Genetic influences on ovulation of primary oocytes in LT/Sv strain mice

A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.     

Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein's movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.     

Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice

Our objective was to improve the developmental ability of oocytes in porcine primordial follicles xenografted to nude mice, by treating the host mice with gonadotrophins to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Gonadotrophin treatments were commenced around 60 days after vaginal cornification in the mice. Ovarian grafts were obtained 2 or 3 days after treatment with equine chorionic gonadotrophin (eCG-2 and eCG-3 groups), after porcine FSH infusion for 7 or 14 days, or after infusion of porcine FSH for 14 days with a single injection of estradiol antiserum (FSH-7, FSH-14 and FSH-14EA groups, respectively). Gonadotrophin treatments accelerated follicular growth within the xenografts compared with that in control mice given no gonadotrophins, consistent with higher (P < 0.05) circulating inhibin levels in the gonadotrophin-treated mice. In contrast, circulating mouse FSH levels were significantly (P < 0.05) depressed. We recovered large numbers of full-sized oocytes with meiotic competence to the mature stage from the eCG-3, FSH-7, and FSH-14EA, unlike in the control group. Moreover, 56% of matured oocytes with the first polar body (n = 39) were fertilized in vitro in the FSH-14EA group. After in vitro fertilization and subsequent culture for 7 days, one blastocyst was obtained from each of the eCG-3, FSH-7 and, FSH-14EA groups, whereas no blastocysts appeared in the other groups. Exogenous gonadotrophins--not mouse FSH--stimulated the growing follicles that had developed from the primordial follicles in the xenografts: the effects were incomplete but improved to some extent the meiotic and developmental abilities of the oocytes.     

Urinary oestradiol and testosterone levels from novel male mice approach values sufficient to disrupt early pregnancy in nearby inseminated females

Previous research has established that exposure to novel male mice can disrupt intrauterine implantation of fertilised ova in inseminated females and that much of this effect is mediated by factors in the male urine. The present studies were designed to examine whether the steroid content of male urine is sufficient to account for this effect. Pregnancy was terminated by exogenous 17beta-oestradiol administered intranasally on days 2-4 after insemination in doses as low as 0.14 microg/day. Enzyme immunoassay indicated that male mouse urine reliably contains unconjugated 17beta-oestradiol and testosterone. A small but significant increase in the amount of urinary oestradiol was observed in males housed nearby previously inseminated females as opposed to those housed in isolation. This influence was absent in the sire and absent in novel males when the sire was also present. The quantity of active steroids in novel male urine approaches the level sufficient to account for the disruption of implantation in nearby inseminated females.     

Oestradiol transmission from males to females in the context of the Bruce and Vandenbergh effects in mice (Mus musculus)

Male mice actively direct their urine at nearby females, and this urine reliably contains unconjugated oestradiol (E(2)) and other steroids. Giving inseminated females minute doses of exogenous E(2), either systemically or intranasally, can cause failure of blastocyst implantation. Giving juvenile females minute doses of exogenous E(2) promotes measures of reproductive maturity such as uterine mass. Here we show that tritium-labelled E(2) ((3)H-E(2)) can be traced from injection into novel male mice to tissues of cohabiting inseminated and juvenile females. We show the presence of (3)H-E(2) in male excretions, transmission to the circulation of females and arrival in the female reproductive tract. In males, (3)H-E(2) given systemically was readily found in reproductive tissues and was especially abundant in bladder urine. In females, (3)H-E(2) was found to enter the system via both nasal and percutaneous routes, and was measurable in the uterus and other tissues. As supraoptimal E(2) levels can both interfere with blastocyst implantation in inseminated females and promote uterine growth in juvenile females, we suggest that absorption of male-excreted E(2) can account for major aspects of the Bruce and Vandenbergh effects.     

Kiwifruit of Actinidia eriantha cv. Bidan has in vitro antioxidative,anti-inflammatory and immunomodulatory effects on macrophages and splenocytes isolated from male BALB/c mice

Food Science and Biotechnology - Kiwifruit is known to contain considerable amount of antioxidative phenolics. The objective of this study was to evaluate the antioxidative, anti-inflammatory and...     

PCR-based procedures in detection and DNA-fingerprinting of Salmonella from samples of animal origin

The usefulness of selected PCR-protocols for the detection of Salmonella in 117 samples of animal origin (17 raw minced meat, 27 raw chicken meat, 8 raw sausages, and 25 egg samples, as well as 18 poultry faecal, and caecal swabs samples) and DNA-fingerprinting typing is shown. To establish an accurate PCR-procedure for Salmonella detection the following parameters were evaluated: two pre-PCR concentration procedures, centrifugation and immunomagnetic separation (IMS) using Dynabeads anti- Salmonella; the specificity and sensitivity of 10 sets of primers; and different conditions of the amplification reaction. In light of the results obtained from the use of PCR-based procedures alone or in combination with conventional methods, the following findings can be underlined: First, IMS is more efficient than centrifugation in the recovery of Salmonella. Second, the selected IMS/PCR-detection protocol is less time-consuming (45 h) than the IMS/culture procedure (90 h), and a good concordance between them was found when the Kappa coefficient was calculated (0·87). Third, PCR-ribotyping technique showed a very low discrimination power, being able to differentiate only three profiles. Fourth, RAPD technique using specific primers supports previous works in which it was proposed as a simple and useful tool for discriminating isolates between and within serotypes. Fifth, The efficiency, rapidity, and flexibility of the PCR-protocols applied were high, and they can be performed using two PCR-programs and the same basic equipment.     

