Spectrophotometric Quantitation of Mammalian Spermatozoon Motility I. Human

Transcription

1 BIOLOGY OF REPRODUCTION 18, (1978) Spectrophotometric Quantitation of Mammalian Spermatozoon Motility I. Human R.W. ATI-IERTON, E.W. RADANY and K.L. POLAKOSKI2 Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming of Obstetrics-Gynecology, Washington University School of Medicine, 4911 Barnes Hospital Plaza, St. Louis, Missouri ABSTRACT Human sperm has been spectrophotometrically quantitated by a technique based upon the optical anisotropic properties of motile sperm. A sperm index was determined and controlled experiments showed that the index (SMI) was positively correlated to the percent of progressively motile cells (r=0.92) and negatively correlated to the percent of heat killed cells in the sample (r=-0.92). In a single dilution of sperm cells with aliquots studied at selected time intervals for 90 mm, the index correlated with a decline in the progressive (r=0.95). The correlation between the index and the percent progressive in the fresh dilution was r= When the index was determined on the same original population of spermatozoa, but experimentally prepared with different concentrations of cells, the index was independent of final cell numbers in a range of X 106 cells/ml. Semen analyses were performed on each ejaculate and a correlation between the SMI and progressive (r=0.85) was observed. INTRODUCTION Sperm has been considered to be an important parameter in determining the quality of human semen (Farris, 1949; MacLeod and Gold, 1951; DaRugna, 1966; Botella-Llusia, 1966). Present procedures for measuring sperm are either subjective or so complex that clinical adaptability may be difficult (Atherton, 1977). However, a spectrophotometric procedure, based upon the optical anisotrop ic characteristic of sperm (Walton, 1952), that overcomes these shortcomings has been utilized for evaluating the of sperm from sea urchins (Timourian and Watchmaker, 1970; Atherton, 1975), roosters (Wall and Boone, 1973), bulls (Atherton, 1975a) and most recently humans (Atherton, et al., 1977). This communication reports the improvement of sensitivity and reproducibility of this spectrophotometric technique for measuring human sperm. Emphasis was placed on the selection of equipment to insure minimal cost, maximum reliability, objectivity and adaptability for both clinical and research requirements. Finally, a sperm index Accepted October 31, Received August 29, (SMI) obtained from semen donated by fertile males was correlated with sperm progressive motilty. MATERIALS AND METHODS After a minimum of 36 h continence, human semen was collected by masturbation into clean glass vials. The semen was allowed to liquify at 20 #{176}Cuntil a homogeneous sample was obtained. The sample was then analyzed for volume, ph, sperm count as determined with a hemacytometer and the percentage of dead cells was quantitated with the eosin staining procedure (Eliasson and Treichl, 1972). The progressively motile sperm, nonprogressively motile sperm and the nonmotile sperm were microscopically determined on a minimum of 200 sperm cells. The percent nonprogressively motile cells are the combined cells that exhibited either wiggling motion or were nonmotile. Semen samples were routinely diluted by the addition of various concentrations of semen:5 ml of Baker s saline (Zaneveld and Polakoski, 1977) at ph 7.6 so that an initial absorbance of was obtained. The sperm was spectrophotometrically quantitated by passing the diluted semen through a 1 mm flow cell with the inflow compartment located next to the light source in either a Bausch and Lomb Spectronic 20 colonmeter or a Perkin-Elmer 124 double beam recording spectrophotometer. A Harvard Infusion Pump (Model 940) equipped with a 5 ml syringe was used to maintain a flow rate of approximately 2 mis/mm. After a constant base line absorbance was obtained, the flow was stopped and the decrease in absorbance was determined after 1 mm. The reported sperm index (SMI) values are an average of at least 3 observa- 624

