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Volume And Shape Of Normal Human Spermatozoa*
Vol. 28, No.4, April 1977 Printed in U.S.A.
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
VOLUME AND SHAPE OF NORMAL HUMAN SPERMATOZOA*
NERI LAUFER, M.D.t SHMUEL SEGAL, M.D.:!: HAIM YAFFE, M.D.:!: HARRY SVAR'fZj§ N. B. GROVER, PH.D.§
Department of Obstetrics and Gynecology, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel
An improved apparatus for measuring the electrical size ofparticles, developed in this laboratory and based on the principle of the Coulter counter, is used to size human spermatozoa. The typical size distribution is unimodel, with a skew to the right. The actual quantity determined by the measuring system is electrical size (i.e., shape factor x volume); in order to extract the volume, it is necessary to obtain an independent measure of particle shape. This is done by estimating the relative contributions of each part of the spermatozoon, and gives a weighted value for the shape factor of 128. The mean volume ofspermatozoa from 25 normal human seminal fluid specimens is found to be 17.4 ± 1.46 cu pm and the modal volume, 152 ± 127 cu ILm. These values are compared with data reported in the literature after correcting the latter for the effects of particle shape. Zaponin does not affect the cell volume distributions, even when used in high concentrations, provided measurements are carried out within the hour.
The most direct technique for measuring the linear dimensions of human spermatozoa is by microscopic micrometry. 1-3 Ways have been sought to calculate the projected area from simple linear measurements. 4 The limitations of the microscopic method have been widely discussed,5 and stem from the fact that this method is ofnecessity based on a very small sample size. The advent of the Coulter counter provided for the first time a rapid and accurate method for counting and sizing large samples of cells. Using various commercial models of this instrument, Accepted December 10,1976. *Supported in part by a grant from the Joint Research Fund of the Hebrew University and Hadassah. tReprint requests: Neri Laufer, M.D., Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel. :j:Department of Obstetrics and Gynecology, Hadassah University Hospital. §Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School.
different investigators have found widely diverging values for the size of normal human sperm: Segal and Laurence 6 reported a range of 28.0 to 78.4 cu /-tm and a mean of 56.0 cu /-tm; Gordon et al.,7 a range of 10 to 22 cu /-tm and a mean of 15 cu /-t m ; and Brotherton and Barnard,8-10 a range of 17.8 to 23.3 cu /-tm, a mean of 25.6 cu /-tm, and a mode of 19.3 cu /-tm. It was demonstrated some time ago l l that the commercially available Coulter counter does not determine volume accurately, nor does it distinguish between actual volume changes and apparent volume changes which are in fact due to particle shape. Using a modified system designed to measure these parameters correctly and precisely,12 we have investigated normal human sperm size and shape. MATERIALS AND METHODS
Seminal fluid from 25 apparently healthy volunteers was collected after masturbation into clean 456
457
VOLUME AND SHAPE OF HUMAN SPERMATOZOA
Vol. 28, No.4
glass bottles. The period of abstinence was 3 to 4 days. The specimens were examined microscopically immediately after liquefaction, and a hemocytometer count was performed following dilution of 1:10. Size distribution was recorded within 2 hours of collection. The suspending medium was Isoton (Coulter Electronics Ltd.), to which about three drops of Zaponin (Coulter Electronics Ltd.) per 15 ml were added a few minutes prior to measurement. The dilution of semen in the suspending medium was approximately 1:200, and about 105 cells were studied in each sample.
FIG. 1. Oscillogram produced by a spermatozoon during its passage through the orifice of a Coulter-type transducer. (Sweep: 5 ILsecondsJdivision.)
The size distribution of each sample was obtained with an electric sizing apparatus based on the Coulter counter and developed by Grover et al. 12 An oscilloscope was used to monitor the pulses generated by the spermatozoa after they had entered the orifice. The stability of the experimental system was checked daily by samples of erythrocytes fixed in glutaraldehyde; absolute calibration was performed as described previously,12 The parameters of the measuring apparatus were selected so as to reduce background noise to a minimum while still remaining within the permissible range of values dictated by theoretical considerations. 13 The optimal conditions were found to be a current of 181 /-tA and a sensitivity of 1314 channels//-tA for an orifice 30 /-tm in diameter by 60 /-tm in length (nominal dimensions); the flow rate was 4 mlsecond.
factor'Y x volume V); in order to extract the volume, it is thus necessary to obtain an independent measure of 'Y. This was done by estimating the relative contribution to cell volume of each part of the spermatozoon, based on the linear measurements made by Van Duijn 4 : head section, 45%; midpiece, 17%; and tail, 38%. The shape factor for each part was then calculated from these same linear dimensions by assuming ideal geometry14: 1.25 for the head, 2.0 for the midpiece, and 1.0 for the tail. Finally, the effective shape factor 'Y for the whole sperm cell was obtained as 0.45 x 1.25 + 0.17 x 2.0 + 0.38 x 1.0, or 1.28. From the mean electrical size of 22.3 ± 1.8 cu /-tm, we can now calculate the mean volume V to be 17.4 ± 1.46 cu /-tm; similarly, the model volume is 15.2 ± 1.27 cu /-tm.
