Procedures

Procedure 1: Body weight and organ weights

Equipment, software, and supplies

• Scales

Steps

1. Mice are euthanized by CO₂ asphyxiation followed by cervical dislocation.
2. Carcasses are weighed.
3. Reproductive organs are harvested and weighed.

Procedure 2: Sperm count and morphology

Equipment, software, and supplies

• Microscope (Olympus, BX51)
• Digital camera (Olympus, DP72)
• Image analysis software (Molecular Devices, Sunnyvale, CA)
• Iris scissors
• Fine forceps
• Microfuge tubes
• Microscope slides
• Hemocytometer

Reagents and solutions

• Phosphate buffered saline (PBS)
• Human tubal fluid medium (HTF), 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM KH₂PO₄, 0.2 mM MgSO₄·7H₂O, 2 mM CaCl₂, 25 mM NaHCO₃, 2.78 mM glucose, 0.33 mM pyruvate, 21.4 mM lactate, 5 mg/mL of bovine serum albumin, 100 U/mL of penicillin G, and 0.1 mg/mL of streptomycin
• Bovine serum albumin (BSA)
• Peanut agglutinin (PNA), conjugated to a fluorescent tag (Invitrogen, Alexa Fluor 488)
• Methanol

Steps

1. The right cauda epididymis is clipped with iris scissors in 500 µL of PBS and incubated for 10 min at 37°C.
2. Sperm are extruded with fine forceps and the suspension is transferred to a microfuge tube; the collection well is rinsed with an additional 500 µL of PBS.
3. Sperm are diluted appropriately and counted using a hemocytometer.
4. The left cauda epididymis is clipped with iris scissors and incubated in HTF at 37°C (5% CO₂) for 10 min, allowing sperm to swim into the medium.
5. Sperm motility is determined (see Procedure 3).
6. Aliquots (10 µL) of the HTF sperm suspension are spread onto positively charged microscope slides and allowed to air dry briefly until moisture just evaporates.
7. Samples are fixed with -20°C methanol for 10 min, air dried and stored at -20°C.
8. Acrosomes are stained with PNA conjugated to a fluorescent tag.
9. Sperm are scored using phase contrast optics (normal morphology, abnormal head shape, abnormal tail bending (≥ 90°), or broken tails (severed at head/neck junction or at more distal locations along the length of the flagellum).

Procedure 3: Sperm motility

Definitions & Abbreviations: CASA: Computer Assisted Sperm Analysis

Equipment, software, and supplies

• CEROS imaging system (Hamilton Thorne)
• CEROS imaging system software, version 12.3 IVOS (Hamilton Thorne)
• CASAnova, a support vector machines program based on CASA parameters of CD1 sperm
• Leja chambers (Leja, Netherlands)
• 5% CO₂ tissue culture incubator

Reagents and solutions

• Human tubal fluid medium (HTF), 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM KH₂PO₄, 0.2 mM MgSO₄·7H₂O, 2 mM CaCl₂, 25 mM NaHCO₃, 2.78 mM glucose, 0.33 mM pyruvate, 21.4 mM lactate, 5 mg/mL of bovine serum albumin, 100 U/mL of penicillin G, and 0.1 mg/mL of streptomycin
• Bovine serum albumin (BSA)

Steps

1. Sperm suspensions from the left cauda epididymis are diluted with HTF (see Procedure 2).
2. Motility is assessed by computer-assisted sperm analysis CASA) using Leja chambers.
3. Sperm tracks (90 frames, 1.5 s) and kinetic parameters for individual sperm are captured at 60 Hz using motility analysis parameters (mouse 2) recommended by Hamilton Thorne Biosciences, except that slow sperm are considered motile.
4. Tracks in 10 fields are typically recorded for each mouse; motility measurements are captured.
5. CASAnova is used to classify individual sperm into one of five motility groups. Classification is based on Support Vector Machines equations incorporating independent CASA parameters for each sperm track. See Goodson et al., 2011 for details.