The chemistry of lipid alkoxyl radicals and their role in metal-amplified lipid peroxidation.

Reaction of polyunsaturated fatty acid hydroperoxides with metal complexes generates lipid alkoxyl radicals and metal-oxo complexes. Lipid alkoxyl radicals are presumed to be the species responsible for metal-amplified lipid peroxidation because of the chemical analogy of simple organic alkoxyl radicals to the hydroxyl radical. However, polyunsaturated fatty acid alkoxyl radicals exhibit a rich and diverse chemistry that is dominated by intramolecular cyclization to epoxyallylic radicals. Studies described herein demonstrate that the equilibrium between cyclization and ring-opening of epoxyallylic radicals lies overwhelmingly toward cyclization. Thus lipid alkoxyl radicals have a steady-state concentration that is so low that their contribution to metal-amplified lipid peroxidation is insignificant. In fact, the species responsible for metal amplification of lipid peroxidation appears to be the epoxyperoxyl radical formed by coupling the epoxyallylic radical to molecular oxygen.

Chromosome 15 aneuploidy in the sperm and conceptus of a sibling with variable familial expression of round-headed sperm syndrome.

OBJECTIVE: To characterize rates of chromosome aneuploidy in sperm from three siblings, one of whom had an IVF/ICSI conceptus with trisomy 15. DESIGN: Blind evaluation of the sperm chromosome aneuploidy rates, semen quality, and sperm ultrastructure. SETTING: IVF clinic and university-based andrology research laboratory. PATIENT(S): Three brothers, two of whom underwent infertility evaluation and therapy. MAIN OUTCOME MEASURE(S): Semen from three siblings was coded and blindly evaluated for standard World Health Organization semen quality variables and sperm ultrastructure. Sperm were decondensed and hybridized with fluorescent probes specific for chromosomes X, Y, 13, 15, 18, and 21, then evaluated microscopically to determine the aneuploidy rate for those chromosomes. RESULT(S): Two siblings had increased round-headed morphology on standard morphology evaluation, which was confirmed using electron microscopy. The sperm aneuploidy rate was significantly increased for chromosome 15 in sibling 1, the father of a conceptus with trisomy 15. Aneuploidy rates were also slightly increased for chromosomes X, Y, and 18 in sibling 1. CONCLUSION(S): This is the second report of increased sperm chromosome aneuploidy in infertile patients with round-headed sperm. Although ICSI is successful in treating this syndrome, the risk for aneuploidy of the conceptus may be increased. Other studies have reported an increased incidence of sperm chromosome aneuploidy in some infertile patients, but this is the first report of aneuploidy in both the sperm and conceptus of a patient undergoing IVF/ICSI.

Hypoxic/ischemic encephalopathy associated with placental insufficiency in a cloned foal.

Hypoxic/ischemic encephalopathy in a cloned American Quarter horse foal was initially associated with placental insufficiency and exacerbated by protracted hypotension during anesthesia for a surgical procedure. The foal, born at the Texas A&M Veterinary Medical Center, was diagnosed at birth with neonatal maladjustment syndrome that was accompanied by dysmaturity, muscle contracture of the front limbs, and a blood clot within the lumen of the urinary bladder. Seizures that developed after anesthesia were attributed to hypoxia/ischemia during anesthesia and culminated in death. Macroscopically, the cerebrum had flattened cerebral gyri with shallow sulci, yellowish cortical discoloration, and apple-green autofluorescence (under 365-nm ultraviolet light) at the cortical/white matter junction. Microscopically, there was laminar cortical necrosis with prominent diffuse ischemic change of neuronal cell bodies. The white matter had prominent rarefaction with focal axonal and myelin degeneration and focal macrophage (gitter cell) accumulation. Additionally, there was astrocytic hypertrophy with gemistocyte formation. The chorioallantois was diffusely thickened in the area corresponding to the uterine horns. Histologically, microcotyledons were markedly attenuated with absence of chorionic villi.

