Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mm or 0.1 mm of each antioxidant, including oxidative stress (Fe²⁺/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.     

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26 306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.     

Studying pressure denaturation of a protein by molecular dynamics simulations

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This “unfolding‐up‐on‐squeezing” is counter‐intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results—that pressure denatured states are water‐swollen, and theoretical results—that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states—their water‐swollen nature, retention of secondary structure, and overall compactness—mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure‐dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately ?60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500–2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water‐swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Gel filtration of bacterial cell extract

Так как от остатков гомогенизированных клеток легко отделить растительные вирусы (Steere и Qckers, 1962; ?ech, 1962) и митохондрии (Hjertén, 1962), мы сделали попытку использовать этот простой метод и для отделения рибосом. Надосадок гомогената растущих клеток Bacillus megaterium 110 наносили на столбец 3% гранулированного агара (Polson, 1961) и промывали 66 мл буферного раствора Tris (pH 7,72, 0,005 M с 0,01 M?MgCl₂) в течение часа при комнатной температуре. В элюате определяли абсорбцию при 260 мμ и белки (рис. 1). При ультрацентрифугировании образца были обнаружены рибосомы с S ₂₀ ⁰ 107, 71, 5, 45 и 30S (рис. 2). Ультрацентрифугированием фракции № 10 были с помощью адсорбции в УФ лучах найдены рибосомы с s ₂₀ ⁰ 107 и 44,6S (рис. 3). Группа 100S перекрывалась абсорбцией рибосом 70S, группа 30 не была обнаружена. Отделение рибосом как самостоятельной вершины не было получено. Однако, судя по соотношению нуклеиновых кислот и белков во главе вымываемой плазмы (где присутствует только РНК), здесь имела место концентрация практически чистых рибосом, как подтверждает и седиментация. ДНК удавалось определить только во фракциях второй половины элюата плазмы (рис. 4).     

Acceleration of flavour formation in cheese by a bacteriocin-producing adjunct lactic culture

Bacteriocin-producing Enterococcus faecalis INIA 4 was used as adjunct lactic culture at inoculum levels ranging from 0.003% to 0.10% in the manufacture of semi-hard cheese. Cheese made from milk with 0.003% adjunct culture had the most pronounced proteolysis and developed a characteristic cheese flavour faster than the rest, reaching in 30.4 days the flavour intensity score of a 45-day control cheese made without adjunct culture.     

The karyotype of Axarus festivus (Say) and a comparison with species of Lipiniella (Chironomidae: Diptera)

Larvae of Axarus andLipiniella share several apparantly apomorphic features of the mouth-part structure, however adults ofLipiniella show possible relationships toDemeijerea andChironomus. In order to assist in refining the relationships ofAxarus toLipiniella the karyotype ofAxarus festivus was determined and compared to the karyotypes ofLipiniella arenicola andL. moderata. Larvae ofA. festivus from a population in Kansas were monomorphous, with 2n=8, the Ist, IInd and IIIrd chromosomes metacentric, and IVth acrocentric.Axarus festivus therefore differs fromL. arenicola in chromosome number (2n=6), however homologous sections of all chromosomes were identified. Inversions were detected in the Ist, IInd and IIIrd chromosomes ofA. festivus relative toL. arenicola. It was determined that both species have high functional activity, as indicated by the presence of three Balbiani rings, and more than one nucleolus per genome. Differing degrees of polyteny, a feature previously described forL. arenicola, were observed in the salivary glands, with highest degrees of polyteny in cells near the salivary duct. These similarities of chromosome structure indicate close genetic relationships betweenA. festivus andL. arenicola. However, we did not find evidence for similarity ofA. festivus toL. moderata, which supports the previous conclusions byKiknadze et al. (1989) regardingL. arenicola andL. moderata.     

Hormonal modulation of biosynthesis of prostatic specific antigen, prostate specific acid phosphatase and prostatic inhibin peptide.

Hormonal modulation of in vitro biosynthesis of three prostatic secretory proteins, prostate specific acid phosphatase (PSAP), prostate specific antigen (PSA) and prostatic inhibin peptide (PIP) by human benign hyperplasia (BPH) tissue was studied. LH and inhibins caused increase in the synthesis of all three proteins whereas FSH enhanced the synthesis of PIP and PSA only but decreased PSAP synthesis. Prolactin and thyroid releasing hormone decreased synthesis of PIP and PSAP. However, PSA synthesis was enhanced by TRH and was decreased by prolactin. Estradiol caused significant increase in PSA and PSAP but no discernible changes in PIP synthesis were noticed. Testosterone caused an increase in PIP, PSA and PSAP. These data indicate that biosynthesis of PIP, PSA and PSAP by BPH tissue is under multihormonal regulation.