Progress 12/15/00 to 12/31/03
Outputs
Sperm DNA fragmentation analysis, which we pioneered with the Sperm Chromatin Structure Assay (SCSA) in 1980, is currently a hot topic in male reproductive biology. Sperm treated with pH 1.2 buffer will produce partial denaturation of DNA, which is measured as green (native DNA) or red (denatured DNA) fluorescence intensity. Two other tests (TUNEL and COMET) for sperm DNA fragmentation were pioneered both by our lab and other laboratories. These latter two tests were considered to be a direct measure of DNA strand breaks while the SCSA data were interpreted, as we wrote in our pioneer paper, as altered chromatin structure. We have accomplished the primary goal of AIM # 1, namely, our data strongly supports the view that the acid treatment is denaturing the DNA at the sites of DNA strand breaks. Whereas the other two assays are adding a fluorescent probe at the end of DNA strand breaks, or allowing migration of small pieces of DNA that can occur only by the presence of
strand breaks, the SCSA must be adding fluorescent acridine orange probes at the sites of DNA strand breaks. Since we have previously shown that the percent of sperm in a semen sample are about the same whether processed by TUNEL, COMET or the SCSA, combined with this work shows that the SCSA- positive sperm are also COMET positive, we conclude that the SCSA is a direct measure of DNA strand breaks. Specifically, four populations of sperm are resolved by the SCSA: 1) normal population consistent with a fertile semen sample, 2) High DNA stainable (HDS) population, 3) Moderate level of DNA fragmentation (DFI-Mod) and 4) High level of DNA fragmentation ((DFI-Hi). These populations were flow cytometry sorted and placed onto glass microscope slides with a cytocentrifuge. Slides were processed either for computer based image analysis of digitally photographed Feulgen stained sperm, or, by computer based image analysis of digitally photographed YOYO stained sperm processed by single cell gel
electrophoresis (COMET analysis). Results: A. Image analysis. Relative to the normal population, the HDS population had larger, rounder and had more irregular shaped sperm heads, the DFI-Mod population had normal head morphology and the DFI-Hi population had more elongated sperm heads. B. COMET analysis: Relative to the normal population, the HDS population did not have broken DNA migrating as COMETS, the DFI-Mod had extensive migration, as did the Hi-DFI. Over 80% of the DFI sperm showed extensive DNA strand breaks. The significant point is that the DFI-Mod population that has normal appearing sperm contains numerous DNA strand breaks. These sperm have been shown to fertilize oocytes, and if so, likely result in early embryo death.
Impacts
The SCSA can now be viewed as the most informative test for sperm nuclear integrity as it is: a) is a flow cytometric, statistically powerful, and analyzes a population of sperm (HDS) that is not resolved by either of the other two tests. Extensive data show its utility in the diagnosis of livestock sperm for breeding efficiency.
Publications
- Virro, M.R. and Evenson, D. P. (2003) Sperm chromatin structure assay (SCSA) related to blastocyst rate, pregnancy rate and spontaneous abortion in IVF and ICSI cycles. (in press).
- Larson-Cook, K., Brannian, J.D., Hansen, K.A., Kasperson, K., Aamoldt, E.T., and Evenson, D.P. (2003) Relationship between assisted reproductive techniques (ART) outcomes and DNA fragmentation (DFI) as measured by the sperm chromatin structure assay (SCSA). Fertil Steril 80:4;895-902.
- Alvarez, J.G., Larson-Cook, K.L., Evenson, D.P. (2003) Selecting cryopreserved semen for assisted reproductive techniques (ART) based on the level of sperm nuclear DNA fragmentation resulted in pregnancy. Fertility and Sterility (in press).
- Penfold, L.M., Jost, L., Evenson, D.P., and Wildt, D.E. (2003) Normospermic versus teratospermic domestic cat chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection. Biol of Reprod 69: 1730-1735.
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Progress 01/01/02 to 12/31/02
Outputs
Aim # 1. A significant amount of time has been spent on flow cytometry sorting of normal sperm and those with sperm chromatin structure assay (SCSA) defined DNA fragmentation. One difficulty is that we have a FACSort flow cytometer that, in contrast to a droplet-in- air sorter, sorts with a mechanical device pulling out the cell of interest. This results in a large volume of liquid containing the cells of interest. To obtain the proper concentration of sperm on a slide we have used a cytocentrifuge that centrifuges the sperm onto a small spot. These cells must be equilibrated in a soft electrophoretic gel before drying. Adding the gel to the sorted population prior to use of the cytocentrifuge did not work as well as putting a gel on the slide before centrifuging the sperm. Thus, we have spun the sperm onto slides and immediately afterwards we have added a small amount of gel to the spot, air dried and then placed a layer of soft gel over the entire slide. We are now
in the process of capturing the images of the DNA stained nuclei after gel electrophoresis. The data obtained so far supports our hypothesis that the sperm showing DNA denaturation on the SCSA cytograms have a higher degree of Comet positive sperm. Aim # 2. Dr. Wright is working on the in vitro fertilization of bovine oocytes with heat-socked bull semen that have normal and abnormal DNA denaturation patterns.
