Hypercholesterolemia impaired sperm functionality in rabbits

Abstract

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a "folded head"-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.

Figure 1. Rabbits fed with fat-rich diet increased plasma cholesterol.

Plasma cholesterol concentration from NCR (•) and HCR (□) during the 11 experimental months. Values are expressed as mean ± SEM. Arrowhead indicates fat intake start in HCR, and arrow indicates the moment from which HCR weight resulted significantly different from NCR, (p<0.001).

Figure 2. Ultrastructural changes.

Transmission-electron micrographs of rabbit sperm heads from NCR (A) and HCR (B to D). Notice the small vesicles in the acrosome region in B and the long side fold of sperm head in D. Some sperm cells show the remaining residual body (white empty arrow, C). A and C, X 12,000; B and D, X 20,000.

Figure 3. Sperm membrane cholesterol was elevated in HCR.

A: Fluorescence micrographs showing cholesterol content in plasma membrane of ejaculated rabbit spermatozoa detected by filipin probe. Images correspond to phase contrast (inset) and filipin-stained sperm cells (X 600) from NCR and HCR at the beginning of the experiment (first row, ED start) and four months later (second row, 4 months ED). B: Bars represent RFI means (± SEM) in sperm cells isolated from NCR and HCR as described in “Materials and Methods”, from both conditions, non capacitated (- BSA/NaCOH₃) and capacitated (+ BSA/NaCOH₃). Asterisks  =  significantly different from control (**, p<0.01, ***, p<0.001).

Figure 4. Saturated-fat consumption damaged sperm plasma membrane in rabbits.

Photographs (X 250) represent sperm cell morphology after hypo-osmotic stress. A (NCR): normal coiled tails and B (HCR): straight tails. C: Bars represent the percentage (means ± SEM) of spermatozoa swollen from NCR (black bar) and HCR (white bar) rabbits. **  =  significantly different from control (p<0.01).

Figure 5. Hypercholesterolemia altered sperm capacitation and acrosome reaction.

Protein tyrosine phosphorylation (A) and acrosomal exocytosis index (B) of spermatozoa from control (NCR) and HCR. Non capacitated: culture medium without (−) and capacitated with (+) BSA 4 (4 mg/ml), BSA 40 (40 mg/ml), IBMX (100 µmol/L)/db-cAMP (1 mmol/L). A: phospho-Y proteins showed different patterns ranging from one band (control-non capacitated, approximately 60 kDa) to many bands (capacitated with IBMX/db-cAMP, from over 20 to 100 kDa). Notice that the p-Y pattern differed between NCR and HCR: Non capacitated sperm presented one/two phosphorylated proteins (figure A, lane 1) but under capacitation conditions NCR showed wider molecular weight range of p-Y proteins compared with HCR (lanes 2 and 6). The experiment was performed at least three times and representative blot is shown. B: Bars represent AR index after 10 µM progesterone sperm incubation. AR index corresponds to normalized data (see Materials and Methods). ***  =  significantly different from control (p<0.001).