Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an α-fetoprotein-secreting hepatoma

The purpose of this work was to study the effect of α-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-17β (E₂)-dependent marker, i.e., progestophilins in E-sensitive cells C₂9RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of E₂, while they proliferate in culture regardless of the presence of E₂. C₂9RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3 × 10⁻¹⁰ M E₂ in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3 × 10⁻⁸ M E₂ in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 11-Methoxy-17β ethynylestradiol (R₂₈₅₈), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E₂ and with R₂₈₅₈.In addition, we compared the free E₂ levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E₂ concentrations above 11 pg E₂/ml, whereas there was no correlation between the free E₂ levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E₂ below 0.4 pg/ml, whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E₂ of 44 pg/ml. We conclude that the effect of hepatoma serum on the E₂ induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than E₂ trapping by AFP.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

Genome-wide analyses of the Jeju,Thoroughbred, and Jeju crossbred horse populations using the high density SNP array

The Jeju horse is an indigenous Korean horse breed that is currently registered with the Food and Agriculture Organization of the United Nations. However, there is severe lack of genomic studies on Jeju horse. This study was conducted to investigate genetic characteristics of horses including Jeju horse, Thoroughbred and Jeju crossbred (Jeju?×?Thoroughbred) populations. We compared the genomes of three horse populations using the Equine SNP70 Beadchip array. Short-range Linkage disequilibrium was the highest in Thoroughbred, whereas r² values were lowest in Jeju horse. Expected heterozygosity was the highest in Jeju crossbred (0.351), followed by the Thoroughbred (0.337) and Jeju horse (0.311). The level of inbreeding was slightly higher in Thoroughbred (??0.009) than in Jeju crossbred (??0.035) and Jeju horse (??0.038). F_ST value was the highest between Jeju horse and Thoroughbred (0.113), whereas Jeju crossbred and Thoroughbred showed the lowest value (0.031). The genetic relationship was further assessed by principal component analysis, suggesting that Jeju crossbred is more genetically similar to Thoroughbred than Jeju horse population. Additionally, we detected potential selection signatures, for example, in loci located on LCORL/NCAPG and PROP1 genes that are known to influence body. Genome-wide analyses of the three horse populations showed that all the breeds had somewhat a low level of inbreeding within each population. In the population structure analysis, we found that Jeju crossbred was genetically closer to Thoroughbred than Jeju horse. Furthermore, we identified several signatures of selection which might be associated with traits of interest. To our current knowledge, this study is the first genomic research, analyzing genetic relationships of Jeju horse, Thoroughbred and Jeju crossbred.     

Maternal and paternal genetic variation in Estonian local horse breeds in the context of geographically adjacent and distant Eurasian breeds

The maternal and paternal genetic variation of horse breeds from the Baltic Sea region, including three local Estonian breeds, was assessed and compared with that of Altai and Yakutian horses. In the mtDNA D‐loop region, 72 haplotypes assigned to 20 haplogroups in the nine breeds were detected. In Estonian local breeds, 38 mtDNA haplotypes were found, and five of them were shared by the three breeds. More than 60% of all identified haplotypes were rare. Compared with the Estonian Native and Estonian Heavy Draught breeds, a higher haplotypic diversity was found in the Tori breed (h = 0.969). Moreover, four haplotypes shared among Finnish and Estonian local horse breeds indicated ancient ancestry, and of these, H30 (haplogroup D3) showed global sharing and genetic links between modern Baltic Sea region and Siberian horses, specifically. The studied breed set showed high variability in maternal inheritance and mixed patterns of the international and native breeds of the Siberian and Baltic regions. No variation was found in paternally inherited markers among horse breeds in the Baltic Sea region.     

PHOTOSYNTHESIS-IRRADIANCE RELATIONSHIPS IN SEA ICE MICROALGAE FROM McMURDO SOUND,ANTARCTICA1

Sea ice microalgae in McMurdo Sound, Antarctica were examined for photosynthesis-irradiance relationships and for the extent and time course of their photoadaptation to a reduction in in situ irradiance. Algae were collected from the bottom centimeter of coarse-grained congelation ice in an area free of natural snow cover. Photosynthetic rate was determined in short term (1 h) incubations at ?2° C over a range of irradiance from 0 to 286 μE·m^?2·s^?1. Assimilation numbers were consistently below 0.1 mg C·mg chl a^?1·h^?1. The I_k's³ averaged only 7 μE·m^?2·s^?1, and photosynthesis was inhibited at irradiances above 25 μE·m^?2·s^?1. Photosynthetic parameters of the ice algal community were examined over a nine day period following the addition of 4 cm of surface snow while a control area remained snow-free. A reduction of 40% in P_m^B relative to the control occurred after two days of snow cover; α, β, I_k, and I_m were not significantly altered. Low assimilation numbers and constant standing crop size, however, suggested that the algal bloom may have already reached stationary growth phase, possibly minimizing their photoadaptive response.     

