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Size-Frequency Distributions in Spermatozoa
Size-Frequency Distributions in Spermatozoa C. VAN DUIJN, JR., CHEM.E., A.M. TECH. I. (GT. BRIT.), F.R.M.S., M.I.P.T., M.I.BIOL.
IN
Shettles27 claims to have demonstrated in preparations of human spermatozoa two populations, distinguished by size and shape of the nuclei and heads, by chromosomal arrangements, and by other characteristics. These assertions are offered as if they were proved facts, referring only to Shettles's preceding papers,2426 and omitting any mention of the fact that these claims have been refuted by Rothschild, Bishop, MacLeod, and van Duijn. 6 It has been shown that the images interpreted as "chromosomes" by Shettles are no more than optical artefacts resulting from failing to observe the principles of critical microscopy and especially of those of phasecontrast microscopy. Types of spurious images similar to those obtained by Shettles have been discussed extenSively by Oettle. 19 , 20. 21 Whereas, according to Shettles,24,25 round-headed spermatozoa should be female and show a globular X chromosome, and elongated types should contain a rod-shaped Y chromosome, no such differentiation is known in cytogenetics. 10,]8 Globular or round chromosomes do not exist in the human species, and the X chromosome is about 3-4 times larger than the ylO and ranks approximately between the sixth and seventh autosomal pairs in descending order of size. 16 The Y chromosome is one of the 5 smallest acrocentrics in the male complement. 16 Further, Shettles's published photographs do not in the least resemble any known human chromosomal pattern. Thus, even if the observed images were not spurious, as indeed they are, the interpretation would still have been at fault and in conflict with all available knowledge concerning the morphologic pattern of human chromosomes. With respect to Shettles's claims 25 -28 concerning the presence of two distinct populations of different shape and size, it has already been emphasized that this should require presentation of bimodal size-frequency distributions with mathematical proof of the significance of data. 6, 22 The only "evidence" given is an unsubstantiated statement: "A definite bimodal distribution of head lengths within a given specimen was found."27 The only real material A RECENT PAPER
From the Biophysics Department, Unit of Reproductive Physiology, Research Institute for Animal Husbandry, "Schoonoord," Hoogt 10, Utrecht, Netherlands.
509
510
VAN DUIJN
FERTILITY
&
STERILITY
offered by Shettles is contained in his Fig. 1 and I shall demonstrate below that the data that can be obtained from it disprove Shettles's assertions as to the existence of any bimodal distribution in his material. Magnification calibration in Shettles's material is extremely in error. '0
N 5
HEAD LENGTH
Fig. 1. Histiograms of size and shape distributions obtained by measurement of the spermatozoa presented in Shettles's Fig. 1, showing that no bimodal distribution exists in size (L, length; Bma " maximum width; and area) or in shape (defined by LI Bmax). The distribution presented here for shape is definitely skew and conforms with the logarithmic distributions found in my investigations, whereas the distribution of area appears to be gaussian. Both are unimodal and homogeneous.
Whereas thus far all authorities agree that the mean head length of normal human spermatozoa is in the range 4-5 jJ-, Shettles's photographs indicate sizes of no less than 30 jJ-,24 of 30-50 jJ-,25, 2'6 and of 11-19 jJ-.27 Consequently, no absolute sizes can be obtained from his photographs, but at least relative measurements can be taken. My Fig. 1 shows the graphic representation of the biometric data derived from Shettles's photomicrograph (his Fig. 1, referred to above), giving the size frequencies for the breadth and length of the head, for the ratio of length to breadth (indicating shape), and for total head area. Length and breadth were measured on the photograph in millimeters, and the total head area was calculated by the formula:
A
= 0.73 LBmax
derived in previous investigations. 4 ,5 None of these distributions is bimodal and their shape and spread are in complete agreement with biometric data obtained in other investigations. 11 -14 , 17, 23, 31 Thus Shettles is contradicted by his own material. Shettles's statement: "The least variable criterion in measurement was found to be the head length"27 is also refuted, since the standard deviation for the head lengths in his photograph is 12.5 per cent as compared with 12.0 per cent for their maximum width. These figures do not differ significantly. The standard deviations for the ratio of length to breadth and area, are respectively 16 and 19 per cent. Really bimodal size distributions have never been found, either in normal
VOL.
