Unusual Features of the Nuclear Envelope in Human Spermatogenic Cells H. E. CHEMES,' D. W. FAWCE?T3 AND M. DYM Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 021 15
ABSTRACT Different types of human germ cells show unusual features of the nuclear envelope. Spermatogonial nuclei demonstrate two kinds of modifications. The first one is a series of intranuclear flattened cisterns, parallel to each other and to the inner aspect of the nuclear envelope. The second one is a nuclear envelope protrusion into the cytoplasm occupied by a double membrane-limited vesicle. Pores are found on the membrane of the vesicle facing the interior of the nucleus. In spermatocytes the nuclear pores are concentrated over certain areas and completely absent from others. In the regions where they are absent a single cytoplasmic cistern of rough endoplasmic reticulum is closely apposed to the outer membrane of the nuclear envelope. Early modifications of the nuclear surface appear in spermatids before the attachment of the acrosomic vesicle and may indicate an active role of the nuclear envelope in the morphogenesis of the acrosome. In round spermatids nuclear pores are absent from the area which is first related to the Golgi and later covered by the acrosomal cap. Single or multiple layers of cytoplasmic annulate lamellae are closely associated with the nuclear envelope over the pore rich areas. Frequently there are intranuclear accumulations of dense material adjacent to the annulate lamellae-nuclear pore complex. The chromatoid body is usually present on the cytoplasmic side of this complex. In the elongating spermatids most annulate lamellae are free in the cytoplasm, often in relation with Golgi and chromatoid body remnants near the axial filament. Few stacks of annulate lamellae are noted adjacent to the pore rich nuclear regions. It is suggested t h a t the described modifications are related to an active nuclear-cytoplasmic interaction. Several types of modifications in the nucleus, the nuclear envelope and the adjacent cytoplasm of spermatogenic cells have been reported in different mammalian species. In spermatocytes and spermatids the nuclear pores are concentrated over certain regions of the nucleus and absent from others (Fawcett, '74; Franke and Scheer, '74; Sandoz, '74; Chemes and Fawcett, '78). Other kinds of modifications such as dilatations andlor narrowings of the perinuclear cistern in Golgi phase spermatids, alignment of chromatin granules on the inner aspect of the nuclear envelope underneath the acrosomal cap, the presence of a "redundant envelope" in late spermatids, and bleb or cistern-like formations in human spermatogonia and rat spermatocytes have also ANAT. REC.
(1978)192: 493-512.
been described (Fawcett and Ito, '65; Franklin, '68; deKretser, '69; Sandoz, '70; Rowley et al., '71; Fawcett, '74; Russell, '76). Furthermore, there are reports of annulate lamellae in human spermatogenic cells and their close associations with the nuclear envelope (Horstmann, '61; Smith and Berlin, '77). During the course of studies on normal and pathological human testis (Chemes et al., '77), we noted t h a t human germ cells are parReceived May 11, '78. Accepted July 27, '78. ' Supported by Research Grants HD 02344 and HD 06969 and by a Public Health Service International Research Fellowship 5-FO5TWO 2223-02 (Dr. H. Chemes). Present address: Division de Endocrinologia, Hospital General de Nirios, Gallo 1330, Buenos Aires, Argentina. 3Address reprint requests to: Don W. Fawcett, Department of Anatomy, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, U S A .
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sumed t o be continuous with t h e nuclear envelope (fig. 1). Serial sections would be required to ascertain whether these inclusions are detached vesicles entirely within the nucleoplasm or cross sections of digitiform invaginations of cytoplasm. There is a dense strip of chromatin-like material interposed between t h e vesicle and t h e nuclear envelope. The contents of t h e vesicle a r e granular, frequently resembling t h e cytoplasm, although in some cases i t is difficult t o distinguish them MATERIALS AND METHODS from nuclear material. Pores a r e found on t h e The source of material for this study has side of t h e vesicle facing t h e nucleus as well as been testicular biopsies from four normal in t h e nuclear envelope near t h e protrusion. young men of proven fertility.4These subjects Either t h e nucleolus or dense irregular clumps had normal urinary and plasma levels of go- of heterochromatin may be closely associated nadotropins and repeatedly normal sperm with these structures. I t is possible to find incounts (Rowley et al., '71). The tissues were stances where t h e flat cisternae described immersed immediately after removal in 3% above and t h e vesicles or invaginations are asglutaraldehyde buffered with 0.1 M cacodylate sociated. Single profiles of annulate lamellae at pH 7.3 and after 30 minutes were cut into a r e occasionally seen in t h e surrounding cyto1-mm blocks and left a n additional 60 minutes plasm. in t h e original fixative. They were subseSpermatocytes quently postfixed in 1.3% OsO, at 4°C for 60 minutes, dehydrated in a graded series of e t h In late zygotene and pachytene spermatoanol followed by propylene oxide and embed- cytes extensive areas of t h e nuclear surface ded in Epon 812. One micron thick sections are covered by closely applied flattened ciswere cut with glass knives in a MT2 Sorvall ternae of t h e rough endoplasmic reticulum ultramicrotome, mounted on glass slides and (RER). A row of ribosomes is located in t h e stained with 1%toluidine blue in 1%sodium narrow 14-20 nm space which separates t h e borate for light microscopic examination. nuclear envelope from the RER cistern; t h e Thin sections exhibiting pale gold t o silver ribosomes are more densely packed in this interference colors were cut with a diamond location than on t h e membranes of the RER knife and mounted in uncoated 200- to 300- cisterns facing t h e cytoplasm (figs. 4, 5). This mesh copper grids. These were double stained is a constant finding and gives t h e specious with uranyl acetate in 50% acetone and lead appearance of a double nuclear envelope on citrate and examined in a Philips 200 electron part of t h e periphery of spermatocyte nuclei. microscope at 60 kv. Although usually single, occasionally, two or more cisternae a r e stacked adjacent to t h e nuRESULTS cleus. It is difficult t o estimate t h e percentage Spermatogonia of t h e nuclear surface which is associated Two types of nuclear specializations a r e with t h e endoplasmic reticulum (ER) in this found in spermatogonia. (1) Intranuclear manner, but assuming t h a t t h e linear extent cisternae: this type of specialization consists of ER in thin sections is representative of its of a series of flattened cisternae parallel t o tridimensional area, a mantle of reticulum each other and to t h e inner aspect of t h e nu- may cover up t o 50% of t h e nuclear surface. clear envelope. An 18-nm thick layer of closely Nuclear pores a r e consistently excluded from spaced dense granules resembling chromatin these areas with associated reticulum, but are surrounds each of t h e cisternae. Usually one present grouped in variable numbers over the or two cisternae, each 1-2 p m long, a r e present rest of t h e nucleus (figs. 3, 4, 8). In some in(fig. 2). There a r e no nuclear pores immediate- stances they seem t o occupy nearly all of t h e ly overlying t h e cisternae, but pores a r e often space which is not covered by t h e RER. noted in t h e adjacent segments of t h e nuclear Clumps of dense chromatin granules and nuenvelope. (2) The second modification is a ve'The biopsies were provided through the kindness of Mans J . sicular inclusion at t h e periphery of t h e nu- Rowley and Carl G . Heller from the Pacific Northwest Research cleus, limited by a double membrane pre- Foundation, Seattle.
ticularly rich in these kinds of modifications and demonstrate a high degree of specificity in the association of a given feature to a particular germ cell type. In t h e present study we document t h e characteristics of several nuclear envelope specializations in human germinal cells, their association with particular organelles and their possible significance in the overall regulation of nuclear-cytoplasmic interaction.
