Formation and function of the manchette and flagellum during spermatogenesis.

The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular-based protein delivery platforms transport proteins to the developing head and tail: the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review, we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra-manchette transport to the basal body at the base of the developing sperm tail and through the intra-flagellar transport to the construction site in the flagellum. This review focuses on the microtubule-based mechanisms involved and the consequences of their disruption in spermatid elongation.

Microdialysis-Assessed Exercised Muscle Reveals Localized and Differential IGFBP Responses to Unilateral Stretch Shortening Cycle Exercise.

Microdialysis allows for a preview into local muscle metabolism and can provide physiological insight that blood measurements cannot. Purpose: To examine the potential differential IGF-I system regulation in interstitial fluid during unilateral stretch shortening cycle exercise. Methods: 10 men (26 ± 7 year) performed unilateral jumping [stretch shortening cycle (SSC) exercise at 50% of optimal jump height] until volitional fatigue on a sled apparatus. Biological sampling took place using a catheter inserted into an antecubital vein (serum), and 100 kDa microdialysis probes inserted into the thigh muscle of each exercise/control leg (dialysate). Serum was drawn before (Pre; -3 h) and after SSC [Post I (+0 h), II (+3 h), or III (+20 h)]; dialysate was sampled for 2 h before (Pre), during/immediately after (Ex), and 3 h into recovery (Rec) following SSC. IGF-I system parameters (free/total IGF-I and IGFBPs 1-6) were measured with immunoassays. Interstitial free IGF-I was estimated from dialysate IGF-I and relative recovery (ethanol) correction. Data were analyzed with repeated measures ANOVA. Results: Serum total IGF-I remained elevated +3 h (Post II: 182.8 ± 37.6 vs. Pre: 168.3 ± 35.0 ng/mL, p < 0.01), but returned to baseline by +20 h (Post III vs. Pre, p = 0.31). No changes in serum free IGF-I were noted. Serum BP-1 and -3 increased over baseline, but not until + 20 h after SSC (Post III vs. Pre: 7.6 ± 4.9 vs. 3.7 ± 2.3 and 1,048.6 ± 269.2 vs. 891.4 ± 171.2 ng/mL, respectively). We observed a decreased serum BP-6 +3 h after SSC (p < 0.01), followed by a return to baseline at +20 h (p = 0.64 vs. Pre). There were no exercise-induced changes in serum BP-2, -4, or -5. Unlike serum, there were no changes in dialysate or interstitial free IGF-I in either leg (p > 0.05). Dialysate BP-1 remained increased in both exercise and control legs through 3 h into recovery (Rec vs. Pre, p < 0.01). Dialysate BP-3 also demonstrated a prolonged elevation over Pre SSC concentrations, but in the exercise leg only (Ex and Rec vs. Pre, p < 0.04). We observed a prolonged decrease in dialysate BP-5 (Ex and Rec vs. Pre, p < 0.03) and an increase in BP-4 IP in the exercise leg only. There were no changes relative to Pre SSC in dialysate BP-2 or -6. Conclusions: Unilateral exercise drives differential regulation of the IGF-I system at both local and systemic levels. More specifically, this is the first study to demonstrate that localized exercise increases IGFBP-3, IGFBP-4 and decreases in IGFBP-5 in muscle interstitial fluid.

Strength, [corrected] endurance or combined training elicit diverse skeletal muscle myosin heavy chain isoform proportion but unaltered androgen receptor concentration in older men.

We investigated whether the myosin heavy chain (MyHC) proportion and androgen receptor (AR) concentration in skeletal muscle differ following 21 weeks of strength, endurance and combined training in untrained older men. Strength (S) and endurance (E) groups trained twice per week and combined (S+E) group trained four times per week (two strength and two endurance). Muscle biopsies were obtained before and after the training period from m. vastus lateralis (VL) and AR mRNA and protein concentration and MyHC proportion were determined. 1RM increased during the training period in S, S+E and E but the changes were greater in S and S+E than in E. Statistically significant increases were observed only in S and S+E in maximal isometric force as well as in VL thickness. VO (2max) increased significantly only in E. MyHCIIa proportion increased in S, while MyHCIIa proportion decreased and MyHCI increased (p<0.05) in E. No statistically significant changes were observed in serum testosterone and in AR mRNA or protein concentrations. The present results indicate that 21 weeks of strength, endurance or combined training changed MyHC proportion according to the training method but did not have an effect on AR mRNA or protein expression in skeletal muscle at rest.

Native tertiary structure in an A-state.

The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, fluctuating tertiary structure. We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state. Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, delta GA reversible D and delta GN reversible D. We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants. The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface. We also examined the pH and sulfate concentration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants. A delta GA reversible D versus-delta GN reversible D plot for the interface variants has a slope near unity and the control variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report. We determined that the heat capacity change for A-state denaturation is approximately 60% of the value for native-state denaturation, indicating that the A-state interior is native-like. We discuss our results in relation to ferricytochrome c folding kinetics.

A hypothesis linking hypoglycemia, hyperuricemia, lactic acidemia, and reduced gluconeogenesis in alcoholics to inactivation of glucose-6-phosphatase activity by acetaldehyde.

Preliminary data have been obtained indicating that glucose-6-phosphatase is inactivated upon preincubation with 447 and 224 mM acetaldehyde for 30 min at room temperature, resulting in a loss of 67% and 33% of the original activity, respectively. The reaction with acetaldehyde is rapid because 44% of the enzymic activity is lost in 5 min. Comparable quantities of ethanol inhibit the enzyme to the extent of 11%, indicating a very slight, statistically insignificant organic solvent effect. Because chronic alcoholics present a clinical picture of hypoglycemia, hyperuricemia, reduced gluconeogenesis, and lactic acidemia, it is hypothesized that glucose-6-phosphatase may be a focal enzyme whose inactivation may be related to each of the disorders. Glucose-6-phosphatase is the terminal key enzyme in the gluconeogenesis pathway leading to increased blood glucose. Inhibition thereof may explain both the alternate reduction of pyruvate with concommittent increased formation of lactic acid, and the increase in the pentose phosphate pathway leading to hyperuricemia (as also observed in von Gierke's disease).

Optical projection tomography as a tool for 3D imaging of hydrogels.

An Optical Projection Tomography (OPT) system was developed and optimized to image 3D tissue engineered products based in hydrogels. We develop pre-reconstruction algorithms to get the best result from the reconstruction procedure, which include correction of the illumination and determination of sample center of rotation (CoR). Existing methods for CoR determination based on the detection of the maximum variance of reconstructed slices failed, so we develop a new CoR search method based in the detection of the variance sharpest local maximum. We show the capabilities of the system to give quantitative information of different types of hydrogels that may be useful in its characterization.

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.