12, No.6, 1961
511
SPERM FORM
spermatozoa or in pathologic specimens from men,ll-14,17 bulls 5 ,31 or boars. 7 Figure 2 gives the size distribution curve for head length of human spermatozoa obtained in my own investigations. Since it might be expected that nuclear size would be more relevant than total head size, an example of 0.15,..--------:":'--------------,
Fig. 2. Normalized size-frequency distribution curve of >head length of living human Z 0.10 spermatozoa, measured by ~ W direct microscopic microm- ...a: etry with an absolute accuracy w per measurement of 0.2 p.. ;0.05 Complete absence of any W-' bimodality is clearly demon- a: strated. N = 1500. (J
I.&J
.
[-
nuclear length distribution is also presented (Fig. 3). All material was obtained from donors of proved high fertility (at least 3 pregnancies, each produced within 3 months of unprotected coitus) and all measurements were taken in the living state by direct microscopic micrometry, using the Baker cross-system, phase-contrast, oil-immersion microscope objective 100, N.A. 1.30, combined with a X 30 compensating micrometer eyepiece, for total head size. The Baker interference microscope and ordinary transmitted illumination were used with vitally stained specimens for determining total head size and nuclear size. The results obtained with these different methods were in complete agreement. The curves (Fig. 2 and Fig. 3) show complete absence of any bimodality and both follow a mathematic equation for logarithmic distributions: N = Nmax expo
[-k( In ~m)
2 ]
where N is the absolute number of spermatozoa of size class L; N max, top value of the distribution curve; and Lm, modal value. Fig. 3. Normalized size frequency distribution curve of nuclear length of human spermatozoa, obtained by direct microscopic micrometry in the living state. Pooled data obtained from measurements with the interference microscope and of vitally stained specimen with ordinary transmitted illumination, pooling having been done only after the analysis of separate data obtained in previous investigations had shown mathematical identity of the results. N 766.
=
0.30 >u
z
UJ
50 .20 UJ
...a:
0.10
o~~~~~~~~~~~-J
1.5
2.0
2.5
NUCLEAR
3.0
3.5
LENGTH
4.0
4.5 5.0 5.5
(MICRONS)
512
VAN DUIJN
FERTILITY
&
STERILITY
A further check on the homogeneity of the distributions is obtained by a straight-line transformation,4 resulting if Vlog (NmaxIN) is plotted against -+- log (LI Lm ). With normal (in the biological sense) material this transformation produces an unbroken straight line. * However, with certain types of pathologic material (infertility cases) the slope constants for the righthand and left-hand sides of the distribution may be different, or one of the lines appears broken (two different slopes with a sudden break). Thus far, this has only been observed in bull sperm. 8 Figure 4 gives the straight-line transformation for the nuclear length
1.5
Fig. 4. Straight-line transformation of the data of the size distribution curve of nuclear length of Fig. 3, showing complete homogeneity and conforming to equations for logarithmic distribution, thereby proving that it has not been produced from two distinctly different populations .
1.0
0.5
•
±logL+logL m
oif---~--~--~~~~--~--~--~
0.10
0.20
0.30
distribution curve (Fig. 3), demonstrating complete homogeneity and proving that it has not been produced by two distinctly different populations. Similar results have been obtained for total head length, head width, nuclear breadth and other parameters in human, bovine and boar sperm atozoa.4, 5,7, 8 Only a few remarks need be given with respect to Shettles's strange ideas about cytochemistry.27 From a chemical point of view it must remain a riddle why one should want to add oil of anise to spermatozoa, and what mysterious "feeling" it is which tells Shettles that this liquid should be a reagent to whatever substance. Oil of anise is a natural product, containing 80-90% anethole, which is almost insoluble in water, and methylchavicol and anisaldehyde, also very slightly soluble in water. The only "reaction" occurring when such a substance is mixed with a protein solution such as seminal plasma, is some emulsification, in which oil droplets may be adsorbed to any surface of suspended particles, a purely physical process that has nothing at all to do with a chemical test. * For size distributions conforming to the Gauss equation, the corresponding transformation is obtained by plotting I L - Lm I on the abscissa instead of ± log (L/Lm).
