Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Body weight and organ weights
• Procedure: Testis histology
• Procedure: Sperm count and morphology
• Procedure: Sperm lactate production
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice euthanized and weighed	Scales	Body weight
2	Reproductive organs harvested and weighed	Scales	Testis, epididymis with attached vas deferens, and seminal vesicle weights
3	Testis fixed, prepared for histology, and imaged	Microscope	Number of seminiferous tubule cross sections, tubule radii, and number of tubules exhibiting defects
4	Sperm harvested from right cauda epididymis and counted	Hemocytometer	Sperm counts
5	Sperm harvested from left cauda epididymis for in vitro capcitation study on sperm motility (see Odet2)	-	-
6	Sperm from left cauda epididymis prepared to assess morphology	Microscope	Sperm morphology
7	Sperm from left cauda epididymis prepared to measure lactate production	Spectrophotometer	Sperm lactate production

Equipment and supplies

• Scales
• Olympus BX51 microscope equipped with an Olympus DP72 digital camera, a motorized stage and MetaMorph automation and image analysis software (Molecular Devices, Sunnyvale CA)
• Iris scissors
• Fine forceps
• Spectrophotometer
• 5% CO₂ tissue culture incubator

Reagents and solutions

• Bouins solution
• Paraffin
• Periodic acid-Schiff reagent
• Hematoxylin
• Lactate dehydrogenase from rabbit muscle 10U/mL (Sigma-Aldrich, St. Louis MO)
• 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 (Goodson et al. 2011).
• Bovine serum albumin (BSA)
• Peanut agglutinin (PNA) conjugated to a fluorescent tag (Alexa Fluor 488, Invitrogen)

Procedure: Body weight and organ weights

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

Procedure: Testis histology

1. One testis per mouse is fixed in Bouins solution, cut in half horizontally and embedded in paraffin.
2. Testis sections (8 µm) are stained with periodic acid-Schiff reagent and counterstained with hematoxylin.
3. Composite images showing a complete transverse section from each testis (20x magnification) are generated using an Olympus BX51 microscope set up.
4. Measurements of height, width, perimeter and area of each section is recorded.
5. Digitized images are annotated using a custom interactive image analysis package (developed by submitting investigators; all images are available at the Systems Genetics website at UNC. Each composite image is examined in detail to determine the number of seminiferous tubule cross sections and the number of tubules exhibiting defects.

Procedure: Sperm count and morphology

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 Odet2).
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: Sperm lactate production

1. Sperm are collected from the left cauda epididymis (above) in HTF without lactate and pyruvate.
2. Sperm are incubated at 37°C; duplicate aliquots are removed at 0 and 2 h to measure lactate accumulation in the medium; sperm counts are obtained as well.
3. Sperm are removed from the suspension by centrifugation and the samples are added to assay buffer (pH 9.0) containing 1 mM NAD⁺ and 10 U/mL lactate dehydrogenase and incubated for 2 h at 25°C (the concentration of lactate in the sample is proportional to the increase in absorbance at 340 nm, as NAD⁺ is reduced to NADH).
4. Lactate production is calculated based on 10⁸ sperm.

Data collected by investigator

• body weight
• testis weight
• epididymis weight with attached vas deferens
• seminal vesicle weight
• number of seminiferous tubules/transverse section
• mean tubule radius
• seminiferous epithelium length/transverse section
• number of tubules with vacuoles
• number of tubules with many vacuoles
• number of tubules with germ cell loss
• number of tubules with abnormal germ cells
• number of tubules with germ cell sloughing
• sperm counts (per mouse, per mg testis, per seminiferous epithelium length)
• percentage of sperm with normal morphology
• percentage of sperm with abnormal head shape
• percentage of sperm with abnormal tail bending
• percentage of sperm with broken tails
• sperm lactate production

Goodson SG, Zhang Z, Tsuruta JK, Wang W, O'Brien DA. Classification of mouse sperm motility patterns using an automated multiclass support vector machines model. Biol Reprod. 2011 84(6): 1207-15.