Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in Mozambique tilapia.

Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ERα, ERß1, and ERß2. A primary function of 17ß-estradiol (E(2)) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg). To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ERα and ERß, and an antagonist for mammalian ERα, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-piperidino-pyrazole (MPP), respectively. ERα was more sensitive and responsive to PPT than ERß1 or ERß2 in transactivation assays. All ER isoforms indicated equivalent responsiveness to DPN compared with E(2), although sensitivity to DPN was lower. MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E(2) effect on Vg and ERα 48h post-injection. DPN increased ERα and Vg expression and plasma Vg post-injection, whereas PPT was without effect; DPN seems to stimulate Vg production through activation of ERα. The ligand binding domain of all tilapia ER forms shares only 60-65% amino acid identity with human ERα and ERß. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs.

Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.

From reptilian phylogenomics to reptilian genomes: analyses of c-Jun and DJ-1 proto-oncogenes.

Genome projects have revolutionized our understanding of both molecular biology and evolution, but there has been a limited collection of genomic data from reptiles. This is surprising given the pivotal position of reptiles in vertebrate phylogeny and the potential utility of information from reptiles for understanding a number of biological phenomena, such as sex determination. Although there are many potential uses for genomic data, one important and useful approach is phylogenomics. Here we report cDNA sequences for the c-Jun(JUN) and DJ-1(PARK7) proto-oncogenes from 3 reptiles (the American alligator, Nile crocodile, and Florida red-belly turtle), show that both genes are expressed in the alligator, and integrate them into analyses of their homologs from other organisms. With these taxa it was possible to conduct analyses that include all major vertebrate lineages. Analyses of c-Jun revealed an unexpected but well-supported frog-turtle clade while analyses of DJ-1 revealed a topology largely congruent with expectation based upon other data. The conflict between the c-Jun topology and expectation appears to reflect the overlap between c-Jun and a CpG island in most taxa, including crocodilians. This CpG island is absent in the frog and turtle, and convergence in base composition appears to be at least partially responsible for the signal uniting these taxa. Noise reduction approaches can eliminate the unexpected frog-turtle clade, demonstrating that multiple signals are present in the c-Jun alignment. We used phylogenetic methods to visualize these signals; we suggest that examining both historical and non-historical signals will prove important for phylogenomic analyses.

Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol.

Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.

The behavior of estrogenic substances in the biological treatment process of sewage.

A study was conducted for about one year on the fate and behavior of estrogens, namely 17beta-estradiol (E2), estrone (E1), and estriol (E3) in an activated sludge process of a pilot scale plant supplied with domestic sewage. A simultaneous analytical method for these three substances using LC-MS/MS was developed and applied to sewage samples. The average removal of E2 was 94.7%, while that of E3 was 96.9%. In contrast, the average removal of E1 was relatively low at 69.2% with a maximum concentration of 55.4 ng/L detected in the treated water. The theoretical values of estrogenic activity calculated from the concentrations of each natural estrogen in treated water were found to correlate with the values of estrogenic activity measured by a yeast estrogen screening assay. The effect of E2 on estrogenic activity in influent was found to be high, while that of E1 in treated water was considerably higher. In batch treatment tests on E2, E2 turned into E1 immediately after being charged. After three hours of aeration, the values of both E1 and E2 were around threshold limits. It was determined from this that E1 and E2 were substances that could be degraded by biological treatment. As the removal of E2 was found to be sufficiently high at times, optimization of operational conditions based on E1 removal should be important for reducing estrogenic activity in treated water.

Dot pattern generation technique using molecular dynamics.

We have developed a new technique for generating homogeneously distributed irregular dot patterns useful for optical devices and digital halftoning technologies. To introduce irregularity, we use elaborately designed sequences called low-discrepancy sequences instead of pseudorandom numbers. We also use a molecular-dynamics redistribution method to improve the distribution of dots. Our method can produce arbitrary density distributions in accordance with a given design. The generated patterns are free from visible roughness as well as any moiré patterns when superimposed on other regular patterns. We demonstrate that our method effectively improves luminance uniformity and eliminates moiré patterns when used for a backlight unit of a liquid-crystal display.

Developmental effects of estrogenic agents on mice, fish, and frogs: a mini-review.

