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Influence of Light on Nuclear Size and Deoxyribonucleic Acid Content of Stored Bovine Spermatozoa
Influence of Light on Nuclear Size and Deoxyribonucleic Acid Content of Stored Bovine Spermatozoa S. K. PAUFLER ~ and R. H. FOOTE
Department of Animal Science, Cornell University, Ithaca, New York Abstract
heads, or the variability in DNA content, was related to differences in fertility (7, 9, 13, 16). Fertility of spermatozoa declines during storage at 5 C, and Hanada et al. (6) and Salisbury et al. (10) have reported that the Feulgenpositive material decreased during storage of spermatozoa at 5 C. The importance of maintaining the integrity of nuclear material of the sperm cell used for artificial insemination is obvious. Our objective was to determine what effect light had upon the Feulgen-positive material in the heads of bull spermatozoa. This material is referred to as DNA.
Feulgen-positive material, assumed to be deoxyribonucleic acid (DNA), was deternfined by mierospeetrophotometry for 790 spermatozoa from 17 bulls stored in Cornell University Extender (CUE) at 5 C in sealed ampules. Factorially arranged treatmerits included light and dark, air and N.. gassing, and addition of fi-mereaptoethylamine (MEA). Light was associated with reduced motility and increased rim.lear area, the correlation between the two variabIes being --0.51 (P < .05). Light also reduced the DNA content from 4.52 to 3.70 relative units (P < .01). The correlation between motility and total DNA content corrected for nuclear area was 0.70 (P ~ .01). N:. was more effective in preserving sperm motility than in preventing a decrease in DNA in sperm exposed to light. Bulls differed in size of the sperm nuclei (range 24.9 to 31.5 ~:), and in DNA content, but there was no significant relationship between either variable and fertility. Addition of 0.5 ~ MEA was toxic to spermatozoa and caused complete disappearance of the nuclei exposed to light in an air atmosphere. I n N~ many nuclei were shrunken. Other nuclei in the presence of MEA were swollen or ruptured, or both, as were the nuclei of spermatozoa stored in the dark with air or N~.
Experimental Procedure
Visible light (8), and particularly the combination of agitation and light under aerobic conditions (5), markedly reduces motility of bovine spermatozoa. This effect can be partially negated by replacement of air with N: and addition of eatalase to the medium (5). Presumably, the harmful effect of light on motility reflects damage to the tail portion of the cell; the effect upon the Feulgen-positive genetic material has not been examined previously. Several workers have noted that the average deoxyribonucleic acid (DNA) content of sperm Received for publication March 28, 1967. ~Present address: Tier~irztliches Institut der Universit~t GSttingen, 3400 GSttingen, West Gernmny.
Bull semen was evaluated and processed as previously described (5). All semen was stored in Cornell University Extender (CUE) in sealed glass ampules and maintained at 5 C, either in the dark or exposed to 2,150 lux of illumination supplied by daylight fluorescent tubes. The stored semen samples were agitated gently by inversion twice daily, to insure a thorough saturation of the medium with the gaseous phase present. Three experiments were conducted, and all three included a comparison of ampuled semen stored in the dark and in the light. Experiment 1 included a comparison of ampuled semen stored with air vs. N.o in the gas phase. I n Experiment 2 catalase was included in half of the replicates exposed to light. The third experiment was a 2 × 2 × 3 factot~ial arrangement with storage in the dark and in the light, ah" o1" N~. in the gas phase, and 0, 0.004, and 0.5 ~ of fi-mercaptoethylamine (MEA), also called cysteamine, added. The highest level of MEA caused disruption of the sperm heads and DNA content could be estimated only in the two lower levels. Arrangement of the experimental treatments is presented subsequently, along with the results in Table 1. The extended semen was stored for varying intervals of time until the spermatozoa in eerrain treatments showed no progressive motility. These times were 8, 12, and 4 days for Experiments 1, 2, and 3, respectively. At this time semen smears were made for estimating the DNA content of sperm. The smears were made
1475
PAUFLER
1476
FOOTE
AND
TABLE 1 Motility, nudearsize, and Feulgen-DNA
Expt. no.
No. of bulls
1
8
2
5
3
4
Over-all
17
eontentofspermatozoa Nuclear
Treatment Light
Gas
Additive
None None Light Light None Light Light None None None None Light Light Light Light None None Light Light
Air N._, Air N~ Air Air Air Air Air N2 N~ Air Air N.o N._. Air N2 Air N~
........ ..... . ....... ........ ........ ........ Catalase b ........ MEA ~ .
.
.
.
.
.
.
MEA ........ MEA ........ MEA ........ e ........ ........
