Male Infertility Clinic Protocols
Sperm Motility Assay:
Sperm are incubated in fresh MEM prepared with Earle’s balanced salt solution, both essential and nonessential amino acids (Gibco, Grand Island, NY, 0.23 mM pyruvic acid, 75 mg/L penicillin G, 50 mg/L streptomycin sulfate, 0.01 mM tetra sodium EDTA (Sigma), 3mg/ml BSA (Sigma), and 5% Fetal Bovine Serum (Sigma). The motility of at least one hundred spermatozoa is determined; a spermatozoon is considered motile when sperm progression or regular movement of the flagellum is observed.
Sperm Morphology Assay:
A slide is prepared by spreading 5-10 ul of a sperm suspension across the surface. After drying, slides are fixed in 5% acetic acid in 95% ethanol for 3 min, air dried, stained in 5% Eosin Y (Sigma) for 4 min, rinsed in 70% ethanol, air dried, and mounted with 30 ml of Permount (Fisher). Slides are scored using bright field optics with a 60X objective. Abnormal sperm are those that exhibit aberration of the head (e.g. length-width ratio or shape), defective attachment of the head to the tail, defects of tail (length, morphology) or persistence of the cytoplasmic droplet.
In vitro Fertilization and Development:
Epididymal sperm from adult male mice are used to fertilize superovulated wild-type oocytes. Fertilization is carried out for 4 hours in minimum essential medium (MEM) prepared with Earle’s balanced salt solution, both essential and nonessential amino acids (Gibco, Grand Island, NY, 0.23 mM pyruvic acid, 75 mg/L penicillin G, 50 mg/L streptomycin sulfate, 0.01 mM tetra sodium EDTA (Sigma) and 3mg/ml BSA (Sigma). This incubation takes place at 37C in a modular incubation chamber (Billups Rothenberg, Del Mar, CA) thoroughly infused with a gas mixture composed of 5% O2:5% CO2:90% N2. After incubation, the oocytes are washed twice in fresh medium and then cultured overnight in 1 ml of the same medium. Embryos that cleave to the 2-cell stage are collected and washed twice in KSOM supplemented with 0.5-strength essential and nonessential MEM amino acids; they are then cultured until the blastocyst stage in this medium.
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