Laboratory experiments have demonstrated that exposure of rodents to sex hormones during prenatal or early postnatal life can cause permanent and irreversible alterations of the endocrine and reproductive organs, such as ovary, fallopian tube, uterus, cervix, vagina, and mammary gland in females; and testis, epididymis, prostate, and seminal vesicle in males; as well as non reproductive organs including bones and muscle and immune and nervous systems in both sexes. Early development of Xenopus laevis into the tadpole and Fundulus heteroclitus goes through a rapid cell division, gastrulation, neurulation, and organogenesis within 1 week after fertilization. The developing embryo is very fragile and sensitive to estrogenic agents. Thus, these animals can be used as a suitable model for examining the effect of endocrine disruptors (hormonally active agents) on the development of aquatic living beings, which are most likely to be exposed to the compounds.

Biochemical identification of Xenopus Pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a Nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein.

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.

Ca2+ is required for phosphorylation of clam p82/CPEB in vitro: implications for dual and independent roles of MAP and Cdc2 kinases.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs which are translationally dormant or masked until meiotic maturation. Fertilisation of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. Here we examine the protein kinase(s) that phosphorylates clam p82/CPEB using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. We show that p82/CPEB phosphorylation requires Ca2+ (<100 microM) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. We propose a model of clam p82/CPEB phosphorylation in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed.

Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype.

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). We reported that expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes were deficient in cultured mast cells (CMC) derived from mutant mice of mi/mi genotype. Despite the reduced expression of both MMCP-5 and MMCP-6, their regulation mechanisms were different. Because MMCP-5 is a chymase and MMCP-6 a tryptase, there was a possibility that the difference in regulation mechanisms was associated with their different characteristics as proteases. We compared the regulation mechanisms of another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The expression of the MMCP-4 gene was also deficient in mi/mi CMC. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-4 gene in mi/mi CMC, indicating the involvement of +-MITF in transactivation of the MMCP-4 gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4 expression was more similar to MMCP-6 than to MMCP-5. We also showed the deficient expression of granzyme B and cathepsin G genes in mi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and MMCP-5 are located on chromosome 14. Because all these genes showed deficient expression in mi/mi CMC, there is a possibility that MITF might regulate the expression of these genes through a locus control region.

Translational regulation of cyclin B mRNA by 17alpha,20beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone) during oocyte maturation in a teleost fish, the goldfish (Carassius auratus).

17Alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was identified as maturation-inducing hormone (MIH) in several teleost fishes. In goldfish (Carassius auratus), 17alpha,20beta-DP induces oocyte maturation by stimulating the de novo synthesis of cyclin B, a regulatory subunit of maturation-promoting factor (MPF). In this study, we examined the control mechanisms of 17alpha,20beta-DP-induced de novo synthesis of cyclin B protein in oocytes, which is a prerequisite step for MPF activation during oocyte maturation in goldfish. Cycloheximide-treated oocytes failed to undergo meiotic maturation in response to 17alpha,20beta-DP; in this group neither cyclin B nor 34-kDa active cdc2 was detectable in oocytes. In contrast, oocytes exposed to actinomycin D plus 17alpha,20beta-DP or 17alpha,20beta-DP underwent maturation; in these groups both cyclin B and 34-kDa cdc2 were present. Northern blotting showed that cyclin B mRNA is present in both immature and mature oocytes. Sequence analysis revealed that goldfish cyclin B mRNA contains four copies of cytoplasmic polyadenylation element (CPE)-like motifs in the 3' noncoding region, suggesting that the initiation of cyclin B synthesis during oocyte maturation may be controlled by the elongation of poly (A) tail. We then examined the polyadenylation state of cyclin B mRNA during 17alpha,20beta-DP-induced oocyte maturation by means of a PCR poly (A) test, and found that cyclin B mRNA is polyadenylated during oocyte maturation. Polyadenylation of cyclin B mRNA occurred at the same time of germinal vesicle breakdown. Furthermore, cordycepin, an inhibitor of poly (A) addition of mRNA, prevented 17alpha,20beta-DP-induced oocyte maturation. These findings suggest that in goldfish oocytes, the synthesis of cyclin B protein is under translational control and that cytoplasmic 3' poly(A) elongation is involved in 17alpha,20beta-DP-induced translation of cyclin B mRNA.

Alteration of protease expression phenotype of mouse peritoneal mast cells by changing the microenvironment as demonstrated by in situ hybridization histochemistry.