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS
cleolar material are frequently attached to or concentrated near the pore-rich regions of the nuclear envelope (fig. 3). Smaller clusters of similar appearance are observed on the cytoplasmic side of the nuclear pores or free in the perinuclear cytoplasm (fig. 8 ) . Other round aggregations of electron opaque material are seen on the periphery of the nucleus of pachytene spermatocytes. Not infrequently there is a local separation of the inner and outer membranes of the nuclear envelope forming a dilatation in which membranebound dense bodies of similar structure are located. The images suggest an intranuclear origin for these granules and their subsequent extrusion into the cytoplasm (figs. 3,6, 7). Spermatids In early round spermatids a number of structural modifications can be seen on the nuclear envelope in the vicinity of the Golgi complex. Some coated and uncoated vesicles t h a t appear to be budding off from the outer membrane of the nuclear envelope appear in the region adjacent to the Golgi complex which, in some cases, shows similar coated surfaces on the side facing the nucleus (fig. 13). In addition, a round local protrusion of the nuclear envelope containing a dense material between the two membranes has been observed in the same region. This is a rare finding but its occurrence may actually be much more frequent if one considers the low probability of inclusion of such a small structure in random thin sections (figs. 10, 11). Occasionally, intranuclear materials in the form of round granules or rod like structures appear to be in a process of emergence into the Golgi area of the cytoplasm (fig. 12). Pores are absent from the nuclear surface adjacent to the Golgi complex in the area of impending attachment of the acrosomal vesicle and its expansion to form the acrosomal cap (fig. 9). However, closely spaced pores are present peripheral to this Golgi associated region and often the cavity of the nuclear envelope in these regions is somewhat dilated to a width of 60-85 nm (fig. 9). The chromatin in round spermatids is dispersed and has a fairly homogenous granular appearance, but there are some local condensations which tend to be associated with the inner side of the nuclear envelope in the porerich areas (figs. 9, 16). Even though spermatids lack a typical nucleolus i t has been suggested t h a t some of the dense material ob-
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served in the nucleoplasm may represent nucleolar material (Burgos et al., '70). In many instances single or multiple layers of cytoplasmic cisternae, sometimes with the typical configuration of annulate lamellae, are apposed to the nuclear envelope over the pore-rich areas. A finely granular dense material is present between the lamellae and the nuclear envelope and between the lamellae themselves (figs. 14-16). Two types of chromatoid body material are clearly discernible in early round spermatids. The first one is a dark dense granular mass of irregular outline; 18- t o 20-nm granules can be seen toward the periphery of the mass but are not resolved within the dense core possibly because of closer packing; however, the possibility of two categories of granules cannot be excluded (figs. 9, 15). The peripheral granules are rather similar in size and density to the ribosomes observed either free in the cytoplasm or associated with membranous elements of the endoplasmic reticulum. The second type of chromatoid body consists of loosely arranged granules intermingled with numerous small membranous vesicles of ER (figs. 9, 16). Both compact and diffuse forms of the chromatoid body are located very near the nucleus and almost always in close association with the nuclear envelope or the annulate lamellae-nuclear pore complex previously described. Membrane bounded dense granules that appear to be contributed to the cytoplasm, can be observed between the two membranes of the nuclear envelope in young spermatids. Dense accumulations of nuclear material on the inner side of the nuclear envelope and similar granules free in the perinuclear cytoplasm are sometimes found in the vicinity (fig. 17). As spermatid maturation proceeds the acrosome spreads over the nucleus, and the porerich areas of the nuclear envelope and the associated chromatoid body move toward the caudal pole of the nucleus where they are concentrated when the acrosomal cap attains its full development (fig. 18). In the elongating spermatids a few stacks of annulate lamellae may remain attached to the posterior part of the nucleus. In this location the nuclear envelope, now very rich in pores, frequently diverges from the condensing chromatin and in some instances shows vesicular protrusions or folds extending toward the neck region. During the process of spermatid elongation all the cytoplasmic constituents move caudally.
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In the caudal region of the cytoplasm free stacks of annulate lamellae and chromatoid body constituents are found in close relation with Golgi remnants (figs. 18-20). The diminution in the number of annulate lamellae apposed t o the nuclear envelope and their simultaneous appearance in the periaxial region suggest that they detach from the nuclear envelope and become free in the caudal cytoplasm. Similar associations between annulate lamellae, chromatoid body material and Golgi remnants can be seen in residual bodies (fig. 21). The annulate lamellae share the same fate as other organelles, undergoing degeneration within the residual bodies. These are very rich in ribosomes and possess structures which resemble the granulo-vesicular components of the chromatoid body (fig. 21). Even though there is extensive degeneration of the membranous organelles in residual bodies, the ribosomes and the granular materials believed to be derived from the chromatoid body seem to persist until the late stages of residual body evolution just prior to their disappearance within the Sertoli cells. DISCUSSION
The presence of several kinds of nuclear envelope anomalies or specializations in human spermatogenic cells raises the question of their possible significance in nuclearcytoplasmic interaction. Briefly summarized the described features include the following: (1) Bleb and cistern-like formations in spermatogonia. (2) Regional aggregation of nuclea r pores in spermatocytes and spermatids. (3) Close apposition of rough endoplasmic reticulum to large areas of the nuclear envelope in spermatocytes and association of annulate lamellae with the nuclear envelope in spermatids. (4)Close association of the chromatoid body t o the nuclear envelope. (5) Modifications of the nuclear envelope adjacent t o the Golgi complex in the early steps of acrosome formation. Some of the nuclear modifications described here have previously been noted in human spermatogonia (Rowley et al., '71) and rat spermatogonia and spermatocytes (Russell, '76; Chemes, unpublished). Similar nuclear configurations have also been noted in neoplastic cells (Anderson, '66; Erlandson, '75). A possible relation of some of these nuclear events to chromatoid body formation was suggested in the case of rat spermatocytes (Russell, '76). In human spermatogonia, however,
there is little morphological evidence t o support a role of the cisternae and vesicles in the nucleo-cytoplasmic transfer of substances. Furthermore, their size and frequency of OCcurrence is not very impressive. A high incidence of spermatogonial degeneration has been reported as a normal feature of spermatogenesis in rodents (Clermont, '62; Oakberg, '56). The possibility exists that some of the nuclear envelope modifications described here may be part of the programmed degeneration of many of these cells. The fact that their incidence seems to be higher in cases of severe germ cell depletion (Chemes, unpublished observation) would tend to lend support t o the latter interpretation. The uneven distribution of nuclear pores in spermatocytes is of interest in relation t o the extensive cisternae of the RER found closely applied to the pore-free regions of the nuclear surface. These features are difficult to interpret but suggest t h a t the nuclear-cytoplasmic interchange mediated through nuclear pores takes part preferentially in certain areas of the nuclear surface. The fact that cytoplasmic organelles are not congregated over the porerich regions suggests that this grouping of pores is related to an inhomogeneity in distribution of nuclear components. In freeze-fracture replicas of guinea pig spermatocytes i t has been shown t h a t nuclear pores are closely clustered in areas of irregular contour separated by large pore-free regions and the suggestion has been made that the localization of nuclear pores in high concentration in certain areas may be related to the disposition of the underlying meiotic chromosomes (Moses, '64; Fawcett and Chemes, '78). The intimate association between the terminal end of synaptinemal complexes, condensed regions of chromosomes and of the sex vesicle with the nuclea r envelope suggest a possible relationship of these structures in the unusual distribution of nuclear pores in spermatocytes. The close association of ribosomes and RER with the nuclear envelope raises the possibility that the perinuclear cisternae may serve as storage site or way station in the translocation of proteins from nucleus to cytoplasm or in the opposite direction (for review see Franke and Scheer, '74). The presence of round fibrillar bodies in the nucleus and within local dilatations of the perinuclear space can be detected in pachytene spermatocytes and round spermatids. Similar structures have been described in
