Cryopreservation increases the rate of apoptotic spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, especially in assisted reproduction the use of apoptotic sperms should be avoided. Early events of apoptosis in cryopreserved spermatozoa are not detectable by conventional methods. However, the surface of apoptotic spermatozoa is characterised by externalisation of phosphatidylserine (PS), which has a high affinity to Annexin V. Therefore, colloid paramagnetic Annexin-V-conjugated microbeads (AN-MB) were tested for their ability to eliminate apoptotic spermatozoa from a total of 40 fresh and in TEST yolk buffer cryopreserved semen samples which were provided by 15 healthy volunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) the sperm suspensions were divided into 2 sperm fractions depending on bound magnetic Annexin V-microbeads (AN-MB) to spermatozoa. As additional markers of apoptosis CD95 (Fas, APO-1) on the sperm surface and activated caspases in the cytosol were detected in both fractions. Supplementary investigations comprised eosin-supravital staining and computer assisted sperm motion analysis. The separation was supervised by flow cytometric analysis of spermatozoa labelled with FITC-conjugated anti Annexin V-antibodies. Analyses of the magnetic inactive sperm fraction (AN-MB-negative) showed CD95 on 0.6 +/- 0.3% (X +/- SEM) of spermatozoa and only 3.2 +/- 0.5% were stainable with eosin, whereas, 40.6 +/- 6.7% of the remaining cells in the column appeared to be CD95 positive and 99.8 +/- 0.1% stainable with eosin after cryopreservation. Indeed the overall amount of CD95 positive spermatozoa did not significantly increase after cryopreservation (2.5 +/- 0.5% vs. 4.3 +/- 1.2%; p > 0.05). Activated caspases were found in 21.8 +/- 2.6% of the spermatozoa in fresh and in 47.7 +/- 5.8% of cryopreserved semen samples (p < 0.01). The separation procedure of the cryopreserved spermatozoa reduced significantly the quantity of those containing activated caspases to 9.3 +/- 2.2% within the AN-MB-negative fraction. In contrast 89.1 +/- 2.3% of AN-MB-positive sperms showed activation of these proteolytic enzymes. Flow cytometric analyses using FITC-conjugated anti Annexin V-antibodies for monitoring of AN-MB-binding to spermatozoa showed 5.2 +/- 1.0% labelled spermatozoa in the AN-MB negative fraction and 72.6 +/- 2.7% labelled spermatozoa in the AN-MB positive one. There was no significant influence of the separation column and the magnetic field on the sperm functions. The passage through the column led to a sperm loss of 0.8 +/- 1.2%. Conclusion: The binding of paramagnetic Annexin V-conjugated microbeads is an excellent method to eliminate spermatozoa at early apoptotic stages from cryopreserved semen samples. A deleterious influence of the separation column and the magnetic field on the spermatozoa was not observed.
"Survival of the spermatozoa after the freezing– thawing process depends on the plasma membrane, as this is the most crucial region and regarded as the primary site of cryoinjury (Söderquist et al., 1997). The AnMACS preparation technique was believed to yield motile, viable, morphologically normal spermatozoa that displayed higher cryosurvival rates as well as higher fertilization potential (Grunewald et al., 2001; Said et al., 2005). Moreover, non-apoptotic sperm separated by AnMACS prior to cryopreservation had significantly higher motility following cryopreservation Faezah et al. "
[Show abstract][Hide abstract] ABSTRACT: Summary Cryopreservation is a technique used to preserve cells for long-time storage. It is widely used in agriculture to store male gametes in liquid nitrogen. The aim of this study was to determine the optimum thawing temperature and time for samples subjected to annexin V magnetic-activated cell sorting (AnMACS) as the sperm preparation technique. Pooled semen samples from three ejaculates were divided into two groups. The treatment group was subjected both to AnMACS and to being cryopreserved, whilst the control group was cryopreserved directly without MACS. Post-thaw analysis was carried out for samples thawed at either 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s or 80°C for 5 s. Sperm kinematics, viability and capacitation status were determined for samples subjected to all thawing temperatures described. Results showed that thawing at 37°C for 13 s for MACS-processed samples was a superior option compared with other thawing procedures; there was a significant difference in P < 0.05 values for curvilinear velocity (VCL μm/s) and sperm straightness (STR %) when samples were thawed at 40°C for 7 s, with fewer capacitated spermatozoa (P < 0.05) when samples were thawed at 37°C for 30 s, 40°C for 7 s or 60°C for 6 s. Hence, we can speculate that the use of AnMACS as the sperm preparation technique can somehow enhance sperm cryosurvival rate after cryopreservation, however the fertilization potential of these cells has yet to be determined.
"Apoptosis signaling in human sperm has been a controversially debated issue for a long time, because sperm are mainly transcriptionally inactive cells (Grunewald et al., 2005) and the knowledge gained from studies of somatic cells cannot be transferred without experimental evidence. Although initial studies denied the presence of apoptosis in ejaculated human sperm at all (Weil et al., 1998), and further studies suggested an abortive apoptosis as a remnant of incomplete spermatogenesis (Sakkas et al., 1999a), recent publications have suggested that human spermatozoa have the ability to undergo apoptosis or apoptosis-like conditions in response to a variety of stimuli (Grunewald et al., 2001; Paasch et al., 2003, Taylor et al., 2004; Eley et al., 2005; Barroso et al., 2006; Martin et al., 2007; Bejarano et al., 2008; Espino et al., 2011). Apoptosis signaling in human sperm is preferentially based on the mitochondria-associated pathway, the main features being activation of the initiator caspase, caspase-9, disruption of the transmembrane mitochondrial potential, activation of the major effector caspase, caspase-3, and consecutive cellular degradation (Paasch et al., 2004; Bejarano et al., 2008). "
"Recent developments in sperm biology have led to the development of novel selection strategies for use in ART. For instance, sperm with phosphatidylserine in the outer leaflet of the plasma membrane (an early apoptotic event in other cell types) can be eliminated by using either annexin-V-conjugated microbeads and Magnetic Cell Sorting (MACS)  or annexin-V labeling and Fluorescence Activated Cell Sorting (FACS) . Annexin-negative subpopulations, enriched in viable sperm, also seem to have more sperm with normal morphology , , as well as more motile cells , although this last result has been questioned , . "
[Show abstract][Hide abstract] ABSTRACT: Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.
PLoS ONE 03/2011; 6(3):e18112. DOI:10.1371/journal.pone.0018112 · 3.23 Impact Factor
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