Water-extractable phytochemicals from Phyllanthus niruri exhibit distinct in vitro antioxidant and in vivo hepatoprotective activity against paracetamol-induced liver damage in mice

The antioxidative and hepatoprotective potential of Phyllanthus niruri, a widely used medicinal plant in Brazil, were investigated. The aqueous extracts of leaves showed inhibition against thiobarbituric acid-reactive species (TBARS), induced by different pro-oxidants (10 μM FeSO₄ and 5 μM sodium nitroprusside) in rat liver, brain and kidney homogenates. The extracts also lowered the formation of TBARS in phospholipids extracted from egg yolk. The plant exhibited strong antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical (IC₅₀, 43.4 ± 1.45 μg/ml) and iron chelation assay. The hepatoprotective activity of the extracts were also demonstrated in vivo against paracetamol-induced liver damage, as evidenced by the decrease in serum glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT), and increased catalase activity in the liver in treatment groups, compared to the control. The results of the present study suggest the potential use of P. niruri in the treatment of various diseases, among which liver disease is the most important, due to its ability to act as an antioxidant. Furthermore, since the treatment of human intoxications with paracetamol is always limited to the administration of N-acetyl-cysteine, additional studies would be important to determine whether aqueous extracts of P. niruri could increase the efficacy of N-acetyl-cysteine against paracetamol acute hepatotoxicity.     

Marine collagen peptide isolated from Chum Salmon (Oncorhynchus keta) skin facilitates learning and memory in aged C57BL/6J mice

To observe the neuroprotective effects of marine collagen peptide (MCP) isolated from Chum Salmon (Oncorhynchus keta) skin by enzymatic hydrolysis, 20-month-old female C57BL/6J mice were fed with chow diet, 0.22%, 0.44% or 1.32% (wt/wt) MCP diet for 3 months. Comparing with aged control group, the abilities of passive avoidance and spatial memory and learning were significantly enhanced evaluated by step-down test and Morris water maze respectively. Furthermore, the abilities of learning and memory had no significant difference between 0.44% and 1.32% MCP treated groups and young control group. The alleviated oxidative stress, reduced apoptotic neurons, up-regulated expression of brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95) were observed in MCP treated groups compared with aged control group. Our research revealed that there were no significant difference between 0.44% and 1.32% MCP treated groups and young control group. These findings suggest that MCP could be a candidate for functional food to relieve memory deficits associated with aging.     

Effects of flavonoid-rich extract from seeds of Eugenia jambolana (L.) on carbohydrate and lipid metabolism in diabetic mice

Eugenia jambolana (EJ) is long known for its antidiabetic activities in traditional medicines. The aim of this study was to determine the probable mechanism of action of a flavonoid-rich extract from seeds of EJ on streptozotocin-induced diabetic mice. Various biochemical parameters, e.g. glucose tolerance, lipid profile, glycogen biosynthesis, glucose uptake and insulin release in vivo and in vitro, were significantly improved in the extract-treated diabetic mice. Similarly, differential regulation and expression of glucose homeostatic enzymes, e.g. glucose-6-phosphatase and hexokinase, were also changed significantly in response to the flavonoid-rich extract, which clearly demonstrated the hypoglycemic and hypolipidemic effects in treated animals. Further, analysis of the extract using HPLC, demonstrated the presence of different flavonoids and their derivatives which are known for their antidiabetic and antioxidant potential. The data showed that this flavonoid-rich seed extract has a remarkable dual hypoglycemic and hypolipidemic effect.     

Lotus (Nelumbo nucifera Gaertn) plumule polysaccharide protects the spleen and liver from spontaneous inflammation in non-obese diabetic mice by modulating pro-/anti-inflammatory cytokine gene expression

A novel lotus plumule polysaccharide (LPPS) was administered to non-obese diabetic (NOD) mice for 15 weeks to evaluate the protective effects of LPPS on type 1 diabetes. After the 15-week feeding experiment, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 expressions in the spleen, liver and kidney of the experimental mice were measured using enzyme-linked immunosorbent assay or real-time quantitative polymerase chain reaction assay. The results showed that LPPS significantly (p < 0.05) decreased the absolute weights of the enlarged spleens in the NOD mice in a dose-dependent manner, inhibited pro-inflammatory TNF-α and IL-6 cytokine production and decreased the secretion ratio of IL-6/IL-10 in splenocyte cultures. LPPS markedly decreased the relative expression of pro-/anti-inflammatory cytokine genes (TNF-α/IL-10 and IL-6/IL-10) in the livers of NOD mice. Our results suggest that LPPS protected the spleen and liver from spontaneous inflammation in NOD mice by modulating pro-/anti-inflammatory cytokine gene expression.     

Gui-ling-gao (turtle jelly), a traditional Chinese functional food,exerts anti-inflammatory effects by inhibiting iNOS and pro-inflammatory cytokine expressions in splenocytes isolated from BALB/c mice

Gui-ling-gao (GLG), also known as turtle jelly, is a popular medicinal Chinese health/functional food prepared from several traditional Chinese medicinal herbs. The present study aimed at examining and explaining the anti-inflammatory properties of GLG by using lipopolysaccharide (LPS)-induced inflammation in splenocytes isolated from BALB/c mice. Additionally, the effects of GLG on mRNA and protein expressions of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in LPS-stimulated splenocyte proliferation were evaluated by real-time RT-PCR and Western blot assays. We demonstrated that GLG significantly inhibited LPS-induced splenocyte proliferation in a concentration-dependent manner. LPS-mediated up-regulations in the gene and protein expressions of iNOS, interleukin-1β (IL-1β) and tumour necrosis factor-alpha (TNF-α) were suppressed by GLG. These findings suggest for the first time that GLG exerts anti-inflammatory effects by inhibiting the expression of iNOS and pro-inflammatory cytokines such as IL-1β and TNF-α. The current study provides strong scientific evidence for the health-beneficial claim that consumption of GLG could help the body to modulate the immune system.