Polyunsaturated fatty acid alkoxyl radicals exist as carbon-centered epoxyallylic radicals: a key step in hydroperoxide-amplified lipid peroxidation.

13-Hydroperoxyoctadeca-9,11,15-trienoic acid was reacted with a catalytic amount of 5,10,15,20-tetraphenyl-21H,23H-porphyrin iron(III) chloride in dichloromethane containing 2,4,6-tri-tert-butylphenol. The principal products were identified as 13-oxooctadeca-9,11,15-trienoic acid, 13-oxotrideca-9,11-dienoic acid, and a series of isomeric epoxyaryl ethers [9-(2,4,6-tri-tert-butylphenoxy)-12,13-epoxyoctadec-10-enoic acids and 11-(2,4,6-tri-tert-butylphenoxy)-12,13-epoxyoctadec-9-enoic acids]. The epoxyaryl ethers are coupling products of 2,4,6-tri-tert-butylphenoxyl radical and an epoxyallylic radical formed by cyclization of an intermediate alkoxyl radical. The high yield of epoxyaryl ethers relative to 13-oxotrideca-9,11-dienoic acid suggests the equilibrium between alkoxyl radical and epoxyallylic radical lies predominantly toward epoxyallylic radical. This cyclization appears to be a key step in the amplification of lipid peroxidation by polyunsaturated fatty acid hydroperoxides.

Effect of fluctuations in temperature encountered during handling and shipment of human cryopreserved semen.

The effect of temperature fluctuations which cryopreserved spermatozoa may undergo during routine shipping and handling was evaluated in sperm frozen with two cryoprotectants. Sperm frozen in TEST-yolk buffer maintained motility better than those frozen in glycerol solution in all studies. Sperm motility was significantly compromised in samples stored more than one day in dry ice, regardless of the cryoprotectant, and more than two days in a liquid nitrogen shipping dewar if frozen in glycerol solution. Sperm motility was not compromised following exposure to room temperature for up to 3 min if TEST-yolk buffer was used as the cryoprotectant, but was compromised following 1 min exposure using glycerol cryoprotectant. This study describes the limits of non-ideal conditions that spermatozoa may undergo during shipping and handling, and demonstrates the effects of the cryoprotectant on those limits.

Pyrroles as effective agents for blocking amine nitrosation.

The ability of 10 pyrroles to block the acidic nitrosation of morpholine has been determined by using an assay that measures their effectiveness in the presence of a 10-fold excess of amine. The log (% N-nitrosomorpholine) formed is a linear function of blocking agent concentrations ranging from 0.0 to 1.5 times the equivalents of initial nitrite. The negative slopes of these plots allow a ranking of the effectiveness of the blocking agent. Several of the pyrroles have been found to be much more effective than established blocking agents such as ascorbic acid. The following order of blocking ability has been determined: 2,5-dimethylpyrrole = 1-benzyl-2,5-dimethylpyrrole much greater than 4-methylcatechol greater than ascorbic acid = 1,2-phenylenediamine = pyrrole greater than 1,2,5-tribenzylpyrrole = 1-benzylpyrrole = octamethylporphine much greater than ammonium sulfamate = hydrazine = 2,5-diphenylpyrrole much much greater than beta-nicotyrine greater than 2-pyrrolecarboxylic acid. Pyrroles give complex mixtures devoid of N-nitroso compounds upon nitrosation.

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.

Mechanism of reaction of fatty acid hydroperoxides with soybean peroxygenase.

