Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa

The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

Improvement of motility of post-thaw Poitou jackass sperm using glutamine

The relative effectiveness of L-glutamine in preserving motility and movement characteristics of Poitou jackass spermatozoa diluted at 60 x 10(6) sperm/ml in INRA 82 medium modified by 4 % (v/v) glycerol and 2 % (v/v) quail's egg yolk during the cooling and freezing-thawing process was studied. After cooling to 4 degrees C, glutamine at 80, 120 or 240 mM did not improve the percentages of motile and progressively undulating spermatozoa or the movement characteristics (VCL = curvilinear velocity, VSL = straight line velocity, VAP = velocity of the average path, LIN = VSL/VCL x 100, ALH = amplitude of the lateral head displacement, BCF = beat cross frequency) assessed by the automated analyzer ATSM. However, after the FT process, 80 mM glutamine significantly improved motility, the percentage of progressively undulating spermatozoa and all the movement characteristics analyzed. The presence of glutamine at 80 mM in a glycerol-FT medium thus improves the motility of Poitou jackass spermatozoa during the freezing-thawing process. The presence of glutamine at 80 mM was not sufficient to offset the need to use glycerol in the freezing-thawing medium. This could indicate that glutamine has a mechanism of cryoprotection for Poitou jackass spermatozoa that is independant of glycerol.     

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.     

A procedure for Poitou jackass sperm cryopreservation

We have tried to establish sperm banking for the endangered Poitou donkeys. No successful cryopreservation technique had been described for spermatozoa of this species; our preliminary work indicated that a particular medium and procedure may be effective for cryopreservation of Poitou jackass spermatozoa as evaluated by sperm motility, membrane integrity and pregnancy rate after AI with frozen-thawed semen. We found that glutamine at 80 mM and 10% (v/v) quail egg yolk in a basal medium containing 4% (v/v) glycerol (T2-94 medium) improved the post-thaw total and progressive motility and velocity assessed with the automated analyzer ATS-M. The T2-94 medium also preserved the sperm nuclear, acrosom, and plasma membrane integrity as assessed with the acridine orange method, fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) lectin procedure, and hypo-osmotic swelling test, respectively. Semen frozen-thawed in T2-94 medium as used to artificially inseminate. 13 Poitou jennies from the beginning of estrus to ovulation during 4 cycles at a rate of one AI per day. Heigh pregnancies and 3 foals were obtained, but only when the glycerol was removed from sperm before AI. We conclude that the cryopreservation of Poitou jackass semen for sperm banking may succeed by using the T2-94 medium and removing the glycerol post-thaw, but before AI.     

Lung epithelium injury biomarkers in workers exposed to sulphur dioxide in a non-ferrous smelter

Serum Clara cell protein (CC16) and surfactant-associated protein D (SP-D) were measured in 161 workers exposed to sulphur dioxide (SO₂) in a non-ferrous smelter. Seventy workers from a blanket manufacture served as referents. Exposure to SO₂ and tobacco smoking were associated with a decrease of CC16 and an increase of SP-D in serum. Tobacco smoking and exposure SO₂ interacted synergistically to decrease serum CC16 but not to increase serum SP-D. While further illustrating the potential of serum CC16 and SP-D, our study confirms that SO₂ can cause airways damage at exposure levels below current occupational exposure limits.     

Contribution of epigenetic alteration of BRCA1 and BRCA2 genes in breast carcinomas in Tunisian patients

Objective: The aim of this study was to evaluate the contribution of the BRCA1 and BRCA2 promoter methylation in the pathogenesis of sporadic breast cancer in Tunisian patients. Methods: Breast carcinoma tissues (n = 117) and available paired normal breast tissues (n = 65) from Tunisian women who had no family history were investigated for the methylation status of BRCA1 and BRCA2 promoters using methylation-specific PCR. Breast specimens from women without carcinoma (16 fibroadenomas and 5 mastopathies) were used as control. Results: Hypermethylation of BRCA1 and BRCA2 promoters was detected respectively in 60.7% and 69.2% of the carcinoma tissues, and in only 7.7% and 4.6% of the paired normal breast tissues. None of the fibroadenomas and mastopathies showed hypermethylation. Correlations were found between BRCA1 and BRCA2 hypermethylation and decrease in their mRNA expression (p = 0.02 and p = 0.009, respectively). Moreover, BRCA1 methylation correlates with patients age (p = 0.01) and triple negative (ER?, PR?, HER2?) tumors (p = 0.01). Patients with methylated BRCA1 and/or BRCA2 had a significant prolonged survivals compared to those with unmethylated tumors (p = 0.002). Conclusion: Our results suggest an important role of BRCA1 and BRCA2 promoter methylation in breast cancer development in the Tunisian population.     

Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret?also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.     

Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen

Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). In recent years, arguments concerning the presence of cryoprotective antagonists in egg yolk, have reinforced interest in the use of only the LDL extracted from egg yolk in the extenders. However, current methods of LDL purification do not support the use of LDL in commercial extenders because they offer a poor recovery rate. Consequently, we have developed an easy method to extract LDL from egg yolk. Several concentrations of purified LDL (between 2.5 and 20%, w/v) were tested in freezing extenders for bull semen, and compared with commercial extenders. Our extraction method reached 97% purity and about 67% yield, and is easily reproducible on an industrial scale. Analysis of sperm motility showed that the motility and characteristics of spermatozoa movement were improved with LDL in the extender, as compared to a commercial extender containing egg yolk. The optimum LDL concentration in the extender was 8%. In conclusion, we propose that an extender containing LDL extracted from egg yolk could be used as cryoprotective media with a better efficiency than present commercial extenders.     

Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.