Two distinct old yellow enzymes are involved in naphthyl ring reduction during anaerobic naphthalene degradation

The 2‐naphthoyl‐coenzyme A (NCoA) reductase (NCR) is so far the only characterized enzyme involved in the anaerobic degradation of the environmentally relevant polycyclic aromatic hydrocarbons. The old yellow enzyme (OYE) family member apparently reduced the nonactivated naphthyl ring to 5,6,7,8‐tetrahydro‐2‐napthoyl‐CoA (THNCoA). In this work, the candidate genes of three NCRs from the sulphate‐reducing, naphthalene‐degrading N47 and NaphS2 cultures were expressed in Escherichia coli. The isolated products contained flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) a [4Fe‐4S] cluster and catalyzed only the two‐electron reduction of NCoA to 5,6‐dihydro‐2‐naphthoyl‐CoA (5,6‐DHNCoA) at a very negative E°′ = ?493 mV. All NCRs exhibited high NCoA‐forming DHNCoA oxidase activities that are proposed to be involved in oxygen‐detoxification during naphthalene degradation. Extracts of N47 and NaphS2 catalyzed the reduction of 5,6‐DHNCoA to THNCoA. Genes putatively coding for 5,6‐DHNCR from N47 and NaphS2 were heterologously expressed in E. coli. The enriched enzyme products specifically catalyzed the reduction of 5,6‐DHNCoA to THNCoA at E°′ = ?375 mV. With the three NCRs and two 5,6‐DHNCRs, five OYEs have been characterized that are involved in the reduction of the nonsubstituted naphthyl‐ring system; these unprecedented enzymatic reactions expand our knowledge of the functional diversity of OYE.     

Influence of energy and nutrient supply pre and post partum on performance of multiparous Simmental,Brown Swiss and Holstein cows in early lactation

A study was conducted to evaluate the effects of pre partum (PRE) and post partum (POST) dietary energy and nutrient supply (E) and their interactions on feed intake, performance and energy status in dairy cows of three breeds. In this experiment, the effects of three energy and nutrient supply levels (low (L), medium (M), high (H)), both pre-calving and post-calving, were investigated, using a 3×3 factorial arrangement of treatments. In both phases (84 days pre- and 105 days post-calving) E levels applied to a total of 81 multiparous cows of breeds Simmental (SI), Brown Swiss (BS) and Holstein–Friesian (HF; n=27 for each breed), were 75%, 100% and 125% of recommendations of the German Society of Nutrition Physiology (GfE). Dry matter intake (DMI) was restricted, if energy intake exceeded target values. Pre partum DMI and energy intake were different as designed, liveweight and body condition score (BCS) of SI cows were higher, but EB was lower, compared to BS and HF cows. Milk yield and composition were influenced by all three main experimental factors (E_PRE, E_POST, breed). Energy-corrected milk yield was 25.6, 28.6 and 30.1 kg/day for L_PRE, M_PRE and H_PRE as well as 21.5, 30.1 and 32.6 kg/day for L_POST, M_POST and H_POST, respectively. Numerically, only for milk protein content the interactions E_PRE×E_POST and E_PRE×breed reached significance. Impact of energy supply pre-calving was more pronounced when cows had lower energy supply post-calving and vice versa. On the other hand, milk yield response of cows to energy supply above requirements was greater for cows that were fed on a low energy level pre partum. Impact of energy level pre partum was higher for HF cows, showing that their milk production relies to a greater extent on mobilization of body reserves. Increasing energy supply pre partum led to a more negative energy balance post partum, mainly by increasing milk yield and content, whereas feed intake was slightly reduced. Increasing energy supply post partum enhanced milk yield as well as milk protein and lactose content. Calculated energy balance corresponded well with liveweight and BCS change. Response of milk yield to increasing energy supply followed the principle of diminishing returns, since energy was increasingly partitioned to body retention. Increasing energy supply pre partum enhances milk yield and content post partum, but exacerbates negative energy balance and its consequences.     

