121 ACROSOME REACTION AS EFFECT OF ADDITION OF CHOLESTEROL TO BOAR SPERM MEMBRANES

Abstract

Altering the lipid composition of sperm plasma membranes not only affects the ability of sperm capacitation and acrosome reaction, it also affects the way sperm respond to cryopreservation. The objective was to determine if increasing sperm membrane cholesterol levels, by adding cholesterolloaded cyclodextrin (CLC) to boar sperm, alter the cryopreservation sperm to undergo acrosome reaction in vitro. The CLC was prepared as described by Purdy and Graham (2004) with some modification: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents removed using a hot plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Ejaculates (n = 5) from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept for 2 h at 22°C. Afterward, the ejaculates were put at 15°C/ for 60 min. Later, the ejaculates were centrifuged at 15°C at 400g/10 min, the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. These treatments were incubated for 15 min at 15°C. The samples were cooled to 5°C/90 min period and diluted 1:1 with freeze diluent (72.5-mL lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). The sperm were packaged into 0.5-mL straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath 37°C/30 s. A 90% Percoll solution was prepared by diluting 1 mL of 10× PBS with 9 mL of Percoll. A 35% Percoll solution was then prepared by diluting 90% Percoll (0.67 mL) with Medium 199 (1.33 mL). Frozen/thawed spermatozoa (2 mL) were then layered onto 2 mL of 35% Percoll solution in a 15-mL conical tube and centrifuged at 400g/5.5 min. The resulting pellet was suspended with Medium 199 to 100 million cells/mL, and the cells were stained with 5 μL of PI (1 mg mL^-1 in water) and 10 μL of FITC-PNA (1 mg mL^-1 in 10× PBS). The cells were incubated for 5 min at room temperature to allow PI and FITC-PNA to become incorporated. The acrosomal status of viable cells for each treatment was then determined by epifluorescence microscope at 400× magnification, and the percentage of acrosome reacted cells was calculated as the proportion of FITC-PNA stained and PI negative cells (acrosome reacted, live)/total live cells (PI negative, FITC-PNA positive and negative). Treatment differences for acrosome reaction were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher acrosome reaction (28%) compared with control cells (22%; P < 0.05). Several studies evaluated the ability of bull and stallion sperm treated with CLC to capacity and acrosome react. Adding cholesterol might alter the plasma membrane structure, improving the acrosome reaction in CLC-treated boar spermatozoa.

FAPEMIG, Piglandia, CNPq, FACEPE.