Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility.

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells.

Spermatogonial stem cells (SSC) have important applications in domestic animal reproduction and advanced biotechnologies. Because differential plating is one of the most common methods used for SSC enrichment, the goal of this study was to compare three differential plating methods for the enrichment of bovine SSC. To achieve this goal, testicular parenchyma from pre-pubertal calves was minced and single cells were obtained after two enzymatic digestions. We compared three coating methods for differential plating: laminin (20 ng/ml), BSA (0.05 mg/ml) and PBS. Cells were incubated at 37°C, 5% CO2 in air for 15 min onto laminin-coated dishes or 2 h onto BSA- or PBS-coated dishes. Cell viability was assessed by trypan blue exclusion method. Recovered cells were analysed for the expression of SSC molecular markers by quantitative RT-PCR (GFRA1, CXCR4, ITGA6, THY1) and flow cytometry (GFRA1, CXCR4 and ITGA6). Cells at time 0, adherent cells on laminin and non-adherent cells from BSA and PBS groups had the same cell viability (p = 0.0655). GFRA1, CXCR4 and THY1 relative gene expression was higher (p = 0.0402, p = 0.0007, p = 0.0117, respectively) for non-adherent cells selected in PBS group. Flow cytometry analysis revealed that the presence of GFRA-positive (GFRA+) cells was higher in non-adherent cells from BSA and PBS groups (p < 0.001). However, laminin-adherent cells had higher number of ITGA6+ cells (p < 0.001) and lower presence of CXCR4+ cells (p = 0.0012). In conclusion, differential plating is an effective method for the enrichment of bovine undifferentiated spermatogonia and higher expression of SSC markers is obtained without laminin or BSA coating.

Effect of chemical or electrical activation of bovine oocytes on blastocyst development and quality.

Activation of in vitro-matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 microM CA, 5 min; n = 88), CA + BSA (5 microM CA, 5 min; BSA, 5 min; n = 90), IO (5 microM IO, 5 min; n = 91), IO + BSA (5 microM IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 micros; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.