Implication of cortisol and 11beta-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts

This study investigated cortisol inactivation by 11beta-hydroxysteroid dehydrogenase (11beta HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP(+)-dependent 11beta-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11beta HSD, this studyalso investigated whether levels of 11beta HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11beta HSD1 activity in rat kidney homogenates decreased progressively from 50 +/- 8% inhibition by FF from small antral follicles (P < 0.001) to 23 +/- 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11beta HSD1 activity by 59 +/- 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35-48% inhibition, P < 0.05) and cyst fluid (45-75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11beta HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11beta HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.     

Differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes

This work describes the differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR targeting sequences from two molecular markers: the mitochondrial displacement loop (D-loop) region and the nuclear melanocortin receptor 1 (MC1R) gene. A polymorphic D-loop fragment (～270bp) was amplified and sequenced in a number of wild and domestic Sus scrofa meat samples, to find a nucleotide region suitable for PCR-RFLP analysis. Sequence data showed the presence of only a few point mutations across Sus scrofa D-loop sequences, not allowing direct discrimination between wild boar and domestic swine meats. Later, the MC1R gene was targeted and Sus scrofa-specific primers designed to amplify a 795bp MC1R fragment. Subsequent RFLP analysis of the MC1R swine-specific amplicons allowed selection of BspHI and BstUI endonucleases to carry out intraspecific Sus scrofa differentiation. Digestion of MC1R amplicons with the chosen enzymes generated characteristic PCR-RFLP profiles that allowed discrimination among meats from wild and domestic swine specimens. The technique also enabled the detection of samples that yielded heterozygous profiles, suggesting hybrids resulting from wild boar and domestic pig breeding. The PCR-RFLP reported here, targeting the MC1R gene may be routinely applied to verify the correct labelling of game products.     

Nutritional evaluation of the lipid fraction of feral wild boar (Sus scrofa scrofa) meat

Consumer increasing demand for wild boar meat and scarceness of data on its lipid fraction justified this study. The psoas major muscle collected from 25 feral wild boars was used to quantify the total lipid, total cholesterol, fatty acid (FA) profile, and vitamin E homologues. Intramuscular fat and total cholesterol contents averaged 4.64 g/100g of meat and 56.9 mg/100g of meat, respectively. No differences were found in FA composition between groups, except for 20:5n-3 that was higher in youngsters. All groups presented small concentrations of rumenic acid in meat (CLA; 0.24% of total FA). FA profile showed considerable resemblance with pork, while the vitamin E profile is marked by high concentrations of both alpha- (17.4 ± 3.3 μg/g meat) and gamma-tocopherols (2.6 ± 1.3 μg/g meat) and by the presence of other vitamin E homologues not previously reported in wild boar meat.     

Changes of testicular aromatase expression during fetal development in male pigs (Sus scrofa)

Male pig fetuses secrete considerable amounts of estrogens, but the location of aromatase activity within the fetal testis is not known. The location of aromatase expression was investigated by immunocytochemistry in fetal testes from week 6 (n = 5), weeks 10, 13, and 15 (each: n = 6) of gestation and additionally in neonates (n = 4). Blood was sampled from the umbilical artery of fetuses and jugular vein of neonates. Histological evaluation of testes involved morphological criteria and counting of Leydig cells, Sertoli cells, and gonocytes. Aromatase activity was localized immunocytochemically and quantified by the percentage of positive stained cells within the same cell type. Aromatase expression was further characterized by quantitative RT-PCR. Concentrations of estrogens, testosterone, FSH, and LH were measured in blood plasma. Total estrogens increased from week 10 to a maximum of 31.03 nmol/l in week 15. Increased testosterone concentrations were only measured at week 6 and were paralleled by slightly elevated estrogens. Thereafter, testosterone dropped and was low throughout. The increase of estrogens was not paralleled by a similar increase of FSH and LH but was related to the increase of the total number of Leydig cells. This increase was also found for mRNA expression. Both Leydig cells and gonocytes were identified as contributors to estrogen formation. Gonocytes were the main source of aromatase at week 10, when gene expression by Leydig cells is low due to the preparation of a wave of Leydig cell mitosis.     

