Donald Evenson
Olson Biochemistry Laboratories
South Dakota State University
Department of Chemistry and Biochemistry
Brookings, SD 57007 USA

email: evensond@mg.sdstate.edu

SPERM CHROMATIN STRUCTURE ASSAY (SCSA;10): A Measure of Nuclear DNA/Protein Structure Related to Fertility Potential

Abnormal chromatin structure is defined by the SCSA as an increased susceptibility to acid (pH 1.2, 30 sec) induced denaturation. When acridine orange (AO) stained sperm are exposed to 488 nm laser light, AO intercalated into ds DNA fluoresces green and AO bound to ss DNA fluoresces red. The SCSA measures the shift from green (native, ds DNA) to red (denatured, ss DNA) fluorescence in each of 5000 cells per sample and the extent of this denaturation is quantified by alpha t [alpha t, = red/(red+green) fluorescence]. Normal chromatin remains structurally sound at low pH, producing minimal red fluorescence and giving a narrow at alpha distribution. Usually, DNA with abnormal chromatin structure partially denatures under the acid conditions of the SCSA, yielding increased red fluorescence and a broader alpha t distribution; i.e. higher mean channel (X alpha t) and increased % of Cells Outside the Main Population (COMP alpha t), The standard deviation of (alpha t (SD alpha t) describes the extent of chromatin structure abnormality (1,2,5,7,9-12). Correlations between at variables and fertility potential have been as high as 0.94 (p -.0l).

Figure 1: Green versus red fluorescence cytograms and corresponding alpha t frequency histograms of Acridine Orange (AO) stained bull semen samples prepared and measured by the Sperm Chromatin Structure Assay (SCSA). In cytograms (A & B), green fluorescence (Y-Axis) corresponds to native, ds DNA and red fluorescence (X-Axis) to denatured, ss DNA. A & C represent semen analyzed from a bull of known high fertility, and B & D from a bull of very low fertility. Note the higher proportion of sperm with increased levels of red fluorescence in cytogram B compared to A and the resulting shift in the alpha t distributions (compare C to D).

Figure 2: The SCSA can be done concomitantly with a sperm count determination by admixing a known number of fluorescent beads with the sperm sample (13). The methodology is simple, highly accurate and a logical extension of this assay. The gate in the bottom left hand corner excludes debris and only sperm and beads are included in the analysis. Since the bead concentration is known, a simple calculation determines the sperm concentration, an important parameter of fertility potential.

SPERM VIABILITY ASSAYS I. RHODAMINE 123/PROPIDIUM IODIDE (Rl23/Pl; 6,8,14,20):

The R123/Pl assay provides a quantitative measurement of mitochondrial membrane potential (cell motility) and cell membrane viability. After staining with R123, fluorescence intensity in the sperm midpiece is related to the mitochondrial membrane potential and motility. Red fluorescence from the PI stained nuclear DNA results from compromised cell membranes and is indicative of dead or dying cells. Bright green fluorescence was correlated with vigorous sperm motility and dull green fluorescence correlated with slow motility. Bright red and bright green fluorescence are mutually exclusive.

Figure 3: Two parameter isometric displays of green and red fluorescence from 5000 bull sperm stained with Rhodamine 123 and Propidium Iodide (RI23/Pl). Sperm samples from known fertile (A) and less fertile (B) donors are shown.

II. SYBR14/Pl (15,16)

Co-staining sperm with SYBRI4 and PI readily identifies cells with intact membranes versus membrane compromised cells. SYBRI4, a membrane permeable DNA stain (green fluorescence) requires an intact cell membrane for optimal fluorescence. PI, a membrane impermeable stain (red fluorescence) stains dead or dying cells with breaks in the cell membranes.

Figure 4: Two parameter green (log) versus red (linear) fluorescence isometric displays of 10,000 cells collected from fertile (A) and less fertile (B) bull sperm stained with SYBRI4 and counterstained with Propidium Iodide (PI). Population I contains all red (PI, dead) sperm and population 3 contains bright green (SYBRI 4, viable) sperm; population 2 are sperm in transition from live to dead.

TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE ASSAY (TdTA;18,19,21,22): Detection of DNA Strand Bmaks in Sperm Nuclei

DNA strand breaks in fertilizing sperm nuclei have potentially serious consequences for developing embryos. DNA strand breaks can be detected by incubating fixed sperm in the presence of avidintagged DUTP and TDTA, which adds the base to the ends of the DNA strand breaks. These tagged additions can be quantitated by incubation in the presence of FITC tagged biotin followed by flow cytometric measurement of the sperm.

Figure 5: Flow cytometric data from TDTA incubated sperm from the same bull collected during a fertile period (A,D) and collected 3 to 3.5 years later (B,C,E,F), during periods of decreasing fertility. The panels on the left (A,C) show green fluorescence frequency histograms from the TdT control samples. The panels on the right (D-F) show green fluorescence frequency histograms depicting the subtraction of the TdT control green (upper curve) from the TdT-positive green (lower curve). Shaded areas in D-F depict sperm with increased presence of endogenous DNA strand breaks. A correlation has been observed (19,21) between percent of sperm with susceptibility to DNA denaturation and percent sperm demonstrating DNA strand breaks.

