Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

Influence of graft size, histocompatibility,and cryopreservation on reproductive outcome following ovary transplantation in mice.

PURPOSE: Transplantation of ovarian tissue is a valuable method to rescue mouse strains with fertility problems and to revitalize archived strains. The purpose of this study was to investigate the effect of (i) different sizes of transplanted ovary pieces on reproductive outcome, (ii) use of immunodeficient recipients to overcome the limitation of histocompatibility, and (iii) to compare different protocols for cryopreservation of ovarian tissue. METHODS: Halves, quarters, and eights of mouse ovaries were transplanted. Half ovaries from B6 donors were transferred into immunodeficient mice. Halves of ovaries were frozen according to four different protocols, thawed and transferred. RESULTS: Pregnancy rate after transplantation of ovarian tissue was high (90-100%) independent of the transplant size. Although, the average litter size was significantly lower for recipients of quarters and eights (4.4 and 4.6 vs. 6.5), the total number of offspring produced per donor ovary was higher compared with recipients of halves. Pregnancy rate of immunodeficient recipients was 40% (mean 4.7 offspring per litter). All four cryopreservation protocols used were able to preserve functionality of the ovarian tissue. CONCLUSIONS: Transplantation of ovarian tissue smaller than halves resulted in reduced litter sizes. The distribution of ovarian tissue of one donor female to 4 or 8 recipients will therefore yield in a higher total number of offspring in a certain time period. The use of immunodeficient recipients is an option for non-histocompatible donors. Cryopreservation of ovarian tissue is generally feasible but the function of frozen-thawed ovary halves after transplantation differs depending on the freezing protocol used.

Influence of hernioplastic implants on male fertility in rats.

This study explored the vulnerability of the ductus deferens due to mesh induced inflammation and shrinkage after hernia repair in the rodent model. Two commonly used types of hernioplastic implants (Prolene and Vypro II) were surgically wrapped around the ductus deferentes on both sides in 20 juvenile and 20 adult Sprague-Dawley rats. Twenty male rats underwent sham surgeries as controls. After 3 months, each male was mated with 2-3 adult females, which were subsequently sacrificed and oocytes or embryos were flushed and counted. Histochemical investigations of the implants and the ductus recovered surgically 4 weeks after implantation (one side) and after the fertility test (second side) were conducted. All groups exhibited 1-3 males with decreased or restricted fertility but there was no difference between groups. Histochemical analysis of the implants and the ductus recovered 4 weeks and 4 months after implantation revealed some sperm granulomes due to lesions of the spermatic cord caused by the implant in the Prolene group. There was no inflammatory reaction in the mucosa or blockage of the spermatic cord visible. Both types of hernioplastic implants tested in this investigation do not give an indication of a negative influence on male fertility in juvenile or adult rats.

Detection of bcrabl fusion mRNA in samples of whole, unfractionated blood.

Using a new strategy, capture-RT-PCR, bcrabl fusion mRNA sequences were specifically and sensitively detected in samples of whole blood from leukemia patients with the Philadelphia chromosome. Sample processing required only mixing blood with the chaotropic salt, GuSCN, and mRNA was captured during a short incubation of prepared blood with an affinity membrane. Immobilized mRNA sequences were amplified without elution. No radioisotopes or Southern transfer were needed.

Birth of a piglet derived from an oocyte fertilized by intracytoplasmic sperm injection (ICSI).

This report is of a live piglet derived from an embryo produced by intracytoplasmic sperm injection and carried to term. In vivo-matured oocytes were injected with in vitro-capacitated spermatozoa and after 40 h of culture, 2 to 4-cell-stage embryos were transferred to the oviducts of oestrus induced gilts 24 h after the expected ovulation time. Pregnancy of the surrogate mothers was maintained by estrogen treatment on days 10 to 16 after transfer. The single piglet that was born to a mother that had received 32 embryos was underweight, though healthy and lively.

Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium-ionophore activation in the pig.

In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitro-matured porcine oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as handling controls. After subsequent in vitro-culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved with fresh in vitro-capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In Experiment 2, in vitro matured oocytes were injected with freshly ejaculated in vitro-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 microM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Ca-ionophore treatment after injection of spermatozoa was ineffective at 100 microM, where at 50 microM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperm-injected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed.

Application of GaN-based ultraviolet-C light emitting diodes--UV LEDs--for water disinfection.

