Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation.

Spermatozoa require the capacitation, a series of biochemical events, to perform fertilization. Many toxic compounds can interfere in this process, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which belong to the perfluoroalkyl substances (PFAS). Since both substances are found in many everyday materials and are highly persistent, they accumulate in organisms where they have been associated with fertility problems. This study analyzes the effects of PFOS and PFOA on the functionality of boar spermatozoa, and changes in the plasma membrane (PM) during capacitation. The median lethal concentrations (LC50) of PFOS and PFOA were 460 and 1894 µM, respectively, while the mean inhibitory concentrations of capacitation (ICC50) were 274 µM and 1458 µM, respectively. The ICC50 of PFOA was insufficient to reduce the capacitation, but 950 µM (½ LC50) of PFOA and the ICC50 of PFOS significantly reduced the number of capacitated spermatozoa. PFOS and PFOA also impeded the progesterone (P4)-induced acrosomal reaction (iAR). These effects occur despite the accumulation of [Ca2+]i under capacitating conditions. The accumulation of [Ca2+]i produces saturation, which prevents its entry through ionophore A23187 and P4 in the presence of PFOS. Membrane potential (Emv) was deregulated. Both PFAS affected lipid membrane conductance mediated by valinomycin. The spermatozoa presented 49% and 47% of membrane dysfunction with PFOS and PFOA, respectively. By causing membrane damage, both substances prevented the release of cholesterol and altered the organization of membrane microdomains (MMDs). Data indicate that both PFAS caused alterations in PM functionality.

Perfluorooctane Sulfonate (PFOS) and Perfluorohexane Sulfonate (PFHxS) Alters Protein Phosphorylation, Increase ROS Levels and DNA Fragmentation during In Vitro Capacitation of Boar Spermatozoa.

Perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) are toxic and bioaccumulative, included in the Stockholm Convention's list as persistent organic pollutants. Due to their toxicity, worldwide distribution, and lack of information in spermatozoa physiology during pre-fertilization processes, the present study seeks to analyze the toxic effects and possible alterations caused by the presence of these compounds in boar sperm during the in vitro capacitation. The spermatozoa capacitation was performed in supplemented TALP-Hepes media and mean lethal concentration values of 460.55 µM for PFOS, and 1930.60 µM for PFHxS were obtained. Results by chlortetracycline staining showed that intracellular Ca2+ patterns bound to membrane proteins were scarcely affected by PFOS. The spontaneous acrosome reaction determined by FITC-PNA was significantly reduced by PFOS and slightly increased by PFHxS. Both toxic compounds significantly alter the normal capacitation process from 30 min of exposure. An increase in ROS production was observed by flow cytometry and considerable DNA fragmentation by the comet assay. The immunocytochemistry showed a decrease of tyrosine phosphorylation in proteins of the equatorial and acrosomal zone of the spermatozoa head. In conclusion, PFOS and PFHxS have toxic effects on the sperm, causing mortality and altering vital parameters for proper sperm capacitation.

The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis.

Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten's). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.

Novel Techniques of Sperm Selection for Improving IVF and ICSI Outcomes.

Almost 50% of the infertility cases are due to male factors. Assisted reproductive technologies (ARTs) allow to overcome the incapacity of these patients' spermatozoa to fertilize the oocyte and produce a viable and healthy offspring, but the efficiency of the different techniques has still the potential to improve. According to the latest reports of the European Society of Human Reproduction and Embryology (ESHRE) and the Centers for Disease Control and Prevention of the United States (CDC), the percentages of deliveries per ART cycle in 2014 and 2016 were 21 and 22%, respectively. Among the reasons for this relatively low efficiency, the quality of the spermatozoa has been pointed out as critical, and the presence of high percentages of DNA-damaged spermatozoa in patients' ejaculates is possibly one of the main factors reducing the ARTs outcomes. Thus, one of the main challenges in reproductive medicine is to ensure the highest quality of the spermatozoa used in ARTs, and specifically, in terms of genetic integrity. The latest techniques for the preparation and selection of human spermatozoa are herein discussed focusing on those proven to improve one or several of the following parameters: sperm genetic integrity, fertilization capacity, embryo production, and in vitro survival, as well as pregnancy and delivery rates following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In addition, we discuss the potential of techniques developed in non-human mammals that could be further transferred to the clinic.