Effect of epinephrine administration on glycogen metabolism in red and white muscle of anaesthetized pigs (Sus scrofa domesticus)

The glycogenolytic effect of exogenous epinephrine was studied in white (Longissimus) and red (Trapezius) muscle of anaesthetized pig. In addition, we assessed the metabolic action of epinephrine during the 3 h following the cessation of perfusion. Twelve purebred Large White pigs, averaging 80 kg live-weight, were used. The animals were anaesthetized and perfused (0–15 min) with 5 μg kg^?1 min^?1 epinephrine or saline (control animals). Blood samples were taken to determine plasma levels of glucose, lactate and non-esterified fatty acids (NEFA). Muscle samples were taken to determine the concentrations of glycogen and related metabolites, and the activity ratio of the enzyme glycogen phosphorylase. Control animals showed stable levels of plasma and muscle metabolites during the 3 h anaesthesia. However, the resting level of the activity ratio of phosphorylase was high (80%). Epinephrine treatment induced significant increases in plasma metabolites and an overall significant glycogen depletion. The extent of epinephrine-induced glycogenolysis was greater in the red Trapezius than in the white Longissimus muscle. This was associated with a greater rise in the muscle lactate content and in the activity ratio of phosphorylase in the Trapezius muscle during epinephrine administration. In both muscles, no significant glycogen depletion was observed during 3 h following the cessation of epinephrine administration. This occurred despite the fact that the activity ratio of phosphorylase remained high in the Longissimus muscle until the end of the experiment.     

Discrimination of two alleles of the melanocortin receptor 1 gene to discern European wild boar (<Emphasis Type="Italic">Sus scrofa scrofa</Emphasis>) and domestic pig (<Emphasis Type="Italic">Sus scrofa domestica</Emphasis>) in meat products by real-time PCR

A duplex real-time polymerase chain reaction (PCR) technique for the discrimination of two subspecies of sus scrofa in meat products has been developed. Primers and probes target at a sequence in the melanocortin receptor 1 (MCR1) gene being associated in the expression of wild-type coat color. Both PCRs amplify a 56-bp product of DNA from wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) likewise. One of the TaqMan probes specifically anneals to the wild boar sequence and the other one to the domestic pig sequence, their base sequence differing only in a single nucleotide (single nucleotide polymorphism, SNP). This qualitative-method allows the detection of genomic DNA from wild boar and domestic pig as low as 25 pg in a 25-μl reaction volume. No cross reactivities were found with either genomic DNA from various other meat species, or with other ingredients of meat products (e.g. spices). The PCR efficiency is >95% for both targets. Although both PCRs are impaired by the presence of bovine and porcine DNA (wild boar detection is impaired by domestic pig DNA and vice versa), the method is applicable for the detection of low levels of wild boar and domestic pig meat simultaneously in commercial meat products. The limit of detection (LOD) in meat samples is 2% for wild boar and 5% for domestic pig.     

Changes in endocrine profile and reproductive organs during puberty in the male marmoset monkey (Callithrix jacchus)

Data on pubertal maturation in male marmoset, a model for human reproduction, are scant and conflicting. We collected data on novel parameters to characterize puberty. Twenty-five marmoset monkeys were assigned to five age groups by weeks (wk): 21 (pre-pubertal), 43 (onset of puberty), 52 (fully pubertal), 70 (mature), and 116 (fully adult). Serum and intratesticular testosterone and pituitary bioactive chorionic gonadotropin (bioCG) were measured. Testicular development was assessed by ultrasonography, histology, and flow cytometry. Three consecutive blood samples revealed extreme fluctuations in testosterone concentrations, suggesting an erratic secretion. Age-related changes in serum testosterone and pituitary bioCG concentrations were observed. Intratesticular androgens (ITAs) showed high fluctuations within groups at all ages and were high in some animals by 21 wk. Unexpectedly, no correlation between pituitary bioCG and serum testosterone or ITAs was found, but these parameters significantly correlated with testicular weight and volume. These observations were consistent a dependence on the testis growth on bioCG. Unfortunately, the low serum levels of bioCG were not measurable in this study. At 43 wk, the animals reached puberty. At 52 wk of age, animals attained maximum body and epididymal weights and qualitatively normal spermatogenesis, but testes continued growing, reaching a maximum of all parameters at 70 wk of age, without further major changes at the age of 116 wk. It is concluded that (1) gonadal activation is evident at wk 21, (2) the male marmoset reaches the pubertal threshold around 43 wk of age, attains qualitative parameters at 52 wk, matures further to sexual maturity at 70 wk, and (3) serum testosterone and ITAs are highly variable without any identifiable correlation with pituitary bioCG.     

