Progress 06/01/02 to 06/30/11
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This proposal was submitted to a different sponsor and progress reports were submitted to the appropriate agency.
Publications
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: N/A PARTICIPANTS: In this third progress report for our renewed R01, the principal investigator will report experimental progress on the above-named project. Dr. Victoria Burruel continues as Co-PI in her appointment as Staff Research Assistant (SRA 2). Dr. Burruel presently has a 100% effort on this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have demonstrated that oxidative stress and its relationship with osmotic stress play strong roles during the cryopreservation process for rhesus sperm. These effects may indicate mechanisms by which these sperm adapt to, or fail to, adapt to environmental stresses leading to sperm cell death. We have also shown that the heat shock proteins, Hsp 70 and 90, may play a role in adaptation to osmotic and oxidative stress.
Publications
- Cole J, Meyers SA. 2009. The Role of Spermatozoal Heat Shock Proteins 70 and 90 in Response to Osmotic Stress and Cryopreservation in the Rhesus Macaque. Submitted, Biology of Reproduction
- Megan McCarthy, Julie Baumber, Philip H Kass, and Stuart Meyers. Osmotic Stress Induces Oxidative Cell Damage to Rhesus Macaque Spermatozoa. Biology of Reproduction, 2010. published ahead of print October 21, 2009,
- Burnaugh L, Ball BA, Sabeur K, Thomas AD, Meyers SA. Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses. Anim Reprod Sci. 2010 Feb;117(3-4):249-60.
- Meyers S, Wen-Li M, Enders AC, Overstreet JW. Rhesus Macaque Blastocysts Resulting from Intracytoplasmic Sperm Injection of Vacuum-Dried Spermatozoa. Journal of Medical Primatology 38(5): 310-317, 2009.
- Correa LM, Thomas A, Meyers SA. Reorganization of the Macaque Sperm Actin Cytoskeleton in Response to Osmotic Stress Contributes to Changes in Motility and Morphology. Biology of Reproduction 77: 942-953, 2007.
- Hung P, Baumber J, Meyers S, VandeVoort CA. Effects of Environmental Tobacco Smoke In Vitro on Rhesus Monkey Sperm Function. Reprod Toxicol. Jun;23(4):499-506, 2007.
- Li MW, Meyers S, Tollner TL, Overstreet JW. Damage to Chromosomes and DNA of Rhesus Monkey Sperm Following Cryopreservation. Journal of Andrology 28: 493-501, 2007.
- Nascimento JM, Shi LZ, Meyers S, Gagneux P, Loskutoff N, Botvinick EL, Berns MW. The use of optical tweezers to study sperm competition and motility in primates. Journal of the Royal Society, published online, 2007.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Hypothesis 1, Experiment set 2: Sperm express quaporin water channels. Objectives: (1a): As a first step in defining their roles in modulating the effects of osmotic stress, we will identify and localize aquaporins in macaque sperm. We have identified aquaporin 8 in sperm protein extracts, by immunoblotting. We have also determined that AQP 1, 7, 8 and 9 are present in sperm using Flow Cytometry with specific antibodies. Using ICC, we have localized all four aquaporins to various sperm domains and detected changes in AQP7 and 8 distribution after osmotic stress Osmotic stress activates signaling pathways involving Rho GTPases and downstream effectors important for regulation of cytoskeleton reorganization. Objectives: to determine if Rho A or other Rho family GTPases and associated signaling cascades are present in macaque sperm, and if they are activated or inactivated during osmotic stress. In preliminary experiments, total Rho A protein in sperm was determined by Flow
Cytometry following osmotic stress incubations. Oxidative Stress: Dihydroethidium (DHE) is oxidized by O2-. to a fluorescent product, previously assumed to be ethidium. Preliminary experiments have demonstrated that oxidation of DHE was prevented by the addition of superoxide dismutase (SOD) and not catalase (CAT), confirming that O2-. and not H2O2, is responsible for the formation of this product, similar to that of human sperm. Macaque Spermatozoa Generate Reactive Oxygen Species: Motile macaque spermatozoa were samples were incubated at room temperature 0.5 mM NADPH for 3hrs. A sample of capacitated spermatozoa was obtained. Fluorescence of oxidized DHE (2 mM) was determined by flow cytometry. Capacitated spermatozoa and those incubated in the presence of NADPH exhibited a dramatic shift in DHE fluorescence when compared to room temperature control. The generation of O2-. by macaque spermatozoa incubated with NADPH suggests the presence of a NADPH oxidase as described in human
spermatozo). Establishment of DHE Flow Cytometry Assay with Rhesus Macaque Spermatozoa: Motile macaque sperm were Percoll washed, resuspended at 25 x 106/mL in BWW supplemented with PVA (0.1%) and incubated at 38 C for 1 hour with or without the xanthine (X; 0.1 mM) - xanthine oxidase (X-XO; 0.01 U/mL) system. At the end of the 1 hour incubation, the samples were diluted to 1 x 106/mL in BWW (0.1% PVA) and analyzed by flow cytometry. DHE fluorescence was expected to be comparable between the control, X only, and SOD only samples. In comparison to the control samples, an increase (shift to the right on x-axis) in DHE fluorescent intensity was anticipated with the addition of the X-XO system. Alternatively, in the presence of the X-XO generating system, the addition of SOD should decrease DHE fluorescent intensity (a percentage of the population that shifted to the right should shift back to the left). The results presented exhibit the anticipated shifts in DHE fluorescence, the next
step is to test the affect osmotic stress and cryopreservation have on the production of the superoxide anion (02-).
