Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm

in Reproduction

Correspondence should be addressed to N C Bernecic; Email: Naomi.Bernecic@ul.ie
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Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20–23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.

Supplementary Materials

    • Supplementary Table 1. Motility and kinematic parameters assessed using computer-assisted sperm analysis (CASA) following exposure to media with or without bicarbonate and cAMP-regulators (BSA present in all conditions) for up to 3 h (180 min).
    • Supplementary Table 2. Motility and kinematic parameters assessed using computer-assisted sperm analysis (CASA) following exposure to media with or without BSA and cAMP-regulators (bicarbonate present in all conditions) for up to 3 h (180 min).
    • Supplementary Figure 1. The percentage of PI- and PI+ acrosome intact (A and B) and PI+ acrosome reacted (C) spermatozoa following exposure to conditions with or without bicarbonate and cAMP up-regulators (db-cAMP, caffeine and theophylline) for up to 3h (180 min). Membrane and acrosome integrity were assessed with flow cytometry using propidium iodide and PNA-FITC, respectively. Data correspond to mean ± S.D. (A and C) or geometric mean ± 95% confidence interval (B) for ten independent samples. **P<0.01 and ***P<0.001 indicate significant differences from TALP (bicarbonate and BSA)
    • Supplementary Figure 2. The percentage of PI- and PI+ acrosome intact (A and B) and PI+ acrosome reacted (C) spermatozoa following exposure to conditions with or without BSA and cAMP up-regulators (db-cAMP, caffeine and theophylline) for up to 3h (180 min). Membrane and acrosome integrity were assessed with flow cytometry using propidium iodide and PNA-FITC, respectively. Data correspond to mean ± S.D. (A and C) or geometric mean ± 95% confidence interval (B) for seven independent samples. *P<0.05, **P<0.01 and ***P<0.001 indicate significant differences from TALP (bicarbonate and BSA).
    • Supplementary Figure 3. The percentage of Yo-Pro-1- spermatozoa with high (A) and low (B) membrane fluidity following exposure to conditions with or without bicarbonate and cAMP up-regulators (db-cAMP, caffeine and theophylline) for up to 3h (180 min). Membrane fluidity and integrity were assessed with flow cytometry using merocyanine 540 (M540) and Yo-Pro, respectively. Data correspond to geometric mean ± 95% confidence interval (A) or mean ± S.D. (B) for ten independent samples. ***P<0.001 indicate significant differences from TALP (bicarbonate and BSA).
    • Supplementary Figure 4. The percentage of Yo-Pro-1- spermatozoa with high (A) and low (B) membrane fluidity following exposure to conditions with or without BSA and cAMP up-regulators (db-cAMP, caffeine and theophylline) for up to 3h (180 min). Membrane fluidity and integrity were assessed with flow cytometry using merocyanine 540 (M540) and Yo-Pro, respectively. Data correspond to geometric mean ± 95% confidence interval (A) or mean ± S.D. (B) for seven independent samples. *P<0.05 and ***P<0.001 indicate significant differences from TALP (bicarbonate and BSA).

 

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