2 QUANTITATION OF HUMAN SPERM MOTILITY 625 tions on each sample and were obtained by the following formula: SMlafAbsorbance (initial)-absorbance (at 1 min)/ Absorbance (initial)i X 100 RESULTS Standardization of the Technique The usefulness of this procedure for quantitating sperm was first described using sperm from sea urchins (Timourian and Watchmaker, 1970). The wavelength which was used in these experiments was 254 nm, however, mammalian seminal plasma contains numerous compounds which interfere with the absorbance readings at that wavelength and have therefore limited the usefulness of this technique. Previous experiments have demonstrated that this obstacle can be overcome by first passing the semen samples through a glass wool column (Paulson and Polakoski, 1977; Atherton, et al., 1977); however, one of the aims of the present research was to minimize the possible variables so as to increase the clinical usefulness of the technique. It was therefore decided that alternate wavelengths, which would have minimal seminal plasma interference, would be investigated. Diluted (1:10 with saline) seminal plasma from 2 vasectomized human donors was each scanned between 250 and 500 nm. The results showed that at 475 nm the absorbance resulting from seminal plasma compounds, cellular debris and nonsperm cells was minimized. A second variable which was examined was the diameter of the flow cell chamber. It was found that more sensitive as well as reliable assays could be obtained with a 1 mm chamber than the previously used 2 mm chamber (Atherton, 1975a). This was presumably the result of decreasing both the turbulence and the formation of air bubbles in the system. In light of the above observations a 1 mm flow chamber at 475 nm was utilized to obtain the presented data. Correlation of the SM! to the Motility of Sperm It has been established that the SM! obtained at 254 nm with sperm from sea urchins (Timourian and Watchmaker, 1970) and humans (Atherton, et al., 1977) is lost in sperm cells that were made nonmotile by heating. These results were confirmed for readings obtained at 475 nm and expanded to show the influence of dead sperm on the SMI (Table 1). The data were obtained by passing a pool of 3 ejaculates through a glass wool column to remove the dead sperm cells. An aliquot was saved and a SMI determined. A second aliquot was heated at 50#{176}for 15 mini to kill all the sperm present and the resulting SMI was 0. These samples were mixed at the various concentrations of dead to live cells with the total cell number remaining constant and the SM! determined. These results show a negative correlation between the SM! and the number of dead sperm cells (r=-0.92). EJfect of Storage on the SM! Two experimental approaches were used to determine the effect storage has on SM!. First a time course study was performed by incubating freshly ejaculated sperm at 23#{176}C and removing aliquots at various time intervals for TABLE 1. Correlation of the sperm cell index (SMI) with progressively motile sperm. % Heat killed sample Human sem en in bakers % Progressive X SMI ± SD % Nonprogressive = ± ± ± ± ± ± Correlation coefficient between SMI and % progressive is Correlation coefficient between SMI and % heat killed cells is

3 626 ATHERTON ET AL. dilution and SM! determination (fresh dilution). After the sample was diluted, the sperm was also evaluated microscopically. A second time course study was performed by initially diluting the semen and determining the and SM! of the specimen at various time intervals (single dilution). The values obtained in these 2 sets of experiments are shown in Table 2. The fresh dilution and single dilution were compared at the same time intervals after the beginning of the experiment. It was found that the SM! of the fresh dilution retained a higher by 2 or 3 times that of the single dilution. This difference was first noted at 15 mm and continued throughout the experimental period. Effect of Sperm Dilution As shown in Table 3, when a particular semen sample was diluted several times in sequence, the SM! of the various dilutions did not change, indicating that the as defined by the present technique was independent of the sperm concentrations utilized. SM! Values Obtained on Semen from Proven Fathers The SMI was compared to various seminal parameters in 8 proven fathers and the results are shown in Table 4. A correlation (r=0.85) was found between the SMI and the percent of progressively motile sperm. Two individuals (1 and 2) had been diagnosed as oligospermic and had exhibitied a delay in achieving fertilization of their respective wives. DISCUSSION This report described the spectrophotometric quantiration of human sperm. In order to overcome interference due to the high absorbance of seminal plasma proteins and cellular debris, a wavelength of 475 nm was used. To reduce turbulence and trapped air bubbles in the flow cell chamber, a 1 mm flow cell chamber was designed. Both of these adaptations increased the sensitivity and reliability of the procedure. Previous studies with sperm from sea urchins (Timourian and Watchmaker, 1970) and roosters (Wall and Boone, 1973) had shown a correlation between the percent of actively motile sperm cells and the SM!. In the present study, using human sperm which exhibited a range from nonmotile to very motile cells, a good correlation (r=0.92) was found between the SM! and progressive sperm. However, this correlation was not seen in a previous study using highly motile bull sperm (Atherton, 1975a). Since this procedure measures the degree of randomness produced by a population of sperm cells, it is possible that other factors such as the velocity of the sperm may also influence the SM!. This could possibly explain why the data in Table 4 show a difference in SM! values of while the progressive sperm differed TABLE 2. Effect of storage and initial dilution on SMI. Time in mm X SMI ± SD % Progressive Fresh dilution ± ± ± ± ± ± 0.68 (r = 0.86) Single dilution ± ± ± ± ± ± 1.06 (r = 0.95)

