Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse

Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium and oocyte survival, as well as the intracellular events involved in transducing the antiapoptotic actions of these cytokines in germ cells, remain to be elucidated. In this report, we investigated the effects of SCF and LIF, alone and in combination, on the survival of oogonia and oocytes, and elaborated on components of the signal transduction pathway used by these molecules, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth factor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 13.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrity. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such that most oogonia and oocytes present after 72 h were apoptotic. Morphometric analysis of serially sectioned ovaries indicated that the numbers of nonapoptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 38%, and 10%, respectively, of the number present before culture (P < 0.05 for all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apoptosis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF) or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml IGF-I maintained survival of approximately two-thirds of the germ cells in cultured ovaries (P < 0.05), whereas a combination of all three growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng/ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, such that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovaries with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K), LY294002 (5 microM) or wortmannin (50 nM), whereas cotreatment with an inhibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These data indicate that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-mediated female germ cell survival. Moreover, TGF-beta can partially override the antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, suggesting the existence of a complex signaling network that ultimately determines fetal ovarian germ cell fate.     

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Retrograde ureteral intussusception: a rare complication

A 45-year-old man with a history of cyclophosphamide exposure underwent repeated ureteroscopy for positive urine cytology findings after resection of a Grade 2 papillary transitional-cell carcinoma of the bladder. Despite careful technique, an intussusception developed in the left ureter, which was repaired by resection and construction of a Boari flap. To our knowledge, this is the first report of retrograde ureteral intussusception caused by ureteroscopy.     

Corneal epithelial recovery following photorefractive keratectomy

AIMS: To further understand the morphological and functional recovery of corneal epithelium following excimer laser photorefractive keratectomy (PRK). METHODS: The right eyes (group 1) of 15 male, New Zealand white rabbits weighing 2-3 kg underwent PRK. The left eye of each rabbit (group 2) underwent simple mechanical de-epithelialisation and were examined as treated controls. Both eyes of another eight rabbits (group 3) served as untreated controls. All eyes underwent a corneal epithelial permeability study by fluorophotometry at 2, 4, and 8 weeks after surgery. Five animals in groups 1 and 2 were sacrificed at 9, 10, and 12 weeks after surgery. The animals in group 3 were sacrificed at the end of the 12 week experimental period. Both eyes of each sacrificed animal were enucleated immediately and processed for both haematoxylin and eosin stain and electron microscopic study. The electron micrograph was magnified to 14,000x and the extent of hemidesmosome formation was quantified and analysed. RESULTS: The corneal epithelial barrier to sodium fluorescein was subnormal and returned to a normal barrier state 4 weeks after PRK in group 1 whereas it was normal in group 2 throughout the examination period. The extent of hemidesmosome formation was abundant yet subnormal in both groups 1 and 2 up to 12 weeks, when compared with that in group 3. CONCLUSION: The corneal epithelium regained its functional barrier 4 weeks after PRK in rabbits while the extent of hemidesmosome formation was still subnormal 12 weeks after mechanical de-epithelialisation, with or without PRK.     

A behavioral medicine intervention in persons with HIV

This study used a pretest-posttest preexperimental design to examine the effect of a 10-week behavioral medicine support group intervention in a sample of persons with HIV. Using Solomon's psychoneuroimmunologic framework, the 10-week behavioral medicine program focused on the mind/body interaction, the relaxation response, coping with illness, hardiness, and nutrition. Pearson correlation coefficients and t tests were performed on the pre- and postintervention measures of hardiness, social support, immune function, and perceived health status. Results of the study indicated that hardiness (preintervention) and CD4 counts (pre- and postintervention) were significantly correlated with health status; however, CD4 counts decreased over the course of the behavioral medicine program. Implications for nursing and recommendations for further research are discussed.