-
VOL.
12, No.6, 1961
SPERM FORM
513
In extensive cytomicrochemical investigations, no evidence for the existence of cytochemical differences between two types of spermatozoa has been found. 3
DISCUSSION
From the foregoing argument it is evident that claims concerning the existence of any bimodal size distribution in spermatozoa cannot be accepted. However, it may be enlightening to discuss briefly whether it is reasonable to expect shape and size variations to correlate with the presence of an X or Y chromosome. The haploid number of chromosomes being 23, and the Y chromosome being conSiderably smaller than the X,l, 10, 16, 18 and not as assumed by Shettles,24-26 the maximum size difference between the two types which may be expected is 1/23, or slightly less than 4.5 per cent. This upper estimate will be reached only if it is assumed that the total nuclear size is exactly proportional to the DNA content and that the over-all head size is strictly proportional to nuclear size, which is highly improbable, the nucleo-plasmatic ratio actually being not constant. 4 Furthermore, in order to get a statistical difference, the DNA content of each type should be constant within significantly smaller limits than the difference between the types; otherwise, no differentiation is possible. Although we know that these conditions are not fulfilled,12, 14 it is valuable to calculate the size difference the presumptions would imply. The results come to 0.2 f.L for the modal head length and 0.1 f.L for the nuclear length. This clearly demonstrates that it is utterly impossible to detect any size difference between X and Y chromosome-bearing spermatozoa by any system of light microscopy. As to direct identification of X and Y chromosomes in spermatozoa, this is prevented by the special condition of the nucleoprotein material in the sperm nucleus, which has been shown to be not only of the compact karyosome type, but, even in a crystalline state, comparable to that of crystallized virus desoxyribonucleoprotein, as found by X-ray diffraction analysis. so This special condition also makes it immune to attack by desoxyribonuclease. 29 SUMMARY
Size-frequency distributions of spermatozoa are shown to be unimodal and to follow an equation for a homogeneous distribution in material from biologically normal individuals. Claims to the contrary made by Shettles are proved to be untrue, using his own published material. It is shown by calculation that detection of any size difference between X and Y chromo-
514
VAN DUIJN
FERTILITY
&
STERILITY
some-bearing spermatozoa by any system of light microscopy is fundamentally impossible. Research Institute for Animal Husbandry "Schoonoord," Hoogt 10, Utrecht, Netherlands