RESULTS
DISCUSSION
The mean volume of 25 semen specimens was 3.8 ml with a standard deviation of 0.3 ml (range, 3.0 to 5.5 ml); the mean sperm count was 101 ± 8 x 106 cells (range, 60 to 150 x 106 ). A photograph of the oscilloscope trace produced by a sperm cell during its passage through the orifice is shown in Figure 1. The predominance of such flat, tableshaped pulses indicates that the particles do not rotate within the orifice but rather maintain a constant shape factorY No difference was observed in the form of the signals generated by untreated cells and by cells treated with Zaponin, even at high concentrations (15 to 20 drops/15 ml of Isoton), if measured within 1 hour. A typical size-distribution curve of normal human sperm cells shows adequate separation between background noise and the true data (Fig. 2). The distribution is unimodal, with a skew to the right. The actual quantity determined by the measuring system is electrical size (i.e., shape
Van Duijn1 has carried out elaborate measurements on the dimensions of human spermatozoa under the light microscope. By investigating scale models, this author derived empirical expressions for the volumes of the different parts of the spermatozoon and estimated the over-all modal volume to be 13 to 16.5 cu /-tm for the living cell in its own seminal fluid. The main disadvan810
S x
(!i8
~6
•
II:: UJ
5;4
~2 II:: UJ III
~°7.10::--::15;-::'20::-::2-=-5-:3~0-:3:!:"5-4""'0-4"'5"--':50~55~60~6-5-7~0-7~5-80~8~5-90~9~5~100 CHANNEL NUMBER
FIG. 2. Size-distribution histogram of normal human spermatozoa.
458
LAUFER ET AL.
tage of this approach lies in the smallness of the sample on which it is based. Use of the Coulter counter avoids this problem. Unfortunately, it gives rise to another: size as measured by a Coulter counter-type transducer is a function of both the volume of the particle and its shape. We have attempted to obtain an independent estimate of effective sperm cell shape by calculating the geometric shape factor for each of the different parts of a spermatozoon and weighting these factors by the relative contribution of the part to the total volume. Our result of 1.28 is probably quite near the true value, despite the necessity of considering each section as an idealized ellipsoid, because shape factors for prolate-like bodies are rather insensitive to the precise dimensions of the particle and because the entire calculation is independent of any systematic errors of measurement in the form of proportionality constants. All of the values of sperm cell volume reported in the literature thus far are based on instrument calibration with standard spherical latex or pollen particles, the nonspherical shape of the spermatowon having been ignored. Since a sphere has a shape factor of 1.5,11 this implies an underestimation of 17%. If we now correct those data, we find that our values are virtually identical with those of Gordon et aJ.7 (corrected mean, 17.6 cu JLm) but considerably below those obtained by Brotherton and Barnard8 -1o (30.0 cu JLm mean and 22.6 cu JLm mode, corrected); the agreement with the geometric measurements of Van Duijn 1 is excellent. Segal and Laurence's results 6 are much too high, probably due to inadequate calibration of the Coulter counter: only one standard particle was used, and such a procedure has been shown to be capable of producing large errors. 15 The positively-skewed volume distribution reported here has been observed before, and it has been suggested that it may be due to a difference in the sizes ofthe X- and Y-bearing spermatozoa; such differences, however, have proved to be insignificant. 16 The presence offully diploid spermatozoa has also been advanced as a likely explanation. 9 Another possibility is that all of the cells