4 QUANTITATION OF HUMAN SPERM MOTILITY 627 TABLE 3. Effect of dilution on the SMI: human semen in bakers. Concentration of sperm5 drops/mi-dilution Absorbance Cell 0b X 106 /ml X SMI ± SD 15/5-1: ± /5-1: ± /5-1: ± /5-1: ± Onigin sample from which dilutions were made contained 1.78 X 108 /celi. bfin concentration analyzed. by less that 2% in the ejaculates from 2 different fertile donors. If this is the case, it is possible that more information may be obtained from the SM! than from the microscopic analysis and is therefore currently being pursued. During the first 15 mm following a single dilution of semen into buffered physiological saline, no differences were observed when comparisons were made to fresh aliquot dilutions. However, as might be expected, the fresh dilutions of semen after 15 mm maintained at levels higher than that observed in the same aged single diituion. These differences may have resulted from undiluted spermatozoa living longer than diluted spermatozoa. The quantitation of such differences becomes important when proposing experiments hypothesized to alter sperm. For example, if a treatment is not effective in stimulating initial, it may become effective in restoring after a certain percentage decline in has occurred. The ability to quickly monitor sperm as a function of time could also be of considerable importance to the interpretation of physiological measurements on spermatozoa. The method used to calculate the SM! inherently reduces variability that might be due to semen samples with different sperm concentrations; that is, the difference in absorbance is divided by the initial absorbance. However, this calculation alone does not prove that the SMI is independent of the sperm concentration. When sperm concentrations between 2.5 and 14.3 X 106/ml were produced by dilution of the same sample originally containing 178 X 106 cells/mi, the SM! remained constant. The ability of these procedures to quantitate in samples with a few million cells/mi expand the adaptability of the procedure to the clinical setting. For the optical anisotropic technique could be of significant value in comparisons between normospermic and oligospermic individuals. In summary, an inexpensive, simple and rapid method for quantitating human sperm has been developed. The derived TABLE 4. Sperm index (SMI) compared to semen characteristics of 8 proven fathers. sperm index (SM!) correlates well with the percent of live cells, percent of pro- % %Non-.... Ejaculate Progressive Sperm progressive SMI X 106/mI Vol. ph Correlation between SMI and % progressive, r = 0.85.

5 628 ATHERTON ET AL. gressively motile cells and exhibits differences based upon the time of storage of sperm in vitro. Since repetitive and statistically analyzable SMI s can be obtained every minute after a 5 mm preparation time, this procedure appears to be of value to both the clinical and research laboratory. ACKNOWLEDGMENTS Support from the Division of Basic Research of the College of Arts and Sciences at the University of Wyoming is gratefully acknowledged. The authors wish to thank also the Rockefeller Foundation for its support to the Department of Obstetrics-Gynecology, Washington University. The technical assistance of Mr. Fred Jackson and Mr. Gerald Bond is gratefully appreciated. REFERENCES Atherton, R. W. (1975). Neurochemical effects on sperm in Strongylocentrotus purpuratus, the purple sea urchin. Comp. Biochem. Physiol. 50C, Atherton, R. W. (1975a). An objective method for evaluating Angus and Hereford sperm. Int. J. Fertil. 20, Atherton, R. W., E. W. Radany and K. L. Polakoski. (1977). Quantitation of human sperm. (E. S. E. Hafez, ed.) Elsevier/North Holland, Botella-Llusia, J. (1966). Methods for determining the type and the degree of spermatic. In: Proc. 5th World Congress Ferti!.-Stenil., June 16-22, Stockholm, Sweden. (Westin and Wiqvist, eds.) Excerpts Media Fdn., Amsterdan (Nov., 1967). DaRugna, D. (1966). The clinical significance of sperm and its relation to the volume of the ejaculate, the number and morphology of sperm cells. In: Proc. 5th World Congress Fertil.-Steril., June 16-22, Stockholm, Sweden. (Westin and Wiqvist, eds.) Excerpts Medica Fdn., Amsterdam (Nov., 1967). Eliasson, R. and B. Treichl. (1972). Supravital staining of human spermatozoa. Fertil. Steril. 22, w Farris, E. J. (1949). The number of motile spermatozoa and an index of fertility in man: A study of 406 semen specimens. J. Urology 61, MacLeod, J. and R. Z. Gold. (1951). The male factor in fertility and infertility. Ill. An analysis of motile activity in spermatozoa of 1000 fertile men and 1000 men in infertile marriage. Fertil.-Steril. 2, Pauson, J. D. and K. L. Polakoski. (1977). A glass wool column procedure for removing extraneous material from the human ejaculate. Fertil. Steril. 28, Timourian, H. and G. Watchmaker. (1970). Determination of spermatozoon. Develop. Biol. 21, Wall, K. A. and M. A. Boone. (1973). Objective measurement of sperm. Poult. Sci. 52, Walton, A. (1952). Flow orientation as a possible explanation of wave-motion and rheotaxis of spermatozoa. J. Exptl. Bmol. 24, Zaneve!d, L. J. D. and K. L. Polakoski. (1977). Collection and physical examination of the ejaculate. (E. S. E. Hafez, ed.) Elsevier/North Holland, RECOMMENDED REVIEWS Atherton, R. W. (1977). Evaluation of sperm. (E. S. E. Hafez, ed.) E!sevier/North Holland, Mitchell, J. A., L. Nelson and E. S. E. Hafez (1977). Motility of spermatozoa, Chapt. 9, In: Human Semen and Fertility Regulation in Men. (E. S. E. Hafez, ed.) Mosby, St. Louis.