REFERENCES 1. BAIKIE, A. G., COURT-BROWN, W. M., BUCKTON, KARIN, E., HARNDEN, D. G., JACOBS, P. A., and TOUGH, M. A possible specific chromosome abnormality in human chronic myeloid leukemia. Nature 188:1165, 1960. 2. BISHOP, D. W. X and Y spermatozoa. Nature 187:255, 1960. 3. DUlJN, C. VAN, JR. Cytomicrochemistry of human spermatozoa. /. Roy. Micr. Soc. 74:69, 1954. 4. DUIJN, C. VAN, JR. Biometry of human spermatozoa. J. Roy. Micr. Soc. 77:12, 1957. 5. DUlJN, C. VAN, JR. Mensuration of the heads of bull spermatozoa. Mikroskopie 14:265, 1960. 6. DUIJN, C. VAN, JR. Nuclear structure of human spermatozoa. Nature 188:916, 1960. 7. DUlJN, C. VAN, JR. Mensuration of the heads of boar spermatozoa. Mikroskopie 15:142, 1961. 8. DUlJN, C. VAN, JR. Unpublished data. 9. FORD, C. E., and JACOBS, P. A. Human somatic chromosomes. Nature 181:1565, 1958. 10. KOLLER, P. C. The sex chromosomes. In Clinical Genetics, ed. by A. Sorsby. Mosby, St. Louis, 1953. 11. LEUCHTENBERGER, C., SCHRADER, F., WEIR, D. R., and GENTILE, D. P. The desoxyribose nucleic acid (DNA) content in spermatozoa of fertile and infertile human males. Chromosoma 6:61, 1953. 12. LEUCHTENBERGER, C., WEIR, D. R., SCHRADER, F., and MURMANIS, D. P. The desoxyribose nucleic acid (DNA) content in spermatozoa of repeated seminal fluids from fertile and infertile men. /. Lab. & Clin. Med. 45:851, 1955. 13. LEUCHTENBERGER, C., MURMANIS, 1., MURMANIS, L., lTo, S., and WEIR, D. R. Interferometric dry mass and microspectrophotometric arginine determinations on bull sperm nuclei with normal and abnormal DNA content. Chromosoma 8:73, 1956. 14. LEUCHTENBERGER, C., and LEUCHTENBERGER, R. Die Desoxyribonucleoproteide im Siiugetiersperma: Eine quantitative Studie an menschlichen und Stierspermatozoen mittels Mikrospektrophotometrie und Interferenzmikroskopie. Hoppe-Seyler's Ztschr. physiol. Chemie 313: 130, 1958. 15. MACLEOD, J. Personal communication, 1961. 16. MAKINO, S., and MOTOMICHI, S. A study of the somatic chromosomes in the Japanese. Jap. J. Genetics 35:228,1960. 17. MOENCH, G. L., and HOLT, H. Biometrical studies of head length of human spermatozoa. /. Lab. & CUn. Med. 17:297, 1932. 18. NATURAL HISTORY MUSEUM, BRITISH MUSEUM, London. Display of human chromosomes. In the exhibition Evolution and Genetics, 1960. 19. OETTLE, A. G. Experiments with a variable amplitude and phase microscope. /. Roy. Micr. Soc. 70:232, 1950. 20. OETTLE, A. G. "Optical membranes": A common artefact. J. Roy. Micr. Soc. 70:255, 1950.
VOL.
12, No.6, 1961
SPERM FORM
515
21. OETTLE, A. C. Personal communication, 1961. 22. LORD ROTHSCHILD. X and Y spermatozoa. Nature 187:254,1960. 23. SAVAGE, A., WILLIAMS, W. W., and FOWLER, N. M. A statistical study of the head length variability of bovine spermatozoa and its application to the determination of fertility. Proc. & Tr. Roy. Soc. Canada 21 :425, 1927. 24. SHETTLES, L. B. Nuclear morphology of human spermatozoa. Nature 186:648, 1960. 25. SHETTLES, L. B. X and Y spermatozoa. Nature 187:254, 1960. 26. SHETTLES, L. B. Nuclear structure of human spermatozoa. Nature 188:918, 1960. 27. SHETI'LES, L. B. Differences in human spermatozoa. Fertil. & Steril. 12:20, 1961. 28. SHETTLES, L. B. Human spermatozoan shapes in relation to sex ratios. Fertil. & Steril. 12:502, 1961. 29. TOR6, I., and P6SALAKY, Z. Histochemische Untersuchung der Spermiogenese. Acta histochem., lena 8:393, 1959. 30. WILKINS, M. H. F., and RANDALL, J. T. Crystallinity in sperm heads: Molecular structure of nucleoprotein in vivo. Biochim. biophys. acta 10:192, 1953. 31. WILLIAMS, W. W., and SAVAGE, A. Observations on the seminal micropathology of bulls. Cornell Vet. 15:353, 1925.