% Motile 42 48 1 ~* 44 52 0"* 0"* 56 5 ** 55 35 *~ 4 ~* 0"* 51 22** 39 46 1"* 39
nleasurements
Area, i*~ 25.6 24.3 *~ 26.4 24.9 27.9 30.3 ~* 30.9** 29.3 31.1" 29.4 30.3 31.9" 31.4" 27.7* 30.5 28.5 28.0 30.2 * 27.7
a
DNA units 4.19 4.37 3.60* 3.46 * 4.70 3.45* 3.86* 4.76 4.28 4.65 4.71 3.29* 3.76* 4.30 3.86* 4.48 4.58 3.59** 3.92 ~*
* P ~ 0.05 when compared with controls. H p ~ 0.01 when compared with controls. Ten nuclei were measured in each bull-treatment subclass. b Catalase activity had been destroyed by light at the time spermatozoa were subsampled for DNA determinations. " MEA concentration = 0.004 )~ ; 0.5 ~t level disrupted nuclei and they could not be measured. d Over-all means included the appropriate light-gas treatments with and without MEA. on glass slides of u n i f o r m thickness, dried, fixed f o r 7 nfin in C a r n o y ' s fixative, a n d stained o n the following day by the F e u l g e n - s t a i n i n g procedure (14). Coverslips were n m u n t e d with Bioloid a n d the slides stored in the dark. The hydrolysis time of 12 rain w i t h x HC1 a t 60 C was rigidly controlled. R e p o r t s have varied on the o p t i m u m hydrolysis time to use with aged s p e r m (3, 6, ]0, 11). This is p a r t i c u l a r l y imp o r t a n t in c o m p a r i n g D N A content of s p e r m a tozoa stored f o r v a w i n g periods. C o m p a r i s o n s here were made on a w i t h i n e x p e r i m e n t basis, with control a n d t r e a t e d s p e r m a t o z o a stored f o r the same l e n g t h of time. C o n t e n t of D N A in i n d i v i d u a l cells was m e a s u r e d with a m i c r o s p e c t r o p h o t m n e t e r assembled f r o m Leitz microscope components, a B a u s c h a n d Lomb m o n o c h r o n m t o r , a n d a P h o t o v o l t p h o t o n m l t i p l i e r (14). The a r r a n g e m e n t of the c o m p o n e n t s is shown in F i g u r e 1. C o m p o n e n t s t h r o u g h which the l i g h t passes or p o i n t s a t which it m a y be deflected f o r locating, aligning, a n d f o c u s i n g the specimen are n u m bered consecutively, s t a r t i n g w i t h the l i g h t source. All c o m p o n e n t s m u s t be carefully a l i g n e d (4, 14), a n d ceuterable clutches, where necessary, were p r o v i d e d f o r t h a t purpose. The p h o t o g r a p h , I n s e t A in F i g u r e 1, shows the movable slide made f o r m e a s u r i n g l i g h t J . ])AIRY SCIENCE VOL. 50, NO. 9
t r a n s m i t t a n e y t h r o u g h s p e r m nuclei. B y simply m o v i n g the slide, light passed e i t h e r t h r o u g h the mask or t h r o u g h the clear hole in the opposite end of the slide. The l a t t e r p e r m i t t e d the r o u n d d i a p h r a g n l (no. 17, Fig. 1) to be used to control the area m e a s u r e d f o r conventional microspectrophotometry. The sperm-shaped mask was completely o p a q u e s u r r o u n d i n g the image of the head. A t the 1,425 magnification used, the clear area of the mask allowed light to pass t h r o u g h a b o u t 25 / o f the s p e r m nucleus, a n area slightly smaller t h a n most nuclei, excepting in E x p e r i m e n t 1. This arr a n g e m e n t pernfitted m e a s u r e n l e n t s of t r a n s m i t t a n c y to be t a k e n on light p a s s i n g t h r o u g h a m a j o r i t y of the area of each nucleus, thus m i n i m i z i n g the effect of the optical density g r a d i e n t s f o u n d in s p e r m nuclei (1, 4). A r o t a t i n g stage, a l o n g with cross-hairs on the f o c u s i n g telescope, allowed the s p e r m nuclei to be positioned exactly in the center of the field a n d rotated, so t h a t the long axis of each was always oriented in the same direction. The slide c o n t a i n i n g the m a s k was moved into position a n d s u p e r i m p o s e d so t h a t the image of the nucleus slightly more t h a n filled the field. T r a n s m i s s i o n of light t h r o u g h the F e u l g e n s t a i n e d nuclei was m e a s u r e d at 560 mt~. T h e e n t r a n c e a n d exit slits were set a t 0.5 mm,
1477
L I G H T E F F E C T S ON SPERZ~IATOZOAN D N A
i •
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BELLOWS
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ULAR
MOVABLE \ !~.'-~JI / L~- 13. P.,S.
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SPEC,MEN
I
7.
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LENS
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:
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2. ENTRANCE EXIT SLIT SL,T
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<~,,.~1. LIGHT SOURCE t~m• 1• Diagram of the equipment used for microspectrophotometric determinations of the FeulgenDNA content of bovine spermatozoa• Inset A shows the movable slide containing the nuclear-shaped mask for measuring light transmittance through sperm nuclei. providing light of high spectral purity. The single wavelength method was used, because previous studies with this instrument by Swierstra (14) showed this procedure to give
as reliable results as the two-wavelength method. Background measurements were made through the mask on clear areas adjacent to the nuclei selected for study• The extinction J. DAIRY SCIEI~O~ ~7OL. 50, NO. 9
1478
PAUFLEa
(E) was calculated by subtracting the log of the light transmittancy through sperm (I~) from the log of the light transmitted through the background (Io). All extinctions were corrected for nuclear area, because it has been shown (11) that as nmeh as 51% of the variation in optical density readings can be removed by this correction. The area of each nucleus was determined by the formula proposed by van Duijn (15). The fornmla is as follows: Area =
1.050 -- 0.225 B=.~
1. B~.~o = width of the base of the nucleus. 2. B~.~ = nmxinmm width of the nucleus. 3. L = total length of the nucleus. These linear measurements were made with a Zeiss ocular micrometer. Salisbury and coworkers (2, 4, 11, 12) have found this formula to give an accurate estimate of the total area of the nuclei of bovine spermatozoa. Optical density and area measurements were made on 790 spermatozoa. Ten sperm cells were measured per slide, representing' a bull X treatment subclass. The bulls used and cells measured were considered to be random variables, but the selected treatments were assumed to be fixed effects. Data were analyzed by conventional analysis of variance techniques.
