Effects of human follicular fluid and high-density lipoproteins on early spermatozoa hyperactivation and cholesterol efflux

Abstract

The preovulatory human follicular fluid contains only HDLs as a lipoprotein class with a typically high proportion of prebeta HDL. We first examined the role of follicular fluid and HDL subfractions on human spermatozoa capacitation, a process characterized by a hyperactivation of the flagellar movement and a depletion of plasma membrane cholesterol. Whole follicular fluid and isolated HDL, used at constant free cholesterol concentration, were both able to promote an early flagellar hyperactivation. Moreover, incubation of [(3)H]cholesterol-labeled spermatozoa with follicular fluid induced a rapid cholesterol efflux from spermatozoa that was confirmed by mass measurements of cholesterol transfer. Using isolated HDL, the cholesterol efflux had a similar time course and represented 70% of that mediated by whole follicular fluid. We then analyzed the time course of radioactive labeling of HDL subfractions. In the first minute of incubation, we found that the prebeta HDL fraction incorporated the main part of the radioactivity (60%), with the rest being found in alpha-HDL, but strikingly, the labeling of alpha-HDL increased with time at the expense of prebeta HDL.Thus, our results indicate that HDLs are involved in both spermatozoa hyperactivation and cholesterol effl ux and suggest the role of prebeta-HDL particles as fi rst cellular cholesterol acceptors.

Fig. 1.

A: Effect of follicular fluid concentration on spermatozoa hyperactivation. Human spermatozoa (1.5 × 10⁷cells/ml) were incubated for 5 min with the indicated dilution (v/v) of FF in B2 medium. Results are expressed as mean ± SE of three experiments. B: Kinetics of spermatozoa hyperactivation. Spermatozoa (1.5 × 10⁷cells/ml) were incubated with or without a 20% dilution of FF from 0–300 min at 37°C. Results represent a typical experiment of two.

Fig. 2.

Comparison of FF and isolated HDL effects on spermatozoa hyperactivation. Hyperactivation measurements were performed after incubation, for 0 to 30 min, of 1.5 × 10⁷ sperm cells with diluted FF and isolated HDL, both used at a constant free cholesterol concentration of 13.6 µM. Results are expressed as mean ± SE of four independent experiments, each performed in duplicate.

Fig. 3.

A: Dose-response curve for the release of labeled cholesterol from sperm in function of FF cholesterol content. Sperm cells (1.2 × 10⁷ cells/ml) were incubated 30 min with the indicated FF concentrations. Experimental values (mean of three experiments ± SE) are expressed as the fractional efflux of [³H]cholesterol from cells versus total cholesterol concentration of FF pool (n = 6). Inset, Lineweaver-Burk plot of dose-response data. Labeling procedure of spermatozoa is detailed in Methods. B: Fractional cholesterol efflux from spermatozoa to isolated HDL compared with whole FF. Experimental values are expressed as the fractional efflux of [³H]cholesterol from spermatozoa (1.2 × 10⁷ cells/ml) versus incubation time with isolated HDL and whole FF, both used at constant free cholesterol concentration (13.6 µM). Results are the mean of two independent experiments, each performed in duplicate. The variation between the two experiments is lower than 10%.

Fig. 4.

Evolution of free cholesterol/apoA-I ratio of HDL subfractions during the incubation of labeled spermatozoa with FF (n = 4). The ratio was calculated using the data from Table 1 and according to FF apoA-I content, apoA-I repartition among HDL subfractions, and specific radioactivity of [³H]cholesterol.