GaN-based ultraviolet-C (UV-C) light emitting diodes (LEDs) are of great interest for water disinfection. They offer significant advantages compared to conventional mercury lamps due to their compact form factor, low power requirements, high efficiency, non-toxicity, and overall robustness. However, despite the significant progress in the performance of semiconductor based UV LEDs that has been achieved in recent years, these devices still suffer from low emission power and relatively short lifetimes. Even the best UV LEDs exhibit external quantum efficiencies of only 1-2%. The objective of this study was to investigate the suitability of GaN-based UV LEDs for water disinfection. The investigation included the evaluation of the performance characteristics of UV LEDs at different operating conditions as well as the design of a UV LED module in view of the requirements for water treatment applications. Bioanalytical testing was conducted using Bacillus subtilis spores as test organism and UV LED modules with emission wavelengths of 269 nm and 282 nm. The results demonstrate the functionality of the developed UV LED disinfection modules. GaN-based UV LEDs effectively inactivated B. subtilis spores during static and flow-through tests applying varying water qualities. The 269 nm LEDs reached a higher level of inactivation than the 282 nm LEDs for the same applied fluence. The lower inactivation achieved by the 282 nm LEDs was compensated by their higher photon flux. First flow-through tests indicate a linear correlation between inactivation and fluence, demonstrating a well designed flow-through reactor. With improved light output and reduced costs, GaN-based UV LEDs can provide a promising alternative for decentralised and mobile water disinfection systems.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. established through micromanipulation.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.

The relationship between critical power and running performance.

Critical power is a theoretical concept that presumes there is a certain work-rate which may be maintained without exhaustion. The extent to which critical power predicts running performance over varying distances has not been determined, and so the aim of this study was to correlate measurements of critical power in the laboratory to running performances in the field at 40 m and 1, 10 and 21.1 km in a group of .17 male long-distance runners (mean +/- S.D. age = 31.7 +/- 7.3 years). Each subject ran to exhaustion on the treadmill in the laboratory at six different speeds, ranging from 17 to 25 km h-1. Least squares analyses were used to fit an exponential decay to the relationship between the running speed (y) versus time to exhaustion (x). Critical power was calculated as the running speed (y) coinciding with the asymptote or C parameter of the y = A.e(-Bx) + C relationship. The VO2 max was also measured in all subjects. For the data in the field, each subject was timed over 40 m and 1 km and participated in 10- and 21.1-km races. The mean critical power of the subjects in this study was 18.5 +/- 1.6 km h-1. The test-retest correlation coefficient for the determination of critical power was r = 0.99. The mean VO2 max, measured in a progressive exercise protocol starting at 13 km h-1 and increasing by 1 km h-1 every minute, was 59.2 +/- 4.6 ml O2 kg-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissolve and capture: a strategy for analysing mRNA in blood.

Messenger RNA (mRNA) sequences were amplified from whole blood prepared in guanidine thiocyanate (GuSCN) after capture of the mRNA with an affinity membrane.

Glutamate-cysteine ligase modulatory subunit in BAL alveolar macrophages of healthy smokers.

The antioxidant glutathione (GSH) is increased in the epithelial lining fluid (ELF) of chronic smokers. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), also known as gamma-glutamylcysteine synthetase, consisting of a heavy, catalytic (GCLC) and a light, modulatory (GCLM) subunit. To determine the contribution of bronchoalveolar lavage (BAL) cells to GSH levels in ELF, BAL was performed in eight smokers and eight never-smokers. Intra- and extracellular total glutathione (GSHt) levels and GCL subunit expression were assessed. GSHt was increased in ELF from smokers (1,090.1 +/- 163.0 microM versus 559.2 +/- 48.2 microM). GSHt content of BAL cells (nmol x mg protein(-1)) was decreased in smokers without differences reaching statistical significance (8.0 +/- 1.4 versus 12.4 +/- 2.6). GCLM expression was also reduced in smokers (0.6 +/- 0.1 versus 2.8 +/- 0.4) and correlated with intracellular GSHt content. There was no significant difference in GCLC expression and in differential cell counts in BAL fluid. The authors conclude that smoking does increase glutathione levels in the epithelial lining fluid but not intracellular levels in bronchoalveolar lavage cells. The data suggest that the intracellular glutathione concentration of bronchoalveolar lavage cells (predominately alveolar macrophages) is regulated by the modulatory glutamate-cysteine ligase subunit rather than the catalytic subunit.

[Selective hematoporphyrin derivative (HMD) application in arterial vessels using a porous balloon catheter results in equivalent levels as compared to high-dose systemic administration]. / Selektive Hämatoporphyrinderivat-(HPD-)Applikation im arteriellen Gefässsystem mittels eines porösen Ballonkatheters erreicht vergleichbare Konzentrationen wie nach hochdosierter systemischer Gabe.

