During sperm capacitation, intracellular signaling leads to protein tyrosine phosphorylation (PTP) of multiple cellular structures. However, the connection of this molecular signaling to the physiology of capacitated spermatozoa is not completely understood. This is the case of the short lifespan of capacitated spermatozoa and their increased susceptibility to initiate acrosomal exocytosis (AE) during incubation. Herein, by employing frozen/thawed bull spermatozoa, we aimed to study the relationship between PTP with AE and with plasma membrane integrity (PMI) at the cellular level. For this, we employed double staining following immunofluorescence for PTP combined with fluorescence probes for the acrosome (PNA-FITC) and PMI (LIVE/DEAD Fixable Dead Cell Stain Kit). Our results revealed that the presence of PTP at sperm head was less abundant in the sperm fraction that triggered the AE after 3 h of incubation under capacitating conditions, or by its induction with calcium ionophore, compared to the unreacted fraction. Furthermore, PTP at the equatorial region of the head (PTP-EQ) was enriched in the fraction showing damaged membrane while induction of AE with calcium ionophore did not alter the PMI and its relation to PTP-EQ. These results suggest that spontaneous AE and induced AE trigger similar cellular events regarding PTP and the spermatozoa showing PTP-EQ are more prone to suffer plasma membrane damage.

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.

In vitro fertilization (IVF) in rabbits is an important biotechnology, but its success remains low due to inefficient sperm capacitation. This study examines relationships between motility parameters (MP), protein tyrosine phosphorylation (PTP) and acrosomal exocytosis (AE) between sperm recovered after direct swim-up (DSU), and after 16 h of in vitro incubation under capacitating conditions, and heterologous IVF capacity. Variables were assessed in fresh ejaculates (ES), and after 2 h of DSU, and 16 h (T16) of incubation. PTP were grouped into four patterns in the different sperm structures according to their time-dependent appearance and AE. Throughout incubation, MP remained stable whereas AE increased after DSU and 16 h of incubation. Mid-piece PTP patterns increased after DSU and persisted until 16 h in reacted (R) and non-reacted spermatozoa (NR). In R and NR, annulus-ring PTP levels were also significantly higher in T16 versus DSU or ES. Flagellum PTP levels rose significantly during incubation such that highest levels were recorded in T16. In R sperm, levels of PTP patterns associated with the equatorial zone in T16 were significantly higher compared to NR and to DSU and ES. Cleavage rates were significantly higher at T16 than DSU and these were similar to rates recorded for homologous IVF used as positive control. Our capacitation protocol and PTP patterns at 16 h rendered the sperm competent to fertilize an oocyte in vitro. This time point matches the physiological timing of fertilization in vivo. We also describe a new annulus ring PTP pattern for this species.