Sociedad Peruana de Fertilidad Matrimonial Officers of the Society, for 1961-1962, are as follows: President Vice President Dr. Rafael de la Puente L. Dr. Javier Hoyle Cox Secy. General Recording Secy. Dr. Roberto Ruiz Gonzalez Dr. Julio Munoz Valdivieso Treasurer Dr. Noe Ramirez Zapata Executive Member Executive Member Dr. Alfonso Zamorano D.D. Dr. Abraham Ludmir Grimberg
IN
Shettles27 claims to have demonstrated in preparations of human spermatozoa two populations, distinguished by size and shape of the nuclei and heads, by chromosomal arrangements, and by other characteristics. These assertions are offered as if they were proved facts, referring only to Shettles's preceding papers,2426 and omitting any mention of the fact that these claims have been refuted by Rothschild, Bishop, MacLeod, and van Duijn. 6 It has been shown that the images interpreted as "chromosomes" by Shettles are no more than optical artefacts resulting from failing to observe the principles of critical microscopy and especially of those of phasecontrast microscopy. Types of spurious images similar to those obtained by Shettles have been discussed extenSively by Oettle. 19 , 20. 21 Whereas, according to Shettles,24,25 round-headed spermatozoa should be female and show a globular X chromosome, and elongated types should contain a rod-shaped Y chromosome, no such differentiation is known in cytogenetics. 10,]8 Globular or round chromosomes do not exist in the human species, and the X chromosome is about 3-4 times larger than the ylO and ranks approximately between the sixth and seventh autosomal pairs in descending order of size. 16 The Y chromosome is one of the 5 smallest acrocentrics in the male complement. 16 Further, Shettles's published photographs do not in the least resemble any known human chromosomal pattern. Thus, even if the observed images were not spurious, as indeed they are, the interpretation would still have been at fault and in conflict with all available knowledge concerning the morphologic pattern of human chromosomes. With respect to Shettles's claims 25 -28 concerning the presence of two distinct populations of different shape and size, it has already been emphasized that this should require presentation of bimodal size-frequency distributions with mathematical proof of the significance of data. 6, 22 The only "evidence" given is an unsubstantiated statement: "A definite bimodal distribution of head lengths within a given specimen was found."27 The only real material A RECENT PAPER
From the Biophysics Department, Unit of Reproductive Physiology, Research Institute for Animal Husbandry, "Schoonoord," Hoogt 10, Utrecht, Netherlands.
509
510
VAN DUIJN
FERTILITY
&
STERILITY
offered by Shettles is contained in his Fig. 1 and I shall demonstrate below that the data that can be obtained from it disprove Shettles's assertions as to the existence of any bimodal distribution in his material. Magnification calibration in Shettles's material is extremely in error. '0
N 5
HEAD LENGTH
Fig. 1. Histiograms of size and shape distributions obtained by measurement of the spermatozoa presented in Shettles's Fig. 1, showing that no bimodal distribution exists in size (L, length; Bma " maximum width; and area) or in shape (defined by LI Bmax). The distribution presented here for shape is definitely skew and conforms with the logarithmic distributions found in my investigations, whereas the distribution of area appears to be gaussian. Both are unimodal and homogeneous.
Whereas thus far all authorities agree that the mean head length of normal human spermatozoa is in the range 4-5 jJ-, Shettles's photographs indicate sizes of no less than 30 jJ-,24 of 30-50 jJ-,25, 2'6 and of 11-19 jJ-.27 Consequently, no absolute sizes can be obtained from his photographs, but at least relative measurements can be taken. My Fig. 1 shows the graphic representation of the biometric data derived from Shettles's photomicrograph (his Fig. 1, referred to above), giving the size frequencies for the breadth and length of the head, for the ratio of length to breadth (indicating shape), and for total head area. Length and breadth were measured on the photograph in millimeters, and the total head area was calculated by the formula:
A
= 0.73 LBmax
derived in previous investigations. 4 ,5 None of these distributions is bimodal and their shape and spread are in complete agreement with biometric data obtained in other investigations. 11 -14 , 17, 23, 31 Thus Shettles is contradicted by his own material. Shettles's statement: "The least variable criterion in measurement was found to be the head length"27 is also refuted, since the standard deviation for the head lengths in his photograph is 12.5 per cent as compared with 12.0 per cent for their maximum width. These figures do not differ significantly. The standard deviations for the ratio of length to breadth and area, are respectively 16 and 19 per cent. Really bimodal size distributions have never been found, either in normal
VOL.
