Estrogen oversees the maintenance of the female genetic program in terminally differentiated gonochorists.

The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17ß (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype.

Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle.

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. At least two protein bands, at 30 kDa (detected by GC3beta antibody) and 62 kDa (detected by 1-4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3beta 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. The 62-kDa protein band detected by 1-4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30-kDa protein was an alpha4 subunit, which is one of the alpha-subunit group of the 20S proteasome, and the 62-kDa protein was a homologue of CCTepsilon, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the alpha4 subunit of goldfish 20S proteasome, alpha4_ca, is phosphorylated in G2-phase and dephosphorylated in M-phase. A binding assay using a recombinant goldfish CCTepsilon revealed that unmodified CCTepsilon interacts with the 26S proteasome. Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3beta cross-reacting upper band reappeared. The 62-kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.

Identification of the Xenopus 20S proteasome alpha4 subunit which is modified in the meiotic cell cycle.

The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation.

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.

Initiation of cyclin B degradation by the 26S proteasome upon egg activation.

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.

Endocrine and hemodynamic changes during liver surgery in patients with compensated liver cirrhosis.

The purpose of this study was to determine hormonal levels in compensated liver cirrhotic patients under general anesthesia before and after liver surgery. We measured plasma norepinephrine, epinephrine, arginine vasopressin, and aldosterone levels and renin activity in non-cirrhotic and compensated cirrhotic patients undergoing liver resection after induction of anesthesia but before skin incision and after the end of operation but before discontinuation of nitrous oxide. We simultaneously measured hemodynamic variables. Plasma levels of norepinephrine (P < 0.001), epinephrine (P < 0.001), arginine vasopressin (P < 0.05), renin (P < 0.05) and aldosterone (P < 0.001) significantly increased after completion of surgery compared with those before incision in both groups. There was a significant positive correlation between plasma renin and aldosterone (r = 0.56, P < 0.01) levels in non-cirrhotics, but no correlation was observed in cirrhotics; and there was a significant positive correlation between plasma norepinephrine and arginine vasopressin (r = 0.45, P < 0.05) levels in non-cirrhotics, but no correlation in cirrhotics. Cardiac index and arterial pressure increased after the end of operation (P < 0.05). This increase after the operation was the same between cirrhotic and non-cirrhotic groups. There were no changes in heart rate, mean pulmonary arterial pressure, and pulmonary capillary wedge pressure after the end of operation. We conclude that hemodynamic and endocrinological changes were similar between compensated cirrhotic patients and non-cirrhotic patients during liver surgery. Endocrine changes might partly explain the hemodynamic changes during surgery.

Effect of combined infusion of nitroglycerin and nicardipine on femoral-to-radial arterial pressure gradient after cardiopulmonary bypass.

Nitrates and calcium channel blockers are frequently administered during cardiac surgery. We simultaneously measured femoral arterial pressure and radial arterial pressure to investigate whether nitrates, in conjunction with calcium channel blockers, would influence the central-to-peripheral arterial pressure gradient. Combined nitroglycerin and nicardipine infusion during cardiac surgery involving coronary artery bypass grafting or valve replacement resulted in a significant increase above baseline levels in the femoral-to-radial arterial pressure gradient at 60 min after cardiopulmonary bypass. In control patients there was no significant increase in the femoral-to-radial arterial pressure gradient at 60 min after completion of cardiopulmonary bypass. A subsequent study in patients given nitroglycerin and nicardipine identified that the difference in the systolic arterial pressure between femoral and radial arteries was observed 15, 60, and 120 min after completion of cardiopulmonary bypass. However, there was no difference in the mean arterial pressure between femoral and radial arteries throughout the same period. We conclude that combined infusion of nitroglycerin and nicardipine, a new calcium channel blocker, intensifies the magnitude and duration of the femoral-to-radial arterial pressure gradient after cardiopulmonary bypass.