Jon W. Gordon

Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

A mouse model of familial amyotrophic lateral sclerosis expressing a mutant superoxide dismutase 1 shows evidence of disordered transport in the vasopressin hypothalamo‐neurohypophysial axis

Amyotrophic lateral sclerosis (ALS) is a fatal, paralytic disorder that primarily affects motoneurons. By combining physiological and morphological approaches, we examined the effect of a murine superoxide dismutase 1 (SOD1) mutation (G86R), which induces neurological disorders resembling human familial ALS (FALS), on the arginine vasopressin (AVP) hypothalamo‐neurohypophysial axis, an unmyelinated tract poor in neurofilaments. First, we observed that G86R mice progressively consumed more water than wild‐type littermates. Furthermore, levels of plasma AVP and neurohypophysial AVP content were decreased in the SOD1 mutant mice, whereas the amount of hypothalamic AVP increased in an age‐dependent manner. However, hypothalamic AVP mRNA levels were not significantly modified in these animals. At the ultrastructural level, we found that the neurohypophysis of G86R mice had a decreased number of neurosecretory axons. Conversely, the presence of large axon swellings was more pronounced in the SOD1 mutant mice. In addition, the size of neurosecretory granules was higher in G86R than in wild‐type animals. All these findings strongly suggest that the FALS‐associated SOD1 mutation injures the hypothalamo‐neurohypophysial axis by provoking early, progressive disturbances in the axonal transport of neurosecretory products from neuronal perikarya to nerve terminals. This blockade could ultimately result in degeneration of the tract, as proposed for the myelinated, neurofilament‐enriched motor axons affected by ALS.

Failed fertilization in vitro: second day micromanipulation of oocytes versus reinsemination

OBJECTIVE To compare routine reinsemination with 2nd day micromanipulation in patients with poor day 1 fertilization. DESIGN A retrospective review of patient records. SETTING The Mount Sinai Medical Center Assisted Reproductive Technologies Program. PARTICIPANTS Patients undergoing IVF-ET who had poor fertilization (< 35%) with standard insemination and underwent second day reinsemination of oocytes (group I, n = 84) compared with patients who underwent 2nd day micromanipulation with subzonal insemination (group II, n = 12). MAIN OUTCOME MEASURES Fertilization rate, cleavage rate, number of embryo transfers, and pregnancy rate. RESULTS Fertilization rate and cleavage rate were significantly higher in group II patients. Pregnancies per transfer were similar between groups I (3/21, 14.3%) and II (0/9, 0%). Second day fertilization was possible in 9 of 12 group II patients, and fertilization rate was higher than day 1 in all nine, however, only 50% achieved cleavage, and none achieved pregnancy. CONCLUSIONS Although micromanipulating oocytes that fail to fertilize may identify occult male factor infertility, may help the clinician plan future cycles, and may result in fertilization and even transfer of embryos in some cycles, there were no pregnancies in our series, and, for now, the clinical efficacy of this procedure remains in question.

Oxidative Stress and a Murine Superoxide Dismutase-1 Mutation Promoting Amyotrophic Lateral Sclerosis Alter Neurosecretion in the Hypothalamo-Neurohypophyseal Axis

In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuroendocrine function, the release of arginine vasopressin (AVP) by the hypothalamo-neurohypophyseal axis. Treatment of mouse-isolated hypothalami and neurointermediate lobes () with H2O2 increased AVP release. This effect was inhibited by copper-zinc superoxide dismutase-1 (SOD1) analogs. By measuring cGMP accumulation as an indicator of biologically active NO, we found that H2O2 treatment decreased cGMP formation in both hypothalami and . We have previously shown that NO inhibits AVP release by a cGMP-independent mechanism. Given that H2O2 stimulated AVP release, while it reduced cGMP production, our findings strongly suggest that oxidative damage affects neurosecretion by reducing NO availability. To test whether such a mechanism may operate under pathological conditions with pronounced oxidative stress, we compared neurosecretion in wild-type and transgenic mice carrying a mutated form of SOD1 associated with human familial amyotrophic lateral sclerosis. Reminiscent of the data obtained from H2O2-treated tissues, hypothalami and from SOD1 mutants displayed decreased cGMP accumulation and increased AVP release, compared with tissues from wild-type littermates. Since neuronal NO synthase expression was not modified, we conclude that the perturbed free radical metabolism associated with the SOD1 mutation is likely to trap NO, and thereby alter neurosecretion, a mechanism that can be exacerbated in specific physiopathological conditions.

Penetration of hamster oocytes by human sperm in an in vitro fertilization microchamber after insemination with unprocessed semen

OBJECTIVE To determine if unprocessed human spermatozoa could capacitate in an IVF microchamber. DESIGN Semen was loaded into an IVF microchamber with zona-free hamster oocytes, and a hamster penetration test was performed. SETTING Medical School basic research laboratory. PATIENTS Men who provided sperm for other laboratory tests. INTERVENTIONS None. MAIN OUTCOME MEASURES Penetration of zona-free hamster oocytes by human sperm. RESULTS In 12 of 19 sperm samples, hamster egg penetration was detected. In most negative cases other measures of sperm function suggested that the SPA might be compromised. CONCLUSIONS Positive hamster oocyte penetration after loading of unprocessed semen into an IVF microchamber indicates that the chamber supports sperm capacitation. Use of such microchambers thus might reduce or eliminate many steps in sperm processing.