Glutathione transferase isoenzymes from human prostate.

By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.     

Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis)

Husbandry and health of the African clawed frog, Xenopus laevis, greatly influences the quality of oocytes produced. One factor affecting oocyte quality is the water conditions in which females are maintained. Dechlorination and adequate salt concentration are known to affect oocytes, but water hardness has not been considered an important factor in Xenopus husbandry by the research community. We found that, when females were kept in soft water or water with marine salts alone, even when it was cooled to 17 to 18 degrees C, the quality of oocytes decreased; only 20 to 25% of resulting embryos developed to tailbud stages. Survival and normal development of embryos increased significantly within one month of addition to the laboratory housing water of salts that mimic conditions in African Rift Valley lakes. These salts greatly increased water hardness; development of embryos to tailbud stages remained high (50 to 70% on average) for more than a year after their addition to the water housing females. Water from South African ponds where X. laevis are collected, and from wells used by the major suppliers of X. laevis, also was moderately to very hard. Our results suggest that X. laevis is naturally adapted to hard water, and indicate that increasing general hardness during laboratory housing is more important for oocyte quality and embryo development than is increasing carbonate hardness (alkalinity) in the water used to house females.     

Cutaneous acariasis in the African clawed frog (Xenopus laevis)

Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Factors affecting oogenesis in the South African clawed frog (Xenopus laevis)

Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.     

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.     

Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Subunit homologies of lactate dehydrogenase (LDH) isoenzymes between amphibians (Xenopus laevis) and mammals (Wistar rat)

• 1.1. The subunit distribution and subunit homologies of LDH isoenzymes were studied in the amphibian Xenopus laevis and in Wistar rats.
• 2.2. Several of the 11–15 isoenzymes of the pattern in Xenopus, separable by vertical starch gel electrophoresis, were purified, hybridized, and the cross-reaction of antibodies against the most positively charged isoenzyme with the isoenzymes present in tissue extracts of both species was tested.
• 3.3. The isoenzyme with the highest positive charge in Xenopus is a M-homotetramer homologous to mammalian LDH₅.
• 4.4. The multibanded pattern of Xenopus LDH isoenzymes is very probably due to heterozygoty of the gene locus controlling the synthesis of M-subunits rather than to an epigenetic subbanding phenomenon.

     

A comparison of Xenopus laevis oocyte and embryo mRNA

Total RNA, extracted from mature oocytes and tadpoles of Xenopus laevis, was used as a template for in vitro protein synthesis. The oocyte RNA is markedly deficient in abundant mRNA species by comparison to tadpole RNA or other somatic RNAs, in agreement with previous experiments using RNA-cDNA hybridization analysis (S. Perlman and M. Rosbash, 1978, Develop. Biol.63, 197–212). Oocyte pA⁺ RNA is also larger than tadpole pA⁺ RNA or other somatic pA⁺ populations. The larger oocyte pA⁺ RNA and smaller oocyte pA⁺ RNA are equally good templates for in vitro protein synthesis, which implies that much, and perhaps all, of the large oocyte pA⁺ RNA is bona fide mRNA. We suggest that the relatively large size of the oocyte pA⁺ RNA population is due, at least in part, to the relative lack of abundant mRNA species in the population. This suggestion follows from the observation of 0. Meyuhas and R. P. Perry (1979, Cell16, 139–148) that L-cell-abundant mRNAs are preferentially small and rare mRNAs preferentially large. Most of the oocyte pA⁺ sequences are also present in tadpoles and are still adenylated at this stage. Oocyte proteins synthesized in vivo do not appear deficient in abundant proteins, suggesting that a translational control mechanism operates to select certain pA⁺ RNAs at higher frequencies than others.     

Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.     

Tradeoffs between somatic and gonadal investments during development in the African clawed frog (Xenopus laevis)

Tradeoffs between time to and size at metamorphosis occur in many organisms with complex life histories. The ability to accelerate metamorphosis can increase survival to the next life stage, but the resulting smaller size at metamorphosis is often associated with lower post-metamorphic survival or reduced fecundity of adults. Reduced fecundity is thought to be because of reduced energy reserves, longer time to maturity, or reduced capacity to carry eggs or compete for mates. This pattern could also be explained by a shift in allocation to somatic growth that further retards the growth or development of reproductive tissues. The main goal of this study was to determine if the relationship between growth and development of somatic and gonadal tissues depends on environmental conditions. We address this question through two experiments in which we quantify the development and growth of the body and gonads of Xenopus laevis reared in different resource environments. First, tadpoles were reared communally and development and growth were evaluated over time. Restricted food reduced somatic and gonadal growth rate, but did not affect the developmental rate of either tissue type. Second, tadpoles were reared individually and evaluated at metamorphosis. Restricted food reduced somatic development and growth, but only influenced size, and not developmental stage of testes at metamorphosis. This work demonstrates that environmental conditions influence tradeoffs between growth and development of somatic and gonadal tissues, apparently in a sex-specific manner. These tradeoffs may contribute to phenotypic correlations between small size and reduced fitness.     

Oogenesis in Xenopus laevis (Daudin)

Summary This study was designed to explore the relationship of estrogen, human chorionic gonadotropin (HCG), and food availability to endocytosis in developing oocytes. When estrogen alone is administered to an animal, large amounts of vitellogenin are synthesized by the liver and secreted into the circulatory system, where it accumulates. Under these conditions there is no evidence of endocytosis at the surface of the oocytes. Other studies have shown that following HCG injection into estrogen-treated animals, vitellogenin is removed from the circulation and the oocyte surface is highly contoured and displays endocytotic activity. Food deprivation has much the same effect on oocyte endocytosis as does estrogen. When animals are given HCG and subsequently starved for 20 days, developing oocytes show little endocytotic activity. We conclude that HCG acts to promote or stimulate endocytosis in developing oocytes while estrogen and/or starvation inhibits this process.Research sponsored by the Energy Research and Development Adiministration under contract with Union Carbide Corporation.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.     

Classification of DNA polymerase activities from ovaries of the frog,Xenopus laevis

Four distinct DNA polymerase activities were isolated from ovaries of the frog Xenopus laevis. Specific assays for each activity were established. The isolated activities were characterized by molecular weight, template-primer preferences, and sensitivity to specific inhibitors as Xenopus laevis ovarian DNA polymerases-α₁, -α₂, -β, and -γ. All previously described Xenopus laevis DNA polymerases were classified using these properties.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.     

Expression of RhoB in the developing Xenopus laevis embryo

Rho GTPases are signaling components that participate to the control of cell morphology, adhesion and motility through the regulation of F-actin cytoskeleton dynamics. In this paper, we report the identification of RhoB in Xenopus laevis (XRhoB) and its expression pattern during early development. Whole-mount in situ hybridization analysis indicated that XrhoB is expressed at high levels in the dorsal marginal zone early in gastrula and in the dorsal midline at later stages. At mid-neurula stages, XrhoB expression extends to the central nervous system, presomitic mesoderm and somites. Later during development, rhoB mRNA is detected in the eyes, the migrating neural crest cells as well as the dorso-lateral part of the somites.