This study demonstrates that sperm DNA fragmentation, as measured by TUNEL assay, is a highly valuable indicator of male fertility.

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), as well as the albumin fraction (d greater than 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60-70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2-3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.

No abstract

Cholesteryl sulfate is a normal constituent of human spermatozoa. The in vitro uptake of tritiated cholesteryl sulfate resulted in the labeling of all spermatozoa as demonstrated by light-microscope radioautography. The binding of the sterol sulfate was localized mainly in the head and midpiece. Radioautography at the level of the electron microscope revealed that the sterol sulfate is localized on the plasma membrane, mostly in the region of the acrosome. Further proof for this localization was obtained by selective dissolution of the plasma membrane and acrosome of the spermatozoa with low concentrations of Triton X-100. This treatment resulted in the simultaneous removal of tritiated cholesteryl sulfate bound to the spermatozoa. A hypothesis is presented concerning the role of cholesteryl sulfate as a membrane stabilizer and enzyme inhibitor during the maturation of spermatozoa in the epididymis. According to this hypothesis, the cleavage ofthe sulfate moiety within the female reproductive tract triggers a cascade of events leading to sperm capacitation and fertilization. One of the challenges remaining in reproductive biology is the elucidation ofthe biochemical events involved in the maturation and capacitation of mammalian spermatozoa. A review of the literature (1)(2)(3)(4)(5) indicates that the following biochemical events occur during capacitation. Stabilizing factor(s), associated with the spermatozoal membrane during transit or storage within the epididymis inhibit the release ofacrosomal enzymes in the male tract, but the factor(s) must be removed during migration in the female tract to allow the contact of the acrosomal enzymes with the investments of the ovum in order to facilitate penetration. Removal ofthe stabilizing substance(s) is thought to be enzymecatalyzed and involves changes in membrane conformation and permeability, ultimately leading to the acrosome reaction.Evidence is accumulating in support of our contention that sterol sulfates play an important role in the biochemistry of sperm maturation and capacitation. Thus, we have reported that cholesteryl sulfate (CholSO4) is an important component of human spermatozoa and is avidly taken up by these cells during in vitro incubation (6, 7). In addition, during transit through the epididymis, hamster spermatozoa exhibit a severalfold increase in desmosteryl sulfate concentration from the caput to the cauda regions (8). The finding of sterol sulfotransferase activity in the hamster epididymis (9) demonstrates that the biosynthesis of sterol sulfates can occur in this tissue.That low concentrations of sterol sulfates can block in vitro capacitation by hamster cumulus cells (10) and also inhibit acrosin (11), the sperm acrosomal proteinase involved in the penetration of the zona pellucida of the ovum (12), provides evidence that sterol-sulfatase (sterol-sulfate sulfohydrolase, EC 3.1.6.2) could be involved in the mechanism of sperm capacitation and ovum penetration. Furthermore, sterol-sulfatase is present in the human female reproduct...