Plasma membrane and acrosomal status in stallion sperm during epididymal transit

Morphological and biochemical changes during epididymal transit are required for development of the fertile spermatozoa. The plasma membrane of the maturing sperm cell is a target for a variety of post-testicular modifications. The remodeling of the molecular structure of the plasma membrane during sperm maturation in epididymis leads to significant consequences for intracellular events, such as the regulation membrane permeability and functionality. This study was design to assess the changes in stallion sperm plasma membrane and acrosome status during epididymal transit. Eighteen epididymides from nine adult (3-4 yr old) stallions were subdivided into 10 regions (E0–E1, proximal caput; E2, middle caput; E3–E4, distal caput; E5–E6, corpus; E7–E9, cauda). Epididymal sperm were obtained by microperfusion with sterile phosphate saline buffer (pH 7.2). Samples were diluted in modified TALP medium to a final concentration of 5 × 106 sperm/mL. Aliquots of 200µL of sperm suspension of each segment from each animal were mixed with 7µM Hoechst 33342 (Sigma), 1.5µM Propidium Iodide (Sigma), 1 ng FITC-PSA (Sigma) and then incubated at 37°C for 15 min in the dark. A total of 10000 gatedevents were analyzed per sample by flow cytometry (BD LSRFortessa). The green fluorescence (FL1) was collected through a 580-nm bandpass filter and the red fluorescence (FL3) through a 635-nm bandpass filter. The mean channel fluorescence was determined for both red and green. Statistical analyses were performed using One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test with a significance level of P ≤ 0.05. Data are presented as means ± standard error (SEM). The percentage of intact plasma membrane and acrosome (PMA) was significant higher in E0 (73.2 ± 2.3) and E9 (74.4 ± 4.1) compared to E5 (58.9 ± 1.8). Similarly, the segment E8 (78.4 ± 3.5) showed higher PMA integrity in comparison with E3 (64.0 ± 2.3), E4 (61.9 ± 2.3) and E5 (P ≤ 0.05). No difference was observed in PMA integrity among other epididymal regions (P ≥ 0.05). Considering the caput (E0-E4), corpus (E5-E6) and cauda (E7-E9) regions, epididymal cauda (74.6 ± 2.0) showed higher percentage of PMA integrity compared to caput (68.1 ± 1.3) and corpus epididymal sperm (62.8 ± 2.4) (P ≤ 0.05) . During epididymal maturation, an important structural change in spermatozoa is the increased adherence of the 'inner' acrosome membrane to the subjacent membranes (nucleus) and the reduction in dimensions of the acrosome. Probably, the lower percentages of sperm with intact PMA in caput and corpus epididymal sperm is due to changes in the permeability and acrosome structure in immature epididymal sperm, which makes it more fragile and susceptible to damage, since the acrosome is not firmly attached to the underlying structures. In conclusion, the population with intact PMA increases along epididymal maturation. The lower population of sperm with intact PMA in caput and corpus epididymal sperm may be associated with the immaturity of equine epididymal sperm, as permeability of plasma membrane and re-structure of acrosome.