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS
rat pronuclei (Szollosi, '65) and in early meiotic plant cells (La Cour and Wells, '72). In the case of the rat pronuclei i t was reported that they originate in the nucleolus and are subsequently extruded to the cytoplasm. Additionally, Bouitelle et al. ('74) described the presence of small spherical structures of proteinaceous nature within the nucleus of very active cells, their numbers being sometimes influenced by hormones. Our findings in spermatocyte and spermatid nuclei closely resemble those in the previous reports. In this respect i t should be mentioned that pachytene spermatocytes are metabolically very active cells and known to be under hormonal influence and early spermatids are cells which undergo a series of elaborate morphogenetic changes that may be associated with high synthetic activity. Recent studies seem to indicate that the RNA synthesized in pachytene spermatocytes and early spermatids is HnRNA which is partially extruded into the cytoplasm (Kierszenbaum and Tres, '74; Soderstrom and Parvinen, '76a). It is precisely in those cellular types that the chromatoid body first appears, annulate lamellae become prominent and numerous indications of nuCleo-cytoplasmic interchange can be detected. It has been proposed that mRNA stored could be used in further steps when transcription has ceased in spermatid nuclei (Fawcett et al., '70; Soderstrom and Parvinen, '76a). The nonrandom distribution of nuclear pores in spermatids is related t o the development of the acrosome (Fawcett, '74). The increasing concentration of the pores toward the caudal pole coincides with the progressive expansion of the acrosomal cap over the anterior pole. It also coincides in time with the general movement of cytoplasmic constituents toward the tail region of the spermatid. The patchy distribution of pores in the caudal hemisphere of spermatid nuclei may be a reflection of their close association with the chromatoid body and with adherent annulate lamellae. An association of the chromatoid body with nuclea r pores has been described in a number of species and a nuclear origin of the chromatoid body has been suggested because of the apparent passage of material through the nuclear pores, but the mode of origin of the chromatoid body is still controversial (Maillet and Gouranton, '65; Fawcett et al., '70; Commings and Okada, '72; Eddy, '74; Soderstrom and Parvinen, '76b). The close association of the chromatoid body with the nuclear envelope
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does not necessarily mean that it arises "in toto" from the nucleus but strongly supports some kind of interaction between chromatoid body and nucleus. This possibility derives support from the presence of dense intranuclear condensations in the region of the chromatoid body. In addition the concentration of nuclear pores and association of annulate lamellae in the same area may be an additional indication of a significant nuclear-cytoplasmic interchange a t this site. A number of modifications of the inner aspect of the nuclear envelope in the region of the acrosome has been described in spermatids (Sandoz, '70, '74; Fawcett, '74). It has been shown that some of these are actually present prior to the development of the acrosome. It has been proposed, therefore, that these nuclear envelope specializations may determine the site of attachment of the acrosomal vesicle and, therefore, the anterior pole of the sperm (Sandoz, '74). In newly formed human spermatids the portion of the nuclear envelope which faces the Golgi complex is characterized by blebbing activity and absence of nuclear pores. Occasionally dense evaginations of the nuclear envelope and granular or rod-like structures apparently being extruded from the nucleus can be observed in this region. It is possible that the nuclear envelope plays a more significant role in the morphogenesis of the acrosome than has previously been attributed to it, not only determining thesite of acrosomal vesicle attachment but possibly also contributing materials to its formation. Spermatid elongation is accompanied by nuclear condensation, the appearance of new types of basic proteins rich in arginine and cysteine, and the exit of somatic type histones and water from the nucleus. Some of the morphological and histochemical changes which characterize this process have been shown to occur in an orderly fashion from the anterior t o the posterior pole of the spermatid nucleus (Courtens and Loir, '75). This particular pattern of nuclear-cytoplasmic interchange could be correlated with the formation of folds of redundant nuclear envelope in the neck region (deKretser, '69)rich in closely spaced pores. The remnants of the chromatoid body and annulate lamellae as well as all the spermatid cytoplasmic components that do not take part in the formation of the mature sperm are cast off in the residual bodies. It is generally assumed that they are phagocytized and disposed of by the Sertoli cells (see review by
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Fawcett, ’75). However, very little is known about t h e biological role, if any, of these structures and their significance, if any, in t h e regulation of seminiferous tubular function. LITERATURE CITED Anderson, D. R. 1966 Ultrastructure of normal and leukemic leucocytes in human peripheral blood. J. Ultrastr. Res. (Suppl.), 9: 5-42. Bouitelle, M., M. Lava1 and A. M. Dupuy Coin 1974 Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In: The Cell Nucleus. Vol. I. Harris Busch, ed. Acad. Press, New YorkLondon. Burgos, M. H., R. Vitale-Calpe and A. Aoki 1970 Fine structure of the testis and its functional significance. In: The Testis. A. D. Johnson, W. R. Gomes and N. L. Vandemark, eds. Acad. Press, New York-London, Vol. I, pp. 551-649. Chemes, H. E., M. Dym, D. W. Fawcett, N. Javadpour and R. J . Sherins 1977 Pathophysiological observations of Sertoli cells in patients with germinal aplasia or severe germ cell depletion. Ultrastructural findings and hormone levels. Biol. Reprod., 17: 108-123. Clermont, Y. 1962 Quantitative analysis of spermatogenesis of the rat: A revised model for t h e renewal of spermatogonia. Am. J. Anat.. 2 1 1 : 111-129. Commings, D. E., and T. A. Okada 1972 The chromatoid body in mouse spermatogenesis: evidence that i t may be formed by the extrusion of nucleolar components. J. Ultrastr. Res., 39: 15-23. Courtens, J. L., and M. Loir 1975 Mise en evidence par cytochimie ultrastructurale de la migration des histones riches en lysine a u cours de la spermiogenese du belier. J. de Microscopie, 24: 249-258. deKretser, D. M. 1969 Ultrastructural features of human spermiogenesis. 2. Zellforsch., 54: 68-89. Eddy, E. M. 1974 Fine structural observations on the form and distribution of nuage in germ cells of the rat. Anat. Rec., 178: 731-758. Erlandson, R. A. 1975 Nuclear envelope alterations in human tumor cells. Anat. Rec., 181: 353. Fawcett, D. W. 1974 Morphogenesis of the mammalian sperm acrosome in new perspective. In: The Functional Anatomy of t h e Spermatozoon. B.A. Azfelius, ed. Pergamon Press, Oxford-New York, pp. 199-210. - 1975 Ultrastructure and function of the Sertoli cell. In: Handbook of Physiology. Vol. V . Endocrinology. Section 7, Male Reproductive System. D. W. Hamilton and R. 0. Greep, eds. Williams and Wilkins Co., Baltimore, pp. 21-55. Fawcett. D. W.. and H. Chemes (1978. submitted) Chanees in distribution of nuclear pores during differentiationof the male germ cells. Cell and Tissue.
