Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described

Sperm membrane integrity (SYBR-14/PI) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>LIVE % (SYBR-14+ sperm), DEAD% (PI+ sperm), DAMAGED, (SYBR-14+/PI+sperm). Within a row values with different superscript differ statistically a-b p<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Mitochondrial membrane potential (Δφm) and lipid peroxidation (LPO) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>(Means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion). High spermatozoa depicting high Δφm, High and Low spermatozoa depicting simultaneously mitochondria with low and high Δφm, Low spermatozoa with low Δφm, LPO spermatozoa showing peroxidation of the lipids of their membranes.</p

Membrane intactness and subtle changes in sperm membrane integrity of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>Intact spermartozoa are those not stained and thus represent spermatozoa with completely intact membranes. YoPro+ are early apoptotic sperm depicting an increase in sperm membrane permeability, YoPro+/Eth+ are late apoptotic and Eth+ are necrotic spermatozoa. Within a row values with different superscript differ statistically a-c p<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Sperm motility and kinematics after computer assisted sperm analysis (CASA) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>TM% total motile sperm, LM% linear motile sperm, RS% rapid sperm, VCL circular velocity, VSL straight line velocity, VAP average velocity, ALH lateral head displacement, BCF beat cross frequency. Within a row values with different superscripts differ statistically a-e, P<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Changes in LC3B processing in stallion spermatozoa stored under refrigeration (5°C) for five days, after single layer centrifugation (Filtrated) or unprocessed (native sperm).

<p>In native sperm storage induced a significant increase in LC₃B processing at day 5 indicating that autophagy was activated during the period of storage. On the other hand, filtration of sperm selected a subpopulation of spermatozoa in which autophagy was already activated at the beginning of the period of storage and did not change over the time. Results are representative of 28 identical experiments (seven stallions, four ejaculates per stallion) * p<0.01.</p