12, No.6, 1961
511
SPERM FORM
spermatozoa or in pathologic specimens from men,ll-14,17 bulls 5 ,31 or boars. 7 Figure 2 gives the size distribution curve for head length of human spermatozoa obtained in my own investigations. Since it might be expected that nuclear size would be more relevant than total head size, an example of 0.15,..--------:":'--------------,
Fig. 2. Normalized size-frequency distribution curve of >head length of living human Z 0.10 spermatozoa, measured by ~ W direct microscopic microm- ...a: etry with an absolute accuracy w per measurement of 0.2 p.. ;0.05 Complete absence of any W-' bimodality is clearly demon- a: strated. N = 1500. (J
I.&J
.
[-
nuclear length distribution is also presented (Fig. 3). All material was obtained from donors of proved high fertility (at least 3 pregnancies, each produced within 3 months of unprotected coitus) and all measurements were taken in the living state by direct microscopic micrometry, using the Baker cross-system, phase-contrast, oil-immersion microscope objective 100, N.A. 1.30, combined with a X 30 compensating micrometer eyepiece, for total head size. The Baker interference microscope and ordinary transmitted illumination were used with vitally stained specimens for determining total head size and nuclear size. The results obtained with these different methods were in complete agreement. The curves (Fig. 2 and Fig. 3) show complete absence of any bimodality and both follow a mathematic equation for logarithmic distributions: N = Nmax expo
[-k( In ~m)
2 ]
where N is the absolute number of spermatozoa of size class L; N max, top value of the distribution curve; and Lm, modal value. Fig. 3. Normalized size frequency distribution curve of nuclear length of human spermatozoa, obtained by direct microscopic micrometry in the living state. Pooled data obtained from measurements with the interference microscope and of vitally stained specimen with ordinary transmitted illumination, pooling having been done only after the analysis of separate data obtained in previous investigations had shown mathematical identity of the results. N 766.
=
0.30 >u
z
UJ
50 .20 UJ
...a:
0.10
o~~~~~~~~~~~-J
1.5
2.0
2.5
NUCLEAR
3.0
3.5
LENGTH
4.0
4.5 5.0 5.5
(MICRONS)
512
VAN DUIJN
FERTILITY
&
STERILITY
A further check on the homogeneity of the distributions is obtained by a straight-line transformation,4 resulting if Vlog (NmaxIN) is plotted against -+- log (LI Lm ). With normal (in the biological sense) material this transformation produces an unbroken straight line. * However, with certain types of pathologic material (infertility cases) the slope constants for the righthand and left-hand sides of the distribution may be different, or one of the lines appears broken (two different slopes with a sudden break). Thus far, this has only been observed in bull sperm. 8 Figure 4 gives the straight-line transformation for the nuclear length
1.5
Fig. 4. Straight-line transformation of the data of the size distribution curve of nuclear length of Fig. 3, showing complete homogeneity and conforming to equations for logarithmic distribution, thereby proving that it has not been produced from two distinctly different populations .
1.0
0.5
•
±logL+logL m
oif---~--~--~~~~--~--~--~
0.10
0.20
0.30
distribution curve (Fig. 3), demonstrating complete homogeneity and proving that it has not been produced by two distinctly different populations. Similar results have been obtained for total head length, head width, nuclear breadth and other parameters in human, bovine and boar sperm atozoa.4, 5,7, 8 Only a few remarks need be given with respect to Shettles's strange ideas about cytochemistry.27 From a chemical point of view it must remain a riddle why one should want to add oil of anise to spermatozoa, and what mysterious "feeling" it is which tells Shettles that this liquid should be a reagent to whatever substance. Oil of anise is a natural product, containing 80-90% anethole, which is almost insoluble in water, and methylchavicol and anisaldehyde, also very slightly soluble in water. The only "reaction" occurring when such a substance is mixed with a protein solution such as seminal plasma, is some emulsification, in which oil droplets may be adsorbed to any surface of suspended particles, a purely physical process that has nothing at all to do with a chemical test. * For size distributions conforming to the Gauss equation, the corresponding transformation is obtained by plotting I L - Lm I on the abscissa instead of ± log (L/Lm).