April 1977 involved in spermatogenesis are ejaculated; if, as in other cell types, size decreases with age, then one would expect a positively-skewed distribution to result. Much additional work will be necessary before this question can be answered with any measure of confidence. REFERENCES 1. Van Duijn C Jr: Biometry of human spermatozoa. J R Microse Soe 77:12, 1957 2. Van Duijn C Jr, Van Voorst C: Precision measurements of dimensions, refractive index and mass of bull spermatozoa in the living state. Mikroskopie 27:142, 1971 3. Van Duijn C Jr, Van Voorst C, Hellinga G: Precision measurements of dimensions, shape, and mass density of spermatozoan heads in normal and subfertile human males. Eur J Obstet Gynecol 2:37, 1972 4. Van Duijn C Jr: Mensuration of spermatozoa. Bibl Reprod 25:121, 1975 5. Adams RB, Gregg EC: Pulse shapes from particles traversing Coulter orifice fields. Phys Med Biol17:830, 1972 6. Segal SJ, Laurence KA: Automatic analysis of particulate matter in human ejaculates. Ann NY Acad Sci 99: 271,1962 7. Gordon DL, Moore DJ, Thorshund T, Paulsen CA: The determination of size and concentration of human sperm with an electronic particle counter. J Lab Clin Med 65: 506, 1965 8. Brotherton J: Estimation of number, mean size and size distribution of human spermatozoa using a Coulter counter (abstr). J Reprod Fertil 35:626, 1973 9. Brotherton J, Barnard G: Estimation of number, mean size and size distribution of human spermatozoa in oligospermia using a Coulter counter. J Reprod Fertil 40: 341, 1974 . 10. Brotherton J: The counting and sizing of spermatozoa from ten animal species using a Coulter counter. Andrologia 7:169, 1975 11. Grover NB, Naaman J, Ben-Sasson S, Doljanski F: Electrical sizing of particles in suspensions. I. Theory. Biophys J 9:1398, 1969 12. Grover NB, Naaman J, Ben-Sasson S, Doljanski F, Nadav E: Electrical sizing of particles in suspensions. II. Experiments with rigid spheres. Biophys J 9:1415, 1969 13. Grover NB, Naaman J, Ben-Sasson S, Doljanski F: Electrical sizing of particles in suspensions. III. Rigid spheroids and red blood cells. Biophys J 12:1099, 1972 14. Velick S, Gorin M: The electrical conductance of suspensions of ellipsoids and its relation to the study of avian erythrocytes. J Gen Physiol23:753, 1940 15. Brotherton J: Calibration of the Coulter counter model A for the size determination of cells. Proc Soc Anal Chern 8: 264, 1971 16. Van Duijn C Jr: Size frequency distribution in spermatozoa. Fertil Steril 12:509, 1961
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
VOLUME AND SHAPE OF NORMAL HUMAN SPERMATOZOA*
NERI LAUFER, M.D.t SHMUEL SEGAL, M.D.:!: HAIM YAFFE, M.D.:!: HARRY SVAR'fZj§ N. B. GROVER, PH.D.§
Department of Obstetrics and Gynecology, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel
An improved apparatus for measuring the electrical size ofparticles, developed in this laboratory and based on the principle of the Coulter counter, is used to size human spermatozoa. The typical size distribution is unimodel, with a skew to the right. The actual quantity determined by the measuring system is electrical size (i.e., shape factor x volume); in order to extract the volume, it is necessary to obtain an independent measure of particle shape. This is done by estimating the relative contributions of each part of the spermatozoon, and gives a weighted value for the shape factor of 128. The mean volume ofspermatozoa from 25 normal human seminal fluid specimens is found to be 17.4 ± 1.46 cu pm and the modal volume, 152 ± 127 cu ILm. These values are compared with data reported in the literature after correcting the latter for the effects of particle shape. Zaponin does not affect the cell volume distributions, even when used in high concentrations, provided measurements are carried out within the hour.
The most direct technique for measuring the linear dimensions of human spermatozoa is by microscopic micrometry. 1-3 Ways have been sought to calculate the projected area from simple linear measurements. 4 The limitations of the microscopic method have been widely discussed,5 and stem from the fact that this method is ofnecessity based on a very small sample size. The advent of the Coulter counter provided for the first time a rapid and accurate method for counting and sizing large samples of cells. Using various commercial models of this instrument, Accepted December 10,1976. *Supported in part by a grant from the Joint Research Fund of the Hebrew University and Hadassah. tReprint requests: Neri Laufer, M.D., Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel. :j:Department of Obstetrics and Gynecology, Hadassah University Hospital. §Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School.