AND FOOTE
Nuclear area of spermatozoa from different bulls differed significantly (P < 0.01 in Experiments 1 and 2, P < 0.10 in Experiment 3). The mean area for sperm nuclei from 17 bulls, based on measurements of the control treatments stored in the dark without additives, ranged from 24.9 ~ to 31.5 ~ . Nuclear area in Experiment 3 also was affected by the lightair-lV[EA combination, as this interaction was significant statistically (P < 0.05). Nuclear area was not related to total DNA, r = --0.09. Large nuclei tended to have lower extinction values per unit area, but the calculated total DNA per nucleus included an adjustment for nuclear size. Light reduced the amount of measurable DNA (P < 0.01). The mean Feulgen-DNA content of sperm stored in the dark was 4.52 ± .24, and of sperm stored in the light, 3.70 ± .32. Frequency distributions of the 790 DNA measurements made on cells stored in light as opposed to the dark are shown in Fig. 2. This figure illustrates that the reduction in :FeulgenDNA following exposure to light was caused by a general shifting of the curve toward the lower values. The correlation between the relative units of DN~I and percentage of motile spermatozoa was r ----0.70 (P < .01). This correlation is of considerable interest, but it cannot be interpreted, under the conditions of the present experiment, to indicate that spermatozoa with 50
Results and Discussion
~[ean nuclear area and DNA content of spermatozoa, along with their statistical significance, is summarized in Table 1. Each of the nuclear measurements given represents means for 80, 50, and 40 determinations in Experiments 1, 2, and 3, respectively. Inspection of Table 1 reveals that exposure of spermatozoa to light, particularly in air, resulted in low motility of spermatozoa and large nuclear size. The correlation between per cent motility aud nuclear area was 0.51 (P < 0.05). These findings are in agreement with the report by Baker and Salisbm3; (2) and Hanada et al. (6), that eosin-positive nuclei from stored spermatozoa are larger than nuclei from unstained spermatozoa. However, the same report by Hanada et al. (6), and an earlier report by SalisbmT e t ah (10), suggest that sperm nuclear areas decrease upon sperm storage. Since the proportion of eosin-positive (dead) spermatozoa increases with storage, one might expect the opposite trend to occur. -
J. DAI]~¥ SeIENe~ VOL. 50, NO. 9
40
~,-
,_20
J
DARK
\
-
10
!
i
|
7
8
FEULGEN-DNAUNITS Pie. 2. Distribution of Feulgen-DNA measurements made on 790 bovine spermatozoa stored in the dark or exposed to light.
LIGHT
EFFECTS
ON
SPERIVIATOZOAN
more initial DNA show greater progressive motility during storage. Rather, it indicates that light reduced both motility and measurable DNA. N~ was much more effective in preventing reduction in sperm motility than it was in preventing a decrease in Feulgen-DNA in the samples exposed to light. This is believed to be the first report that visible light reduces the Feulgen-DNA of bovine spermatozoa. Wu and Prince (17) reported that 320,000 r but not 30,000 r of X-irradiation reduced the FeulgenDNA of bovine sperm cells. The mechanisms by which light damages spermatozoa are unknown. Sufficient energy is provided by light to break covalent bonds. Also, secondary photochemical reactions may take place. Hydrogen peroxide could have accunmluted, even in the treatment containing eatalase, because light destroyed the activity of the added heine protein eatalase (5). Twelve bulls on which DNA measurements were made were used regularly for artificial breeding at the time of the study. Their fertility based upon 27,383 first inseminations performed within six months of this study ranged from 69 to 76% 60- to 90-day nonreturns. The among-bull correlation between the relative units of Feulgen-DNA and the per cent nonreturns was --0.16 (P > 0.10). The lack of a significant bull DNA-fertility relationship within this narrow range of fertility is sinfilar to results obtained by Smmuerhill and Olds (13). Likewise, mean nuclear area was not correlated with fertility (r = 0.10). No data were given in Table 1 for the 0.5 level of M E A tested~ because all spermatozoa were killed by this treatment within a few hours, and many appeared literally to have undergone a nuclear explosion. Accurate measurements could not be made. The appearance of some of these nuclei exposed to M E A is shown in Figure 3, and the frequency of the respective types based on classification of 1,200 nuclei is sununarized in Table 2. The shrunken nuclei shown in Figure 3 were distorted in shape. The base width was similar to normal bull sperm nuclei and the maximum width and length
1479
DNA
Fro. 3. Effect of 0.5 M fl-mereaptoethylamine (MEA) on the nuclei of bull sperm. (Feulgen stain, X 2,500.) A. Shrunken nucleus and one of nearly normal size, but distorted in shape and stained faintly. B. Nucleus classified as swollen, beginning to rupture. C. Nucleus classified as ruptured. D. Nucleus classified as variegated, because it was one of many forms of Feulgenpositive material found which apparently represents greatly distorted nuclei. A shrunken nucleus also is shown. were reduced. These nuclei did not fill the area of the 25 / mask in the microspectrophotometer. Note also the relatively deep homogeneous staining of the nucleus, which normally stains more faintly at the apex (4). The other nuclei in Figure 3 represent varying degrees of distortion. Whether this alteration of the nuclei is related to the effect of M E A upon sulfhydlT1 bonds is unknown. No nuclei were found following exposure to light in an air atmosphere (Table 2). Likewise, no spermatozoa were found in unstained smears made from extended semen processed this way. When spermatozoa were gassed with N~ and exposed to light, many nuclei became
TABLE 2 Proportion of morphologically abnormal nuclei following storage of spermatozoa for 24 hr in exteuder containing 0.5 ~ fl-mereaptoethylamlne (MEA) Treatment
Proportion of nuclear types (%)
Light
Gas
Shrunken
Light Light Dark Dark
Air N2 Air N~
52 0 0
Swollen
Ruptured
No visible nuclei 14 14 15 65 10 70
Variegated 20 20 20
J-. DAIRY SCIENCE VOL. 50, NO. 9
1480
PAUFLER
shrunken, whereas the m a j o r i t y of the s p e r m a tozoa stored u n d e r air and N~ in the dark were ruptured. S p e r m nuclei are unusually resistant to deg r a d a t i o n by physical and chemical t r e a t m e n t s often used f o r other tissues. Therefore, the dramatic effects and bizarre shapes p r o d u c e d are surprising, and w o r t h y of f u r t h e r investigation to elucidate the mechanisms involved.
