Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunogenetic cell sorting
University of Leipzig, Department of Dermatology/Andrology Unit, Liebigstrasse 21, D-04103, Leipzig, Germany. Cell and Tissue Banking
(Impact Factor: 1.25).
09/2001; 2(3):127-33. DOI: 10.1023/A:1020188913551
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.
Available from: actavet.vfu.cz
- "Therefore, externalization of PS could be used to remove apoptotic-like spermatozoa from sperm preparations to enhance sperm quality in assisted reproduction technologies. Phospholipid phosphatidylserine has a high and selective affinity for the phospholipid-binding protein annexin V (van Heerde and de Groot 1995), therefore magnetic-activated cell sorting (MACS) using annexin V-conjugated superparamagnetic microbeads was established to separate non-apoptotic human spermatozoa from apoptotic ones (Grunewald et al. 2001). "
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ABSTRACT: One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16-17 degrees C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P < 0.05) in bound (14.1 +/- 10.6% and 24.1 +/- 10.2%, respectively) than in unbound fractions (3.4 +/- 2.1% and 12.7 +/- 3.1%) and control (3.5 +/- 1.6% and 12.0 +/- 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 +/- 8.0 %), which differed significantly (P < 0.05) from the control. In unbound fractions there was a significantly higher concentration (P < 0.05) of morphologically normal spermatozoa (31.8 +/- 12.6%) compared to bound ones (5.9 +/- 7.3%). A significantly (P < 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 +/- 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions.
Available from: Siti Fatimah Ibrahim
- "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. "
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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.
Available from: Javier Espino
- "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). "
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