Electronic and steric control in regioselective addition reactions of organolithium reagents with enaldimines.

A reaction mode of imines derived from naphthalene-1-carbaldehyde and acyclic alpha,beta-unsaturated aldehydes with organolitium reagents was dependent on the characteristic nature of a substituent on the imine nitrogen atom. An imine having an electron-withdrawing aryl group on the nitrogen atom behaves as a 1,2-directing imine toward organolithium reagents. In contrast, an imine bearing an alkyl or a bulky aryl group favors 1,4-addition of organolithium reagents. Electronic and steric tuning of a substituent on the imine nitrogen atom for a reaction mode was rationalized on the basis of molecular orbital calculations.

Superwind Model of Extended Lyalpha Emitters at High Redshift.

We propose a new model for the extended Lyalpha blobs found recently at high redshift (z approximately 3). The observational properties of these blobs are as follows: (1) the observed Lyalpha luminosities are approximately 1043 h-2 ergs s-1, (2) they appear elongated morphologically, (3) their sizes amount to approximately 100 kpc, (4) the observed line widths amount to approximately 1000 km s-1, and (5) they are not associated with strong radio continuum sources. All these observational properties seem to be explained in terms of galactic winds driven by successive supernova explosions shortly after the initial burst of massive star formation in the galactic centers. The observed number density of Lyalpha blobs ( approximately 3.4x10-5 h3 Mpc-3) may be explained if their present-day counterparts are elliptical galaxies with a luminosity above approximately 1L*.

[A case report of primary hemangiopericytoma of the chest wall].

A 35-year-male was found to have an abnormal shadow with an extrapleural sign located in the right lower lung field by a chest X-ray. Chest CT revealed a well demarcated tumor in the chest wall adjacent to the 4th rib. Chest MRI showed that the tumor contained punctate or linear low-intensity areas, which were considered to be small blood vessels. A diagnosis of hemangiopericytoma was established by percutaneous needle biopsy. Under the definite diagnosis, extended resection of the chest wall was performed to remove the tumor with a surgical margin of more than 5 cm, corresponding to surgery for other malignant soft-tissue neoplasmas. Hemangiopericytomas rarely arise in the chest wall, although they can be found in any region which contains pericytes. Preoperative definite diagnoses of hemangiopericytoma have rarely been reported. However, preoperative diagnosis is an important factor in deciding the operative procedure for hemangiopericytoma. It has been reported that hemangiopericytomas show local recurrences and distant metastases, although they are histologically benign. We consider that hemangiopericytomas in the chest wall should be treated with extensive resection corresponding to surgery for other malignant soft-tissue neoplasmas.

Extraskeletal Ewing's sarcoma mimicking traumatic hematoma.

We describe the clinical course of a 16-year-old baseball player with a history of recurrent hematoma of the thigh. The lesion was aspirated percutaneously several times and curetted under the diagnosis of traumatic hematoma. Microscopical examination revealed massive hemorrhage, necrosis, and a small number of Ewing's sarcoma cells. He died of multiple metastases. With recurrent hematoma in the soft tissue, neoplastic lesions should be ruled out.

Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation.

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22 h in vitro, 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro, only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs.

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca(2+)-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.

Quantified analysis of cortical granule distribution and exocytosis of porcine oocytes during meiotic maturation and activation.

Polyspermy is one of the unresolved problems that exist regarding pig oocytes matured and inseminated in vitro. Quantitative study of the changes in the cortical granule (CG) population in oocytes is essential for understanding the mechanism of how oocytes block polyspermic penetration and for developing the optimum conditions for in vitro maturation (IVM) and in vitro fertilization (IVF). The present study was conducted to quantify the CG distribution in pig oocytes during IVM and IVF by using fluorescein isothiocyanate-labeled peanut agglutinin with laser confocal microscopy. The results indicate that CGs are distributed in the cortex cytoplasm of oocytes at the germinal vesicle (GV) stage with a mean number of 33.8 +/- 7.3 CGs/100 microm2 of cortex. As nuclear maturation proceeded to metaphase I and metaphase II, CGs migrated to the cortex and formed a continuous monolayer under the oolemma. No distinct CG-free domain was observed in oocytes during maturation. The migration of CGs to the cortex continued during maturation, with an increased CG density after the GV stage. All oocytes penetrated by spermatozoa were activated and released CGs from ooplasm with an average residual number of 3.5 +/- 4.6 CGs/100 microm2 of cortex at 18 h after insemination. Complete CG exocytosis was observed in 45% of oocytes. Calcium ionophore did not induce oocyte nuclear activation, but CGs were released from oocytes with an average of 7.1 +/- 4.5 CGs/100 microm2 of cortex still present when examined 18 h after treatment. An electrical pulse induced 89% of nuclear activation in matured oocytes, and CG exocytosis was observed only in nuclear-activated oocytes with an average residual number of 6.4 +/- 9.4 CGs/100 microm2 of cortex. Complete CG exocytosis was induced by ionophore and electrical pulse in 10% and 25% of the oocytes, respectively. These results indicate that CGs migrate to the cortex in pig oocytes during IVM and that the matured oocytes obtained under these maturation conditions possess the ability to release CGs upon sperm penetration, ionophore treatment, and electrical pulse. However, a functional block to polyspermic penetration in oocytes after CG exocytosis was not fully established in these studies. The present methods and results provide the approach for further investigation of the reasons for polyspermy in pig oocytes matured and inseminated in vitro.

Induction of cortical granule exocytosis of pig oocytes by spermatozoa during meiotic maturation.

