Identification of a protein kinase which phosphorylate α4 subunit of the 26S proteasome in goldfish oocytes

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3β (anti-goldfish 20S proteasome component 3β) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the α4 subunit of goldfish 20S proteasome (α4 _ca ) were isolated by an immuno-screening method using GC3β. Phosphatase treatment of the 26S proteasome revealed that a part of α4 _ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant α4 _ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1α (CK-1α). The result suggests that CK-1α phosphorylate α4 subunit of the 26S proteasome in immature oocyte of goldfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

Comparative observations of the growth changes of the histochemical properties and collagen architecture of the iliotibialis lateralis muscle from Silky, layer and meat type cockerels

Growth‐related changes in the histochemical property and collagen architecture of the iliotibialis lateralis muscle were compared among Silky, layer and meat cockerels. Histochemical and immunohistochemical methods were employed to observe the collagen architecture. The total intramuscular collagen was also determined. The muscle consisted of type IIA, IIB and IIC myofibers, of which type IIB occurred at the highest frequency. The diameter of type IIB myofibers in each week was largest in the layer, followed by the meat, and was smallest in the Silky. The total amount of collagen reached 3.38 mg/g in the meat bird, 3.03 mg/g in the layer and 2.71 mg/g in the Silky by 30 weeks of age, respectively. In the perimysium, the collagen bundles increased in size and density of fibrils with growth. At 30 weeks of age the layer had compact collagen platelets while the Silky had loose collagen bundles. In the meat bird, the collagen bundles were moderately compact. The endomysial collagen network had a large mesh size at 1 week and thereafter accumulated many collagen fibrils to form a felt‐like fabric of fibrils at 30 weeks of age. From these results it appears that growth‐related changes in the iliotibialis lateralis muscle are not necessarily causally affected by the different growth rates of chicken breeds.     

Impact of bark stripping by sika deer, Cervus nippon, on subalpine coniferous forests in central Japan

We investigated the effect of bark stripping by sika deer, Cervus nippon, on forest regeneration in subalpine coniferous forests on Mt. Ohdaigahara and in the Ohmine Mountains of central Japan. Bark stripping by sika deer occurred in five major tree species: Abies homolepis; Abies Veitchii; Tsuga diversifolia; Picea jezoensis var. hondoensis; and Chamaecyparis obtusa. The percentage of damaged trees on Mt. Ohdaigahara was higher than in the Ohmine Mountains, probably because of the higher deer density. On Mt. Ohdaigahara, the DBH distributions of stems for P. jezoensis var. hondoensis, A. homolepis, T. diversifolia andC. obtusa were bell-shaped with fewer smaller and larger trees. On the other hand, in the Ohmine Mountains the distributions for P. jezoensis var. hondoensis and A. Veitchii showed a reverse-J shaped with more smaller trees. Larger overstory conifers on Mt. Ohdaigahara were killed by bark stripping when 100% barked, although in the Ohmine Mountains ca. 50% of the trees survived even when 100% barked. After the disappearance of the overstory conifers on Mt. Ohdaigahara, the dwarf bamboo, Sasa nipponica, expanded into the forest floor due to changes in light reaching the forest floor. Since S. nipponica is the main forage of deer in this area, this increase caused a corresponding increase in the deer population, which in turn, could cause a further decline in the coniferous forests.     

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T₄) ‘overdose’ were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T₄ treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol–17β levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.     

Expression of cytochrome P-450aromatases in the sex-reversed Nile tilapia

Present study revealed the expression of ovarian (oP450arom) and brain (bP450arom) type cytochrome P-450 aromatases in the gonads and brains of sex reversed Nile tilapia.     

Differentiation and development of Leydig cells,and changes of testosterone levels during testicular differentiation in tilapiaOreochromis niloticus

Initial appearance and development of Leydig cells (LCs) during testicular differentiation in tilapia,Oreochromis niloticus, were investigated histologically. In addition, changes of testosterone levels in gonadal tissue and serum were examined by radioimmunoassay. In the gonads of fry at 23–26 days after hatching, initial testicular differentiation was confirmed by the observation of the differentiation of connective tissues into tissues which are characteristic of the adult testis. LCs, which were identified by the ultrastructural features (a moderate number of mitochondria with tubular cristae, well developed smooth endoplasmic reticulum and many free ribosomes) appeared initially at the time of testicular differentiation. LCs increased in number rapidly in the testes of fish at 70 days after hatching. Concomitant with this increase, spermatogonia increased in number. Testosterone was detectable in the fish at 40–50 days after hatching, but levels in tissue and serum were low. Testosterone levels increased gradually in the fish beginning at 70 days after hatching and increased still more at 100–150 days accompanying active spermatogenesis.