Genetic characterization and population bottleneck in the Hucul horse based on microsatellite and mitochondrial data

The aim of this work was to gather information about the origin and genetic characterization of the Central European Hucul horse based on 71 horses using 17 microsatellites and the D‐loop region of mtDNA. Their genetic relationship to the Polish Konik (N = 7), German (N = 4) and Hungarian wild Przewalski horses (N = 4) and 200 horse sequences from GenBank was also analysed. Both microsatellite and mtDNA analysis showed a high genetic variation in the Hucul. A total of 130 alleles were detected, the mean number of observed alleles per microsatellite was 7.647, and the number of effective alleles was 4.401. The average observed and expected heterozygosity were 0.706 and 0.747, respectively. The high heterozygosity values and Wright's fixation index (F_IS) (?0.128) indicated a low level of inbreeding, low or no selection pressure, and large number of alleles. mtDNA analysis revealed 18 haplotypes for the Hucul population with a total of 23 variable sites. Haplotype and nucleotide diversities were 0.935 ± 0.011 and 0.022 ± 0.012, respectively. Neutrality tests (Tajima's D and Fu's Fs) were non‐significant, and mismatch distribution was ragged, indicating that the Hucul population is in genetic equilibrium. The most frequent mtDNA D‐loop region belonged to haplogroup A (48%), which was also present in Przewalski Wild horse samples, while Polish Konik samples belonged to three haplotypes and C, F, and G haplogroups. Large and significant pairwise Φ_ST values along with a small number of common haplotypes indicated a low level of gene flow and lack of genetic structure among the three studied breeds (Hucul, Konik, and Przewalski Wild horse). The present work contributes to our knowledge of the genetic diversity of the Hucul horse and helps to define its genetic conservation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 54–65.     

Genetic profiling of thoroughbred racehorses by microsatellite marker analysis

The thoroughbred (TB) horse breed has exceptional physiological traits and is well-known in horse racing. It has been bred for endurance, strength, and speed for 300 years. Recently, there have been many studies on the molecular characteristics or mechanisms associated with elite athletic performance of the TB breed, and sequence variants in exercise-related genes have also been reported. Here, using 12 microsatellite markers, we assessed genetic diversity and relationships among two groups of TB racehorses (registered by the Seoul Race Park of the Korea Racing Authority) that showed the 48 highest and lowest scores in races. When comparing allelic variation between the highest- and lowest-scoring TB horse groups, a total of 53 different alleles were detected by genotyping with 6′FAM-labelled primers. The genetic variation between the two groups was similar [mean number of alleles (N _A ) = 4.00 and 4.08; allelic richness (A _R ) = 3.95 and 4.01; observed heterozygosity (H _O ) = 0.53 and 0.56, respectively]. However, among the 53 alleles that we assessed, four alleles were detected only in the highest-scoring and five alleles only in the lowest-scoring racing horse group. The group-specific unique alleles indicate that the microsatellite markers could distinguish horses that showed high or low scores in a race. This study demonstrates the feasibility of microsatellite analyses for selection of superior racehorses and suggests further study to understand the relationship between molecular features and phenotype (racing performance).     

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.     

A New Brassinolide-related Steroid in the Leaves of Thea sinensis

Microscopic and dynamic mechanical properties for mixed aqueous gels of agar and gelatin have been studied. The microscopic observation showed formation of micro granules in the gels under the phase-contrast visual field. The constituent was recognized as agar by metachroma tic staining using a microspectrophotometer. Dynamic moduli of the gels were measured from 0.01 to 200 Hz by phase difference and by resonance. A minimum E' value was obtained for the mixed gel at a volume fraction of agar of 0.6. E′ of all gels and E″ of mixed gels were frequency dependent above 30 Hz.     

Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol-17beta.

Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [³H]estradiol-17β (E₂β) by isolated liver cells was specific for E₂β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10^?9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E₂β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E₂β (× 10^?8 M), but not to the relatively inert estradiol-17α (E₂α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10^?7 M E₂β or diethylstilbestrol, but not E₂α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E₂β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol by E₂β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10^?9 M estrogens or to 1 × 10^?4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E₂β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E₂α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E₂β, as well as to other known hepatocarcinogens.     

Genetic Structure and Gene Flows within Horses: A Genealogical Study at the French Population Scale

Since horse breeds constitute populations submitted to variable and multiple outcrossing events, we analyzed the genetic structure and gene flows considering horses raised in France. We used genealogical data, with a reference population of 547,620 horses born in France between 2002 and 2011, grouped according to 55 breed origins. On average, individuals had 6.3 equivalent generations known. Considering different population levels, fixation index decreased from an overall species F_IT of 1.37%, to an average of −0.07% when considering the 55 origins, showing that most horse breeds constitute populations without genetic structure. We illustrate the complexity of gene flows existing among horse breeds, a few populations being closed to foreign influence, most, however, being submitted to various levels of introgression. In particular, Thoroughbred and Arab breeds are largely used as introgression sources, since those two populations explain together 26% of founder origins within the overall horse population. When compared with molecular data, breeds with a small level of coancestry also showed low genetic distance; the gene pool of the breeds was probably impacted by their reproducer exchanges.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

Estrogenic and Antiestrogenic Down-regulation of Estrogen Receptor Levels: Evidence for two Different Mechanisms