PROJECT MODIFICATIONS: Dr. Liane Correa, originally listed as a co-PI on this project was dropped from the project to pursue other research directions. Dr. Victoria Burruel has taken on Dr. Correa's responsibilities in her appointment as Staff Research Assistant (SRA 2). Dr. Burruel presently has a 100% effort on this project. At this time, I have also removed Drs. Frederick Kleinhans and Mary Hagadorn from the "Key Personnel" list as they were inappropriately listed. More correctly, these two consultants will be listed as "Additional Personnel" in their capacity of "Consultant" for this project.
Impacts
This project has been funded by NIH for an additional 5 years, through June 30, 2011. The major goal of this project is to investigate the controlling mechanisms of cell cryodamage and to determine potential sources of individual male variation in cryosurvival and fertility.
Publications
- Correa LM, Thomas A, Meyers SA. 2007. Reorganization of the Macaque Sperm Actin Cytoskeleton in Response to Osmotic Stress Contributes to Changes in Motility and Morphology. Biology of Reproduction 77: 942-953.
- Hung P, Baumber J, Meyers S, VandeVoort CA. 2007. Effects of Environmental Tobacco Smoke In Vitro on Rhesus Monkey Sperm Function. Reprod Toxicol. Jun;23(4):499-506.
- Nascimento JM, Shi LZ, Meyers S, Gagneux P, Loskutoff N, Botvinick EL, Berns MW. 2007. The use of optical tweezers to study sperm competition and motility in primates. Journal of the Royal Society, published online, doi:10.1098/rsif.2007.1118.
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Progress 01/01/06 to 12/31/06
Outputs
Our studies have demonstrated that rhesus sperm have unique biophysical characteristics including lipid composition and membrane phase transitions. In addition, ice formation does not appear to be a significant factor in reduced post-thaw sperm function, primarily because we have not seen evidence that cell integrity is damaged or that the cells are disrupted as part of the cryopreservation process. Freezing macaque sperm at vastly different cooling rates in the subzero range has not resulted in significant loss of sperm cell function. Thus, the loss of function that we have observed at each cooling rate is likely due to factors other than IIF. We have also demonstrated that standard cryopreservatives have led to only mild broadening of osmotic tolerance limits in these cells. Differential scanning calorimetry demonstrated a method to model various freezing rate curves for rhesus spermatozoa. Interestingly, the optimal predicted freezing rate was 10oC/min and this
rate is commonly used by researchers in this field. However, these data suggest that optimal cooling rate, choice of CPA, and mathematical modeling of a predictable cooling curve may not allow us to optimally predict or modify spermatozoal treatments to adequately survive freezing.
Impacts
We have made significant advances in understanding the role of the sperm's actin cytoskeleton in response to anisosmolality and signaling events that regulate this response.
Publications
- 1. Rutllant JL, Pommer AC, Meyers SA. Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa. Journal of Andrology 24: 534-541, 2003
- 2. Meyers S. Spermatozoal response to osmotic stress. Animal Reprod Sci 89: 57-64, 2005.
- 3. Baumber J, Meyers SA. Changes in Membrane Lipid Order With Capacitation in Rhesus Macaque (Macaca mulatta) Spermatozoa . J Androl 27: 578-587. (2006a)
- 4. Meyers S. Dry storage of sperm, applications in primates and domestic animals. Reproduction, Fertility, and Development 18 (1,2): 1-5, 2006.