Fawcett, D. W., E. M. Eddy and D. M. Phillips 1970 Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis. Biol. Reprod., 2: 129-153. Fawcett, D. W., and S. Ito 1965 The fine structure of bat spermatozoa. Am. J. Anat., 216: 567-609. Franke, W. W., and U. Scheer 1974 Structure and functions of the nuclear envelope. In: The Cell Nucleus. Vol. I . H. Busch, ed. Acad. Press, New York-London. Franklin, L. E. 1968 Formation of the redundant nuclear envelope in monkey spermatids. Anat. Rec., 161: 149-162. Horstmann, E. 1961 Elektronenmikroskopiscbe untersuchungen zur spermiohistogenese beim menschen. 2. Zellforsch., 54: 68-89. Kierszenbaum, A. L., and L. L. Tres 1974 Nucleolar and perichromosomal RNA synthesis during meiotic prophase in t h e mouse testis. J. Cell Biol., 60: 39-53. La Cour, L. F., and B. Wells 1972 The nuclear pores of early meiotic prophase nuclei of plants. 2. Zellforsch., 123: 178-194. Maillet, P. L., and J. Gouranton 1965 Sur I’expulsion de I’acide ribonucleique nucleaire par les spermatides de Philaenus Spumarius (Hornoptera Cercopidae). C. R. Acad. Sc. Paris, 261: 1417-1419. Moses, M. J . 1964 The nucleus and chromosomes: a cytological perspective. In: Cytology and Cell Physiology. G. H. Bourne, ed. Acad. Press, New York-London, pp. 424-558. Oakberg, E. 1956 A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99: 391-413. Rowley, M. J., J. D. Berlin andC. G. Heller 1971 The ultrastructure of four types of human spermatogonia. Z. Zellforsch., 122: 139-157. Russell, L. 1976 Membrane modifications within the nucleus of r a t spermatocytes. Anat. Rec., 184: 519. Sandoz, D. 1970 Evolution des ultrastructures au cours de la formation de l’acrosome du spermatozoide chez la souris. J. Microscopie, 9: 535-558. 1974 Modifications in t h e nuclear envelope during spermiogenesis of Discoglossus Pictus (Anuran Amphibia). J. Submicr. Cytol., 6: 399-419. Smith, F. E., and J. D. Berlin 1977 Cytoplasmic annulate lamellae in human spermatogenesis. Cell Tiss. Res., 176: 235-242. Soderstrom, K. O., and M. Parvinen 1976a Incorporation of (3H) uridine by the chromatoid body during rat spermatogenesis. J. Cell Biol., 70: 239-246. 1976b Transport of material between the nucleus, the chromatoid body and t h e Golgi complex in the early spermatids of t h e rat. Cell Tiss. Res., 168: 335-342. szollosi, D. 1965 Extrusion of nucleoli from pronuclei of the rat. J. Cell Biol., 25: 545-562.
PLATES
PLATE 1 EXPLANATION OF FIGURES
1 Periphery of the nucleus of a human spermatogonium. There is a rounded protrusion enclosing a double membrane-limited vesicle with granulo-vesicular contents. Some dense aggregations (arrowheads) appear to be connected to the pores (NP) located on the internal side of the vesicle. X 24,300.
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2
Two parallel intranuclear flattened cisternae in a spermatogonium (C). A dense layer of chromatin-like granules surrounds each of the cisternae. There are no pores on the overlying nuclear envelope. X 70,200.
3
Pachytene spermatocyte showing a pore-rich area of the nuclear envelope with the Two dense close association of nucleolar (NUC) and chromosomal material (0. round structures, one connected to t h e nuclear envelope and the other one apparently free can be noted in the perinuclear cytoplasm (arrowheads). X 16,110.
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS H. E. Chemes. D. W. Fawcett and M. Dym
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
Figs. 4-8 Pachytene spermatocytes. 4
Pachytene spermatocyte. A major part of the nuclear surface is covered by single flattened cisterna of the rough endoplasmic reticulum (arrowheads, see also fig. 5). Nuclear pores are grouped over t h e remaining areas. The nucleolus (NUC), synaptinemal complexes (SC) and the disposition of the chromatin are characteristic of this cell type. X 12,150.
5 Higher magnifications of the perinuclear endoplasmic reticulum cisternae in pachytene spermatocytes. Note absence of pores on the nuclear envelope and presence of ribosomes on both sides of the cisternae. N, nucleus; Cyt, cytoplasm. Figure 5a X 20,970, figure 5b X 59,940.
6, 7 Two examples of membrane bounded dense bodies within local dilatations of the perinuclear space. In figure 6 the connection with the internal membrane of the nuclear envelope is clearly visible. The granular contents of these bodies is very similar to t h e dense aggregations of intranuclear material present in the vicinity. Figure 6 X 23,400, figure 7 X 24,210. 8 In this pachytene spermatocyte, a dense mass of chromatin (asterisk) is apposed to the inner aspect of the nuclear envelope in the vicinity of the nuclear pores. Dense granulo-filamentous material can be seen either free in the perinuclear cytoplasm or in connection with the pores (arrowheads). x 27,000.
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PLATE 2
PLATE 3 EXPLANATION OF FIGURE
9 Golgi phase spermatid. The nuclear envelope facing t h e Golgi complex shows absence of nuclear pores and a narrowing of the perinuclear space (between arrows). Patches of closely spaced nuclear pores and a dilatation of the perinuclear cistern are present peripherally to this zone (arrowheads). A dense cluster of intranuclear material (asterisk) and the chromatoid body (cb) are applied over the pore-rich areas on either side of the nuclear envelope. Note the other component of t h e chromatoid body (cb') near t h e Golgi complex. X 15,030.
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PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
Figs. 10-13 Golgi phase spermatids. 10 A small local protrusion of the nuclear envelope showing a dense content (arrow) is
apposed to t h e Golgi vesicles. The arrowheads indicate t h e outer and inner membranes of the nuclear envelope. x 35,550. 11 A section tangential to the nucleus near the Golgi complex of a spermatid. A cross section of the structure mentioned in figure 10 is clearly seen here (arrowhead). X 13,590. 12 The nuclear envelope facing the Golgi complex (G) shows absence of nuclear pores.
Intranuclear material appears to be emerging into t h e Golgi area of the cytoplasm (arrowhead). X 27,810. 13 A coated vesicle (arrowhead) is apparently fusing with a Golgi vesicle near a site of extrusion of nuclear material. X 27,630.
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PLATE 4
PLATE 5 EXPLANATION OF FIGURES
Figs. 14-17 Cap phase spermatids. 14 At the postacrosomal region of the nuclear envelope there is an area rich in nuclear pores with a cytoplasmic cistern of endoplasmic reticulum attached to it (arrows). Chromatoid body material (CB) can. be seen in t h e adjacent cytoplasm. X 8,910. 15 Higher magnification of a similar region to t h a t described in figure 14. Note the presence of numerous nuclear pores (arrowheads). A cytoplasmic cistern of endoplasmic reticulum covers the nuclear surface and a dense material is interspaced between them. Part of the chromatoid body (CB) is closely associated with the cytoplasmic side of the cistern. X 44,910. 16 A stack of annulate lamellae (AL) is attached to a pore-rich area of the nuclear
envelope. On the nuclear side a cluster of dense material is applied (asterisk). The granulo-vesicular component of the chromatoid body (CB') is noted nearby in t h e cytoplasm. X 18,720. 17 Three membrane bounded granular bodies (asterisks) are present in a local dilatation of the perinuclear space. Note the dense round condensation of material within t h e nucleus (arrowhead). X 39,870.
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PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
18 In this elongating spermatid a stack of annulate lamellae (all is attached to the posterior pole of the nucleus. The Golgi complex (g) and chromatoid body-like material (cb) are no longer related to the nucleus and can be seen in the residual cytoplasm. X 9,720. 19 Annulate lamellae (all and Golgi remnants ( g ) are present in the postnuclear residual cytoplasm. Only one cistern of endoplasmic reticulum remains attached to the posterior pole of the nucleus (arrows) over t h e pore-rich area. X 12,150. 20 Two chromatoid body remnants (CB) can be seen in the residual cytoplasm of a spermatid in advanced maturation. X 17,370. 21 Annulate lamellae (AL) and chromatoid body (CB) remnants are clearly discerned in this newly formed residual body. X 13,860.
