Category Archives: Spermatozoa - Part 2

A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(5)

They served to determine the levels of passive diffusion and possible spontaneous breakdown of the labeled glucose as well as the background of the radioactivity counter. At the end of incubation, the NaHCO3 fraction was collected quickly in scintillation vials containing 200 ^l of 0.1 M NaOH, which promoted the conversion of dissolved CO2 and bicarbonate into carbonate. After an overnight incubation, 10 ml of … Continue reading

A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(4)

Glucose metabolism via the PPP was assessed by measuring the 14CO2 produced from [1-14C]glucose (55 mCi/ mmol; Amersham). [1-14C]Glucose (55 ^Ci/ml; 1 mM) was added to sperm suspensions containing no unlabeled glucose. In addition, some experiments were performed with [6-14C]glucose (55 mCi/mmol; Amersham) to evaluate the oxidation of labeled glucose by the Krebs cycle. buy flovent inhaler

A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(3)

Gamete Fusion Assay Zona-free oocytes were incubated in insemination drops for 20 min to allow sperm binding to the oolemma. They were then transferred into 25-^l droplets of M16 for 45 min to allow bound sperm to enter the oocytes and to de-condense. The oocytes were then washed as described by Wolf and Hamada to remove loosely attached sperm. For assessment of sperm binding to … Continue reading

A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(2)

Sperm Preparation Spermatozoa were obtained from the cauda epididymidis and the vas deferens of 10- to 16-wk-old OF1 males (BRL, Fullinsdorf, Switzerland). In all experiments, spermatozoa were incubated in 200 ^l of M16 under oil (light white mineral oil; Sigma) at 37°C for 3 h to achieve capacitation and to maximize the proportion of acrosome-reacted sperm. The concentration of motile sperm at the end of … Continue reading

A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(1)

Chemicals and Media The basic culture medium used in all experiments was M16 containing 23.3 mM lactate, 0.33 mM pyruvate, and 5.56 mM glucose, and supplemented with 15 mg/ml type V BSA. Cytochalasin B, phloretin, and iodoacetate were dissolved as stock solutions (1000 X concentrated in dimethyl sulfoxide) and stored at -20°C. Dilutions in culture medium were made before use. All chemicals were obtained from … Continue reading

A Possible Role for the Pentose Phosphate Pathway of Spermatozoa in Gamete Fusion in the Mouse(2)

To permit gamete fusion, glucose must be metabolized because non-metabolizable glucose analogues (L-glucose, 3-O-methylglucose, 2-deoxyglucose) are unable to support this process. In contrast to the metabolism of oocytes, which does not appear necessary for this event, the metabolism of spermatozoa may be linked to the fusion step. While it is well accepted that glucose metabolism through glycolysis provides energy to spermatozoa, the existence and the … Continue reading

A Possible Role for the Pentose Phosphate Pathway of Spermatozoa in Gamete Fusion in the Mouse(1)

The fertilization process requires glucose in a number of species (mouse; rat; human ). In the mouse, the analysis of the sequence of events leading to fertilization in vitro has indicated that glucose is essential for the penetration of the zona pellucida and for the fusion of the gametes. The failure to penetrate the zona pellucida in the absence of glucose has been attributed to … Continue reading

Determination of Plasma Membrane Characteristics: DISCUSSION(9)

Then, on the basis of selection of CPA type and concentration, the procedure should predict an optimal cooling and warming rate based upon: LpCPA, PCPA, cr, and their activation energies. By establishing this order, one is able to work within the cells’ osmotic tolerance limits (which are currently more difficult to modify) and subsequently adjust the cooling and warming rates (which are easily adjustable using … Continue reading

Determination of Plasma Membrane Characteristics: DISCUSSION(8)

Although boar spermatozoa have a relatively low water permeability in the presence of CPAs (0.120 |xm/min/atm, in the presence of DMSO), compared to other cell types such as murine oocytes (0.80 (xm/min/atm in the presence of DMSO) regarding intracellular ice formation, the cells also have a counterbalancing low intracellular water volume (31%) , and a very high surface area:volume ratio (surface area:volume = 156 |xm2:26.3 … Continue reading

Determination of Plasma Membrane Characteristics: DISCUSSION(7)

These data were later applied to resolve previous apparent discrepancies between theoretical predictions and empirical outcomes. They were then used to develop more optimal cryopreservation protocols for human spermatozoa . The same approach to fundamental cryobiology should be used as a paradigm for the optimization of boar spermatozoa cryopreservation.