- 5. Baumber J, Meyers SA. Hyperactivated Motility in Rhesus Macaque (Macaca mulatta) Spermatozoa. Journal of Andrology 27(3): 458-468, 2006b
- 6. Correa L, Thomas A, McCarthy M, Meyers, S. 2006. Reorganization of the actin cytoskeleton in macaque sperm and changes in motility during osmotic stress and cytochalasin treatment. Biology of Reproduction.
- 7. Damage to Chromosomes and DNA of Rhesus Monkey Sperm Following Cryopreservation. Ming Wen Li, Stuart Meyers, Theodore L Tollner, and James W Overstreet J Androl published January 24, 2007 as doi:10.2164/jandrol.106.000869
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Progress 01/01/05 to 12/31/05
Outputs
Our studies have demonstrated that rhesus sperm have unique biophysical characteristics including lipid composition and membrane phase transitions. We have also demonstrated that standard cryopreservatives have led to only mild broadening of osmotic tolerance limits in these cells. Differential scanning calorimetry demonstrated a method to model various freezing rate curves for rhesus spermatozoa. Interestingly, the optimal predicted freezing rate was 10oC/min and this rate is commonly used by researchers in this field. However, these data suggest that optimal cooling rate, choice of CPA, and mathematical modeling of a predictable cooling curve may not allow us to optimally predict or modify spermatozoal treatments to adequately survive freezing. We have made significant advances in understanding the role of the sperm's actin cytoskeleton in response to anisosmolality and signaling events that regulate this response. Consequently, a thorough understanding of
regulation of water transport, CPA transport, and cell-signaling physiology is necessary in order to alter cellular responses for maximum cell survival of rhesus sperm.
Impacts
The development of efficient macaque sperm cryopreservation will aid in successful propagation of germplasm through artificial insemination (AI) and in vitro fertilization (IVF) programs.
Publications
- Meyers S. Spermatozoal response to osmotic stress. 2005. Animal Reprod Sci 89: 57-64.
- Baumber J, Meyers SA. 2005. Hyperactivated motility in rhesus macaque (macaca mulatta) spermatozoa. Journal of Andrology 27(3): published Ahead-of-Print (12/08/05)
- Meyers S. 2006. Dry storage of sperm, applications in primates and domestic animals. Reproduction, Fertility, and Development 18 (1,2): 1-5, 2006.
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Progress 01/01/04 to 12/31/04
Outputs
In studies focusing on osmotic and temperature-related stresses to sperm cells, we have determined that NHP spermatozoa undergo numerous membrane related changes as a result of low temperature storage. Temperature scans of monkey sperm in phosphate buffered saline using Fourier transform infrared spectroscopy (FTIR) reveals three main lipid phase transitions (Tm) after the CH2 stretching frequency is analyzed. These Tm indicate at what temperature(s) different lipid species go from the gel to liquid crystalline phase. The multiple transitions seen in the profiles likely represent various lipid classes in the sperm membrane. At lower temperatures, the transitions may be due to melting of lipids with highly unsaturated acyl chains, which are very fluid at physiological temperatures. The higher temperature events may be assigned to longer chain, saturated lipids and sphingolipids, such as sphingomyelin, as well as glycolipids. When sperm are treated with methyl-beta
cyclodextrin, there is a sharpening of these transitions, which is indicative of cholesterol loss. When sperm are treated with sphingomyelinase, there is a shift towards higher temperatures in the profile, which suggests an increase in the high melting lipid population. This shift is likely due to the cleaving of the sphingomyelin, causing the formation of free fatty acids, which pass through their Tm at elevated temperatures. Cholesterol efflux is essential for sperm to achieve the membrane fluidity required for fertilization and cholesterol efflux during sperm capacitation could be mimicked during cryopreservation resulting in membrane destabilization, changes in fluidity, and leakiness of membranes. The lipophilic fluorescent dye, Merocyanine 540, is believed to stain cell membranes with increasing affinity as their lipid components become more disordered and can be related to changes in membrane fluidity. Experiments have determined that capacitatation of macaque spermatozoa is
associated with a significant increase in the intensity of merocyanine fluorescence, together with a significant (P < 0.0001) increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins and sperm motility parameters indicative of sperm hyperactivation. The translocation of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of the earliest events of cells undergoing apoptosis. Annexin V staining enables identification of cells with deteriorated membrane integrity at an earlier stage than staining with supravital stains. The observed decrease in membrane lipid order with capacitation in our experiments was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with Annexin V-FITC.