Acknowledgments This investigation was supported in part by Public Health Servlee Research Grant GM10263 from The National Institute of General Medical Sciences and by a grant from Eastern Artificial Insemination Cooperative, Inc. The authors are grateful to Dr. JSrg Steinbaeh and Mrs. Valerie Turner for technical assistance.
References (1) Baker, F. N., Bouters, R., and Salisbury, G. W. 1964. Optical Density Profiles in Feulgen-Stained B o v i n e Spermatozoan Nuclei. J. Dairy Sci., 47: 1104. (2) Baker, F. N., and Salisbury, G. W. 1963. Nuclear Size of Live and Dead Bovine Spermatozoa. Nature, 197: 820. (3) Birge, W. J., Anklesaria, G., and Tibbitts, F. D. 1961. Differential Response of DNA of Fresh and Stored Bovine Spermatozoa to Feulgen (HC1) Hydrolysis. Trans. Illinois Acad. Sci., 54: 107. (4) de la Torre, L., and Salisbury, G. W. 1964. Feulgen-DNA Cytophotometry of Bovine Spermatozoa. J. Dairy Sci., 47: 284. (5) Foote, R. H. 1967. Influence of Light and Agitation on Bovine Spermatozoa Stored with Protective Agents. J. Dairy Sci., 50 : 1468. (6) Hanada, A., Hiroe, K., and Tomizuka, T. 1965. DNA Content in Bull Spermatozoa During Storage in Yolk-Citrate Diluent at 4°C. Japanese J. Anim. Reprod., 10: 109. (7) Leuchtenberger, C., Murmanis, I., Murmanis, L., Ito, S., and Weir, D. R. 1956. Interferometric Dry Mass and Microspectrophotometric Arginine Determinations on Bull Sperm Nuclei with Normal and Abnormal DNA Content. Chromosoma, 8: 73.
J. DAIRY SCIENCE VOL, 50, NO. 9
A N D FOOT~
(8) Norman, C., Goldberg, E., and Porterfield, I. D. 1962. The Effect of Visible Radiation on the Visible Life-Span of Mammalian and Avian Spermatozoa. Exptl. Ceil Res., 28 : 69. (9) Parez, M., Petel, J. P., and Vendrely, C. 1960. Sur la Teneur en Acide d~soxyrlbonuel~ique des Spermatozo~des de Taureaux Pr~sentant diff6rents Degr~s de F6condit6. C. R. Acad. Sci., 251: 2581. (10) Salisbury, G. W., Birge, W. J., de la Torre, L., and Lodge, J. R. 1961. Decrease in Nuclear Feulgen-Positive Material (DNA) upon Aging in in Vitro Storage of Bovine Spermatozoa. J. Biophys. Biochem. Cytol., 10 : 353. (11) Salisbury, G. W., Lodge, J. R., and Baker, F. N. 1964. Effects of Age of Stain, Hydrolysis Time, and Freezing of the Cells on the Feulgen-DNA Content of Bovine Spermatozoa. J. Dairy Sci., 47: 165. (12) Salisbury, G. W., and van Dongen, C. G. 1964. A Comparison of Several Methods of Estimating Nuclear Size of Bovine Spermatozoa. J. Anim. Sci., 23: 1098. (13) Summerhill, W. R., and Olds, Durward. 1961. Levels of Deoxyribonucleic Acid in Bovine Spermatozoa and Their Relationship to Fertility. J. Dairy Sci., 44: 548. (14) Swierstra, E. E. 1962. The Cytology and Kinetics of Spermatogenesis in the Rabbit, and the Desoxyribonucleic Acid Content of Spermatogenic Cells. Ph.D. thesis, Cornell University. (15) van DuJjn, C. 1960. Mensuration of the Heads of Bull Spermatozoa. Mikroskopie, 14 : 265. (16) Welch, R. M., and Hanly, E. R. 1960. The Experimental Error of Feulgen Cytophotometry in the Analysis of Bull Spermatozoa Over an Extended Period of Time. I~ A Cytophotometric Analysis of the Deoxyribonucleic Acid (DNA) Content in Germ Cells from Santa Gertrudis Bulls. M. R. Wheeler, ed. University of Texas Publ. 6014. (17) Wu, S. H., and t~rince, J. R. 1963. Effect of Ionizing Radiation on Spermatozoa Metabolism. I n ]~ffects of Ionizing Radiation in the Reproductive System. Carlsson, W. D., and Gassner, F. X., eds. Perganion Press, New York.