Impacts
This research will give insight into the effect of spermatozoa with DNA defects (fragmentation)on embryo quality and impact the cattle breeding industry.
Publications
- Evenson, D.P. and Spano, M. 2002. Flow Cytometry analysis of mammalian sperm. Proceedings of the 9th International Symposium on Spermatology, Oct 6-12, Capetown, South Africa.
- Evenson, D.P. 2002. Assessment of sperm using the sperm chromatin structure assay. Proceedings UCLA 15th Annual In Vitro Insemination and Embryo Transfer Course, July 14-17, UCLA, 2002.
- Evenson, D.P. 2002. Role of sperm chromatin damage in abnormal reproductive outcomes. Society for Theriogeneology /ACT Annual Conference & Symposia Proceedings. Published by SFT. Colorado Springs, CO, Aug 7-11, 2002.
- Virro, M., and Evenson, D. P. 2003. Sperm Chromatin Structure Assay (SCSAr) Related to Blastocyst Rate, Pregnancy Rate and Spontaneous Abortion in IVF and ICSI Cycles. Pacific Coast Reproductive Society. April 23-27, Rancho Mirage, CA.
- Evenson, D.P., Steiner, J.V., and Jost, L.K. 2002. Diagnostic and prognostic value of the sperm chromatin structure assay for stallion sub/infertility. Eighth International Symposium on Equine Reproduction. July 21-26, Fort Collins, CO. (abst)
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Progress 01/01/01 to 12/31/01
Outputs
Bull testes were subjected to an elevated temperature by a 48 hr application of wool heat socks. Semen samples were collected (N. Acavedo and R. Saacke) and sent on liquid nitrogen to SDSU. Spermatogenesis and spermiogenesis were dramatically affected by the mild heat stress, as determined by the Sperm Chromatin Structure Assay (SCSA). Two bulls responded very well to the treatment, 1 bull somewhat and 3 bulls did not show a response to the heat socks. Baseline DFI (DNA Fragmentation Index) for days preceding the application of the sock up to 9 days after placing the socks on the scrotum was between 3 and 8% for all six bulls in the study. Nine days after scrotal sock application, the responders showed significant increases in % DFI. One bull displayed increased DFI of 14%, 23%, 21% and 20% on 12, 16, 20 and 28 days after sock application before returning to baseline values at day 34. The other responder showed elevated DFI of 11%, 10%, 19%, 40% and 24% on 12, 16, 20,
28, and 34 days after heat sock application. After SCSA analysis, the affected samples were sent to Drs. J. Ellington and R. Wright at Washington State University. They are in the process of doing IVF and embryo quality assessments from control and DNA damaged sperm. When finished, the data will be statistically analyzed for the influence of sperm DNA fragmentation and loss of embryo quality. Work is in progress in the SDSU laboratory on the relationship between between SCSA defined DNA fragmentation and DNA strand breaks as measured by the COMET assay. This assay is not as repeatable as the SCSA.
Impacts
This research will give insight into the effect of spermatozoa with DNA defects (fragmentation)on embryo quality and impact the cattle breeding industry.
Publications
- Ostermeier, G. C., Sargeant, G. A., Yandell, B. S., Evenson, D.P., and Parrish, J.J. (2001) The relationship of bull fertility to sperm nuclear shape. J. Andrology 22: 595-603.
- Krzyzosiak, J., Evenson, D., Pitt, C., Jost, L., Molan, P., and Vishwanath, R. (2001) Changes in susceptibility of bovine sperm to in situ DNA denaturation during prolonged storage at ambient temperature under conditions of exposure to reactive oxygen species and nuclease inhibitor. Reproduction, Fertility and Development 12: 251-261.
- Perreault, S.D., Aitken R.J., Baker, H.W.G., Evenson, D.P., Huszar, G., Irvine D.S., Morris, R.A., Robbins W.A., Sakkas D., Spano, M., and Wyrobek, A.J. (2002) Integrating new tests of sperm genetic integrity into semen analysis: summary of breakout group discussion. Proceedings of 2nd International Conference on Male-Mediated Developmental Toxicity.
- Ellington, J.E., Oliver, S.A, Evenson, D.E., and Fleming, J. (2001) Polysaccharides containing arabinose and galactose decrease oxidative damage of bull sperm during in vitro handling. . International Society of Andrology. Montreal, June 15-23.(ABSTRACT)
- Tritle, D.J., Evenson, D. (2002) Correlation between two tests of DNA damage in spermatozoa: Sperm Chromatin Structure Assay and Comet Assay. EPSCoR Conference in Pierre, SD. Feb 15, 2002. (abstract)
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