13(S)-Hydroperoxyoctadeca-9(Z),11(E),15(Z)-trienoic acid (13-HPOT) was used to probe the mechanism of the hydroperoxide O-O bond cleavage catalyzed by solubilized and partially purified soybean peroxygenase. When reacted with this ferrihemoprotein, it was converted to 13(S)-hydroxyoctadeca-9(Z),11(E), 15(Z)-trienoic acid (13-HOT) and a single epoxide regio-isomer, i.e. 15,16-cis-epoxy-13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid (15,16-EHOD). In the absence of co-oxidizable substrates, such as oleic acid or thiobenzamide, this latter compound accounted for about two-thirds of the reaction products. 13-HOT and 15,16-EHOD are products of heterolytic scission of the O-O bond of 13-HPOT; no products arising by homolytic scission could be detected. Therefore, soybean peroxygenase catalyzes hydroperoxide reduction exclusively by a heterolytic mechanism leading to a ferryl-oxo complex analogous to peroxidase compound I. In similar experiments, 13(S)-hydroperoxyoctadeca-9(Z),11(E)-dienoic acid gave 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid and 9,10 epoxy-13(S)-hydroxyoctadec-11(E)-enoic acid. Experiments with 18O-labeled 13-HPOT indicated that about 83% of the oxygen atom incorporated into the epoxide group of 15,16-EHOD, originated from the hydroperoxide group. Moreover, using mixtures of unlabeled and 18O-labeled 13-HPOT, it was established that this transfer takes place predominantly (about 3:1) by an intramolecular process. In the intermolecular reaction 13-HOT, formed after reduction of the hydroperoxide, diffuses from the active site and, after reassociation, is epoxidized at the 15,16-double bond. A unifying mechanistic scheme, which takes into account all of the reactions catalyzed by the peroxygenase, is proposed.

Early and selective pathology of light chain neurofilament in the spinal cord and sciatic nerve of G86R mutant superoxide dismutase transgenic mice.

Pathologic accumulation of neurofilament protein (NF), both within spheroids of the proximal axon and within inclusions of motor neuron somata, is a hallmark of neurodegeneration in amyotrophic lateral sclerosis (ALS). Transgenic mice that express mutations in superoxide dismutase (SOD-1), which were genetically linked to familial ALS, develop symptomatology and pathology that strongly resemble ALS and therefore provide a useful model for studying the disease. Examining NF in the G86R mutant SOD-1 transgenic mice, we previously demonstrated that phosphorylated NF accumulates in motor neuron somata of symptomatic transgenic mice. In the present study, we expand these results by examining the immunocytochemical distribution of the three subunits of NF (i.e., light, medium, and heavy chains) as well as tubulin in presymptomatic and symptomatic SOD-1 transgenic mice. Although all NF subunits, but not tubulin, accumulate along with phosphorylated NF in the spinal cord inclusions of symptomatic mice, numerous inclusions containing only light chain NF are found in the spinal cord of presymptomatic SOD-1 transgenic mice. In addition to these results in the spinal cord, intensely immunoreactive aggregates of NF-L, but not the other NF subunits or tubulin, were observed in the sciatic nerve of both symptomatic and presymptomatic mutant SOD-1 transgenic mice. These results suggest that the mechanism of NF alteration in SOD-1 transgenic mice, and also perhaps in ALS patients, originates with the disruption of NF-L, only later involving the other subunits.

Sperm chromosome aneuploidy as related to male factor infertility and some ultrastructure defects.

Some men have elevated levels of sperm chromosome aneuploidy. In this study, we have evaluated and summarized sperm aneuploidy rates in male infertility patients and control groups. The mean aneuploidy rate for five chromosomes (X, Y, 13, 18, 21) was 1.2 +/- 0.1 for fertile controls, 1.4 +/- 0.1 for a general population control group, and 5.8 +/- 1.14 for the patients. When the patients were classified by the type of male factor infertility, the total aneuploidy rate was 2.6 +/- 0.3 in men with moderately diminished semen quality (n = 7), 4.0 +/- 0.3 patients with severe teratoasthenooligozoospermia, and 15.9 +/- 3.8 for men with rare ultrastructure defects such as round head only syndrome or severe tail agenesis. Some infertility patients have a severely elevated level of sperm chromosome aneuploidy, which may contribute to infertility or diminish the likelihood of a successful outcome from IVF/ICSI. The severity of sperm chromosome aneuploidy appears to be proportional to the severity of abnormal semen quality: in particular, abnormal morphology. The high rates of aneuploidy in patients with severe ultrastructure defects suggest that caution should be employed in counseling those patients prior to IVF/ICSI.