The treatment of atherosclerotic vascular stenosis with percutaneous angioplasty is limited by a rate of restenosis of about 20-40%, in spite of new angioplasty devices. Histological and immune histological examinations of restenosed material obtained by coronary atherectomy indicate that cellular proliferation is an important determinant of restenosis. With the use of photodynamic therapy (PDT), it might be possible to selectively impair proliferating tissue by the application of the photosensitizer Photofrin II (a hematoporphyrin-derivative, HPD) followed by localized laser-light radiation. With the knowledge of the success of PDT in tumor therapy, the extension of the application of PDT in prophylaxis of restenosis should be examined. The technique used up to now works with the systemic application of the sensitizer. By applying HPD locally, however, one might be able to reduce the amount of the photosensitizer, but still achieve an equally cytotoxic effect. A recently developed catheter with a porous balloon enables local application of HPD. The following study describes the uptake and distribution of the hematoporphyrin-derivative Photofrin II within the walls of elastic and muscular type vessels after systemic and selective application. In 20 rabbits and seven pigs, Photofrin II was applied systemically (5 mg/kg i.v.) and locally (5 ml of 2.5 mg/ml). From each animal 12 vascular specimens (six arterial segments of either muscular and elastic type) were removed at a definite time within a defined period of 5 min to 24 h after application. To quantify the uptake of Photofrin II, we used fluorescence microscopy with digital image processing. After systemic application there was an increase of Photofrin II over a 4-h period. In contrast, a maximum concentration of Photofrin II was measured immediately after local application and found to be decreasing over a period of 4 h. The intima showed the highest uptake of HPD, both after local and systemic applications, as compared to uptake by the media and adventitia. The intimal uptake was significantly higher after local than after systemic application. Media and the adventitia showed, respectively, only one-half and one-fifth of the intima's intake. The rapid increase of the HPD concentration after local application would make PDT feasible in restenosis prophylaxis immediately after angioplasty without systemic side-effects of the photosensitizer.

Running intensity as determined by heart rate is the same in fast and slow runners in both the 10- and 21-km races.

The aims of this study were to determine (1) whether running speed is directly proportional to heart rate (HR) during field testing and during 10- and 21-km races, and (2) whether running intensity, as estimated from HR measurements, differs in 10- and 21-km races and between slow and fast runners at those running distances. Male runners were divided into a fast (65-80 min for 21 km; n = 8) or slow (85-110 min for 21 km; n = 8) group. They then competed in 10- and 21-km races while wearing HR monitors. All subjects also ran in a field test in which HR was measured while they ran at predetermined speeds. The 10-km time was significantly less in the fast compared with the slow group (33:15 +/- 1:42 vs 40:07 +/- 3:01 min:s; mean +/- S.D.), as was 21-km time (74:19 +/- 4:30 vs 94:13 +/- 9:54 min:s) (P < 0.01). Despite the differences in running speed, the average running intensity (%HRmax) for the fast and slow groups in the 10-km race was 90 +/- 1 vs 89 +/- 3% and in the 21-km race 91 +/- 1 vs 89 +/- 2%, respectively. In addition, %HRmax was consistently lower in the field test at the comparative average running speeds sustained in the 10-km (P < 0.01) and 21-km (P < 0.001) races. Hence, factors in addition to work rate or running speed influence the HR response during competitive racing. This finding must be considered when running intensity for competitive events is prescribed on the basis of field testing performed under non-competitive conditions in fast and slow runners.

Waist circumference predicts clustering of cardiovascular risk factors in older South Africans.

OBJECTIVE AND DESIGN: A cross-sectional analytical study to determine the cardiovascular risk factor profile of older residents of fishing villages on the West Coast of South Africa, and to determine which anthropometric measures are associated with risk factors. SUBJECTS: A convenient community-based sample of 152 subjects of mixed ancestry aged 55 years and over was recruited door-to-door using an address list of age-eligible subjects provided by the local public health care clinics. METHODS: Cardiovascular relationships were investigated between (i) number of risk factors (hypertension, hypercholesterolaemia, diabetes) and body mass index (BMI), waist-to-hip ratio (WHR), and waist circumference; and (ii) continuous cardiovascular risk factor variables and physical activity, smoking, dietary intake, and 24-hour urinary sodium and potassium concentrations. RESULTS: The prevalence of hypertension ( > or =160/95 mmHg) was 74.3% (95% CI: 67.2 - 81.4%). Neither 24-hour urinary sodium nor potassium concentrations was associated with blood pressure (BP). Past, but not present, moderate-intensity physical activity, particularly that associated with occupation, was negatively associated with systolic BP (r = -0.24, P < 0.05). The prevalence of diabetes and hypercholesterolaemia (serum cholesterol > or = 6.5 mmol/l) was 24.6% (95% CI: 17.2 - 32%) and 40% (95% CI: 31.8 - 48.2%), respectively. The percentage of subjects with 0, 1, or 2 or more cardiovascular risk factors was 13.4%, 44.1% and 42.5%, respectively. Subjects with a waist circumference > or = 92 cm had a significantly higher number of cardiovascular risk factors than those with a waist circumference < 92 cm ( chi(2) = 9.29, P < 0.01), and this association remained significant even after controlling for age, sex and smoking (P < 0.05). Neither BMI tertiles according to sex, nor a BMI cut-point > or =30, was significantly associated with a clustering of risk factors. CONCLUSION: In a sample of older South Africans of mixed ancestry at high risk of cardiovascular disease, waist circumference measuring > or = 92 cm predicts clustering of risks factors, independently of BMI. This simple, population-specific reference value may provide a useful screening tool to identify at-risk individuals for targeted prevention for coronary heart disease and associated metabolic disorders.