A new combined in-vitro test model for the identification of substances affecting essential sperm functions [published erratum appears in Hum Reprod 1997 Nov;12(11):2580]

Abstract

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.

Reprotoxicity of intrauterine insemination and in vitro fertilization-embryo transfer disposables and products: a 4-year survey

“…This survey confirms that the SpST is an easy and inexpensive method that can detect possible reprotoxicity of materials used in ART laboratories (22,27,30,31,35). Human sperm diagnosis and preparation techniques are standardized in house procedures for each ART laboratory, therefore no additional technique has to be mastered by the laboratory technicians to perform the SpST.…”

“…Table 1 summarizes the range of in vitro and in vivo bioassays currently available for reprotoxicity testing of ART products. These reprotoxicity bioassays test the possible negative influence on the reproductive mechanisms and processes like maturation, motility, and fertilization of mammalian spermatozoa and oocytes of mice, hamsters, rabbits, and bovine (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Exposed mouse embryos are being cultured in vitro or transplanted in vivo to test their developmental potential (14,18).…”

Localisation and function of voltage-dependent anion channels (VDAC) in bovine spermatozoa

“…The numbers of live and dead spermatozoa and their acrosomal status after incubation with the anti-VDAC antibodies were evaluated using the double-staining Hoechst 33258 and Pisum sativum agglutinin (PSA)-fluorescein isothiocyanate (FITC) as previously described [18].…”

Human Sperm Bioassay for Reprotoxicity Testing in Embryo Culture Media: Some Practical Considerations in Reducing the Assay Time

“…Alvarez proposed 4 hours for the sperm stress test, while in regular sperm bioassays, assay times of 24 to 96 hours have been used [15–20]. The American Association of Bioanalysts (AAB), the largest PT provider for fertility laboratories in USA, chose an assay time of 48 hours for identifying the quality of the embryo culture media [3, 20–22]. It is possible that if an assay is extended beyond the required time, its sensitivity and specificity can be compromised [10, 12, 17, 23–25].…”