Activation of both Mos and Cdc25 is required for G2-M transition in perch oocyte.

Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.

Mitochondrial NAD+-linked State 3 respiration and complex-I activity are compromised in the cerebral cortex of 3-nitropropionic acid-induced rat model of Huntington's disease.

Mitochondrial complex-I dysfunction has been observed in patients of Huntington's disease (HD). We assessed whether such a defect is present in the 3-nitropropionic acid (3-NP) model of HD. Rats treated with 3-NP (10-20 mg/kg i.p., for 4 days) exhibited weight loss, gait abnormalities, and striatal lesions with increased glial fibrillary acidic protein immunostaining on fifth and ninth days, while increase in striatal dopamine and loss of tyrosine hydroxylase immunoreactivity were observed on fifth day following treatment. We report for the first time a dose-dependent reduction in complex-I activity in the cerebral cortex when analyzed spectrophotometrically and by blue native-polyacrylamide gel electrophoresis following 3-NP treatment. The citrate synthase normalized activities of mitochondrial complex-I, -II, -(I + III) and -IV were decreased in the cortex of 3-NP treated rats. In addition, succinate driven State 3 respiration was also significantly inhibited in vivo and in the isolated mitochondria. These findings taken together with the observation of a significant decrease in vivo but not in vitro of State 3 respiration with NAD(+)-linked substrates, suggest complex-I dysfunction in addition to irreversible inhibition of complex-II and succinate dehydrogenase activity as a contributing factor in 3-NP-induced cortico-striatal lesion.

Taurine fails to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced striatal dopamine depletion in mice.

Taurine, a known antioxidant and neuroprotector has been investigated for its free radical scavenging action in vitro in isolated mitochondria, and tested whether it protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in mice. Taurine (0.1-10 mM) did not affect 1-methyl-4-phenyl pyridinium-induced hydroxyl radical production in isolated mitochondria. Systemic administration of taurine (250 mg/kg, i.p.) caused a small, but significant loss of dopamine levels in the striatum of mice. Taurine failed to reverse MPTP-induced striatal dopamine depletion, but caused significant increase in dopamine turnover in these animals. In the light of the present study it may be suggested that consumption of taurine may neither help in scavenging of neurotoxic hydroxyl radicals in the brain mitochondria, nor would it help in blocking the process of neurodegeneration.

Profilin-2 increased expression and its altered interaction with ß-actin in the striatum of 3-nitropropionic acid-induced Huntington's disease in rats.

Subacute systemic treatment with 3-nitropropionic acid (3-NP) causes specific lesions in the cortex and the striatum, and Huntington's disease behavioral phenotypes in rats. We investigated differentially expressed genes in the striatum, and examined status of a highly expressed huntingtin interacting protein, profilin 2 (Pfn2) in relation to 3-NP-induced striatal neurodegeneration, employing both in vivo animal model and in vitro primary striatal neuronal cultures. Golgi staining of 3-NP-treated rat brain revealed significantly altered dendritic spine morphology and decreased spine density in the cortex and the striatum, as compared to the control. We employed suppression subtractive hybridization (SSH) method to screen differentially expressed genes during striatal neurodegeneration in these animals. Forward and reverse SSH provided a library of 188 clones, which were used for reverse northern dot blot analysis to identify greatly altered striatal-specific genes. Sequence analysis of the clones identified 23 genes, expressions of which were ⩾1.5-fold changed (16 up-regulated) in the striatum of 3-NP-treated rats. Immunoprecipitation assay showed decreased binding of Pfn2 with ß-actin, the level of which remained unaffected in the striata and cortices of 3-NP-treated rats. Primary cultures of striatal glutamic acid decarboxylase-65/67 immunopositive GABAergic neurons revealed loss of co-existence of Pfn2 and ß-actin in fluorescence imaging studies following 3-NP treatment for 24h. Since Pfn2 is known to regulate dendritic spine dynamics by interacting with ß-actin, the reduction in its binding affinity to Pfn2 following 3-NP neurotoxic insult, and the accompanying aberrations of the dendritic spine structure and loss of spine density in striatal neurons suggest that Pfn2 may be involved in neurodegeneration in 3-NP-treated rat model of HD.

Cdc2-cyclin B-induced G2 to M transition in perch oocyte is dependent on Cdc25.

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.

Thyroid hormone-induced protein (TIP) gene expression by 3,5,3(')-triiodothyronine in the ovarian follicle of perch (Anabas testudineus, Bloch): modulation of 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase enzyme by TIP.

Our previous reports had shown that 3,5,3'-triiodothyronine (T(3)) induced the generation of a 52-kDa monomer protein, i.e., TIP (thyroid hormone-induced protein) in the perch ovarian follicle. TIP, in turn, increased progesterone formation by stimulating Delta(5)-3beta-HSD activity (3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase) [Eur. J. Endocrinol. 134 (1996) 128-135; Gen. Comp. Endocrinol. 113 (1999) 212-220]. In the present investigation, perch ovarian follicles were incubated in the absence (control) or the presence of T(3) or gonadotropin (GTH) or human chorionic gonadotropin (hCG). RNAs were isolated and allowed to hybridize with a radiolabeled TIP oligonucleotide probe prepared on the basis of the N-terminal 17-amino-acid sequence of TIP. Only RNA from T(3)-incubated follicles hybridized with the probe, while RNA from control or GTH- or hCG-incubated follicles did not hybridize with the probe. The transcript size of TIP mRNA was approximately 1.8 kb. mRNA isolated from T(3)-incubated ovarian follicles subjected to in vitro translation and Western blot analysis clearly identified a 52-kDa protein which was not found with the mRNA from the control follicles. However, both TIP and GTH stimulated progesterone secretion from perch ovarian follicles in vitro. GTH stimulation of Delta(5)-3beta-HSD was due to the stimulation of enzyme protein synthesis as a more than twofold increase in Delta(5)-3beta-HSD occurred in response to GTH. But TIP did not stimulate synthesis of Delta(5)-3beta-HSD protein. However, in vitro incubation of Delta(5)-3beta-HSD enzyme with TIP in the presence of NAD and substrate (pregnenolone) greatly stimulated enzyme activity, while incubation with GTH had no effect, indicating a modulation of Delta(5)-3beta-HSD protein from a less active to a more active state by TIP. This has been supported by another observation, in which TIP (52 kDa) and Delta(5)-3beta-HSD (45 kDa) incubation resulted in a complex of 99 kDa. This suggests a protein-protein interaction in the process of Delta(5)-3beta-HSD activation by TIP. The present work, therefore, shows some new and interesting aspects of thyroid hormone regulation of the reproductive control mechanism.