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NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS H . E. Chemes, D. W. Fawcett and M. Dym
PLATE 6
ABSTRACT Different types of human germ cells show unusual features of the nuclear envelope. Spermatogonial nuclei demonstrate two kinds of modifications. The first one is a series of intranuclear flattened cisterns, parallel to each other and to the inner aspect of the nuclear envelope. The second one is a nuclear envelope protrusion into the cytoplasm occupied by a double membrane-limited vesicle. Pores are found on the membrane of the vesicle facing the interior of the nucleus. In spermatocytes the nuclear pores are concentrated over certain areas and completely absent from others. In the regions where they are absent a single cytoplasmic cistern of rough endoplasmic reticulum is closely apposed to the outer membrane of the nuclear envelope. Early modifications of the nuclear surface appear in spermatids before the attachment of the acrosomic vesicle and may indicate an active role of the nuclear envelope in the morphogenesis of the acrosome. In round spermatids nuclear pores are absent from the area which is first related to the Golgi and later covered by the acrosomal cap. Single or multiple layers of cytoplasmic annulate lamellae are closely associated with the nuclear envelope over the pore rich areas. Frequently there are intranuclear accumulations of dense material adjacent to the annulate lamellae-nuclear pore complex. The chromatoid body is usually present on the cytoplasmic side of this complex. In the elongating spermatids most annulate lamellae are free in the cytoplasm, often in relation with Golgi and chromatoid body remnants near the axial filament. Few stacks of annulate lamellae are noted adjacent to the pore rich nuclear regions. It is suggested t h a t the described modifications are related to an active nuclear-cytoplasmic interaction. Several types of modifications in the nucleus, the nuclear envelope and the adjacent cytoplasm of spermatogenic cells have been reported in different mammalian species. In spermatocytes and spermatids the nuclear pores are concentrated over certain regions of the nucleus and absent from others (Fawcett, '74; Franke and Scheer, '74; Sandoz, '74; Chemes and Fawcett, '78). Other kinds of modifications such as dilatations andlor narrowings of the perinuclear cistern in Golgi phase spermatids, alignment of chromatin granules on the inner aspect of the nuclear envelope underneath the acrosomal cap, the presence of a "redundant envelope" in late spermatids, and bleb or cistern-like formations in human spermatogonia and rat spermatocytes have also ANAT. REC.
(1978)192: 493-512.
been described (Fawcett and Ito, '65; Franklin, '68; deKretser, '69; Sandoz, '70; Rowley et al., '71; Fawcett, '74; Russell, '76). Furthermore, there are reports of annulate lamellae in human spermatogenic cells and their close associations with the nuclear envelope (Horstmann, '61; Smith and Berlin, '77). During the course of studies on normal and pathological human testis (Chemes et al., '77), we noted t h a t human germ cells are parReceived May 11, '78. Accepted July 27, '78. ' Supported by Research Grants HD 02344 and HD 06969 and by a Public Health Service International Research Fellowship 5-FO5TWO 2223-02 (Dr. H. Chemes). Present address: Division de Endocrinologia, Hospital General de Nirios, Gallo 1330, Buenos Aires, Argentina. 3Address reprint requests to: Don W. Fawcett, Department of Anatomy, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, U S A .
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sumed t o be continuous with t h e nuclear envelope (fig. 1). Serial sections would be required to ascertain whether these inclusions are detached vesicles entirely within the nucleoplasm or cross sections of digitiform invaginations of cytoplasm. There is a dense strip of chromatin-like material interposed between t h e vesicle and t h e nuclear envelope. The contents of t h e vesicle a r e granular, frequently resembling t h e cytoplasm, although in some cases i t is difficult t o distinguish them MATERIALS AND METHODS from nuclear material. Pores a r e found on t h e The source of material for this study has side of t h e vesicle facing t h e nucleus as well as been testicular biopsies from four normal in t h e nuclear envelope near t h e protrusion. young men of proven fertility.4These subjects Either t h e nucleolus or dense irregular clumps had normal urinary and plasma levels of go- of heterochromatin may be closely associated nadotropins and repeatedly normal sperm with these structures. I t is possible to find incounts (Rowley et al., '71). The tissues were stances where t h e flat cisternae described immersed immediately after removal in 3% above and t h e vesicles or invaginations are asglutaraldehyde buffered with 0.1 M cacodylate sociated. Single profiles of annulate lamellae at pH 7.3 and after 30 minutes were cut into a r e occasionally seen in t h e surrounding cyto1-mm blocks and left a n additional 60 minutes plasm. in t h e original fixative. They were subseSpermatocytes quently postfixed in 1.3% OsO, at 4°C for 60 minutes, dehydrated in a graded series of e t h In late zygotene and pachytene spermatoanol followed by propylene oxide and embed- cytes extensive areas of t h e nuclear surface ded in Epon 812. One micron thick sections are covered by closely applied flattened ciswere cut with glass knives in a MT2 Sorvall ternae of t h e rough endoplasmic reticulum ultramicrotome, mounted on glass slides and (RER). A row of ribosomes is located in t h e stained with 1%toluidine blue in 1%sodium narrow 14-20 nm space which separates t h e borate for light microscopic examination. nuclear envelope from the RER cistern; t h e Thin sections exhibiting pale gold t o silver ribosomes are more densely packed in this interference colors were cut with a diamond location than on t h e membranes of the RER knife and mounted in uncoated 200- to 300- cisterns facing t h e cytoplasm (figs. 4, 5). This mesh copper grids. These were double stained is a constant finding and gives t h e specious with uranyl acetate in 50% acetone and lead appearance of a double nuclear envelope on citrate and examined in a Philips 200 electron part of t h e periphery of spermatocyte nuclei. microscope at 60 kv. Although usually single, occasionally, two or more cisternae a r e stacked adjacent to t h e nuRESULTS cleus. It is difficult t o estimate t h e percentage Spermatogonia of t h e nuclear surface which is associated Two types of nuclear specializations a r e with t h e endoplasmic reticulum (ER) in this found in spermatogonia. (1) Intranuclear manner, but assuming t h a t t h e linear extent cisternae: this type of specialization consists of ER in thin sections is representative of its of a series of flattened cisternae parallel t o tridimensional area, a mantle of reticulum each other and to t h e inner aspect of t h e nu- may cover up t o 50% of t h e nuclear surface. clear envelope. An 18-nm thick layer of closely Nuclear pores a r e consistently excluded from spaced dense granules resembling chromatin these areas with associated reticulum, but are surrounds each of t h e cisternae. Usually one present grouped in variable numbers over the or two cisternae, each 1-2 p m long, a r e present rest of t h e nucleus (figs. 3, 4, 8). In some in(fig. 2). There a r e no nuclear pores immediate- stances they seem t o occupy nearly all of t h e ly overlying t h e cisternae, but pores a r e often space which is not covered by t h e RER. noted in t h e adjacent segments of t h e nuclear Clumps of dense chromatin granules and nuenvelope. (2) The second modification is a ve'The biopsies were provided through the kindness of Mans J . sicular inclusion at t h e periphery of t h e nu- Rowley and Carl G . Heller from the Pacific Northwest Research cleus, limited by a double membrane pre- Foundation, Seattle.
ticularly rich in these kinds of modifications and demonstrate a high degree of specificity in the association of a given feature to a particular germ cell type. In t h e present study we document t h e characteristics of several nuclear envelope specializations in human germinal cells, their association with particular organelles and their possible significance in the overall regulation of nuclear-cytoplasmic interaction.
