Time course of cortical and zona reactions of pig oocytes upon intracellular calcium increase induced by thimerosal

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 microM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 +/- 1.14, was observed 509.64 +/- 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 +/- 11.7 CGs/100 micron 2 of cortex in control oocytes to 25.7 +/- 19.2 CGs/100 micron 2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 +/- 10.4 CGs/100 micron 2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 +/- 5.1 CGs/100 micron 2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 +/- 14.8 CGs/100 micron 2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.     

Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro

This study evaluated the effect of adding reduced glutathione (GSH) during sperm washing and insemination on the subsequent fertilization dynamics and development of IVM porcine oocytes. Follicular oocytes were matured in vitro in NCSU 23 medium with porcine follicular fluid, cysteine and hormone supplements for 22 h. They were then matured in the same medium but without hormones for another 22 h. Matured oocytes were stripped of cumulus cells and co-incubated with frozen-thawed spermatozoa for 5 h. Putative embryos were cultured in NCSU 23 with BSA for either 7 h to examine fertilization parameters or 6 d to evaluate cleavage (2 d) and blastocyst rates. In Experiment 1, GSH was added to the insemination medium at 0, 0.125, 0.25 or 0.5 mM. The presence of GSH during insemination did not affect (P>0.05) rates of penetration, polyspermy, male pronuclear formation or cleavage, but did increase (P<0.05) blastocyst formation rates when added at concentrations of 0.125 (36%) and 0.25 mM (34%) compared with that of the control (0 mM; 19%). However, the numbers of inner cell mass and trophectoderm cells of blastocysts were unaffected by GSH treatment (P>0.05). The presence of GSH during insemination was found not to significantly increase intracellular glutathione concentrations of oocytes (P>0.05). In Experiment 2, addition of GSH (0.25 mM) during sperm washing did not affect cleavage or blastocyst formation rates or cell numbers (P>0.05). In conclusion, the presence of GSH during insemination improves the developmental competence of IVM pig oocytes in a dose-dependent manner.     

Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.     

A maize thiamine auxotroph is defective in shoot meristem maintenance

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.     

Intravenously administered cloxacillin-induced neutropenia with eosinophilia in a patient with infective endocarditis: a case report

Background

Bacteremia following Staphylococcus aureus is a serious clinical condition which is often associated with distant metastatic infections. One of the most dreaded complications of Staphylococcus aureus bacteremia is infective endocarditis. Cloxacillin is a common antibiotic prescribed for suspected staphylococcal infections and confirmed methicillin-sensitive Staphylococcus aureus infections. Prolonged use of cloxacillin may lead to neutropenia.

Case presentation

A 38-year-old Sinhalese man presented to Teaching Hospital Kurunegala, Sri Lanka, complaining of a 3-week history of fever; he was found to have a pansystolic murmur over the apex of his heart. He had leukocytosis with predominant neutrocytosis. His C-reactive protein was 231?mg/l and erythrocyte sedimentation rate was 100?mm/first hour. Transthoracic two-dimensional echocardiography revealed prolapsed mitral valve with 7?×?13?mm vegetation over the posterior mitral valve. On the following day, three blood cultures became positive and were subsequently identified as Staphylococcus aureus. Intravenously administered cloxacillin 3?g 6 hourly was started. Following day 24 of intravenously administered cloxacillin, our patient developed high spike fever. His total white blood cells were: 990/mm³ with 34% neutrophils and 22% eosinophils. His hemoglobin concentration was 9.5?g/dL and platelet count remained normal (202?×?10⁶/mm³). His C-reactive protein was 78?mg/l, erythrocyte sedimentation rate was 95?mm/first hour, and he was otherwise comfortable, showing no signs of sepsis beside the high grade fever. His serum was negative for filarial and Toxoplasma antibodies while stool was negative for oocytes and amoebic cysts. Further, his serum was negative for dengue virus, Epstein–Barr virus, cytomegalovirus, and hepatitis B antibodies. He was clinically well on day 6 after stopping cloxacillin with 44% neutrophils and 18% eosinophils. His C-reactive protein and erythrocyte sedimentation rate became normal, and there was no further plan for cardiothoracic intervention or administration of antimicrobials. He was discharged from hospital and remained well 6?months later.

Conclusion

This case report signifies the potential fatal adverse effect of cloxacillin in methicillin-sensitive Staphylococcus aureus infections. Leukopenia is associated with prolonged use of high doses of cloxacillin. In addition to transthoracic two-dimensional echocardiography and inflammatory markers, sequential white blood cells and differential counts would help clinicians to assess the prognosis of patients with infective endocarditis.

     

Morphologic comparison of ovulated and in vitro–matured porcine oocytes,with particular reference to polyspermy after in vitro fertilization

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo–matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro–matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro–matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro–matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro–matured oocytes were dissolved within 131.7 ± 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro–matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for β-D-Gal(1–3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro–matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro–matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro–matured and in vivo–matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional block to polyspermy in pig oocytes. Mol. Reprod. Dev. 49:308–316, 1998. © 1998 Wiley-Liss, Inc.     

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.     

Activation of porcine oocytes with calcium ionophore: effects of extracellular calcium

The present study examined the mechanism of A23187-induced activation in pig oocytes, with special reference to the effects of extracellular calcium on oocyte activation. The following endpoints were evaluated: intracellular free calcium concentration ([Ca2+]i), intracellular pH ([pH]i), cortical granule (CG) exocytosis, pronuclear formation, and blastocyst development. In experiment one, when oocytes were exposed to 50 microM A23187 for 5 min in a medium with, or without, calcium, a significant (P < 0.004) increase in the [Ca2+]i was observed in medium with calcium but not in medium without calcium. An increased [pH]i (0.08 unit in medium with calcium and 0.13 unit in medium without calcium), cortical granule exocytosis and pronuclear formation were observed in oocytes treated with A23187 irrespective of the presence or absence of calcium in the medium. In experiment two, the effects of treatment time (0, 0.5, 1, 2, and 5 min) on nuclear activation of oocytes with A23187 were further examined in medium with, or without, calcium. It was found that a 2 min treatment activated more (71-74%) oocytes than the other treatments. Treatment for 5 min in medium without calcium resulted in chromatin condensation in some oocytes. Microtubules were not found in these oocytes. In experiment three, developmental ability was examined of the oocytes treated with A23187 in medium with, or without, calcium. In vitro fertilized oocytes were used as a positive control. It was found that 16%, 6% and 38% of the oocytes treated with A23187 in medium with calcium, in medium without calcium, and in vitro fertilized oocytes developed to blastocysts after culture for 7 days, respectively. These results indicate that A23187 can induce pig oocyte activation in calcium-free medium without a typical increase in the [Ca2+]i and that A23187-induced pig oocyte activation is accompanied by an increase in [pH]i. Oocytes activated with A23187 can develop to blastocysts regardless of activation in medium with, or without, calcium.