Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3

Abstract

Background: The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature.

Objectives: Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation.

Materials and methods: Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor).

Results: Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01).

Conclusion: We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology.

Fig 1. Effect of rosiglitazone added to the thawing media on stallion sperm motility.

Samples were processed as described in the Materials and Methods and supplemented in the thawing media with rosiglitazone (0, 50, 75 and 100 μM) and then incubated at 37°C for two hours; then the motility was evaluated using a computer assisted system (CASA). Rosiglitazone at 75 μM increased the percentage of total (A) linearly motile (B) spermatozoa (P<0.01) (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 2. Effects of rosiglitazone added to the thawing media on mitochondrial function of stallion spermatozoa after thawing.

Frozen stallion semen was thawed and processed as described in the Materials and Methods. Split samples were supplemented with rosiglitazone (0 and 75 μM) and mitochondrial functionality was investigated after 1 and 2 hours of incubation. A and C, percentage of spermatozoa showing orange fluorescence after JC-1 staining, B and D, mitochondrial functionality expressed as the mean fluorescence intensity in the PE channel indicative of JC-1 aggregates (high mitochondrial potential), E and F production of superoxide after 1 and 2 hours of incubation at 37°C. The results are presented as the means ± SEM. * P<0.05, ** P<0.01. In a and b, the t-SNE map after computational analysis is shown; in the t-SNE map, each point represents individual spermatozoa in the sample, and the heat map applied to the t-SNE map shows increased PE fluorescence (jc-1 aggregates) in the rosiglitazone treated samples. Circles identify the populations of spermatozoa showing high ΔΨm. In c a representative 2D plot after JC-1/H33342 is presented, A live spermatozoa with high ΔΨm, B live spermatozoa, C dead spermatozoa. In d the same plot is presented but a heat map overlay of the APC channel (production of superoxide) is shown over the 2D plot depicted in c, maximum production of superoxide is present in live sperm showing high ΔΨm (orange events in the plot), and this population is also circled (black arrow). Controls for the JC-1 are presented; G negative controls that are samples treated with the mitochondrial uncoupler CCCP 5 μM. Positive controls are presented in H; they are samples treated with oligomycin 10 μM to inhibit the passage of H⁺ to the mitochondrial matrix (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 3

Effect of rosiglitazone added to the thawing media on sORP (mV/10⁶ sperm) (A) that is the integrated measure of the existing balance between oxidants and reductants and (B) antioxidant capacity reserve cORP (μC) (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 4. Changes in caspase 3 and phosphatidylserine (PS) transposition after rosiglitazone supplementation of the thawing media.

Commercial frozen doses of stallion sperm were thawed and processed as described in the Material and Methods. Split samples were incubated in the presence of rosiglitazone 0 and 75 μM and caspase 3 activity was determined by flow cytometry. Data represent percent changes with respect the controls after 1 hour (A) and two hours (B) of incubation and are expressed as the means ± SEM, observed (* P<0.05, results are derived from three independent frozen ejaculates from 6 different stallions n = 18). F) Western blot (WB) controls for caspase 3 using frozen and thawed stallion spermatozoa as positive controls; semen was processed and analyzed as described in reference 50. (G) Further controls were obtained after incubating stallion spermatozoa at 37°C for 3 hours in the presence of three known inductors of apoptosis, staurosporine 10 μM, thapsigargin 50 μM and betulinic acid 50 μM. In C and D, representative cytograms of the simultaneous detection of active caspase 3 and PS transposition are presented where Q2 and Q3 represent events positive both for caspase 3 and Annexin-V. Q2 represents events with higher caspase 3 expression. No significant changes were detected. In E a heat map showing the intensity of Annexin-V staining demonstrates that PS is preferentially expressed in caspase 3 positive cells.

Fig 5. Computational cytometry analysis (t-SNE) graphics.

Heat maps are presented showing the effect of 75 μM rosiglitazone supplementation on caspase 3 activity in thawed stallion spermatozoa. A, Control samples, each point represents an individual spermatozoa, and as seen in the heat map, almost half of the population shows high caspase 3 expression (orange color). B, Samples supplemented with 75 μM rosiglitazone, and as seen in the heat map, caspase 3 expression is reduced with only a small population with high caspase 3 (red circle) (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 6. Effect of rosiglitazone on Akt phosphorylation (Ser ⁴⁷³) on stallion spermatozoa and on the percentage of live non apoptotic spermatozoa (caspase 3 negative).

Commercial frozen doses of stallion sperm were thawed and processed as described in the Materials and Methods. Split samples were incubated in the presence of rosiglitazone 0 and 75 μM and Akt phosphorylation was measured after 1 (A) and 2 hours (B) of incubation at 37°C. Data represent percent changes with respect to the controls and are expressed as the means ± SEM * P<0.05. In C, a representative cytogram showing the identification of live non apoptotic spermatozoa is shown. Live spermatozoa are identified by the red circle. In D the effect of the incubation of stallion spermatozoa in the presence of rosiglitazone 75 μM is presented. Data represent percent changes with respect to controls and are expressed as the means ± SEM * P<0.05 (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 7. Representative overlay cytograms of the p-Akt assay after 1 hour (A) and 2 hours of incubation (B).

To calculate the expression of the different germ cell markers we used the population comparison analysis available in FlowJo, version 10.4.1 (TreeStar, OR, USA). This analysis uses the Overton cumulative histogram subtraction algorithm (Overton, 1988) and overlaps histograms of the control (isotype control) and sample, allowing for subtraction of the control to calculate the percentage of positive cells in the sample (percentage of cells showing increased expression with respect to the controls) (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).

Fig 8. Effects of the Akt1/2 kinase inhibitor, dorsomorphin (AMPK inhibitor) and GW9662 (PPAR γ inhibitor) on caspase 3 inhibition after rosiglitazone treatment.

Thawed semen doses were processed as described in the Materials and Methods and were incubated in the presence of rosiglitazone (0 and 75 μM) or rosiglitazone 75 μM plus an Akt kinase inhibitor 30 μM, rosiglitazone 75 μM plus GW9662 10 μM or rosiglitazone 75 μM plus dorsomorphin 100 μM. After 1 and 2 hours of incubation caspase 3 activity was determined using flow cytometry. The results are presented as the means ± SEM. * P<0.05 A) changes after 1 hour of incubation, B) Changes after 2 hours of incubation (results are derived from three independent frozen ejaculates from 6 different stallions n = 18).