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS
cleolar material are frequently attached to or concentrated near the pore-rich regions of the nuclear envelope (fig. 3). Smaller clusters of similar appearance are observed on the cytoplasmic side of the nuclear pores or free in the perinuclear cytoplasm (fig. 8 ) . Other round aggregations of electron opaque material are seen on the periphery of the nucleus of pachytene spermatocytes. Not infrequently there is a local separation of the inner and outer membranes of the nuclear envelope forming a dilatation in which membranebound dense bodies of similar structure are located. The images suggest an intranuclear origin for these granules and their subsequent extrusion into the cytoplasm (figs. 3,6, 7). Spermatids In early round spermatids a number of structural modifications can be seen on the nuclear envelope in the vicinity of the Golgi complex. Some coated and uncoated vesicles t h a t appear to be budding off from the outer membrane of the nuclear envelope appear in the region adjacent to the Golgi complex which, in some cases, shows similar coated surfaces on the side facing the nucleus (fig. 13). In addition, a round local protrusion of the nuclear envelope containing a dense material between the two membranes has been observed in the same region. This is a rare finding but its occurrence may actually be much more frequent if one considers the low probability of inclusion of such a small structure in random thin sections (figs. 10, 11). Occasionally, intranuclear materials in the form of round granules or rod like structures appear to be in a process of emergence into the Golgi area of the cytoplasm (fig. 12). Pores are absent from the nuclear surface adjacent to the Golgi complex in the area of impending attachment of the acrosomal vesicle and its expansion to form the acrosomal cap (fig. 9). However, closely spaced pores are present peripheral to this Golgi associated region and often the cavity of the nuclear envelope in these regions is somewhat dilated to a width of 60-85 nm (fig. 9). The chromatin in round spermatids is dispersed and has a fairly homogenous granular appearance, but there are some local condensations which tend to be associated with the inner side of the nuclear envelope in the porerich areas (figs. 9, 16). Even though spermatids lack a typical nucleolus i t has been suggested t h a t some of the dense material ob-
495
served in the nucleoplasm may represent nucleolar material (Burgos et al., '70). In many instances single or multiple layers of cytoplasmic cisternae, sometimes with the typical configuration of annulate lamellae, are apposed to the nuclear envelope over the pore-rich areas. A finely granular dense material is present between the lamellae and the nuclear envelope and between the lamellae themselves (figs. 14-16). Two types of chromatoid body material are clearly discernible in early round spermatids. The first one is a dark dense granular mass of irregular outline; 18- t o 20-nm granules can be seen toward the periphery of the mass but are not resolved within the dense core possibly because of closer packing; however, the possibility of two categories of granules cannot be excluded (figs. 9, 15). The peripheral granules are rather similar in size and density to the ribosomes observed either free in the cytoplasm or associated with membranous elements of the endoplasmic reticulum. The second type of chromatoid body consists of loosely arranged granules intermingled with numerous small membranous vesicles of ER (figs. 9, 16). Both compact and diffuse forms of the chromatoid body are located very near the nucleus and almost always in close association with the nuclear envelope or the annulate lamellae-nuclear pore complex previously described. Membrane bounded dense granules that appear to be contributed to the cytoplasm, can be observed between the two membranes of the nuclear envelope in young spermatids. Dense accumulations of nuclear material on the inner side of the nuclear envelope and similar granules free in the perinuclear cytoplasm are sometimes found in the vicinity (fig. 17). As spermatid maturation proceeds the acrosome spreads over the nucleus, and the porerich areas of the nuclear envelope and the associated chromatoid body move toward the caudal pole of the nucleus where they are concentrated when the acrosomal cap attains its full development (fig. 18). In the elongating spermatids a few stacks of annulate lamellae may remain attached to the posterior part of the nucleus. In this location the nuclear envelope, now very rich in pores, frequently diverges from the condensing chromatin and in some instances shows vesicular protrusions or folds extending toward the neck region. During the process of spermatid elongation all the cytoplasmic constituents move caudally.
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In the caudal region of the cytoplasm free stacks of annulate lamellae and chromatoid body constituents are found in close relation with Golgi remnants (figs. 18-20). The diminution in the number of annulate lamellae apposed t o the nuclear envelope and their simultaneous appearance in the periaxial region suggest that they detach from the nuclear envelope and become free in the caudal cytoplasm. Similar associations between annulate lamellae, chromatoid body material and Golgi remnants can be seen in residual bodies (fig. 21). The annulate lamellae share the same fate as other organelles, undergoing degeneration within the residual bodies. These are very rich in ribosomes and possess structures which resemble the granulo-vesicular components of the chromatoid body (fig. 21). Even though there is extensive degeneration of the membranous organelles in residual bodies, the ribosomes and the granular materials believed to be derived from the chromatoid body seem to persist until the late stages of residual body evolution just prior to their disappearance within the Sertoli cells. DISCUSSION
The presence of several kinds of nuclear envelope anomalies or specializations in human spermatogenic cells raises the question of their possible significance in nuclearcytoplasmic interaction. Briefly summarized the described features include the following: (1) Bleb and cistern-like formations in spermatogonia. (2) Regional aggregation of nuclea r pores in spermatocytes and spermatids. (3) Close apposition of rough endoplasmic reticulum to large areas of the nuclear envelope in spermatocytes and association of annulate lamellae with the nuclear envelope in spermatids. (4)Close association of the chromatoid body t o the nuclear envelope. (5) Modifications of the nuclear envelope adjacent t o the Golgi complex in the early steps of acrosome formation. Some of the nuclear modifications described here have previously been noted in human spermatogonia (Rowley et al., '71) and rat spermatogonia and spermatocytes (Russell, '76; Chemes, unpublished). Similar nuclear configurations have also been noted in neoplastic cells (Anderson, '66; Erlandson, '75). A possible relation of some of these nuclear events to chromatoid body formation was suggested in the case of rat spermatocytes (Russell, '76). In human spermatogonia, however,
there is little morphological evidence t o support a role of the cisternae and vesicles in the nucleo-cytoplasmic transfer of substances. Furthermore, their size and frequency of OCcurrence is not very impressive. A high incidence of spermatogonial degeneration has been reported as a normal feature of spermatogenesis in rodents (Clermont, '62; Oakberg, '56). The possibility exists that some of the nuclear envelope modifications described here may be part of the programmed degeneration of many of these cells. The fact that their incidence seems to be higher in cases of severe germ cell depletion (Chemes, unpublished observation) would tend to lend support t o the latter interpretation. The uneven distribution of nuclear pores in spermatocytes is of interest in relation t o the extensive cisternae of the RER found closely applied to the pore-free regions of the nuclear surface. These features are difficult to interpret but suggest t h a t the nuclear-cytoplasmic interchange mediated through nuclear pores takes part preferentially in certain areas of the nuclear surface. The fact that cytoplasmic organelles are not congregated over the porerich regions suggests that this grouping of pores is related to an inhomogeneity in distribution of nuclear components. In freeze-fracture replicas of guinea pig spermatocytes i t has been shown t h a t nuclear pores are closely clustered in areas of irregular contour separated by large pore-free regions and the suggestion has been made that the localization of nuclear pores in high concentration in certain areas may be related to the disposition of the underlying meiotic chromosomes (Moses, '64; Fawcett and Chemes, '78). The intimate association between the terminal end of synaptinemal complexes, condensed regions of chromosomes and of the sex vesicle with the nuclea r envelope suggest a possible relationship of these structures in the unusual distribution of nuclear pores in spermatocytes. The close association of ribosomes and RER with the nuclear envelope raises the possibility that the perinuclear cisternae may serve as storage site or way station in the translocation of proteins from nucleus to cytoplasm or in the opposite direction (for review see Franke and Scheer, '74). The presence of round fibrillar bodies in the nucleus and within local dilatations of the perinuclear space can be detected in pachytene spermatocytes and round spermatids. Similar structures have been described in
