Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes

E.C.C Celeghini,R.P Arruda,C.F Raphael,A.F.C Andrade,J Nascimento

doi:10.1590/s0102-09352010000300006

Abstract

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80% and abnormal morphology <10%, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

Highlights

IntroductionThe maintenance of the sperm fertilizing potential depend on the integrity and functionality of the different cellular structures, which in the practice, reflects in the difficulty to develop an isolate laboratorial test to predict with accuracy the semen fertility potential (Graham et al, 1990; Andrade et al, 2007; Celeghini et al, 2007)
The most applied is that use glycoprotein markers, as lectins (Graham et al, 1990), among them, the Pisum sativum agglutinin (PSA) has been widely used (Cross and Meizel, 1989), which is fluorescein-conjugate, as fluorescein isothiocyanate (FITC) (Holden et al, 1990)
Linear analysis regression of different treatments (T0, T50, and T100) in the semen allowed to trace a line of regression to sperm characteristics evaluated by each probe (Figure 2)

Summary

Introduction

The maintenance of the sperm fertilizing potential depend on the integrity and functionality of the different cellular structures, which in the practice, reflects in the difficulty to develop an isolate laboratorial test to predict with accuracy the semen fertility potential (Graham et al, 1990; Andrade et al, 2007; Celeghini et al, 2007). The PSA is an agglutinin from edible pea and binds to glycoconjugates of acrosomal matrix (Cross and Meizel, 1989); it has affinity for terminal D-glucosyl and -D-mannosyl residues of glycoproteins, and binds to the sugar -mannoside found in the acrosomal contents (Cross et al, 1986) This agglutinin, when bound to FITC, marks damaged sperm acrosome in yellow-green (Cross et al, 1986; Graham et al, 1990; Celeghini et al, 2007)

Methods

Results

Conclusion