THE ANATOMICAL RECORD 229:315-320 (1991) Spermiation and Sperm Maturation in the Marmoset R. ASOK KUMAR AND DAVID M. PHILLIPS The Population Council, New York,New York 10021 ABSTRACT The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostra1 edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hookshaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is a n Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates. The release of the sperm from the seminiferous tubule epithelium is termed spermiation. During this process, Sertoli cells undergo a series of morphological transformations. Although spermiation has been studied in several mammalian species (Fawcett and Phillips, 1969; Sapsford and Rae, 1968; Fawcett, 1975; Morales and Clermont, 1982; Wong and Russell, 1983; Weber et al., 1983; Vogl et al., 1985), the release of spermatozoa has not been examined in monkeys. We describe here the processes of spermiation in marmosets (Callithrixjacchus). The spermatozoa released from the seminiferous epithelium are functionally immature and have not yet developed the capacity to fertilize. They undergo structural, biochemical, and functional changes during epididymal transit (for reviews, see Bedford, 1975; Orgebin-Crist, 1981). Fawcett and Hollenberg (1963) observed remarkable change in the morphology of the guinea pig acrosome during epididymal transit. Since then, morphological changes have been reported in other eutherian mammals, including rabbit (Bedford, 1963, 1965), chinchilla (Fawcett and Phillips, 19691, monkey (Bedford and Nicander, 1971), elephant (Jones et al., 1974), musk shrew (Cooper and Bedford, 19761, hyrax and armadillo (Bedford and Millard, 1978), hamster (de Rosas and Burgos, 1987), and loris (Phillips and Bedford, 1987). We describe here marked structural changes in the common marmoset, C .jacchus, a New World monkey, which have not been previously noted. pH 7.2. For scanning electron microscopy (SEM), the fixed tissue was cut into small pieces. The tissue was postfixed in 1% osmium tetroxide buffered with 0.1 M cacodylate buffer, dehydrated in a graded series of acetone infiltrated in Freon, and critical-point dried from liquid CO,. The dried specimens were coated with gold in a n Edwards 150 sputtercoater and examined with a n ETEC Autoscan. For transmission electron microscopy (TEM), testes and epididymides were carefully cut into small pieces, postfixed in 1% OsO,, and dehydrated in alcohol followed by propylene oxide. Tissues were embedded in Epon. Sections were cut with a diamond knife on a Reichart OmU2 microtome, stained with 3% aqueous uranyl acetate (no lead staining was used), and examined in a Philips 300 electron microscope. OBSERVATIONS Sperrniation Viewed with SEM, heads of the maturation phase spermatids were observed to be embedded in Sertoli cells. Some Sertoli cells displayed funnel-shaped, sleeve-like processes. Some spermatids had presumably fallen out of these Sertoli cells during processing (Fig. 1). We also observed finger-like extensions of the Sertoli cells associated with late spermatids. These finger-like protuberances of the apical region of the Sertoli cells assumed different shapes and sizes (Figs. 2,3). MATERIALS AND METHODS Testes and epididymides of adult marmosets (C.jacchus) were fixed by perfusion in 1% glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium cacodylate buffer a t 0 1991 WILEY-LISS. INC. Received May 29, 1990; accepted July 31, 1990. Address reprint requests to Dr. David M. Phillips, The Population Council, 1230 York Avenue, New York, NY 10021. 316 R. ASOK KUMAR AND D.M. PHILLIPS Fig. 1. SEM showing the cellular association of Sertoli cells and spermatids. Arrows indicate the sleeve-like processes of the Sertoli cells. Spermatids presumably fell out of the sleeves during processing. x 2,000. Fig. 2. During spermiogenesis, the sleeve-like processes (arrows) of Sertoli cells are in close association with spermatids. Tubular cytoplasmic specialization of the Sertoli cells (arrowhead) are observed among the developing spermatids. x 3,300. 