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS
rat pronuclei (Szollosi, '65) and in early meiotic plant cells (La Cour and Wells, '72). In the case of the rat pronuclei i t was reported that they originate in the nucleolus and are subsequently extruded to the cytoplasm. Additionally, Bouitelle et al. ('74) described the presence of small spherical structures of proteinaceous nature within the nucleus of very active cells, their numbers being sometimes influenced by hormones. Our findings in spermatocyte and spermatid nuclei closely resemble those in the previous reports. In this respect i t should be mentioned that pachytene spermatocytes are metabolically very active cells and known to be under hormonal influence and early spermatids are cells which undergo a series of elaborate morphogenetic changes that may be associated with high synthetic activity. Recent studies seem to indicate that the RNA synthesized in pachytene spermatocytes and early spermatids is HnRNA which is partially extruded into the cytoplasm (Kierszenbaum and Tres, '74; Soderstrom and Parvinen, '76a). It is precisely in those cellular types that the chromatoid body first appears, annulate lamellae become prominent and numerous indications of nuCleo-cytoplasmic interchange can be detected. It has been proposed that mRNA stored could be used in further steps when transcription has ceased in spermatid nuclei (Fawcett et al., '70; Soderstrom and Parvinen, '76a). The nonrandom distribution of nuclear pores in spermatids is related t o the development of the acrosome (Fawcett, '74). The increasing concentration of the pores toward the caudal pole coincides with the progressive expansion of the acrosomal cap over the anterior pole. It also coincides in time with the general movement of cytoplasmic constituents toward the tail region of the spermatid. The patchy distribution of pores in the caudal hemisphere of spermatid nuclei may be a reflection of their close association with the chromatoid body and with adherent annulate lamellae. An association of the chromatoid body with nuclea r pores has been described in a number of species and a nuclear origin of the chromatoid body has been suggested because of the apparent passage of material through the nuclear pores, but the mode of origin of the chromatoid body is still controversial (Maillet and Gouranton, '65; Fawcett et al., '70; Commings and Okada, '72; Eddy, '74; Soderstrom and Parvinen, '76b). The close association of the chromatoid body with the nuclear envelope
497
does not necessarily mean that it arises "in toto" from the nucleus but strongly supports some kind of interaction between chromatoid body and nucleus. This possibility derives support from the presence of dense intranuclear condensations in the region of the chromatoid body. In addition the concentration of nuclear pores and association of annulate lamellae in the same area may be an additional indication of a significant nuclear-cytoplasmic interchange a t this site. A number of modifications of the inner aspect of the nuclear envelope in the region of the acrosome has been described in spermatids (Sandoz, '70, '74; Fawcett, '74). It has been shown that some of these are actually present prior to the development of the acrosome. It has been proposed, therefore, that these nuclear envelope specializations may determine the site of attachment of the acrosomal vesicle and, therefore, the anterior pole of the sperm (Sandoz, '74). In newly formed human spermatids the portion of the nuclear envelope which faces the Golgi complex is characterized by blebbing activity and absence of nuclear pores. Occasionally dense evaginations of the nuclear envelope and granular or rod-like structures apparently being extruded from the nucleus can be observed in this region. It is possible that the nuclear envelope plays a more significant role in the morphogenesis of the acrosome than has previously been attributed to it, not only determining thesite of acrosomal vesicle attachment but possibly also contributing materials to its formation. Spermatid elongation is accompanied by nuclear condensation, the appearance of new types of basic proteins rich in arginine and cysteine, and the exit of somatic type histones and water from the nucleus. Some of the morphological and histochemical changes which characterize this process have been shown to occur in an orderly fashion from the anterior t o the posterior pole of the spermatid nucleus (Courtens and Loir, '75). This particular pattern of nuclear-cytoplasmic interchange could be correlated with the formation of folds of redundant nuclear envelope in the neck region (deKretser, '69)rich in closely spaced pores. The remnants of the chromatoid body and annulate lamellae as well as all the spermatid cytoplasmic components that do not take part in the formation of the mature sperm are cast off in the residual bodies. It is generally assumed that they are phagocytized and disposed of by the Sertoli cells (see review by
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Fawcett, ’75). However, very little is known about t h e biological role, if any, of these structures and their significance, if any, in t h e regulation of seminiferous tubular function. LITERATURE CITED Anderson, D. R. 1966 Ultrastructure of normal and leukemic leucocytes in human peripheral blood. J. Ultrastr. Res. (Suppl.), 9: 5-42. Bouitelle, M., M. Lava1 and A. M. Dupuy Coin 1974 Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In: The Cell Nucleus. Vol. I. Harris Busch, ed. Acad. Press, New YorkLondon. Burgos, M. H., R. Vitale-Calpe and A. Aoki 1970 Fine structure of the testis and its functional significance. In: The Testis. A. D. Johnson, W. R. Gomes and N. L. Vandemark, eds. Acad. Press, New York-London, Vol. I, pp. 551-649. Chemes, H. E., M. Dym, D. W. Fawcett, N. Javadpour and R. J . Sherins 1977 Pathophysiological observations of Sertoli cells in patients with germinal aplasia or severe germ cell depletion. Ultrastructural findings and hormone levels. Biol. Reprod., 17: 108-123. Clermont, Y. 1962 Quantitative analysis of spermatogenesis of the rat: A revised model for t h e renewal of spermatogonia. Am. J. Anat.. 2 1 1 : 111-129. Commings, D. E., and T. A. Okada 1972 The chromatoid body in mouse spermatogenesis: evidence that i t may be formed by the extrusion of nucleolar components. J. Ultrastr. Res., 39: 15-23. Courtens, J. L., and M. Loir 1975 Mise en evidence par cytochimie ultrastructurale de la migration des histones riches en lysine a u cours de la spermiogenese du belier. J. de Microscopie, 24: 249-258. deKretser, D. M. 1969 Ultrastructural features of human spermiogenesis. 2. Zellforsch., 54: 68-89. Eddy, E. M. 1974 Fine structural observations on the form and distribution of nuage in germ cells of the rat. Anat. Rec., 178: 731-758. Erlandson, R. A. 1975 Nuclear envelope alterations in human tumor cells. Anat. Rec., 181: 353. Fawcett, D. W. 1974 Morphogenesis of the mammalian sperm acrosome in new perspective. In: The Functional Anatomy of t h e Spermatozoon. B.A. Azfelius, ed. Pergamon Press, Oxford-New York, pp. 199-210. - 1975 Ultrastructure and function of the Sertoli cell. In: Handbook of Physiology. Vol. V . Endocrinology. Section 7, Male Reproductive System. D. W. Hamilton and R. 0. Greep, eds. Williams and Wilkins Co., Baltimore, pp. 21-55. Fawcett. D. W.. and H. Chemes (1978. submitted) Chanees in distribution of nuclear pores during differentiationof the male germ cells. Cell and Tissue.