different investigators have found widely diverging values for the size of normal human sperm: Segal and Laurence 6 reported a range of 28.0 to 78.4 cu /-tm and a mean of 56.0 cu /-tm; Gordon et al.,7 a range of 10 to 22 cu /-tm and a mean of 15 cu /-t m ; and Brotherton and Barnard,8-10 a range of 17.8 to 23.3 cu /-tm, a mean of 25.6 cu /-tm, and a mode of 19.3 cu /-tm. It was demonstrated some time ago l l that the commercially available Coulter counter does not determine volume accurately, nor does it distinguish between actual volume changes and apparent volume changes which are in fact due to particle shape. Using a modified system designed to measure these parameters correctly and precisely,12 we have investigated normal human sperm size and shape. MATERIALS AND METHODS
Seminal fluid from 25 apparently healthy volunteers was collected after masturbation into clean 456
457
VOLUME AND SHAPE OF HUMAN SPERMATOZOA
Vol. 28, No.4
glass bottles. The period of abstinence was 3 to 4 days. The specimens were examined microscopically immediately after liquefaction, and a hemocytometer count was performed following dilution of 1:10. Size distribution was recorded within 2 hours of collection. The suspending medium was Isoton (Coulter Electronics Ltd.), to which about three drops of Zaponin (Coulter Electronics Ltd.) per 15 ml were added a few minutes prior to measurement. The dilution of semen in the suspending medium was approximately 1:200, and about 105 cells were studied in each sample.
FIG. 1. Oscillogram produced by a spermatozoon during its passage through the orifice of a Coulter-type transducer. (Sweep: 5 ILsecondsJdivision.)
The size distribution of each sample was obtained with an electric sizing apparatus based on the Coulter counter and developed by Grover et al. 12 An oscilloscope was used to monitor the pulses generated by the spermatozoa after they had entered the orifice. The stability of the experimental system was checked daily by samples of erythrocytes fixed in glutaraldehyde; absolute calibration was performed as described previously,12 The parameters of the measuring apparatus were selected so as to reduce background noise to a minimum while still remaining within the permissible range of values dictated by theoretical considerations. 13 The optimal conditions were found to be a current of 181 /-tA and a sensitivity of 1314 channels//-tA for an orifice 30 /-tm in diameter by 60 /-tm in length (nominal dimensions); the flow rate was 4 mlsecond.
factor'Y x volume V); in order to extract the volume, it is thus necessary to obtain an independent measure of 'Y. This was done by estimating the relative contribution to cell volume of each part of the spermatozoon, based on the linear measurements made by Van Duijn 4 : head section, 45%; midpiece, 17%; and tail, 38%. The shape factor for each part was then calculated from these same linear dimensions by assuming ideal geometry14: 1.25 for the head, 2.0 for the midpiece, and 1.0 for the tail. Finally, the effective shape factor 'Y for the whole sperm cell was obtained as 0.45 x 1.25 + 0.17 x 2.0 + 0.38 x 1.0, or 1.28. From the mean electrical size of 22.3 ± 1.8 cu /-tm, we can now calculate the mean volume V to be 17.4 ± 1.46 cu /-tm; similarly, the model volume is 15.2 ± 1.27 cu /-tm.
RESULTS
DISCUSSION
The mean volume of 25 semen specimens was 3.8 ml with a standard deviation of 0.3 ml (range, 3.0 to 5.5 ml); the mean sperm count was 101 ± 8 x 106 cells (range, 60 to 150 x 106 ). A photograph of the oscilloscope trace produced by a sperm cell during its passage through the orifice is shown in Figure 1. The predominance of such flat, tableshaped pulses indicates that the particles do not rotate within the orifice but rather maintain a constant shape factorY No difference was observed in the form of the signals generated by untreated cells and by cells treated with Zaponin, even at high concentrations (15 to 20 drops/15 ml of Isoton), if measured within 1 hour. A typical size-distribution curve of normal human sperm cells shows adequate separation between background noise and the true data (Fig. 2). The distribution is unimodal, with a skew to the right. The actual quantity determined by the measuring system is electrical size (i.e., shape
Van Duijn1 has carried out elaborate measurements on the dimensions of human spermatozoa under the light microscope. By investigating scale models, this author derived empirical expressions for the volumes of the different parts of the spermatozoon and estimated the over-all modal volume to be 13 to 16.5 cu /-tm for the living cell in its own seminal fluid. The main disadvan810
S x
(!i8
~6
•
II:: UJ
5;4
~2 II:: UJ III
~°7.10::--::15;-::'20::-::2-=-5-:3~0-:3:!:"5-4""'0-4"'5"--':50~55~60~6-5-7~0-7~5-80~8~5-90~9~5~100 CHANNEL NUMBER
FIG. 2. Size-distribution histogram of normal human spermatozoa.