SPERM IMAGING (17,23,24)

Sperm morphology and morphometry have been shown to relate to fertility potential.(23). Most fertility clinics utilize light microscopy of stained semen smears on slides for assessment of sperm morphology. Recently, some sperm motion analyzers have been fitted with very basic morphology measures, e.g., length, width, circumference. Our laboratory uses a cooled CCD camera and ONCOR imaging software to measure a variety of morphology and morphometry parameters. Sixteen parameters of Feulgen stained sperm were utilized in a recent study. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs and dark blobs was highly significant (r 2=0.999, P- 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure (23).

Figure 6: Display of sperm nuclei, illustrating various states of normal/abnormal head morphology. Nuclei A & B are slightly different from each other, both in length and width, yet both would be visually classified as being morphologically normal. Nucleus C is shorter and wider than normal. D & E are tapered near their base which will alter the curvature measurements and length and width of the nuclei. F is wider than normal and illustrates staining alterations seen in several samples. Dark staining covers the base of the nucleus in most examples but in F, is skewed to one side. Nucleus G is smaller than normal in all respects, and H is highly rounded, indicative of immature sperm. Nucleus I is representative of a special case of sperm head abnormality; the ring of lightly stained areas midway across the nucleus represent "crater" or "diadem" defects.

REFERENCES

1. Ballacbey BE, Hobenboken WD, Evenson DP: Heterogeneity of sperm nuclear chromatin structure and its relationship to fertility of bulls. Biol Reprod 1987; 36:915-925.

2. Ballacbey BE, Evenson DP, Saacke R: The sperm chromatin structure assay relationship with alternate tests of semen quality and heterospermic performance of bulls. J Androl 1988; 9:109-115.

3. Darzynkiewiez Z, Traganos F, Sharpless T, Melamed M: Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 1975; 90:411-428.

4. Darzynkiewicz Z, Traganos F, Sharpless R, Melamed MR: Lymphocyte stimulation: A rapid multiparameter analysis. Proc Natl Acad Sci, USA 1976; 73:2881-2884.

5. Evenson DP: Flow cytometric analysis of male germ cell quality. In: Methods in Cell Biology, Volume 33, Flow Cytometry, Darynkiewiez Z, Crissman H (eds). Academic Press, San Diego, CA 1990; 401410.

6. Evenson D, Ballachey B: Flow cytometric evaluation of bull sperm chromatin structure, mitochondrial activity, viability and concentration. Proc Ilth Tech Conf AI Rep, NAAB. Milwaukee, WI, 1986; 109.

7. Evenson DP, Darynkiewicz Z, Melamed MR: Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980; 240:1131-1133.

8. Evenson DP, Darzynkiewicz Z, Melamed MR: Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J Histochem Cytochem 1982; 30:279-280.

9. Evenson DP, Higgins pH, Grueneberg D, Ballachey B: Flow cytometric analysis of mouse sperm atogenic function following exposure to ethylnitrosourea. Cytometry 1985; 6:238-253.

10. Evenson DP, Jost LK: Sperm chromatin structure assay: DNA denaturability. In: Methods in Cell Biology, Vol 42: Flow Cytometry, Darzynkiewicz Z, Robinson JP, Crissman HA (eds). Academic Press, Inc., Orlando, FL, 1994; 159-176.

11. Evenson D, Jost L, Gandour D, Rhodes L, Stanton B, Clausen OP, De Angelis P, Coico R, Daley A, Becker K, Yopp T: Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers. Cytometry 1995; 19:195-204.

12. Evenson DP, Molamed MR: Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem 1983; 31:248-253.

13. Evenson DP, Parks JE, Kaproth MT, Jost LK: Rapid determination of sperm cell concentration in bovine semen by flow cytometry. J Dairy Sci 1993; 76: 86-94.

14. Garner DL, Pinkel D, Johnson LA, Pace MM: Assessment of spermatozoal functions using dual fluorescent staining and flow cytometric analysis. Biol Reprod 1986; 34:127.

15. Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP: Dual DNA staining assessment of bovine sperm viability using SYBRI4 and propidium iodide. J Androl 1994; 15:620-629.

16. Garner DL, Johnson LA: Viability assessment of mammalian sperm using SYBRI4 and propidium iodide. Biol Reprod 1995; 53:276-284.

17. Garvin AJ, Hall BJ, Brissie RM, Spicer SS: Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence. J Histochem Cytochem 1976;24:587-590.

18. Gorezyca W, Bignian K, Mittelman A, Ahmed P, Gong J, Melamed MR, Darzynkiewicz Z: Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 1993;7:659-670.

19, Gorczyca W, Gong J, Darzynkiewicz Z: Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993;53:1945-1951.

20. Karabinus DS, Evenson DP, Kaproth MT: Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm. J Dairy Sci 1991; 74:3836-3848.

21. Sailer BL, Jost LK, Evenson DP: Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the Terminal Deoxynucleotidyl Transferase Assay. J Androl 1995;16:80-87.

22. Evenson DP, Sailer BL, Jost LK: Relationship between stallion sperm deoxyribonucleic acid (DNA) susceptibility to denaturation in situ and presence of DNA strand breaks: Implications for fertility and embryo viability. Biol Reprod Monog 1: Equine Reproduction VI, 1995; 655-659.

23. Sailer BL, Jost LK, Evenson DP: Bull sperm head morphology related to abnormal chromatin structure and fertility. Cytometry 1996;24:167-173.

24. Moruzzi JF, Wyrobek AJ, Mayall B.H, Gledhill BL: Quantification and classification of human sperm morphology by computer-assisted image analysis. Fert Steril 1988;50:142-152.