Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Familial adenomatous polyposis-associated desmoids display significantly more genetic changes than sporadic desmoids

Desmoid tumours (also called deep or aggressive fibromatoses) are potentially life-threatening fibromatous lesions. Hereditary desmoid tumours arise in individuals affected by either familial adenomatous polyposis (FAP) or hereditary desmoid disease (HDD) carrying germline mutations in APC. Most sporadic desmoids carry somatic mutations in CTNNB1. Previous studies identified losses on 5q and 6q, and gains on 8q and 20q as recurrent genetic changes in desmoids. However, virtually all genetic changes were derived from sporadic tumours. To investigate the somatic alterations in FAP-associated desmoids and to compare them with changes occurring in sporadic tumours, we analysed 17 FAP-associated and 38 sporadic desmoids by array comparative genomic hybridisation and multiple ligation-dependent probe amplification. Overall, the desmoids displayed only a limited number of genetic changes, occurring in 44% of cases. Recurrent gains at 8q (7%) and 20q (5%) were almost exclusively found in sporadic tumours. Recurrent losses were observed for a 700 kb region at 5q22.2, comprising the APC gene (11%), a 2 Mb region at 6p21.2-p21.1 (15%), and a relatively large region at 6q15-q23.3 (20%). The FAP-associated desmoids displayed a significantly higher frequency of copy number abnormalities (59%) than the sporadic tumours (37%). As predicted by the APC germline mutations among these patients, a high percentage (29%) of FAP-associated desmoids showed loss of the APC region at 5q22.2, which was infrequently (3%) seen among sporadic tumours. Our data suggest that loss of region 6q15-q16.2 is an important event in FAP-associated as well as sporadic desmoids, most likely of relevance for desmoid tumour progression.     

Effects of group size and personality on social foraging: the distribution of sheep across patches

Group cohesion in social animals foraging in patchy environmentsdepends on 2 opposite forces, intraspecific competition, andattraction. The decision to leave or stay in a group may varyaccording to the individual personality. We investigated therole of personality when feeding competition increases as aresult of increasing group size. Individuals referred as "bold"and "shy" were identified from an indoor exploration test accordingto their propensity to leave the group to explore a novel environment,using 12 novel objects placed at increasing distances from thegroup. Groups of 2, 4, 6, or 8 shy or bold sheep were testedin 45 x 5–m grass arenas, with one 5 x 5–m patchof preferred vegetation at each end. Sheep grazed on or closeto these patches, but the number grazing the patches seldomreached more than 4–5 individuals, suggesting that crowdingmight affect foraging at the highest densities. The smallestgroups grazed together on the same patch, but there was an increasinglikelihood of splitting into subgroups as group size increased,with equal-sized subgroups most commonly grazing the 2 patchessimultaneously. This effect was greatest in bold sheep, whichtended to split into subgroups at smaller group sizes than shysheep. This study provides new insight into the mechanisms bywhich group-living herbivores distribute themselves across patchyresources in a way that minimizes interference competition anddemonstrates the importance of individual variability for spatialorganization at the level of the group.     

Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.     

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility,Gating Residues and Multiple Binding Pockets

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.     

Genetic characterization and structure of the endemic Colombian silvery brown bare-face tamarin,Saguinus leucopus (Callitrichinae,Cebidae, Primates)

We analyzed 115 Saguinus leucopus, from four Colombian departments (Antioquia, Bolivar, Caldas and Tolima ), for 701 bp of the mt COII gene and at 10 microsatellite loci to estimate gene diversity levels, possible molecular subspecies and historical demographic changes in this species. This endemic Colombian species showed an elevated gene diversity in this gene, although its geographical distribution is very restrictive and extremely threatened by habitat fragmentation. The mt COII gene did not show any geographical structure in the distribution of the haplotypes within this species, but it did show a noteworthy population expansion throughout the history of this species. A Bayesian analysis showed that the haplotype diversification of this species began around 1.6 million years ago (MYA), whilst a haplotype network gave the beginning of this diversification at around 0.5–0.6 MYA. Forty-seven individuals out of the 115 were analyzed for 10 DNA microsatellites. The genetic diversity was relatively elevated for this kind of marker too, and comparable to that found in other Neotropical monkeys with a wider geographical distribution. Two gene pools were detected with the microsatellites, one in the northern distribution area (Antioquia) and the other in the southern distribution area (Tolima). No tests detected any bottleneck affecting this population; however, two procedures (k test and Kimmel et al. 1998 test) detected significant population expansion for the microsatellite markers, like that seen with the mt COII gene.     

Experimental evidence of habitat selection and territoriality in the Amazonian whip spider Heterophrynus longicornis (Arachnida, Amblypygi)

Animals that select and defend suitable habitats against conspecifics may be favored by maximizing prey encounter rate, gaining protection, or securing matings. However, the identification of habitat selection and territoriality may be hindered in observational studies in sedentary species with low-density populations, such as the whip spider Heterophrynus longicornis. To circumvent such difficulties, we adopted an experimental field approach to evaluate if H. longicornis selects and defends habitat in Central Amazon. To evaluate whether individuals perform habitat selection, we monitored the permanence of 29 experimentally released individuals in buttressed trees with a wide variation in diameter at breast height (DBH), with and without burrows at their bases. To evaluate whether individuals are territorial, we experimentally removed 21 individuals from their previously occupied trees and monitored the recolonization of these trees. If H. longicornis were territorial, we predicted that the recolonizers would be smaller than the removed individuals. We found 12 individuals in the trees where they were released on subsequent days, none of them in trees without burrows. The individual permanence was related to the presence of burrows, and not to DBH. There was recolonization by smaller males and females on ten trees from which the amblypygids were removed. In combination, the permanence of individuals in trees with burrows, the rapid recolonization of experimentally vacated trees by smaller individuals, and the preponderance of one individual per tree, suggest that both male and female H. longicornis perform habitat selection and also that after selecting a site, they defend it against conspecifics.