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The effects of progesterone and cholesterol on sperm capacitation, acrosome reaction, and in vitro embryo production in cattle

University of British Columbia

Progesterone (P₄) is known to induce mammalian sperm acrosome reaction (AR) in vitro, whereas cholesterol (CH) one of the major components in seminal plasma, inhibits AR. The objectives of this study were: 1) to elucidate the in vitro effects of P₄, 17 β-estradiol (E₂), CH, and their combination on cryopreserved bovine sperm A R , 2) to study the mechanisms o f CH action on P₄-induced A R , and 3) to determine the effects of exogenous P₄ and CH on zonabinding ability of bovine sperm and in vitro fertilization (IVF). For the first two objectives, swim-up separated sperm from six bulls were incubated in modified Tyrodes* medium for 0-6 h (38.5°C, 5% CO₂). Acrosome reaction was determined following addition of P4 (10 μg/m1), E₂ (10 μg/ml), db-cAMP (1 mM), forskolin (10 μM), and Ca²⁺-ionophore (10 μm), either in the presence or absence of CH (1 (μg/ml). The effects of β-sitosterol (1 μg/ml), a CH plant analog, and CH on P₄-induced AR were also compared. For zona-binding assay, thawed sperm were washed in modified Tyrodes' medium. Sperm (0.2 x 106 sperm/ml) were pre-treated with P₄ (10 pig/ml), either in the presence or absence o f CH (1μg/mi) at 38.5°C, 5% CO₂. Sperm were co-incubated with cumulus-free oocytes for 18 nr. Following washing, fixing, and staining, the number of sperm attached to oocytes was counted under a fluorescent microscope. For IVF, 30 min prior to the addition of matured oocytes, sperm (0.2 x 10⁶ sperm/ml) were pre-treated with P₄ (10 μg/ml), CH (1 μg/ml), combination of P₄ (10 μg/ml) and CH (1 μg/ml), and cyclodextrin (10 μg/ml). Sperm exposed oocytes were then incubated in modified Tyrodes' medium (supplemented with 10% serum) at 38.5°C and 5% CO₂. Cleavage rates, as an indicator of fertilization, were recorded after 72 nr. Acrosome reaction was induced by P₄ (34.4 ± 2.5%), E₂ (4.5 ± 0.7%), db-cAMP (49.3 ± 2.0%), forskolin (81.8 ± 1.2%), and Ca²⁺-ionophore (95.7 ± 1.0%) immediately after-thawing (p < 0.05 for all). Cholesterol and β-sitosterol inhibited P₄- induced A R in an identical manner (95.6% and 95.4% inhibition, respectively). DibutyrylcAMP- induced A R was also inhibited by CH but this was not the case for forskolin and Ca²⁺ - ionophore. Progesterone increased both the number of spermatozoa bound to zona (73.9 ±3.7 compared to 45.8 ± 2.8 for control, 46.2 ± 3.3 for CH , and 45.3 ± 1.8 for P₄ + CH) and the percentage of cleavage rate (42.5 ± 3.8% compared to 22.5 ± 2.1% for control, 18.3 + 2.8% for CH, 20.8 ± 2.0% for p-cyclodextrin, and 17.5 ± 1.7% for P₄ + CH). From this study it is concluded that a) CH inhibits AR induced by P₄as well as by db-cAMP, b) cholesterol inhibits AR possibly by modifying the structure of the sperm plasma membrane, and c) progesterone treatment of sperm immediately prior to fertilization increases cleavage rate and may improve mammalian IVF success rate.

Thesis/Dissertation

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2009-06-16

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http://hdl.handle.net/2429/9338

Animal Science

University of British Columbia

UBCV

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