-
VOL.
12, No.6, 1961
SPERM FORM
513
In extensive cytomicrochemical investigations, no evidence for the existence of cytochemical differences between two types of spermatozoa has been found. 3
DISCUSSION
From the foregoing argument it is evident that claims concerning the existence of any bimodal size distribution in spermatozoa cannot be accepted. However, it may be enlightening to discuss briefly whether it is reasonable to expect shape and size variations to correlate with the presence of an X or Y chromosome. The haploid number of chromosomes being 23, and the Y chromosome being conSiderably smaller than the X,l, 10, 16, 18 and not as assumed by Shettles,24-26 the maximum size difference between the two types which may be expected is 1/23, or slightly less than 4.5 per cent. This upper estimate will be reached only if it is assumed that the total nuclear size is exactly proportional to the DNA content and that the over-all head size is strictly proportional to nuclear size, which is highly improbable, the nucleo-plasmatic ratio actually being not constant. 4 Furthermore, in order to get a statistical difference, the DNA content of each type should be constant within significantly smaller limits than the difference between the types; otherwise, no differentiation is possible. Although we know that these conditions are not fulfilled,12, 14 it is valuable to calculate the size difference the presumptions would imply. The results come to 0.2 f.L for the modal head length and 0.1 f.L for the nuclear length. This clearly demonstrates that it is utterly impossible to detect any size difference between X and Y chromosome-bearing spermatozoa by any system of light microscopy. As to direct identification of X and Y chromosomes in spermatozoa, this is prevented by the special condition of the nucleoprotein material in the sperm nucleus, which has been shown to be not only of the compact karyosome type, but, even in a crystalline state, comparable to that of crystallized virus desoxyribonucleoprotein, as found by X-ray diffraction analysis. so This special condition also makes it immune to attack by desoxyribonuclease. 29 SUMMARY
Size-frequency distributions of spermatozoa are shown to be unimodal and to follow an equation for a homogeneous distribution in material from biologically normal individuals. Claims to the contrary made by Shettles are proved to be untrue, using his own published material. It is shown by calculation that detection of any size difference between X and Y chromo-
514
VAN DUIJN
FERTILITY
&
STERILITY
some-bearing spermatozoa by any system of light microscopy is fundamentally impossible. Research Institute for Animal Husbandry "Schoonoord," Hoogt 10, Utrecht, Netherlands
REFERENCES 1. BAIKIE, A. G., COURT-BROWN, W. M., BUCKTON, KARIN, E., HARNDEN, D. G., JACOBS, P. A., and TOUGH, M. A possible specific chromosome abnormality in human chronic myeloid leukemia. Nature 188:1165, 1960. 2. BISHOP, D. W. X and Y spermatozoa. Nature 187:255, 1960. 3. DUlJN, C. VAN, JR. Cytomicrochemistry of human spermatozoa. /. Roy. Micr. Soc. 74:69, 1954. 4. DUIJN, C. VAN, JR. Biometry of human spermatozoa. J. Roy. Micr. Soc. 77:12, 1957. 5. DUlJN, C. VAN, JR. Mensuration of the heads of bull spermatozoa. Mikroskopie 14:265, 1960. 6. DUIJN, C. VAN, JR. Nuclear structure of human spermatozoa. Nature 188:916, 1960. 