Abstract

Preincubation of MCF-7 cells with estradiol (E₂) produces a decrease of H-E₂ binding capacity (“processing”); the strong antiestrogen methylhydroxytamoxifen (MHT) is also effective but with a ～ 100 fold lower efficiency. Parallel immunological measurement of estrogen receptor contents of the cells (ER-EIA from Abbott) revealed that the mechanisms by which these ligands operate are not of the same nature. Thus, while E₂ produced a loss of the ER peptide, MHT increased it; indicating an accumulation of a non-binding form of the receptor under its treatment. Measurement of the binding capacity of the cells for ³H-ORG 2058 showed a decrease of PgR concentration after preincubation with MHT which contrasted with the classical E₂-induced increase of the receptor. MHT at relatively low concentrations also antagonised the E₂-induced decrease of ³H₂ binding capacity; this property did not result from a difference in chemical structure between the ligands since bisphenol a weak estrogenic analogue of MHT failed to show a similar antagonistic activity. This property conferres to MHT the ability to reduce the efficiency of E₂ to induce PgR. Finally, actinomycin D a known antagonist of the E₂-induced processing was found to be totally ineffective towards the MHT processing. This clearly confirmed that the term “processing” covers at least two distinct mechanisms.     

The effects of dietary protein:energy ratio and amino acid pattern on nitrogen utilization and excretion of rainbow trout Oncorhynchus mykiss (Walbaum)

This study was undertaken to investigate: (1) the effects of both deficiencies and excesses in essential amino acids (EAAs) from an estimated optimum dietary EAA pattern on nitrogen (N) utilization and excretion of rainbow trout Oncorhynchus mykiss, (2) the effects of dietary digestible protein (P_D): digestible energy (E_D) ratio (P_D:E_D) on N utilization and excretion of O. mykiss and (3) the potential interaction of these two factors. A 3 × 3 factorial experiment was conducted, with the two factors EAA pattern and P_D:E_D ratio. The three levels of EAA pattern were: (1) optimum EAA pattern, (2) 60% deficiencies in the three amino acids arginine, histidine and lysine, and (3) 60% excesses in the three amino acids arginine, histidine and leucine. The three levels of P_D:E_D ratio were 18, 21 and 24 g MJ^?1. Amino acid deficiencies from an optimum amino acid pattern caused reductions in mean N retention of 29 to 37%, with the greatest reduction associated with the lowest P_D:E_D ratio, and similar substantial increases in total N and ammonia‐N excretion at all of the dietary P_D:E_D ratios investigated. Amino acid excesses, however, did not negatively affect N retention or excretion. Increasing P_D:E_D ratio was associated with decreasing N retention and increasing N excretion over the range of dietary protein and lipid levels tested. Results of this study showed that a diet with optimum dietary amino acid pattern and lowest P_D:E_D ratio produced the highest N retention (47% of ingested N) and the lowest total N and ammonia‐N excretion of O. mykiss.     

Effect of estrogen-treated porcine ampulla oviductal epithelial cells on early embryonic development in vitro and characterization of their protein synthetic activity

Recent studies by Buhi et al. have demonstrated that estrogen (E₂) is responsible for the induction of de novo synthesis and secretion of certain oviductal secretory proteins (OSP) and inhibition of other OSP in porcine oviductal explant cultures. The present work was undertaken to evaluate the effect of E₂-treated oviductal epithelial cell coculture on the development of early porcine embryos derived from in vitro matured and fertilized oocytes. In vitro synthesis of secretory proteins by E₂-treated oviductal cells used for coculture was also investigated by one-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The results showed that the cleavage rate was significantly enhanced by coculturing fertilized eggs with E₂-treated oviductal epithelial cells. The in vitro protein synthetic pattern of oviductal secretory proteins was influenced by E₂ treatment. These variations included the disappearance of one protein (82 000 M_r) and the appearance of another (33 000 M_r) in the E₂-treated group as assessed by 1D-SDS-PAGE. Additional proteins of M_r 97 000 and an M_r 36 000–45 000 complex were increased in abundance by the E₂ treatment. Analyses by 2D-SDS-PAGE revealed three major E₂-dependent proteins, of M_r 45 000 (pI 5.5), 43 000 (pI 5.5) and a 36 000–45 000 M_r (pI 4.8) protein complex, whereas polypeptides of M_r 97 000 (pI 5.1), 36 000 (pI 8.0) and 25 000 (pI 6.8) were inhibited by E₂ treatment. The results demonstrated that porcine epithelial cell protein synthetic patterns are influenced by E₂ treatment and that estradiol treatment of oviductal cells may increase the rate of zygote cleavage during early development in vitro in pigs.