F4-Neuroprostanes: A Role in Sperm Capacitation

F4-neuroprostanes (F4-NeuroPs), derived from the oxidative metabolization of docosahexaenoic acid (DHA), are considered biomarkers of oxidative stress in neurodegenerative diseases. Neurons and spermatozoa display a high DHA content. NeuroPs might possess biological activities. The aim of this in vitro study was to investigate the biological effects of chemically synthetized 4-F4t-NeuroP and 10-F4t-NeuroP in human sperm. Total progressive sperm motility (p < 0.05) and linearity (p = 0.016), evaluated by a computer-assisted sperm analyzer, were significantly increased in samples incubated with 7 ng F4-NeuroPs compared to non-supplemented controls. Sperm capacitation was tested in rabbit and swim-up-selected human sperm by chlortetracycline fluorescence assay. A higher percentage of capacitated sperm (p < 0.01) was observed in samples incubated in F4-NeuroPs than in the controls. However, the percentage of capacitated sperm was not different in F4-NeuroPs and calcium ionophore treatments at 2 h incubation. The phosphorylated form of AMPKα was detected by immunofluorescence analysis; after 2 h F4-NeuroP incubation, a dotted signal appeared in the entire sperm tail, and in controls, sperm were labeled in the mid-piece. A defined level of seminal F4-NeuroPs (7 ng) showed a biological activity in sperm function; its addition in sperm suspensions stimulated capacitation, increasing the number of sperm able to fertilize.

224 SILDENAFIL ACCELERATES SPERM PENETRATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

Sperm capacitation, a cyclic-adenosine monophosphate-dependent phenomenon, is an important initiation step for penetration into oocytes. In porcine IVF, the use of caffeine, as a nonspecific phosphodiesterase (PDE) inhibitor, is common to accelerate sperm capacitation and penetration. The objective of this study was to examine the effects of PDE inhibitors (cilostamide, rolipram, and sildenafil as PDE type 3-, type 4-, and type 5-specific inhibitors, respectively) on the capacitation of boar sperm and the penetration into porcine oocytes in the absence of caffeine and other capacitation inducers in a chemically defined medium. After washing sperm samples collected from an ejaculated sperm-rich fraction of different individual Berkshires, the sperm were resuspended in capacitation inducer-free (theophylline- and adenosine-free) PGM-tac4 (mPGM-tac) at 5 × 105 cells mL–1. The suspension was cultured in mPGM-tac nonsupplemented or supplemented with 2.5 mM cilostamide, rolipram, or sildenafil for 90 min at 39°C in an atmosphere of 5% CO2 in air, and then the capacitation status was assessed by chlortetracycline fluorescence assay. Other sperm suspensions were used to co-culture with denuded in vitro-matured oocytes in the same medium for 8 h in an atmosphere of 5% CO2 in air and fixed, and sperm penetration was then examined. Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In our result from the chlortetracycline fluorescence assay, although the incidence of intact sperm was significantly reduced in the presence of rolipram (54.3%) and sildenafil (52.7%) as compared with controls (66.7%), there were no differences in capacitated sperm among experimental groups (24.3 to 34.3%). The incidence of acrosome-reacted sperm was higher in the presence of cilostamide (17.3%) than in the others (9.0 to 13.0%). High sperm penetration was observed only in the presence of sildenafil (76.6%) as compared with the control (0%) or the presence of rolipram (4.4%) or cilostamide (1.8%). These results demonstrate that inhibition of PDE type 5, but not PDE type 3 and type 4, significantly accelerates the penetration of boar sperm into the oocytes in a capacitation inducer-free chemically defined medium, whereas inhibition of PDE type 3 may induce an acrosome reaction.