Department of Animal Science, Cornell University, Ithaca, New York Abstract
heads, or the variability in DNA content, was related to differences in fertility (7, 9, 13, 16). Fertility of spermatozoa declines during storage at 5 C, and Hanada et al. (6) and Salisbury et al. (10) have reported that the Feulgenpositive material decreased during storage of spermatozoa at 5 C. The importance of maintaining the integrity of nuclear material of the sperm cell used for artificial insemination is obvious. Our objective was to determine what effect light had upon the Feulgen-positive material in the heads of bull spermatozoa. This material is referred to as DNA.
Feulgen-positive material, assumed to be deoxyribonucleic acid (DNA), was deternfined by mierospeetrophotometry for 790 spermatozoa from 17 bulls stored in Cornell University Extender (CUE) at 5 C in sealed ampules. Factorially arranged treatmerits included light and dark, air and N.. gassing, and addition of fi-mereaptoethylamine (MEA). Light was associated with reduced motility and increased rim.lear area, the correlation between the two variabIes being --0.51 (P < .05). Light also reduced the DNA content from 4.52 to 3.70 relative units (P < .01). The correlation between motility and total DNA content corrected for nuclear area was 0.70 (P ~ .01). N:. was more effective in preserving sperm motility than in preventing a decrease in DNA in sperm exposed to light. Bulls differed in size of the sperm nuclei (range 24.9 to 31.5 ~:), and in DNA content, but there was no significant relationship between either variable and fertility. Addition of 0.5 ~ MEA was toxic to spermatozoa and caused complete disappearance of the nuclei exposed to light in an air atmosphere. I n N~ many nuclei were shrunken. Other nuclei in the presence of MEA were swollen or ruptured, or both, as were the nuclei of spermatozoa stored in the dark with air or N~.
Experimental Procedure
Visible light (8), and particularly the combination of agitation and light under aerobic conditions (5), markedly reduces motility of bovine spermatozoa. This effect can be partially negated by replacement of air with N: and addition of eatalase to the medium (5). Presumably, the harmful effect of light on motility reflects damage to the tail portion of the cell; the effect upon the Feulgen-positive genetic material has not been examined previously. Several workers have noted that the average deoxyribonucleic acid (DNA) content of sperm Received for publication March 28, 1967. ~Present address: Tier~irztliches Institut der Universit~t GSttingen, 3400 GSttingen, West Gernmny.
Bull semen was evaluated and processed as previously described (5). All semen was stored in Cornell University Extender (CUE) in sealed glass ampules and maintained at 5 C, either in the dark or exposed to 2,150 lux of illumination supplied by daylight fluorescent tubes. The stored semen samples were agitated gently by inversion twice daily, to insure a thorough saturation of the medium with the gaseous phase present. Three experiments were conducted, and all three included a comparison of ampuled semen stored in the dark and in the light. Experiment 1 included a comparison of ampuled semen stored with air vs. N.o in the gas phase. I n Experiment 2 catalase was included in half of the replicates exposed to light. The third experiment was a 2 × 2 × 3 factot~ial arrangement with storage in the dark and in the light, ah" o1" N~. in the gas phase, and 0, 0.004, and 0.5 ~ of fi-mercaptoethylamine (MEA), also called cysteamine, added. The highest level of MEA caused disruption of the sperm heads and DNA content could be estimated only in the two lower levels. Arrangement of the experimental treatments is presented subsequently, along with the results in Table 1. The extended semen was stored for varying intervals of time until the spermatozoa in eerrain treatments showed no progressive motility. These times were 8, 12, and 4 days for Experiments 1, 2, and 3, respectively. At this time semen smears were made for estimating the DNA content of sperm. The smears were made
1475
PAUFLER
1476
FOOTE
AND
TABLE 1 Motility, nudearsize, and Feulgen-DNA
Expt. no.
No. of bulls
1
8
2
5
3
4
Over-all
17
eontentofspermatozoa Nuclear
Treatment Light
Gas
Additive
None None Light Light None Light Light None None None None Light Light Light Light None None Light Light
Air N._, Air N~ Air Air Air Air Air N2 N~ Air Air N.o N._. Air N2 Air N~
........ ..... . ....... ........ ........ ........ Catalase b ........ MEA ~ .
.
.
.
.
.
.
MEA ........ MEA ........ MEA ........ e ........ ........
