A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo.

PURPOSE: Currently it is thought to take 60 to 70 days to produce and ejaculate human sperm. This estimate is derived mainly from a single older, descriptive, kinetic analysis of spermatogenesis. We developed a noninvasive method to assess germ cell turnover time accurately in vivo using stable isotope labeling and gas chromatography/mass spectrometry analyses. We confirmed the postulated length of a normal cycle of spermatogenesis. MATERIALS AND METHODS: A total of 11 men with normal sperm concentrations ingested (2)H(2)O daily for 3 weeks. Semen samples were collected every 2 weeks for up to 90 days. Label incorporation into sperm DNA was quantified by gas chromatography/mass spectrometry, allowing calculation of the percent of new cells present. A cycle of sperm production was determined as the lag time until labeled sperm appeared in the ejaculate. RESULTS: Labeled sperm were detected after a mean +/- SD of 64 +/- 8 days (range 42 to 76). In 1 subject the time lag was 42 days but it was at least 60 in all other subjects. In most subjects plateau labeling in sperm was not attained. In 2 subjects the rise and fall of the labeling curve was steep and reached greater than 85% new cells, suggesting rapid washout of old sperm in the epididymal reservoir. CONCLUSIONS: This direct kinetic assessment confirms a course of spermatogenesis that is on the shorter side of traditional estimates based on prior analyses. In addition, the variability observed in healthy men suggests that characteristics such as the epididymal reservoir effect may influence the modeling of in vivo spermatogenesis.

Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA.

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of (2)H(2)O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% (2)H(2)O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0-200 microg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose-response relationship up to the 100 microg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose-response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the (2)H(2)O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270-400 days and die-away values after (2)H(2)O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1-1.5% new cells per day, whereas obese ad libitum-fed obob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term (2)H(2)O enrichments in body water were achieved by daily (2)H(2)O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body (2)H(2)O enrichment ( approximately 3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with (2)H(2)O was 0.056 (CD4(+)) and 0.043 (CD8(+)) (replacement rate <0.1% per day). In summary, (2)H(2)O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.

Chronic medium-chain triacylglycerol consumption and endurance performance in trained runners.

BACKGROUND: This study was designed to assess the effects of chronic consumption of medium-chain triacylglycerols (MCT) on endurance running performance. EXPERIMENTAL DESIGN: participants completed the study in a randomized, cross-over, placebo-controlled, double-blind fashion. SETTING: participants were recruited from the general community to participate in this university based study. PARTICIPANTS: twelve trained male endurance runners (30.5+/-5.2 years of age) completed the study. INTERVENTIONS: participants consumed dietary supplements containing either 56 g of corn oil (LCT) or 60 g of MCT oil daily for 2 weeks. Following each dietary phase, participants completed a maximal treadmill test followed by an endurance treadmill test in which participants ran at 85% VO2max for 30 min proceeded by 75% VO2max until exhaustion. MEASURES: blood was taken at rest and after 45 min of exercise to assess concentrations of lactate, glucose, beta-hydroxybutyrate (beta-HBA), free fatty acids (FFA), glycerol and triacylglycerols (TG). Performance was assessed as time to exhaustion. RESULTS: VO2max (72.0+/-8.0 vs 72.4+/- 9.0 ml x kg(-1) x min(-1)) and endurance time (99.8+/-23.5 vs 106.5+/-29.4 min) did not differ (p>0.05) between MCT and LCT trials, respectively. No differences (p>0.05) in lactate, glucose, beta-HBA, FFA, glycerol or TG were detected between trials. Respiratory exchange ratio (RER) was higher (p<0.05) at 15 min for the MCT trial (0.97+/-0.10) versus the LCT trial (0.90+/-0.20), but was similar between trials at other timepoints. CONCLUSIONS: Results indicate that chronic MCT consumption neither enhances endurance nor significantly alters performance-related metabolism in trained male runners.

The effect of medium-chain triacylglycerols on the blood lipid profile of male endurance runners.

Medium-chain triacylglycerol (MCT) oil is currently marketed for athletes as an ergogenic aid for optimal performance. Research assessing the blood lipid response of humans to MCT consumption is very limited and inconclusive. In this randomized cross-over study, male endurance runners (aged 30.5 +/- 5.5 years) were instructed to consume a low-fat diet (approximately 15% of energy) and consume either supplemental MCT oil (30 g twice each day) or long-chain triacylglycerol (LCT) oil (28 g corn oil twice each day) for 14 days. Each dietary trial was separated by at least 3 weeks. At the end of each trial, fasting blood samples were collected and analyzed for serum concentrations of total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and triacylglycerol (TG). Concentrations of TC (3.83 +/- 0.12 vs. 3.41 +/- 0.15 mmol/L, P = 0.004), LDL-C (1.76 +/- 0.12 vs. 1.51 +/- 0.14 mmol/L, P = 0.033), and TG (1.26 +/- 0.14 vs. 0.98 +/- 0.12 mmol/L, P = 0.006) were higher following the MCT trial than following the LCT trial, respectively. HDL-C concentration did not differ significantly between trials (MCT 1.48 +/- 0.05 mmol/L vs. LCT 1.45 +/- 0.04 mmol/L, P = 0.465). Although blood lipids remained within desirable ranges established by the National Cholesterol Education Program, these results suggest that consumption of MCT oil for 2 weeks negatively alters the blood lipid profile of athletes. Future studies should determine the effects of longer periods of MCT supplementation on serum lipids of exercisers and other groups of individuals. With little data suggesting that MCT are ergogenic, the adverse effects of MCT on blood lipid concentrations may outweigh any proposed benefits for athletes.

Behavioral hierarchy in the medicinal leech, Hirudo medicinalis: feeding as a dominant behavior.

The effect of feeding behavior on other behaviors (swimming, crawling and shortening) was investigated in the leech, Hirudo medicinalis. The stimulus locations and intensities required to produce mechanically elicited behaviors were first determined in the non-feeding leech. Stimuli were delivered while the leech was in various body positions to determine whether stimulus location affected behavioral response. Response thresholds were determined for the mechanically elicited behaviors. The same stimuli were then applied to feeding leeches to determine if response thresholds had changed. A solution with NaCl and arginine was used to elicit feeding. The same sets of stimuli were applied at intervals for an hour after feeding, to determine the duration of feeding-induced changes in behavior. Depending on the body position and stimulus location, stimuli produced different combinations of behaviors that included shortening, swimming and crawling. Anterior stimuli generally elicited shortening, whereas posterior stimuli generally elicited crawling and swimming, with swimming more likely to ventral stimulation than to dorsal stimulation. Having the front sucker attached changed these behavioral patterns. During feeding, the response thresholds changed dramatically, from 3-5 V to greater than 9 V. This increase in threshold began with the start of feeding, even before ingestion commenced. Suppression of the behaviors lasted up to 1 h after the end of feeding, with the effect on swimming being the most pronounced and longest lasting.