitized animals to the sensitizing antigen (0.01-10 micrograms/ml) for 5 min produced a sustained increase in the magnitude of the evoked CAP unaccompanied by a change in the preganglionic volley. Nonsensitizing antigens were ineffective. Also ineffective were antigens applied to nonsensitized SCG. This persistent antigen-induced increase in synaptic transmission was designated antigen-induced long-term potentiation (LTP) (A-LTP) because its duration (> 30 min) greatly outlasted posttetanic potentiation (PTP) in this ganglion. 3. A-LTP and neurogenic LTP (N-LTP) were observed to coexist in the same ganglion; the presence of one form of synaptic plasticity did not preclude the development of the other. Both phenomena were influenced by presynaptic factors: prolonged (2 h, 40 Hz) repetitive presynaptic stimulation abolished A-LTP or N-LTP but did not affect PTP. 4. By contrast to N-LTP, which requires a brief presynaptic tetanus, A-LTP could be triggered over a wide range of presynaptic stimulation (0.016-3 Hz) or even in the absence of presynaptic stimulation. 5. The amplitude and duration of A-LTP were not significantly affected by 1) H1, H2, or H3 histamine receptor antagonists added before or after antigen challenge; 2) the presence of saturating concentrations of histamine (100-300 microM); 3) the presence of specific or nonspecific lipoxygenase inhibitors or a selective cyclooxygenase inhibitor; or 4) blockade of alpha- or beta-adrenergic receptors, 5-HT3 receptors, muscarinic receptors, or glutamate receptors, or inhibition of acetylcholinesterase or protein synthesis. 6. Our results indicate that specific antigen challenge of isolated sympathetic ganglia activates resident mast cells to release substances that initiate a novel form of synaptic plasticity, an activity-independent and long-lasting increase in synaptic efficacy.