% Motile 42 48 1 ~* 44 52 0"* 0"* 56 5 ** 55 35 *~ 4 ~* 0"* 51 22** 39 46 1"* 39
nleasurements
Area, i*~ 25.6 24.3 *~ 26.4 24.9 27.9 30.3 ~* 30.9** 29.3 31.1" 29.4 30.3 31.9" 31.4" 27.7* 30.5 28.5 28.0 30.2 * 27.7
a
DNA units 4.19 4.37 3.60* 3.46 * 4.70 3.45* 3.86* 4.76 4.28 4.65 4.71 3.29* 3.76* 4.30 3.86* 4.48 4.58 3.59** 3.92 ~*
* P ~ 0.05 when compared with controls. H p ~ 0.01 when compared with controls. Ten nuclei were measured in each bull-treatment subclass. b Catalase activity had been destroyed by light at the time spermatozoa were subsampled for DNA determinations. " MEA concentration = 0.004 )~ ; 0.5 ~t level disrupted nuclei and they could not be measured. d Over-all means included the appropriate light-gas treatments with and without MEA. on glass slides of u n i f o r m thickness, dried, fixed f o r 7 nfin in C a r n o y ' s fixative, a n d stained o n the following day by the F e u l g e n - s t a i n i n g procedure (14). Coverslips were n m u n t e d with Bioloid a n d the slides stored in the dark. The hydrolysis time of 12 rain w i t h x HC1 a t 60 C was rigidly controlled. R e p o r t s have varied on the o p t i m u m hydrolysis time to use with aged s p e r m (3, 6, ]0, 11). This is p a r t i c u l a r l y imp o r t a n t in c o m p a r i n g D N A content of s p e r m a tozoa stored f o r v a w i n g periods. C o m p a r i s o n s here were made on a w i t h i n e x p e r i m e n t basis, with control a n d t r e a t e d s p e r m a t o z o a stored f o r the same l e n g t h of time. C o n t e n t of D N A in i n d i v i d u a l cells was m e a s u r e d with a m i c r o s p e c t r o p h o t m n e t e r assembled f r o m Leitz microscope components, a B a u s c h a n d Lomb m o n o c h r o n m t o r , a n d a P h o t o v o l t p h o t o n m l t i p l i e r (14). The a r r a n g e m e n t of the c o m p o n e n t s is shown in F i g u r e 1. C o m p o n e n t s t h r o u g h which the l i g h t passes or p o i n t s a t which it m a y be deflected f o r locating, aligning, a n d f o c u s i n g the specimen are n u m bered consecutively, s t a r t i n g w i t h the l i g h t source. All c o m p o n e n t s m u s t be carefully a l i g n e d (4, 14), a n d ceuterable clutches, where necessary, were p r o v i d e d f o r t h a t purpose. The p h o t o g r a p h , I n s e t A in F i g u r e 1, shows the movable slide made f o r m e a s u r i n g l i g h t J . ])AIRY SCIENCE VOL. 50, NO. 9
t r a n s m i t t a n e y t h r o u g h s p e r m nuclei. B y simply m o v i n g the slide, light passed e i t h e r t h r o u g h the mask or t h r o u g h the clear hole in the opposite end of the slide. The l a t t e r p e r m i t t e d the r o u n d d i a p h r a g n l (no. 17, Fig. 1) to be used to control the area m e a s u r e d f o r conventional microspectrophotometry. The sperm-shaped mask was completely o p a q u e s u r r o u n d i n g the image of the head. A t the 1,425 magnification used, the clear area of the mask allowed light to pass t h r o u g h a b o u t 25 / o f the s p e r m nucleus, a n area slightly smaller t h a n most nuclei, excepting in E x p e r i m e n t 1. This arr a n g e m e n t pernfitted m e a s u r e n l e n t s of t r a n s m i t t a n c y to be t a k e n on light p a s s i n g t h r o u g h a m a j o r i t y of the area of each nucleus, thus m i n i m i z i n g the effect of the optical density g r a d i e n t s f o u n d in s p e r m nuclei (1, 4). A r o t a t i n g stage, a l o n g with cross-hairs on the f o c u s i n g telescope, allowed the s p e r m nuclei to be positioned exactly in the center of the field a n d rotated, so t h a t the long axis of each was always oriented in the same direction. The slide c o n t a i n i n g the m a s k was moved into position a n d s u p e r i m p o s e d so t h a t the image of the nucleus slightly more t h a n filled the field. T r a n s m i s s i o n of light t h r o u g h the F e u l g e n s t a i n e d nuclei was m e a s u r e d at 560 mt~. T h e e n t r a n c e a n d exit slits were set a t 0.5 mm,
1477
L I G H T E F F E C T S ON SPERZ~IATOZOAN D N A
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<~,,.~1. LIGHT SOURCE t~m• 1• Diagram of the equipment used for microspectrophotometric determinations of the FeulgenDNA content of bovine spermatozoa• Inset A shows the movable slide containing the nuclear-shaped mask for measuring light transmittance through sperm nuclei. providing light of high spectral purity. The single wavelength method was used, because previous studies with this instrument by Swierstra (14) showed this procedure to give
as reliable results as the two-wavelength method. Background measurements were made through the mask on clear areas adjacent to the nuclei selected for study• The extinction J. DAIRY SCIEI~O~ ~7OL. 50, NO. 9
1478
PAUFLEa
(E) was calculated by subtracting the log of the light transmittancy through sperm (I~) from the log of the light transmitted through the background (Io). All extinctions were corrected for nuclear area, because it has been shown (11) that as nmeh as 51% of the variation in optical density readings can be removed by this correction. The area of each nucleus was determined by the formula proposed by van Duijn (15). The fornmla is as follows: Area =
1.050 -- 0.225 B=.~
1. B~.~o = width of the base of the nucleus. 2. B~.~ = nmxinmm width of the nucleus. 3. L = total length of the nucleus. These linear measurements were made with a Zeiss ocular micrometer. Salisbury and coworkers (2, 4, 11, 12) have found this formula to give an accurate estimate of the total area of the nuclei of bovine spermatozoa. Optical density and area measurements were made on 790 spermatozoa. Ten sperm cells were measured per slide, representing' a bull X treatment subclass. The bulls used and cells measured were considered to be random variables, but the selected treatments were assumed to be fixed effects. Data were analyzed by conventional analysis of variance techniques.
