Differences in Human Spermatozoa

Transcription

1 Differences in Human Spermatozoa Landrum B. Shettles, M.D. RECENT OBSERVATIONS show that thin, dried, unstained smears of human spermatozoa viewed through the dry objectives of the phase-contrast microscope reveal two populations, distinguished by size and shape of the nuclei. and heads, diffraction of light from the heads, diffraction of light by the discrete chromosomes, the chromosomal arrangements, and the size and shape of the most centrally located chromosome. The relative sizes and shapes of the most central chromosome in each type indicate that the smaller-headed spermatozoa contain the Y and the larger the X chromosome. When normally formed spermatozoa lying horizontally are studied no intermediate types are seen. The characteristics of the dried heads within each population are remarkably constant; however, spermatozoa from one individual may be much larger or smaller than those from another. There is individual variation in the ratio of the two populations. 1-5 This article reports further observations on human spermatozoa regarding head length and the extent of their reaction with volatile oil of anise. MATERIALS AND METHODS Fresh, living spermatozoa were used. Morphologic differences in their heads were investigated as follows: A small drop of normal semen placed on a glass slide was made very thin, and a petrolatum ring was made around the drop. When the slide was placed in an atmosphere of carbon dioxide, all spermatozoa ceased moving within a few seconds, as a result of the intrinsic effect of the gas. 6 The drop was then sealed with a cover glass placed on the petrolatum ring. After the spermatozoa were observed in phase con- From the Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, and the Sloane Hospital for Women, New York. 20

2 Vol. 12. No HUMA~ SPERMATOZOA DIFFERENCES 21 trast, they were photographed. Soon after removal of the cover glass, motility was restored. The extent of any possible cytochemical difference in the cells was tested by placing a drop of fresh semen and a drop of volatile oil of anise (Pimpinella anisum), side by side on a glass slide; currents and evaporation were prevented by a ring of petrolatum around the preparation and a cover glass sealing the top. A definite reaction of the spermatozoa with the oil of anise occurred invariably. The reaction of the spermatozoa as they attempted to traverse the interface was observed using a Zeiss phase-contrast microscope with a built-in accessory microscope. A Leica camera* with an Ibso attachment and microfile film was employed for photography. Spermatozoa from 40 individuals were studied. RESULTS When seen in the living state by means of phase-contrast microscopy, the spermatozoa showed significant differences in the size and shape of their heads in front and side views (Fig. 1). Specimens judged to be normal by the criteria used in semen analysis, and of proved fertility, fell into two categories of spermatozoa with respect to head size, shape, and length. The least variable criterion in measurement was found to be the head length. Checked by caliper in a given photograph, the head length of the larger spermatozoa measured very nearly the same; the same degree of uniformity in measurement was true of the smaller heads. The sizes of sperm heads often varied from one individual to another; however, within a given man there was a high degree of constancy in the length of the larger and smaller heads. A definite bimodal distribution of head lengths within a given specimen was found. Microscopic fields containing a greater number of smallerheaded spermatozoa were more frequently seen. A specific phenomenon, which confirmed the difference in sperm head size in a given individual, occurred at the interface of the oil of anise and the spermatozoa and became discernible in a few seconds as the spermatozoa came in contact with the oil. Within just a few minutes the larger-headed spermatozoa were heavily laden with small, spherical blebs, located along the tail and neck and over the head (Fig. 2). These spermatozoa continued to move, often forward, and rotation on their longitudinal axes was all the more evident. The reaction of the smaller-headed spermatozoa was far less '" E. Leitz, Inc., New York.

3 ". 22 SHETTLES Fertility & Sterility Fig. 1. Living human spennatozoa immobilized with carbon dioixde and photographed in phase contrast. (X 1000) obvious; the head and neck reacted only slightly, the blebs forming mainly along the tail. In time-i.e., min.-many of the blebs became spontaneously dislodged and gave rise to Brownian movement, which occurred to a far greater degree close to the larger-headed spermatozoa. The move-

4 Fig. 2. Reaction of living human spermatozoa with oil of anise, viewed in phase contrast. Note extent of bleb formation in larger and smaller heads. (A, X 400; B, X 640; C, X 1000)

5 24 SHETTLES Fertility & Sterility ment of the spermatozoa treated with oil of anise ceased long before that of untreated ones whose movement was arrested temporarily, with C02. COMMENT Even though the desoxyribose nucleic acid content of spermatozoa obtained at different periods from fertile men has been reported as constant for each individual,7 a difference in head size of cytologically normal, living spermatozoa has been observed. The difference in extent of reaction with oil of anise confirms the existence of large and small heads. It is felt that the oil of anise most likely reacts with lipid in the head, neck, and tail. In support of the thesis that the head sizes of X- and Y-chromosome-containing spermatozoa differ is the manner of their separation when subjected to centrifugal force counteracted by a flow of fluid. 8 Since the characteristics of the two populations of human spermatozoa are now recognizable, the efficacy of methods attempting to separate the X and Y-chromosome-containing spermatozoa can be ascertained. SUMMARY Living human spermatozoa fall into two populations distinguished by head size, shape, length, and the extent of reaction of each type with the volatile oil of anise. These findings are in agreement with recent observations on dried, unstained smears of human spermatozoa in phase contrast showing two distinct populations iii regard to nuclear morphology. REFERENCES 1. SHETTLES, L. B. Nuclear morphology of human spermatozoa. Med. Circle Bull. 7:8, SHETTLES, L. B. Observation~ on human spermatozoa. Bull. Sloane Hasp. for Women 6:48, SHETTLES, L. B. Nuclear morphology of human spermatozoa. Obst. & Gynec. 16:10, SHETTLES, L. B. Nuclear morphology of human spermatozoa. Nature 186:648, 1960, 5. SHETTLES, L. B. X and Y spermatozoa. Nature 187:254, SHETTLES, L. B. Carbon dioxide tension and its relation to quiescence of spermatozoa in vivo. Proc. Soc.,Exper. BioI. & Med. 45:318, LEUC;;HTENBERGER, C., et al. The desoxyribose nucleic acid (DNA) content in spermatozoa of repeated seminal fluid from fertile and infertile men. ]. Lab. & Glin. Med. 45:851, LINDAHL, P. E. Separation of bull spermatozoa carrying X and Y chromosomes by counter-streaming centrifugation. Nature J8J :784,