Impacts
The development of efficient macaque sperm cryopreservation would aid in successful propagation of germplasm through artificial insemination (AI) and in vitro fertilization (IVF) programs.
Publications
- Rutllant JL, Pommer AC, Meyers SA. Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa. Journal of Andrology24: 534-541, 2003.
- Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) Spermatozoa. J Baumber and SA Meyers. . Society for the Study of Reproduction, 37th Annual Meeting, Vancouver, British Columbia, Canada, July 2004
- Enrichment of Rhesus Macaque Spermatogonial Stem Cells. AC Pommer, I Dobrinski, and SA Meyers. Society for the Study of Reproduction, 37th Annual Meeting, Vancouver, British Columbia, Canada, July 2004
- Changes in membrane lipid order during capacitation of rhesus macaque (Macaca mulatta) spermatozoa. Baumber J and Meyers, SA. American Society for Cell Biology Annual Conference, 2003.
- Rutllant J, Pommer A, Linfor J, Gousset K, Walker N, Ollero M, Tablin F, Meyers S. Evidence for Membrane Raft Aggregation during in vitro Capacitation of Macaque (Macaca mulatta) Spermatozoa. American Society for Cell Biology Annual Conference, 2002.
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Progress 01/01/03 to 12/31/03
Outputs
This year we have completed most of the work for specific aims #1 and #2 and studies for #3 are currently being initiated. Temperature scans of monkey sperm in phosphate buffered saline using Fourier transform infrared spectroscopy (FTIR) reveals three main lipid phase transitions (Tms) after the CH2 stretching frequency is analyzed. These Tms indicate at what temperature(s) different lipid species go from the gel to liquid crystalline phase. Transitions occur at lower temperatures, (10-12oC), a major transition at around 30oC, and at higher temperatures (45-50oC). This profile is similar to what has been observed in our lab numerous times with equine sperm. The multiple transitions seen in the profiles likely represent various lipid classes in the sperm membrane. At lower temperatures, the transitions may be due to melting of lipids with highly unsaturated acyl chains, which are very fluid at physiological temperatures. The higher temperature events may be assigned
to longer chain, saturated lipids and sphingolipids, such as sphingomyelin, as well as glycolipids. When sperm are treated with methyl-beta cyclodextrin, there is a sharpening of these transitions, which is indicative of cholesterol loss. When sperm are treated with sphingomyelinase, there is a shift towards higher temperatures in the profile, which suggests an increase in the high melting lipid population. This shift is likely due to the cleaving of the sphingomyelin, causing the formation of free fatty acids, which pass through their Tm at elevated temperatures. Spermatozoa have a high degree of lipid asymmetry of the plasma membrane, with phosphatidylethanolamine and phosphatidyl serine in the inner leaflet and phospholipids with choline as the head group (e.g. phosphatidylcholine and sphinogomyelin) in the outer leaflet. Capacitation of porcine spermatozoa has been associated with a collapse of plasma membrane asymmetry and this was associated with a decrease in membrane lipid
order as determined by merocyanine 540. However, the observed decrease in membrane lipid order with capacitation in our experiments was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with Annexin V-FITC as has been reported in other species. Early phases of disturbed membrane functions are also associated with a loss of phospholipid asymmetry (Vermes et al., 1995). The translocation of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of the earliest events of cells undergoing apoptosis. Sperm cryopreservation has been reported to result in an increase in phosphatidyl exposure. Annexin V staining enables identification of cells with deteriorated membrane integrity at an earlier stage than staining with supravital stains and may be another useful probe in our investigation of osmotic effects on macaque spermatozoa. We have also begun studies with our collaborator Dr. John Bischoff at University of Minnesota in
which Dr. Julie Baumber will be trained using the differential scanning calorimeter at Minnesota.
Impacts
The development of efficient macaque sperm cryopreservation would aid in successful propagation of germplasm through artificial insemination (AI) and in vitro fertilization (IVF) programs.
Publications
- Rutllant JL, Pommer AC, Meyers SA. 2003. Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa. Journal of Andrology24: 534-541, 2003.
- Rutllant J, Meyers SA. Cellular Distribution of Caveolin-1 in Macaque Spermatozoa. 2001. Society for the Study of Reproduction, Proceedings of the Annual Meeting, Ottawa, Canada, P. 196, 2001.
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