AND FOOTE
Nuclear area of spermatozoa from different bulls differed significantly (P < 0.01 in Experiments 1 and 2, P < 0.10 in Experiment 3). The mean area for sperm nuclei from 17 bulls, based on measurements of the control treatments stored in the dark without additives, ranged from 24.9 ~ to 31.5 ~ . Nuclear area in Experiment 3 also was affected by the lightair-lV[EA combination, as this interaction was significant statistically (P < 0.05). Nuclear area was not related to total DNA, r = --0.09. Large nuclei tended to have lower extinction values per unit area, but the calculated total DNA per nucleus included an adjustment for nuclear size. Light reduced the amount of measurable DNA (P < 0.01). The mean Feulgen-DNA content of sperm stored in the dark was 4.52 ± .24, and of sperm stored in the light, 3.70 ± .32. Frequency distributions of the 790 DNA measurements made on cells stored in light as opposed to the dark are shown in Fig. 2. This figure illustrates that the reduction in :FeulgenDNA following exposure to light was caused by a general shifting of the curve toward the lower values. The correlation between the relative units of DN~I and percentage of motile spermatozoa was r ----0.70 (P < .01). This correlation is of considerable interest, but it cannot be interpreted, under the conditions of the present experiment, to indicate that spermatozoa with 50
Results and Discussion
~[ean nuclear area and DNA content of spermatozoa, along with their statistical significance, is summarized in Table 1. Each of the nuclear measurements given represents means for 80, 50, and 40 determinations in Experiments 1, 2, and 3, respectively. Inspection of Table 1 reveals that exposure of spermatozoa to light, particularly in air, resulted in low motility of spermatozoa and large nuclear size. The correlation between per cent motility aud nuclear area was 0.51 (P < 0.05). These findings are in agreement with the report by Baker and Salisbm3; (2) and Hanada et al. (6), that eosin-positive nuclei from stored spermatozoa are larger than nuclei from unstained spermatozoa. However, the same report by Hanada et al. (6), and an earlier report by SalisbmT e t ah (10), suggest that sperm nuclear areas decrease upon sperm storage. Since the proportion of eosin-positive (dead) spermatozoa increases with storage, one might expect the opposite trend to occur. -
J. DAI]~¥ SeIENe~ VOL. 50, NO. 9
40
~,-
,_20
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DARK
\
-
10
!
i
|
7
8
FEULGEN-DNAUNITS Pie. 2. Distribution of Feulgen-DNA measurements made on 790 bovine spermatozoa stored in the dark or exposed to light.
LIGHT
EFFECTS
ON
SPERIVIATOZOAN
more initial DNA show greater progressive motility during storage. Rather, it indicates that light reduced both motility and measurable DNA. N~ was much more effective in preventing reduction in sperm motility than it was in preventing a decrease in Feulgen-DNA in the samples exposed to light. This is believed to be the first report that visible light reduces the Feulgen-DNA of bovine spermatozoa. Wu and Prince (17) reported that 320,000 r but not 30,000 r of X-irradiation reduced the FeulgenDNA of bovine sperm cells. The mechanisms by which light damages spermatozoa are unknown. Sufficient energy is provided by light to break covalent bonds. Also, secondary photochemical reactions may take place. Hydrogen peroxide could have accunmluted, even in the treatment containing eatalase, because light destroyed the activity of the added heine protein eatalase (5). Twelve bulls on which DNA measurements were made were used regularly for artificial breeding at the time of the study. Their fertility based upon 27,383 first inseminations performed within six months of this study ranged from 69 to 76% 60- to 90-day nonreturns. The among-bull correlation between the relative units of Feulgen-DNA and the per cent nonreturns was --0.16 (P > 0.10). The lack of a significant bull DNA-fertility relationship within this narrow range of fertility is sinfilar to results obtained by Smmuerhill and Olds (13). Likewise, mean nuclear area was not correlated with fertility (r = 0.10). No data were given in Table 1 for the 0.5 level of M E A tested~ because all spermatozoa were killed by this treatment within a few hours, and many appeared literally to have undergone a nuclear explosion. Accurate measurements could not be made. The appearance of some of these nuclei exposed to M E A is shown in Figure 3, and the frequency of the respective types based on classification of 1,200 nuclei is sununarized in Table 2. The shrunken nuclei shown in Figure 3 were distorted in shape. The base width was similar to normal bull sperm nuclei and the maximum width and length
1479
DNA
Fro. 3. Effect of 0.5 M fl-mereaptoethylamine (MEA) on the nuclei of bull sperm. (Feulgen stain, X 2,500.) A. Shrunken nucleus and one of nearly normal size, but distorted in shape and stained faintly. B. Nucleus classified as swollen, beginning to rupture. C. Nucleus classified as ruptured. D. Nucleus classified as variegated, because it was one of many forms of Feulgenpositive material found which apparently represents greatly distorted nuclei. A shrunken nucleus also is shown. were reduced. These nuclei did not fill the area of the 25 / mask in the microspectrophotometer. Note also the relatively deep homogeneous staining of the nucleus, which normally stains more faintly at the apex (4). The other nuclei in Figure 3 represent varying degrees of distortion. Whether this alteration of the nuclei is related to the effect of M E A upon sulfhydlT1 bonds is unknown. No nuclei were found following exposure to light in an air atmosphere (Table 2). Likewise, no spermatozoa were found in unstained smears made from extended semen processed this way. When spermatozoa were gassed with N~ and exposed to light, many nuclei became
TABLE 2 Proportion of morphologically abnormal nuclei following storage of spermatozoa for 24 hr in exteuder containing 0.5 ~ fl-mereaptoethylamlne (MEA) Treatment
Proportion of nuclear types (%)
Light
Gas
Shrunken
Light Light Dark Dark
Air N2 Air N~
52 0 0
Swollen
Ruptured
No visible nuclei 14 14 15 65 10 70
Variegated 20 20 20
J-. DAIRY SCIENCE VOL. 50, NO. 9
1480
PAUFLER
shrunken, whereas the m a j o r i t y of the s p e r m a tozoa stored u n d e r air and N~ in the dark were ruptured. S p e r m nuclei are unusually resistant to deg r a d a t i o n by physical and chemical t r e a t m e n t s often used f o r other tissues. Therefore, the dramatic effects and bizarre shapes p r o d u c e d are surprising, and w o r t h y of f u r t h e r investigation to elucidate the mechanisms involved.
