During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HAbound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% 6 2.0% and 45.0% 6 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.
We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.
In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles.
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