There is a fundamental body of evidence suggesting that activated apoptosis signaling in ejaculated human sperm negatively influences their fertilization potential. However, it is still controversial whether this apoptotic signaling is a relic of an abortive apoptosis related to spermatogenesis or if it should be regarded as a functional preformed pathway in mature sperm leading to stereotypical morphological changes reflecting nuclear disassembly. To address this question, apoptosis was induced using betulinic acid in mature and immature ejaculated human sperm enriched by density gradient centrifugation. Execution of apoptosis was monitored by observing ultra-morphological changes via transmission electron microscopy. Typical morphological signs of apoptosis in somatic cells include plasma membrane blebbing with the formation of apoptotic bodies, impaired mitochondrial integrity, defects of the nuclear envelope, and nuclear fragmentation; these morphologies have also been observed in human sperm. In addition, these apoptotic characteristics were more frequent in immature sperm compared to mature sperm. Following betulinic acid treatment, apoptosis-related morphological changes were induced in mature sperm from healthy donors. This effect was much less pronounced in immature sperm. Moreover, in both fractions, the betulinic acid treatment increased the percentage of acrosome-reacted sperm. The results of our ultra-morphological study prove the functional competence of apoptosis in mature ejaculated human sperm. The theory of a sole abortive process may be valid only for immature sperm. The induction of the acrosome reaction by stimulating apoptosis might shed light on the biological relevance of sperm apoptosis.

AIMTo estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding.

METHODSThe mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB).

RESULTSThe cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions.

CONCLUSIONThe cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Apoptosis ; Blotting, Western ; Caspases ; metabolism ; Cold Temperature ; adverse effects ; Cryopreservation ; Humans ; Immunomagnetic Separation ; Intracellular Membranes ; enzymology ; physiology ; Male ; Microscopy, Fluorescence ; Spermatozoa ; enzymology ; physiology

AIMTo evaluate the long-term stability of the fluorescence signals of new fluorescence-based semen analysis assays for clinical application.

METHODSSemen samples from 87 unselected infertile patients were used to perform the following assays: (i) detection of active caspase-3 (n=17); (ii) integrity of the mitochondrial membrane potential (MMP) (n=17); (iii) externalization of phosphatidylserine (EPS) (n=16); and (iv) detection of intact acrosomes via CD46 (n=37). After the assays, 4% paraformaldehyde was added to all aliquots. The fluorescence intensity of each sample was evaluated by flow cytometry on days 0, 3, 7, 10 and 14.

RESULTSDifferences of up to +/-5% positive spermatozoa from the value measured at day 0 were estimated as acceptable deviation. The Caspase-3 FLICA showed mean differences<5% at day 3, 7 and 10. At day 14 the mean difference was 7.6%. In contrast, the disrupted MMP and the EPS detection showed differences>5% at day 3. The CD46-FITC labeling displayed absolute differences<5% CD46-positive spermatozoa at days 3, 7, 10 and 14.

CONCLUSIONAlthough immediate analysis of the fluorescence signals is recommended, it is possible to evaluate caspase-3 activation up to 10 days and CD46 up to 14 days after staining of sperm. The FACS evaluation of MMP and EPS detection should be conducted on the same day.

Fluorescent Dyes ; Humans ; Male ; Spermatozoa ; physiology