Acknowledgments This investigation was supported in part by Public Health Servlee Research Grant GM10263 from The National Institute of General Medical Sciences and by a grant from Eastern Artificial Insemination Cooperative, Inc. The authors are grateful to Dr. JSrg Steinbaeh and Mrs. Valerie Turner for technical assistance.
References (1) Baker, F. N., Bouters, R., and Salisbury, G. W. 1964. Optical Density Profiles in Feulgen-Stained B o v i n e Spermatozoan Nuclei. J. Dairy Sci., 47: 1104. (2) Baker, F. N., and Salisbury, G. W. 1963. Nuclear Size of Live and Dead Bovine Spermatozoa. Nature, 197: 820. (3) Birge, W. J., Anklesaria, G., and Tibbitts, F. D. 1961. Differential Response of DNA of Fresh and Stored Bovine Spermatozoa to Feulgen (HC1) Hydrolysis. Trans. Illinois Acad. Sci., 54: 107. (4) de la Torre, L., and Salisbury, G. W. 1964. Feulgen-DNA Cytophotometry of Bovine Spermatozoa. J. Dairy Sci., 47: 284. (5) Foote, R. H. 1967. Influence of Light and Agitation on Bovine Spermatozoa Stored with Protective Agents. J. Dairy Sci., 50 : 1468. (6) Hanada, A., Hiroe, K., and Tomizuka, T. 1965. DNA Content in Bull Spermatozoa During Storage in Yolk-Citrate Diluent at 4°C. Japanese J. Anim. Reprod., 10: 109. (7) Leuchtenberger, C., Murmanis, I., Murmanis, L., Ito, S., and Weir, D. R. 1956. Interferometric Dry Mass and Microspectrophotometric Arginine Determinations on Bull Sperm Nuclei with Normal and Abnormal DNA Content. Chromosoma, 8: 73.
J. DAIRY SCIENCE VOL, 50, NO. 9
A N D FOOT~
(8) Norman, C., Goldberg, E., and Porterfield, I. D. 1962. The Effect of Visible Radiation on the Visible Life-Span of Mammalian and Avian Spermatozoa. Exptl. Ceil Res., 28 : 69. (9) Parez, M., Petel, J. P., and Vendrely, C. 1960. Sur la Teneur en Acide d~soxyrlbonuel~ique des Spermatozo~des de Taureaux Pr~sentant diff6rents Degr~s de F6condit6. C. R. Acad. Sci., 251: 2581. (10) Salisbury, G. W., Birge, W. J., de la Torre, L., and Lodge, J. R. 1961. Decrease in Nuclear Feulgen-Positive Material (DNA) upon Aging in in Vitro Storage of Bovine Spermatozoa. J. Biophys. Biochem. Cytol., 10 : 353. (11) Salisbury, G. W., Lodge, J. R., and Baker, F. N. 1964. Effects of Age of Stain, Hydrolysis Time, and Freezing of the Cells on the Feulgen-DNA Content of Bovine Spermatozoa. J. Dairy Sci., 47: 165. (12) Salisbury, G. W., and van Dongen, C. G. 1964. A Comparison of Several Methods of Estimating Nuclear Size of Bovine Spermatozoa. J. Anim. Sci., 23: 1098. (13) Summerhill, W. R., and Olds, Durward. 1961. Levels of Deoxyribonucleic Acid in Bovine Spermatozoa and Their Relationship to Fertility. J. Dairy Sci., 44: 548. (14) Swierstra, E. E. 1962. The Cytology and Kinetics of Spermatogenesis in the Rabbit, and the Desoxyribonucleic Acid Content of Spermatogenic Cells. Ph.D. thesis, Cornell University. (15) van DuJjn, C. 1960. Mensuration of the Heads of Bull Spermatozoa. Mikroskopie, 14 : 265. (16) Welch, R. M., and Hanly, E. R. 1960. The Experimental Error of Feulgen Cytophotometry in the Analysis of Bull Spermatozoa Over an Extended Period of Time. I~ A Cytophotometric Analysis of the Deoxyribonucleic Acid (DNA) Content in Germ Cells from Santa Gertrudis Bulls. M. R. Wheeler, ed. University of Texas Publ. 6014. (17) Wu, S. H., and t~rince, J. R. 1963. Effect of Ionizing Radiation on Spermatozoa Metabolism. I n ]~ffects of Ionizing Radiation in the Reproductive System. Carlsson, W. D., and Gassner, F. X., eds. Perganion Press, New York.