317 MARMOSET SPERM Fig. 3. SEM showing Sertoli cell-spermatid association a t spermiation. Several luminal cytoplasmic protuberances of the Sertoli cells are associated with the spermatozoa. x 6,000. Sperm Maturation Events The SEM revealed the caudal migration of the cytoplasmic droplet along the midpiece during epididymal transit. Spermatozoa lying in the caput epididymis were usually bent at their midpiece region (Fig. 4).The bent portion of the spermatozoa was often enveloped by the protoplasmic droplet. Spermatozoa from the corpus epididymis were generally slightly bent at the annulus, where the protoplasmic droplet was observed (Fig. 6). In the cauda epididymis, spermatozoa were straight and protoplasmic droplets were generally not observed (Fig. 8). Maturational changes were not visible in the sperm head with SEM. With TEM, we observed maturational changes in the sperm head that were not revealed by the SEM. In the caput epididymis, the heads of spermatozoa were asymmetric because the apical segment of the acrosome was extended well beyond the rostra1 edge of the nucleus and folded under it. In sagittal sections, the acrosome exhibited a distinct hook-shaped apical segment (Fig. 5). The occurrence of a hook-shaped apical segment of the acroSome was evident in the 'Perm cut sagittally. In the 'OrPUS, the distinctive hook-shaPed segment of the acrOSOme was observed in Some sPermatobut not in Others. the protuberance was smaller than that observed in spermatozoa of the caput epididymis (Fig. 7). In the cauda epididymis, no hooksegment Of the acrosome was Observed (Fig. 9). DISCUSSION We have shown that late spermatids of the marmoset are embedded in sleeve-like processes of Sertoli cells. 318 R. ASOK KUMAR AND D.M. PHILLIPS Fig. 4. SEM of the spermatozoa in the caput epididymis. The bent portion of the neck region is enveloped by a cytoplasmic droplet (D). x 4,600. Fig. 5. Sagittal section of the heads of two spermatozoa from the caput epididymis. The acrosome elongates and bends back towards the nucleus. x 11,000. Fig. 6. SEM showing the spermatozoa in the corpus epididymis. The cytoplasmic droplet is situated in the anterior of the midpiece region of some spermatozoa and in the posterior of the midpiece in others. x 4,600. Fig. 7. Sagittal section of the head of two spermatozoa from corpus epididymis. The rostra1 elongation is observed in only one of the two spermatozoa. x 11,000. MARMOSET SPERM 319 Fig. 8. SEM of the spermatozoa in the cauda epididymis. Most of the spermatozoa have straight tails. The cytoplasmic droplet (D) has been moved to the annulus. x 4,600. Fig. 9. TEM of spermatozoa in the cauda epididymis. x 11,000. We have also observed that the apical end of the Sertoli World monkeys. In fact, a similar phenomenon has cell forms numerous short processes of various shapes, been described in pigtailed monkey (Hoffer et al., which closely associate with the spermatids. These ob- 1981), an Old World species. Thus, although the priservations are basically similar to those of other inves- mate acrosome is not considered to undergo acrosomal tigators working with a variety of mammalian species morphogenesis as occurs in certain other mammals, (Fawcett and Phillips, 1969; Fawcett, 1975, 1979; Fou- further ultrastructural studies may reveal that matuquet, 1972, 1974; Phillips, 1980; Russell, 1984; Gravis, rational changes in the acrosome are more common in 1978; Sakai et al., 1988; Vogl et al., 1983,1985;Dietert, primates than was previously believed. 1966; Nicander, 1967; Sapsford and Rae, 1969; Morales ACKNOWLEDGMENTS and Clermont, 1982). Although the relationship between Sertoli cells and This research was supported in part by a research late spermatids is similar among different species of training grant from WHO to R.A.K. eutherian mammals, there have been various interpreLITERATURE CITED tations of the morphological observations with respect Bedford, J.M. 1963 Morphological changes in rabbit spermatozoa durto the role of the Sertoli cell in spermiation. The variing passage through epididymis. J. Reprod. Fertil., 5t169-173. ety of different interpretations may in part reflect the J.M. 1965 Changes in fine structure of the rabbit sperm head difficulties in determining the events that take place in Bedford, during passage through the epididymis. J. Anat., 99r891-906. a dynamic process from static electron micrographs. Bedford, J.M. 1975 Maturation, transport and fate of spermatozoa in the epididymis. In: Handbook of Physiology. Endocrinology V. We have observed that spermatozoa of the marmoset E.B. Astwood and R.O. Greep, eds. American Physiological Sociundergo unusual structural changes as they mature. ety, Bethesda, Maryland, p. 303-317. The anterior portion of the acrosome, which is folded Bedford, J.M., and Millard, R. 1978 The character of the sperm matback on itself in immature spermatozoa, gradually disuration in the epididymis of the ascrotal hyrak, Proucauca capensis, and armadillo, Dadypus nouemicimctus. Biol. Reprod., 19: appears during sperm development. This acrosomal re396-406. arrangement is obvious with TEM but undetectable J.M., and Nicander, L.N. 1971 