Changes in semen quality and morphology of the reproductive tract of the male tammar wallaby parallel seasonal breeding activity in the female

Changes in semen quality and morphology of the male reproductive tract were studied throughout the year in the highly promiscuous tammar wallaby. Body size, semen quality and gross morphology of the reproductive organs were assessed in adult males each month from January to November. The mean weight of males was similar in most periods sampled, but males were slightly heavier in the minor (P < 0.05) than the non-breeding season. Since body weight was correlated with weights of the testes, epididymides and accessory sex glands, organ weights were adjusted for body weight in subsequent analyses. In the major breeding season (late January/early February), when most females go through a brief, highly synchronized oestrus, the testes, prostate, Cowper's glands, crus penis and urethral bulb were heaviest, volume and coagulation of ejaculates were greatest, and sperm motility had increased. Semen samples collected by electroejaculation at this time contained low numbers of spermatozoa, possibly as a result of dilution and entrapment by the seminal coagulum or depletion of epididymal stores during intense multiple mating activity. In the non-breeding season (late May-July), when mating does not normally occur in the wild, there was a significant decrease in the relative weight of nearly all male reproductive organs and a decline in most semen parameters. In the minor breeding season (September-November), when pubertal females undergo their first oestrus and mating, the weights of testes, epididymides and most accessory sex glands had significantly increased similar to those of males in the major breeding season. The total number and motility of ejaculated spermatozoa were highest during this period, but the volume and coagulation of ejaculates and weight of the prostate had only increased to levels that were intermediate between the major and non-breeding seasons. Ejaculate volume was strongly correlated with prostate weight, and % motile spermatozoa was strongly correlated with epididymis weight. Semen quality thus varied seasonally with changes in androgen-dependent reproductive organs in the male tammar wallaby and appeared to be influenced by the seasonal timing of oestrus in females. Semen quality may also improve in response to an increase in the number of available oestrous females.     

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.     

Effects of larval crowding and nutrient limitation on male phenotype,reproductive investment and strategy in Ephestia kuehniella Zeller (Insecta: Lepidoptera)

Food shortage experienced by juvenile insects affects adult morphology and life-history traits. Developmental plasticity and trade-off between ecological and sexual traits helps maximise individual fitness. Ephestia kuehniella were reared at different larval densities to investigate phenotypic shifts in adult males. Variation in ecological traits (sizes of forewing, head and thorax and adult longevity) and sexual traits (valva and aedeagus, sperm number, mating frequency) were compared. Males that emerged from highest density population (800) had lower body mass and small forewings, head and thorax, suggesting that they could not completely compensate for food shortage. The allometric relationship between body mass and forewing length also changed, and these males had relatively longer wings. This arrangement may enhance dispersal and assist in mate-searching at higher densities. Males from all larval densities achieved similar mating frequency but those from higher density produced fewer eupyrene sperm and had shorter adult lifespan. By mating more frequently and maintaining apyrene sperm production, males increase their reproductive success at sperm competition observed at higher densities. Food stress associated with high density populations did not affect valva and aedeagus size indicating that these traits may be insensitive to external environmental changes because they incur fitness costs to males.     

