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.

References:

Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biology of Reproduction (1996) 54: 197-207

O’Brien MJ, Pendola JK, Eppig JJ. A Revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biology of Reproduction (2003) 68:1682-1686.

Eppig JJ, Wigglesworth K Atypical maturation of oocytes of strain I/LnJ mice. Molecular Reproduction and Development (1994) 9:1136-1142.

Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Molecular Reproduction and Development (1995) 41:232-238.