Ultrastructural changes in with light microscopy or SEM. Since sperm maturation Bedford, the acrosome and sperm membranes during maturation of sperhas been studied in relatively few New World monkeys matozoa in the testis and epididymis of the rabbit and monkey. J. Anat., 108: 527- 543. with TEM, similar changes may occur in other New 320 R. ASOK KUMAR AND D.M. PHILLIPS Cooper, G.W., and Bedford, J.M. 1976 Asymmetry of spermiation and sperm surface charge patterns over the giant acrosome in the musk shrew, Suncus murinus. J . Cell Biol., 69:415-428. de Rosas, J., and Burgos, M. 1987 Epididymal transit and morphology of the hamster sperm head. Microsc. Elec. Biol. Cell., 11:67-69. Dietert, S.E. 1966 Fine structure of the formation of and fate of the residual bodies of mouse spermatozoa with evidence for the participation of lysomes. J. Morphol., 120:317-346. Fawcett, D.W. 1975 Ultrastructure and function of the Sertoli cell. In: Handbook of Physiology. Endocrinology V. American Physiological Society, Bethesda, Maryland. Fawcett, D.W. 1979 Comparative aspects of the organization of the testis and spermatogenesis. In: Animal Models for Research on Contraception and Fertility. Harper and Row, New York, pp. 84104. Fawcett, D.W., and Hollenberg, R. 1963 Changes in the acrosome of guinea pig spermatozoa during passage through epididymis. Z. Zellforsch. Mikrosk. Anat., 60t276-292. Fawcett, D.W., and Phillips, D.M. 1969 Observations on the release of spermatozoa and on the changes in the head during passage through the epididymis. J. Reprod. Fertil., 6[suppl.1:405-418. Fouquet, J.P. 1974 La spermiation et la formation des corps residuels chez le hamster: Role des cellules de Sertoli. J. Microsc., 19t161168. Fouquet, J.P. 1972 Le mecanisme de la spermiation chez le hamster: Signification des relations entre cellules de Sertoli et spermatides. C.R. Acad. Sci. D., 275t2025-2028. Gravis, C.J. 1978 A scanning electronmicroscopic study of the Sertoli cell and spermiation in the syrian hamster. Am. J. Anat., 151: 21-38. Hoffer, A.P., Shalev, M., and Frisch, D.H. 1981 Ultrastructure of and maturational changes in spermatozoa in the epididymis of the pig-tailed monkey, Macaca nemestrina. J. Androl., 3t140-146. Jones, R.S., Rowlands, I.W., and Skinner, J.D. 1974 Spermatozoa in the genital ducts of the African elephant Loxodonta africana. J. Reprod. Fertil., 41:189-192. Morales, C., and Clermont, X. 1982 Evolution of Sertoli cell processes invading the cytoplasm of rat spermatids. Anat. Rec., 203:233244. Nicander, L. 1967 An electronmicroscopical study of cell contacts in the seminiferous tubules of same mammals. Z. Zellforsch. Mikrosk. Anat., 83t375-397. Orgebin-Crist, M.L. 1981 Epididymal physiology and sperm maturation. Prog. Reprod. Biol., 8:80-89. Phillips, D.M. 1980 Observations on mammalian spermiogenesis using surface replicas. J. Ultrastruct. Res., 72:103-111. Phillips, D.M., and Bedford, J.M. 1987 Sperm-sperm associations in loris epididymis. Gamete Res., 18:17-25. Russell, L.D. 1984 SpermiationSperm release process: Ultrastructural observations and unresolved problems. In: Ultrastructure of reproduction. J . Van Blerkom and P.M. Motta, eds. Martinus Nijoof, Boston, pp. 46-66. Sakai, Y., Nakamoto, T., and Yamashina, S. 1988 Dynamic changes in Sertoli cell processes invading spermatid cytoplasm during mouse spermatogenesis. Anat. Rec., 220.5-57. Sapsford, E.S., and Rae, C.A. 1968 Sertoli cell-Spermatid relationships ultrastructural studies of the movements of mature spermatids into the lumen of the seminiferous tubule. J. Anat., 103: 214-215. Sapsford, C.S., and Rae, C.A. 1969 Ultrastructural studies on Sertoli cells and spermatids in the bandicoot and ram during the movement of mature spermatids into the lumen of the seminiferous tubule. Aust. J . Zool., 17:415-445. Vogl, A.W., Lin, Y.C., Dym, M., and Fawcett, D.W. 1983 Sertoli cells of the golden mantled ground squirrel (Sperrnophilus Lateralis): A model system for the study of shape change. Am. J. Anat., 168t83-98. Vogl, A.W., Souzy, L.J., and Foo, V. 1985 Ultrastructure of Sertoli cell penetrating processes found in germ cells of the Golden-mantled ground squirrel (Spermaphilus lateralis). Am. J . Anat., 172:7586. Weber, J.E., Russell, L.D., Wong, V., and Peterson, R.N. 1983 Three dimensional reconstruction of a rat stage V Sertoli cell. 11. Morphometry of Sertoli-Sertoli and Sertoli-germcell relationships. Am. J. Anat., 167r163-179. Wong, V., and Russell, L.D. 1983 Three dimensional reconstruction of a rat stage V Sertoli cell: I. Methods, basic configuration, and dimensions. Am. J. Anat., 167:143-161.