Alterations in placental 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) activities and fetal cortisol:cortisone ratios induced by nutritional restriction prior to conception and at defined stages of gestation in ewes

In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD(+)-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.     

染整用酶制剂的研究与开发现状(Ⅰ)

对酶制剂的发展、作用原理和催化机理作了详细阐述,并介绍了通过基因工程和DNA编码技术生产新的第三代酶制剂的情况.从原理上分析了酶在染整中的新应用领域,如酶洗涤、酶的还原染料隐色体氧化、漆酶的棉漂白和新果胶裂解酶的棉煮练与漂白.     

染整用酶制剂的研究与开发现状(Ⅱ)

4染整用新型酶制剂的开发与应用染整工业中使用酶制剂己有相当长时间,目前已广泛使用的酶制剂:(1)淀粉酶,广泛用于纺织物的退浆;(2)纤维素酶,广泛用于纤维素纤维的减量、柔软、抛光和减少起毛起球,以及用于牛仔服的石磨;(3)果胶酶,用于亚麻和苎麻的脱胶,还有人试用于棉的精练;(4)蛋白酶,用于丝的脱胶,羊毛的减量、柔软、减少起毛起球和防毡缩;(5)过氧化氢酶,用于双氧水漂白后的脱除残余的双氧水.本文将叙述上述工艺以外的新型酶制剂和新的应用领域,以提请印染技术人员进行探索,并在印染工艺中加以采用,以节约成本、减少能源和水的消耗,减轻…     

Comparison of heating methods and the use of different tissues for sensory assessment of abnormal odours (boar taint) in pig meat

Five heating methods (microwave, hotwire, boiling at 25 °C and 75 °C and melting) were used to generate cooking odours from backfat of entire male pigs and a 'composite' sample consisting of fat and muscle from the head along with salivary glands. The methods elicited significantly different scores for odours from 4 groups of 10 samples differing in their concentrations and ratios of skatole and androstenone. The odours (pork odour, abnormal odour, skatole odour and androstenone odour) were assessed by 3 experienced assessors. Correlations between skatole and androstenone concentrations and abnormal odour score in backfat were higher for skatole, suggesting it is the more important boar taint compound. In the composite sample, androstenone concentration was much higher than in backfat and androstenone was a more important contributor to boar taint. The microwave, hotwire and boiling (75 °C) methods produced the clearest separation between samples and the microwave method was considered the most suitable for on-line use.     

Species differences in the ovarian distribution of 3beta-hydroxysteroid dehydrogenase/delta5-->4 isomerase (3beta-HSD) in two marsupials: the brushtail possum Trichosurus vulpecula and the grey,short-tailed opossum Monodelphis domestica

The ovarian distribution of the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase/delta(5-->4) isomerase (3beta-HSD) was investigated by immunocytochemistry in two marsupial species throughout the reproductive cycle, using a rabbit polyclonal antibody raised against human placental 3beta-HSD. In the polyoestrous and polyovular South American opossum Monodelphis domestica, immunostaining was positive for 3beta-HSD in the adrenal cortex, the ovarian interstitial tissue, the corpus luteum and the granulosa cells of antral and atretic follicles. The theca interna was weakly positive for 3beta-HSD, but only in late preantral to early antral stages of follicular development. The adrenal medulla and smaller preantral follicles were completely negative for 3beta-HSD. In contrast, in the polyoestrous and monovular Australian brushtail possum Trichosurus vulpecula, immunostaining showed a strong positive reaction for 3beta-HSD in the theca, whereas the granulosa layer remained predominantly negative for 3beta-HSD except in the largest follicles. The atretic follicles were completely negative for 3beta-HSD. The ovaries of pregnant animals contained grossly enlarged, persistent, antral follicles, which reacted positively for 3beta-HSD. The function of these follicles in T. vulpecula and the 3beta-HSD-positive atretic follicles in M. domestica has not been determined. The differences between the two marsupials represent species variations. The situation in M. domestica does not represent a marsupial-eutherian dichotomy as previously conjectured.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45 degrees C and 55 degrees C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.