Fawcett, D. W., E. M. Eddy and D. M. Phillips 1970 Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis. Biol. Reprod., 2: 129-153. Fawcett, D. W., and S. Ito 1965 The fine structure of bat spermatozoa. Am. J. Anat., 216: 567-609. Franke, W. W., and U. Scheer 1974 Structure and functions of the nuclear envelope. In: The Cell Nucleus. Vol. I . H. Busch, ed. Acad. Press, New York-London. Franklin, L. E. 1968 Formation of the redundant nuclear envelope in monkey spermatids. Anat. Rec., 161: 149-162. Horstmann, E. 1961 Elektronenmikroskopiscbe untersuchungen zur spermiohistogenese beim menschen. 2. Zellforsch., 54: 68-89. Kierszenbaum, A. L., and L. L. Tres 1974 Nucleolar and perichromosomal RNA synthesis during meiotic prophase in t h e mouse testis. J. Cell Biol., 60: 39-53. La Cour, L. F., and B. Wells 1972 The nuclear pores of early meiotic prophase nuclei of plants. 2. Zellforsch., 123: 178-194. Maillet, P. L., and J. Gouranton 1965 Sur I’expulsion de I’acide ribonucleique nucleaire par les spermatides de Philaenus Spumarius (Hornoptera Cercopidae). C. R. Acad. Sc. Paris, 261: 1417-1419. Moses, M. J . 1964 The nucleus and chromosomes: a cytological perspective. In: Cytology and Cell Physiology. G. H. Bourne, ed. Acad. Press, New York-London, pp. 424-558. Oakberg, E. 1956 A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99: 391-413. Rowley, M. J., J. D. Berlin andC. G. Heller 1971 The ultrastructure of four types of human spermatogonia. Z. Zellforsch., 122: 139-157. Russell, L. 1976 Membrane modifications within the nucleus of r a t spermatocytes. Anat. Rec., 184: 519. Sandoz, D. 1970 Evolution des ultrastructures au cours de la formation de l’acrosome du spermatozoide chez la souris. J. Microscopie, 9: 535-558. 1974 Modifications in t h e nuclear envelope during spermiogenesis of Discoglossus Pictus (Anuran Amphibia). J. Submicr. Cytol., 6: 399-419. Smith, F. E., and J. D. Berlin 1977 Cytoplasmic annulate lamellae in human spermatogenesis. Cell Tiss. Res., 176: 235-242. Soderstrom, K. O., and M. Parvinen 1976a Incorporation of (3H) uridine by the chromatoid body during rat spermatogenesis. J. Cell Biol., 70: 239-246. 1976b Transport of material between the nucleus, the chromatoid body and t h e Golgi complex in the early spermatids of t h e rat. Cell Tiss. Res., 168: 335-342. szollosi, D. 1965 Extrusion of nucleoli from pronuclei of the rat. J. Cell Biol., 25: 545-562.
PLATES
PLATE 1 EXPLANATION OF FIGURES
1 Periphery of the nucleus of a human spermatogonium. There is a rounded protrusion enclosing a double membrane-limited vesicle with granulo-vesicular contents. Some dense aggregations (arrowheads) appear to be connected to the pores (NP) located on the internal side of the vesicle. X 24,300.
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2
Two parallel intranuclear flattened cisternae in a spermatogonium (C). A dense layer of chromatin-like granules surrounds each of the cisternae. There are no pores on the overlying nuclear envelope. X 70,200.
3
Pachytene spermatocyte showing a pore-rich area of the nuclear envelope with the Two dense close association of nucleolar (NUC) and chromosomal material (0. round structures, one connected to t h e nuclear envelope and the other one apparently free can be noted in the perinuclear cytoplasm (arrowheads). X 16,110.
NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS H. E. Chemes. D. W. Fawcett and M. Dym
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
Figs. 4-8 Pachytene spermatocytes. 4
Pachytene spermatocyte. A major part of the nuclear surface is covered by single flattened cisterna of the rough endoplasmic reticulum (arrowheads, see also fig. 5). Nuclear pores are grouped over t h e remaining areas. The nucleolus (NUC), synaptinemal complexes (SC) and the disposition of the chromatin are characteristic of this cell type. X 12,150.
5 Higher magnifications of the perinuclear endoplasmic reticulum cisternae in pachytene spermatocytes. Note absence of pores on the nuclear envelope and presence of ribosomes on both sides of the cisternae. N, nucleus; Cyt, cytoplasm. Figure 5a X 20,970, figure 5b X 59,940.
6, 7 Two examples of membrane bounded dense bodies within local dilatations of the perinuclear space. In figure 6 the connection with the internal membrane of the nuclear envelope is clearly visible. The granular contents of these bodies is very similar to t h e dense aggregations of intranuclear material present in the vicinity. Figure 6 X 23,400, figure 7 X 24,210. 8 In this pachytene spermatocyte, a dense mass of chromatin (asterisk) is apposed to the inner aspect of the nuclear envelope in the vicinity of the nuclear pores. Dense granulo-filamentous material can be seen either free in the perinuclear cytoplasm or in connection with the pores (arrowheads). x 27,000.
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PLATE 2
PLATE 3 EXPLANATION OF FIGURE
9 Golgi phase spermatid. The nuclear envelope facing t h e Golgi complex shows absence of nuclear pores and a narrowing of the perinuclear space (between arrows). Patches of closely spaced nuclear pores and a dilatation of the perinuclear cistern are present peripherally to this zone (arrowheads). A dense cluster of intranuclear material (asterisk) and the chromatoid body (cb) are applied over the pore-rich areas on either side of the nuclear envelope. Note the other component of t h e chromatoid body (cb') near t h e Golgi complex. X 15,030.
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NUCLEAR ENVELOPE I N HUMAN SPERMATOGENIC CELLS H E Chemes, D W Fawcett and M Dym
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
Figs. 10-13 Golgi phase spermatids. 10 A small local protrusion of the nuclear envelope showing a dense content (arrow) is
apposed to t h e Golgi vesicles. The arrowheads indicate t h e outer and inner membranes of the nuclear envelope. x 35,550. 11 A section tangential to the nucleus near the Golgi complex of a spermatid. A cross section of the structure mentioned in figure 10 is clearly seen here (arrowhead). X 13,590. 12 The nuclear envelope facing the Golgi complex (G) shows absence of nuclear pores.
Intranuclear material appears to be emerging into t h e Golgi area of the cytoplasm (arrowhead). X 27,810. 13 A coated vesicle (arrowhead) is apparently fusing with a Golgi vesicle near a site of extrusion of nuclear material. X 27,630.
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NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS H. E . Chemes, D. W. Fawcett and M. Dym
PLATE 4
PLATE 5 EXPLANATION OF FIGURES
Figs. 14-17 Cap phase spermatids. 14 At the postacrosomal region of the nuclear envelope there is an area rich in nuclear pores with a cytoplasmic cistern of endoplasmic reticulum attached to it (arrows). Chromatoid body material (CB) can. be seen in t h e adjacent cytoplasm. X 8,910. 15 Higher magnification of a similar region to t h a t described in figure 14. Note the presence of numerous nuclear pores (arrowheads). A cytoplasmic cistern of endoplasmic reticulum covers the nuclear surface and a dense material is interspaced between them. Part of the chromatoid body (CB) is closely associated with the cytoplasmic side of the cistern. X 44,910. 16 A stack of annulate lamellae (AL) is attached to a pore-rich area of the nuclear
envelope. On the nuclear side a cluster of dense material is applied (asterisk). The granulo-vesicular component of the chromatoid body (CB') is noted nearby in t h e cytoplasm. X 18,720. 17 Three membrane bounded granular bodies (asterisks) are present in a local dilatation of the perinuclear space. Note the dense round condensation of material within t h e nucleus (arrowhead). X 39,870.
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PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
18 In this elongating spermatid a stack of annulate lamellae (all is attached to the posterior pole of the nucleus. The Golgi complex (g) and chromatoid body-like material (cb) are no longer related to the nucleus and can be seen in the residual cytoplasm. X 9,720. 19 Annulate lamellae (all and Golgi remnants ( g ) are present in the postnuclear residual cytoplasm. Only one cistern of endoplasmic reticulum remains attached to the posterior pole of the nucleus (arrows) over t h e pore-rich area. X 12,150. 20 Two chromatoid body remnants (CB) can be seen in the residual cytoplasm of a spermatid in advanced maturation. X 17,370. 21 Annulate lamellae (AL) and chromatoid body (CB) remnants are clearly discerned in this newly formed residual body. X 13,860.
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NUCLEAR ENVELOPE IN HUMAN SPERMATOGENIC CELLS H . E. Chemes, D. W. Fawcett and M. Dym
PLATE 6
