Protein tyrosine phosphorylation of the human sperm head during capacitation: immunolocalization and relationship with acquisition of sperm-fertilizing ability

Abstract

The occurrence of tyrosine phosphorylation (TP) in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone (P)-stimulated acrosome reactions (ARs) and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreactivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP (N,2-O-dibutyryladenosine 3',5'-cyclic monophosphate). In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.

Figure 1

Immunoreactivity of monoclonal antibody pY20 by an immunofluorescence test on 5-h-capacitated human spermatozoa using (A) formaldehyde-fixed/unpermeabilized, (B) methanol-fixed and (C) formaldehyde-fixed/permeabilized spermatozoa. No fluorescent labelling was detected with unfixed spermatozoa (D). There was no fluorescent labelling when formaldehyde-fixed/unpermeabilized (E) and fixed/permeabilized (F) spermatozoa were exposed to pY20 mAb pre-incubated with a saturated solution of O-phospho-L-tyrosine. (G): Immunoreactivity of formaldehyde-fixed/unpermeabilized spermatozoa was unchanged after 5 h of incubation under capacitating conditions in the presence of the protein kinase A inhibitor H89 (50 μmmol L⁻¹). Phase contrast (left) and corresponding immunofluorescence photographs (right) (Bars = 5 μm).

Figure 2

Transmission electron micrographs of longitudinal sections of human sperm heads after incubation for 5 h under capacitating conditions. (A): Immunoelectron microscopic peroxidase labelling on fixed/unpermeabilized spermatozoa incubated with monoclonal antibody pY20. Tyrosine phosphorylation (TP) immunoreactivity is localized in the anterior region of the head (detail is showed in the upper panel), external to the acrosome. (B): A swollen acrosome in which phosphotyrosine immunoreactivity was retained along the swollen membranes. (C): No immunoperoxidase labelling was detected on capacitated spermatozoa when the MOPC-21 antibody was used as a control for non-specific binding.

Figure 3

Time course of tyrosine phosphorylation (TP) of head proteins in human spermatozoa (A), spontaneous and progesterone (P)-induced acrosome reactions (ARs) (B) and P-stimulated sperm–oocyte fusion during capacitation (C). Results are from five experiments with different donor semen. (A): Overall significance: P < 0.0001 by ANOVA; ^*P < 0.05, compared with 1 h and 5 h. (B): Overall significance: P < 0.0001 with ANOVA; ^*P < 0.05, compared with 1 h and 5 h. (C): Overall significance: P < 0.0001 with PROC GLM. For this experiment 215 oocytes were used; ^*P < 0.05, compared with 0 h and 1 h.

Figure 4

The effect of the presence of 50% (v/v) seminal plasma (SP) and SP with the cAMP analogue, dbcAMP (5 mmol L⁻¹), during sperm processing and during a 5-h capacitation on sperm head tyrosine phosphorylation (TP) (A), spontaneous and progesterone (P)-induced acrosome reactions (ARs) (B) and P-stimulated sperm–oocyte fusion (C). The results are from five experiments using different donor semen. (A): Overall significance: P < 0.0001 with ANOVA; ^*P < 0.05, compared with the control and ^**P < 0.05 compared with the control and SP + dbcAMP. (B): Overall significance: P < 0.0001 with ANOVA; ^*P < 0.05, compared with SP and SP + dbcAMP. (C): Overall significance: P < 0.0001 with PROC GLM. For this experiment, 220 oocytes were used; ^*P < 0.05, compared with SP and SP + dbcAMP.