Mouse mast cell protease (MMCP) mRNA expression was examined by in situ hybridization histochemistry. Peritoneal mast cells (PMCs) of WBB6F1-(+/+) mice expressed MMCP-2, MMCP-4, MMCP-5, and MMCP-6 mRNAs, but did not express MMCP-1 mRNA. When proliferation of PMCs was induced by culturing them in methylcellulose with T cell-derived cytokines, cells in mast cell colonies expressed MMCP-1 mRNA. These mast cells were transferred to a suspension culture to induce further proliferation. The phenotype of the resulting PMC-derived cultured mast cells was similar to that of bone marrow-derived cultured mast cells. When 10(5) PMC-derived cultured mast cells or 10(5) bone marrow-derived cultured mast cells were injected into the stomach wall of mast cell-deficient WBB6F1-W/Wv mice, mast cells that appeared in the mucosa and muscularis propria were similar to mast cells in the stomach of intact WBB6F1-(+/+) mice, indicating the injected cells adapted to a new tissue environment. In contrast, when 10(5) PMCs were injected into the stomach wall of WBB6F1-W/Wv mice, the injected PMCs did not adapt to the mucosa. When 20 PMCs were injected, they proliferated and adapted to the mucosal environment. The present results suggest that PMCs adapt to new environments when proliferation occurs before redifferentiation.

Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA-binding protein.

Cyclin B is a regulatory subunit of maturation-promoting factor. In goldfish (Carassius auratus) oocytes, cyclin B is synthesized de novo after stimulation by 17alpha,20beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone). In this study, we examined goldfish oocyte proteins bound to cyclin B mRNA. Using oligo(dT)-cellulose affinity chromatography and northwestern blotting analysis, we identified a 54-kDa cyclin B mRNA-binding protein (p54). Southwestern blotting analysis showed the binding of p54 to the Y box DNA element (CTGATTGGCCAA), suggesting that p54 is a Y box protein in goldfish. We isolated two cDNA clones, GFYP1 and GFYP2, the latter of which encodes a germ-cell-specific Y box protein. An antibody against a GFYP2 protein recognized p54, suggesting that p54 is identical or highly similar to GFYP2 protein. This is also supported by the finding that a recombinant GFYP2 expressed in bacteria bound to both the Y box DNA element and the goldfish cyclin B mRNA synthesized in vitro. These results suggest that p54 is a germ-cell-specific Y box protein and is a potential masking protein of cyclin B mRNA in goldfish oocytes.

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.

Molecular cloning and immunological analysis of goldfish cyclin A during oocyte maturation.

Cyclin A belongs to a family of proteins involved in the regulation of the eukaryotic cell cycle. Although cyclin A is thought to be involved in the regulation of both S and M phase, its exact role in the cell cycle, especially in the meiotic cycle (oocyte maturation), is uncertain. We isolated cyclin A cDNA clones from a goldfish oocyte cDNA library. Monoclonal antibody raised against bacterially produced goldfish cyclin A recognized a 47-kDa protein that disappeared after egg activation. Unlike goldfish cyclin B, which is absent in immature oocytes, cyclin A was already present in immature oocytes and its protein level did not change remarkably during oocyte maturation. These results differ from the finding in Xenopus, in which cyclin A is absent, but cyclin B is present, in immature oocytes. Goldfish cyclin A was associated with cdc2 kinase in mature oocytes, but not with cdk2. Recombinant cyclin A bound to and activated cdc2 in a cell-free system, but cyclin A and cdk2 binding was not observed. The kinase activity of cyclin A-cdc2 was undetectable in immature oocytes and first appeared at about the time of germinal vesicle breakdown (GVBD). In contrast to the cyclin B-cdc2 activity that corresponded to the occurrence of GVBD, cyclin A-cdc2 activity increased only slightly until GVBD was completed and increased drastically after the completion of the first meiotic division. Furthermore, microinjection of cyclin A mRNA into immature oocytes did not cause GVBD; however, microinjection of cyclin B mRNA did. These results suggest that cyclin A-cdc2 kinase and cyclin B-cdc2 kinase play different roles in controlling oocyte maturation. The roles of cyclin A in the rapid activation of cyclin B-cdc2 kinase at meiosis I and II transition and in the maintenance of high maturation-promoting factor activity in mature unfertilized eggs are discussed.

Regulation of oocyte growth and maturation in fish.