458
LAUFER ET AL.
tage of this approach lies in the smallness of the sample on which it is based. Use of the Coulter counter avoids this problem. Unfortunately, it gives rise to another: size as measured by a Coulter counter-type transducer is a function of both the volume of the particle and its shape. We have attempted to obtain an independent estimate of effective sperm cell shape by calculating the geometric shape factor for each of the different parts of a spermatozoon and weighting these factors by the relative contribution of the part to the total volume. Our result of 1.28 is probably quite near the true value, despite the necessity of considering each section as an idealized ellipsoid, because shape factors for prolate-like bodies are rather insensitive to the precise dimensions of the particle and because the entire calculation is independent of any systematic errors of measurement in the form of proportionality constants. All of the values of sperm cell volume reported in the literature thus far are based on instrument calibration with standard spherical latex or pollen particles, the nonspherical shape of the spermatowon having been ignored. Since a sphere has a shape factor of 1.5,11 this implies an underestimation of 17%. If we now correct those data, we find that our values are virtually identical with those of Gordon et aJ.7 (corrected mean, 17.6 cu JLm) but considerably below those obtained by Brotherton and Barnard8 -1o (30.0 cu JLm mean and 22.6 cu JLm mode, corrected); the agreement with the geometric measurements of Van Duijn 1 is excellent. Segal and Laurence's results 6 are much too high, probably due to inadequate calibration of the Coulter counter: only one standard particle was used, and such a procedure has been shown to be capable of producing large errors. 15 The positively-skewed volume distribution reported here has been observed before, and it has been suggested that it may be due to a difference in the sizes ofthe X- and Y-bearing spermatozoa; such differences, however, have proved to be insignificant. 16 The presence offully diploid spermatozoa has also been advanced as a likely explanation. 9 Another possibility is that all of the cells
April 1977 involved in spermatogenesis are ejaculated; if, as in other cell types, size decreases with age, then one would expect a positively-skewed distribution to result. Much additional work will be necessary before this question can be answered with any measure of confidence. REFERENCES 1. Van Duijn C Jr: Biometry of human spermatozoa. J R Microse Soe 77:12, 1957 2. Van Duijn C Jr, Van Voorst C: Precision measurements of dimensions, refractive index and mass of bull spermatozoa in the living state. Mikroskopie 27:142, 1971 3. Van Duijn C Jr, Van Voorst C, Hellinga G: Precision measurements of dimensions, shape, and mass density of spermatozoan heads in normal and subfertile human males. Eur J Obstet Gynecol 2:37, 1972 4. Van Duijn C Jr: Mensuration of spermatozoa. Bibl Reprod 25:121, 1975 5. Adams RB, Gregg EC: Pulse shapes from particles traversing Coulter orifice fields. Phys Med Biol17:830, 1972 6. Segal SJ, Laurence KA: Automatic analysis of particulate matter in human ejaculates. Ann NY Acad Sci 99: 271,1962 7. Gordon DL, Moore DJ, Thorshund T, Paulsen CA: The determination of size and concentration of human sperm with an electronic particle counter. J Lab Clin Med 65: 506, 1965 8. Brotherton J: Estimation of number, mean size and size distribution of human spermatozoa using a Coulter counter (abstr). J Reprod Fertil 35:626, 1973 9. Brotherton J, Barnard G: Estimation of number, mean size and size distribution of human spermatozoa in oligospermia using a Coulter counter. J Reprod Fertil 40: 341, 1974 . 10. Brotherton J: The counting and sizing of spermatozoa from ten animal species using a Coulter counter. Andrologia 7:169, 1975 11. Grover NB, Naaman J, Ben-Sasson S, Doljanski F: Electrical sizing of particles in suspensions. I. Theory. Biophys J 9:1398, 1969 12. Grover NB, Naaman J, Ben-Sasson S, Doljanski F, Nadav E: Electrical sizing of particles in suspensions. II. Experiments with rigid spheres. Biophys J 9:1415, 1969 13. Grover NB, Naaman J, Ben-Sasson S, Doljanski F: Electrical sizing of particles in suspensions. III. Rigid spheroids and red blood cells. Biophys J 12:1099, 1972 14. Velick S, Gorin M: The electrical conductance of suspensions of ellipsoids and its relation to the study of avian erythrocytes. J Gen Physiol23:753, 1940 15. Brotherton J: Calibration of the Coulter counter model A for the size determination of cells. Proc Soc Anal Chern 8: 264, 1971 16. Van Duijn C Jr: Size frequency distribution in spermatozoa. Fertil Steril 12:509, 1961