7. DUlJN, C. VAN, JR. Mensuration of the heads of boar spermatozoa. Mikroskopie 15:142, 1961. 8. DUlJN, C. VAN, JR. Unpublished data. 9. FORD, C. E., and JACOBS, P. A. Human somatic chromosomes. Nature 181:1565, 1958. 10. KOLLER, P. C. The sex chromosomes. In Clinical Genetics, ed. by A. Sorsby. Mosby, St. Louis, 1953. 11. LEUCHTENBERGER, C., SCHRADER, F., WEIR, D. R., and GENTILE, D. P. The desoxyribose nucleic acid (DNA) content in spermatozoa of fertile and infertile human males. Chromosoma 6:61, 1953. 12. LEUCHTENBERGER, C., WEIR, D. R., SCHRADER, F., and MURMANIS, D. P. The desoxyribose nucleic acid (DNA) content in spermatozoa of repeated seminal fluids from fertile and infertile men. /. Lab. & Clin. Med. 45:851, 1955. 13. LEUCHTENBERGER, C., MURMANIS, 1., MURMANIS, L., lTo, S., and WEIR, D. R. Interferometric dry mass and microspectrophotometric arginine determinations on bull sperm nuclei with normal and abnormal DNA content. Chromosoma 8:73, 1956. 14. LEUCHTENBERGER, C., and LEUCHTENBERGER, R. Die Desoxyribonucleoproteide im Siiugetiersperma: Eine quantitative Studie an menschlichen und Stierspermatozoen mittels Mikrospektrophotometrie und Interferenzmikroskopie. Hoppe-Seyler's Ztschr. physiol. Chemie 313: 130, 1958. 15. MACLEOD, J. Personal communication, 1961. 16. MAKINO, S., and MOTOMICHI, S. A study of the somatic chromosomes in the Japanese. Jap. J. Genetics 35:228,1960. 17. MOENCH, G. L., and HOLT, H. Biometrical studies of head length of human spermatozoa. /. Lab. & CUn. Med. 17:297, 1932. 18. NATURAL HISTORY MUSEUM, BRITISH MUSEUM, London. Display of human chromosomes. In the exhibition Evolution and Genetics, 1960. 19. OETTLE, A. G. Experiments with a variable amplitude and phase microscope. /. Roy. Micr. Soc. 70:232, 1950. 20. OETTLE, A. G. "Optical membranes": A common artefact. J. Roy. Micr. Soc. 70:255, 1950.
VOL.
12, No.6, 1961
SPERM FORM
515
21. OETTLE, A. C. Personal communication, 1961. 22. LORD ROTHSCHILD. X and Y spermatozoa. Nature 187:254,1960. 23. SAVAGE, A., WILLIAMS, W. W., and FOWLER, N. M. A statistical study of the head length variability of bovine spermatozoa and its application to the determination of fertility. Proc. & Tr. Roy. Soc. Canada 21 :425, 1927. 24. SHETTLES, L. B. Nuclear morphology of human spermatozoa. Nature 186:648, 1960. 25. SHETTLES, L. B. X and Y spermatozoa. Nature 187:254, 1960. 26. SHETTLES, L. B. Nuclear structure of human spermatozoa. Nature 188:918, 1960. 27. SHETI'LES, L. B. Differences in human spermatozoa. Fertil. & Steril. 12:20, 1961. 28. SHETTLES, L. B. Human spermatozoan shapes in relation to sex ratios. Fertil. & Steril. 12:502, 1961. 29. TOR6, I., and P6SALAKY, Z. Histochemische Untersuchung der Spermiogenese. Acta histochem., lena 8:393, 1959. 30. WILKINS, M. H. F., and RANDALL, J. T. Crystallinity in sperm heads: Molecular structure of nucleoprotein in vivo. Biochim. biophys. acta 10:192, 1953. 31. WILLIAMS, W. W., and SAVAGE, A. Observations on the seminal micropathology of bulls. Cornell Vet. 15:353, 1925.
Sociedad Peruana de Fertilidad Matrimonial Officers of the Society, for 1961-1962, are as follows: President Vice President Dr. Rafael de la Puente L. Dr. Javier Hoyle Cox Secy. General Recording Secy. Dr. Roberto Ruiz Gonzalez Dr. Julio Munoz Valdivieso Treasurer Dr. Noe Ramirez Zapata Executive Member Executive Member Dr. Alfonso Zamorano D.D. Dr. Abraham Ludmir Grimberg