Pig oocytes were examined to test their ability to undergo cortical granule exocytosis upon penetration by spermatozoa during meiotic maturation. Immature or maturing oocytes (cultured in vitro for 0 h, 26 h and 46 h) were inseminated with ejaculated boar spermatozoa in vitro. Before and after insemination, oocytes were stained with peanut agglutinin labelled with fluorescein isothiocyanate and the cortical granule distributions were examined under the fluorescent microscope and the laser confocal microscope. Before insemination, all the oocytes at the germinal vesicle stage showed a uniform distribution of cortical granules throughout the cortical cytoplasm. The granules migrated centrifugally during maturation and were distributed just beneath the oolemma in the oocytes after germinal vesicle breakdown, forming a monolayer in metaphase I or metaphase II. Cortical granules were still present in all penetrated oocytes at the germinal vesicle stage 18 h after insemination; in contrast, 26% and 84% of the oocytes inseminated at the stages of germinal vesicle breakdown or at metaphase I and II, respectively, completely released their cortical granules. Nuclear activation rates of penetrated oocytes were 0%, 38% and 96% in oocytes cultured for 0 h, 26 h and 46 h, respectively. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 88% (oocytes cultured for 46 h) released cortical granules completely. Complete cortical granule exocytosis was not observed in nuclear-inactivated oocytes. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 80% (oocytes cultured for 46 h) of monospermic oocytes and 67% (oocytes cultured for 26 h) and 91% (oocytes cultured for 46 h) of polyspermic oocytes released cortical granules, and no statistical difference was observed between oocytes cultured for 26 h or 46 h, or between monospermic and polyspermic oocytes. The proportion of oocytes with cortical granule exocytosis increased as insemination time increased and was greatest 18 h after insemination in oocytes cultured for 26 h and 46 h; no obvious changes were observed when the insemination time was prolonged to 24 h. These results indicate that pig oocytes develop the ability to release cortical granules after penetration by spermatozoa following germinal vesicle breakdown, and that this ability is not fully developed until metaphase II. Cortical granule exocytosis is accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic maturation are responsible for the cortical reaction. Polyspermy may be a result of a complete failure of cortical granule exocytosis in immature oocytes and delayed CG exocytosis in matured oocytes.

Calcium- and meiotic-spindle-independent activation of pig oocytes by the inhibition of staurosporine-sensitive protein kinases.

The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3 microM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3 microM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, respectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.

Distribution of cortical granules in bovine oocytes classified by cumulus complex.

The present study was conducted to examine distributional changes of cortical granules (CGs) during meiotic maturation and fertilisation in vitro and the developmental ability in bovine oocytes classified by cumulus cells. The oocytes were classified by the morphology of their cumulus cell layers as follows: class A, compact and thick; class B, compact but thin; class C, naked; and class D, expanded. Some of the oocytes were stained with Lens curinalis agglutinin (LCA) before and after maturation in vitro and after insemination, and then stained with orcein to observe their nuclear stages. The others were left in culture. Distributional patterns of the CGs were classified into four types: type I, CGs distributed in clusters; type II, CGs dispersed and partly clustered; type III, all CGs dispersed; and type IV, no CGs. Most of the oocytes before culture showed a type I pattern, but this decreased after maturation culture, whereas type III increased in class A. The oocytes of class B showed similar changes while the oocytes of class C did not. In class C, many oocytes showed type I after culture, indicating that cytoplasmic maturation was not completed. In class D, 80.4% of the oocytes exhibited type III before maturation culture, indicating that their cytoplasmic maturation was different from classes A-C. And about 70% of the class D oocytes were at the nuclear stage of germinal vesicle breakdown (GVBD) before culture. The developmental rates to blastocysts in classes A-D were 28.7%, 23.1%, 0.5% and 3.4% respectively.

Effect of follicle cells on in vitro fertilization of pig follicular oocytes.

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.

Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes.

Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with 3H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of 3H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.

Two sensitivity levels of cattle oocytes to puromycin.

Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.

Maturation and fertilization of pig follicular oocytes cultured in pig amniotic fluid.

Three experiments were conducted to assess the ability of pig amniotic fluid to support oocyte maturation and developmental competence. In Experiment 1, pig follicular oocytes were cultured in pig amniotic fluid (PAF) containing luteinizing hormone (LH) and estradiol-17beta for 28 to 48 h, during which time the maturational stages of the oocytes were observed. While the maturation rates to the second metaphase were high (62%) after 33 h of culture, the rates decreased (24 to 30%) when oocytes were cultured in PAF without LH. In Experiment 2, oocytes matured in PAF were inseminated in vitro with fresh semen from three boars. The spermatozoal penetration rate ranged from 42 to 100%, and 15 to 40% of the penetrated oocytes had both male and female pronuclei. In Experiment 3, oocytes were cultured for 46 to 47 h in PAF and transferred to the oviducts of inseminated gilts. Eighteen of 136 embryos recovered from the uteri 128 h after oocyte transfer developed to the morula and blastocyst stages. The embryos were transferred to a recipient, and two piglets were born (one live and one dead) of the resultant pregnancy. These results indicate that PAF can be used for maturation of pig follicular oocytes and that oocytes cultured in PAF have the developmental capacity to become piglets.

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization.

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P less than 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll.

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32 46 ) in Bull A and 54.2% (58 107 ) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 degrees C; (II) 30 to 80%, 10 degrees C; (III) 30 to 80% 20 degrees C; (IV) 30 to 90%, 20 degrees C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV) 66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.

In-vitro fertilization of pig oocytes by frozen boar spermatozoa.

In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37 degrees C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0-40%. In contrast, none of the oocytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.