This chapter has briefly reviewed the current status of investigations on the hormonal regulation of oocyte growth and maturation in fish (see Figs. 4 and 9). Pituitary gonadotropins are of primary importance in triggering these processes in fish oocytes. In both cases, however, the actions of gonadotropins are not direct, but are mediated by the follicular production of steroidal mediators, estradiol-17 beta (oocyte growth) and 17 alpha,20 beta-DP or 20 beta-S (oocyte maturation). Investigators have established that both estradiol-17 beta and 17 alpha,20 beta-DP are biosynthesized by salmonid ovarian follicles via an interaction of two cell layers, the thecal and granulosa cell layers (two-cell-type model). The granulosa cell layers are the site of production of these two steroidal mediators, but their production depends on the provision of precursor steroids by the thecal cell layers. A distinct steroidogenic shift from estradiol-17 beta to 17 alpha,20 beta-DP, occurring in salmonid ovarian follicles immediately prior to oocyte maturation, is a prerequisite for the growing oocytes to enter the maturation stage, and requires a complex and integrated network of gene regulation involving cell specificity, hormonal regulation, and developmental patterning. The cDNAs for most of the steroidogenic enzymes responsible for estradiol-17 beta and 17 alpha,20 beta-DP biosynthesis have been cloned from rainbow trout ovaries. Our next task is to determine how gonadotropin and other factors act on ovarian follicle cells to turn the expression of these specific genes on and off at specific times during oocyte growth and maturation. Increasing evidence now suggests that a variety of neuromodulatory, autocrine, and paracrine factors may also be involved in the regulation of steroidogenesis in fish ovarian follicles. Molecular biological technologies should be applied to identify these substances. Of considerable interest is the finding that MIH, unlike most steroid hormones, acts on its receptors at the surface of oocytes. Further studies of the association of the MIH-MIH receptor complex with a Gi protein, probably resulting in the inactivation of adenylate cyclase, should lead to a discovery of a new mechanism of steroid hormone action. The early steps following MIH action involve the formation of the major cytoplasmic mediator of MIH, MPF. Fish MPF, like that of Xenopus and starfish, consists of two components: cdc2 kinase and cyclin B. Nevertheless, the mechanism of MIH-induced MPF activation in fish oocytes differs from that in Xenopus and starfish because the appearance of cyclin B protein is a crucial step for 17 alpha,20 beta-DP-induced oocyte maturation in fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavior of the components of maturation-promoting factor, cdc2 kinase and cyclin B, during oocyte maturation of goldfish.

We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.

Meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor.

Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.

Induction of reinitiation of meiosis in amphibian Bufo and Xenopus oocytes by injection of M-phase extracts of ciliate Tetrahymena needs the recipient protein synthesis.

We show here that germinal vesicle breakdown of amphibian Bufo and Xenopus oocytes can be induced if ciliate Tetrahymena extracts are injected into them. The activity of meiosis-reinitiation-inducing factor (MRIF) appeared only a M-phase of a synchronously dividing culture, indicating that this MRIF has an important function for induction of M-phase in the mitotic cell cycle. MRIF of Tetrahymena differed from MPF (M-phase-promoting factor), because its action on the induction of GVBD was inhibited by cycloheximide and it could not induce GVBD in starfish oocytes by microinjection. MPF activity was not detected in extracts of vegetatively growing Tetrahymena. Preliminary experiments showed that MRIF was a heat-labile, Ca2(+)-sensitive, and trypsin-sensitive soluble protein.

[Posterior chamber lens implantation concurrent with vitrectomy for proliferative diabetic retinopathy].

Combined operation of cataract removal with posterior chamber intraocular lens implantation and pars plana vitrectomy were performed on 25 eyes in 22 patients with cataract concurrent with diabetic retinopathy. In 21 eyes, extracapsular cataract extraction followed by intraocular lens insertion, aiming at in-the-bag fixation, was performed, and in 4 eyes pars plana lensectomy with anterior capsule left and intraocular lens insertion between the iris and anterior capsule was carried out. Mean postoperative follow-up period was 14 months, ranging from 3 to 32 months. Visual acuity on the last examination was 0.5 or better in 2 eyes (8%), 0.1 or better in 12 eyes (48%), and worse than 0.05 in 9 eyes (36%). Major postoperative complications were fibrin reaction (20 eyes, 80%), pupillary deformation (19 eyes, 76%), pupil capture by intraocular lens (3 eyes, 12%), rhegmatogenous retinal detachment (1 eye, 4%), neovascular glaucoma (2 eyes, 8%), and recurrent vitreous hemorrhage (13 eyes, 52%). Intraocular pressure was well controlled in neovascular glaucoma cases. At the last examination ocular fundus was invisible due to vitreous hemorrhage in two eyes.