Mitochondrial transmembrane potential and plasma membrane translocation of phosphatidylserine (PS) as early apoptosis markers: analysis of fractionated human spermatozoa.

Abstract

Objective: To detect and quantify early stages of apoptosis at the plasma and mitochondrial membrane levels in separated fractions of high and low sperm motility.Design: Prospective, ongoing study.Materials/Methods: Semen samples from donor subjects (n = 10) were subjected to processing by gradient centrifugation (Percoll™). The high (90% layer, HMF) and low motility (40% layer. LMF) sperm fractions were evaluated. Sperm concentration, motility (%) and (%) morphology (strict criteria) were analyzed. Changes in mitochondrial transmembrane potential, which typically occur during early apoptosis, were detected using the Apoalert™ mitochondrial membrane sensor kit (MM). Plasma membrane PS translocation was monitored using annexin V (AnV) binding with the simultaneous application of propidium iodide to identify live cells/necrosis. Apoptosis was detected and quantified (as % spermatozoa) in single sperm cells using indirect immunofluorescence.Results: The early apoptosis was significantly correlated in LMF when AnV and MM were analyzed (r = 0.68), (p = 0.05).Early apoptosis outcome in fractionated human spermatozoa. Tabled 1MotilityNecrosisLive, AnVMMHMF (90%)88.2 ± 3.110.1 ± 5.48.0 ± 5.210.5 ± 5.0LMF (45%)6.2 ± 2.127.5 ± 9.512.1 ± 7.129.0 ± 9.7P-value0.0050.00060.020.0002 Open table in a new tab Conclusions: These preliminary data show that early apoptosis changes of plasma and mitochondrial membranes are present in ejaculated human spermatozoa. The highly motile sperm fractions have significantly lower number of spermatozoa with early signs of apoptosis as well as necrosis when compared to the low motility sperm fractions. We postulate that sperm from infertile men may have increased levels of such apoptotic markers. The identification and separation of non-apoptotic cells could be of significance when preparing spermatozoa to be used in assisted reproductive technologies.Supported by: Serono de Mexico. Objective: To detect and quantify early stages of apoptosis at the plasma and mitochondrial membrane levels in separated fractions of high and low sperm motility. Design: Prospective, ongoing study. Materials/Methods: Semen samples from donor subjects (n = 10) were subjected to processing by gradient centrifugation (Percoll™). The high (90% layer, HMF) and low motility (40% layer. LMF) sperm fractions were evaluated. Sperm concentration, motility (%) and (%) morphology (strict criteria) were analyzed. Changes in mitochondrial transmembrane potential, which typically occur during early apoptosis, were detected using the Apoalert™ mitochondrial membrane sensor kit (MM). Plasma membrane PS translocation was monitored using annexin V (AnV) binding with the simultaneous application of propidium iodide to identify live cells/necrosis. Apoptosis was detected and quantified (as % spermatozoa) in single sperm cells using indirect immunofluorescence. Results: The early apoptosis was significantly correlated in LMF when AnV and MM were analyzed (r = 0.68), (p = 0.05). Early apoptosis outcome in fractionated human spermatozoa. Tabled 1MotilityNecrosisLive, AnVMMHMF (90%)88.2 ± 3.110.1 ± 5.48.0 ± 5.210.5 ± 5.0LMF (45%)6.2 ± 2.127.5 ± 9.512.1 ± 7.129.0 ± 9.7P-value0.0050.00060.020.0002 Open table in a new tab Conclusions: These preliminary data show that early apoptosis changes of plasma and mitochondrial membranes are present in ejaculated human spermatozoa. The highly motile sperm fractions have significantly lower number of spermatozoa with early signs of apoptosis as well as necrosis when compared to the low motility sperm fractions. We postulate that sperm from infertile men may have increased levels of such apoptotic markers. The identification and separation of non-apoptotic cells could be of significance when preparing spermatozoa to be used in assisted reproductive technologies. Supported by: Serono de Mexico.