Chromosoma (Berl.) 53, 141--151 (1975) 9 by Springer-Verlag 1975
Nucleolar RNA Synthesis of Meiotic Prophase Spermatocytes in the Human Testis Laura L. Tres The Laboratories for Reproductive Biology, Division of Health Affairs, 111 Swing Building, University of North Carolina at Chapel Hill, Chapel Hill, N. C. 27514, U.S.A. Abstract. Human meiotic prophase spermatocyte nuclei were studied by electron microscope autoradiography after a 3 hours aH-uridine labeling pulse, followed by postincubation in non-radioactive medium. In autosomes, 3tt-uridine nucleolar labeling reaches a peak during early-middle zygotene prior to the peak labeling of chromosomal RNA species at middle pachytene. Transcription activities of sex chromosomes are inconspicuous when compared with that of autosomes. An increasing condensation of nucleolar-associated chromatin in acrocentric bivalents contributes to the formation of basal knobs in human pachytene spermatocytes. Upon completion of knob formation, nucleolar components segregate and the uptake of aH-uridine decreases. These findings suggest that the template capability of ribosomal DI~A cistrons, located next to the basal knob region, is largely associated with a dispersed state of chromatin whereas increased chromatin condensation is correlated with a restriction of ribosomal RNA transcription.
Introduction Previous studies have indicated that uucleolar and chromosomal R N A synthesis takes place in mouse spermatocytes during meiotic prophase (Kierszenbaum and Tres, 1974a, b). The studies indicated further that nucleolar organizers may be located next to paracentromeric heterochromatic regions or basal knobs (Woollam and Ford, 1964) of some autosomal bivalents, whereas ribonucleoprotein complexes (presumably heterogeneous nuclear RNA) are transcribed at the ehromatin loops of all autosomes (Kierszenbaum and Tres, 1974b). These findings are based on light and electron microscope autoradiography and on visualization of structures displaying transcription relationships. Besides providing original evidences for a nucleolar RNA synthesis during meiotic prophase in mouse spermatocytes, we advanced an interpretation (Kierszenbaum and Tres, 1974a) of a puzzling fact described by Monesi (1965) and Utakoji (1966), namely that nucleoli, especially those associated with the sex chromosomes, do not incorporate labeled precursors for RNA. The association of nucleoli with specific terminally-placed chromosomal regions ("nucleolar chromomeres") of certain bivalents in human spermatocytes was described by Ferguson-Smith (1964)on the basis of light microscopy. Solari and Tres (1967, 1970a, b) have pointed out some fine structural aspects of nueleoli associated with autosomal bivalents in human pachytene spermatocytes. In this study, the nucleolar and chromosomal SH-uridine-labeling patterns seen at leptotene, zygotene, and pachytene stages in human spermatocytes are described using an electron microscope autoradiographie technique. Structural features of the sex chromosomes as related to nucleolar formation by autosomal
142
L.L. Tres
b i v a l e n t s are also reported since t h e y facilitates t h e i d e n t i f i c a t i o n of meiotic prophase stages i n the man.
Material and Hethods Biopsy samples from testes of 8 normal, untreated patients attending infertility clinics were examined. Selection of subjects was based on clinical and laboratory tests. Upon removal, a few seminiferous tubules were separated from the biopsies and the remaining portions were immediately fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 6.9) for 2 hours, washed in buffer, and postfixed in 1% buffered osmium tetroxide for 1 hour. Tissues were embedded in Maraglas (Polyseiences, Inc., Rydal, Pa. USA) according to the usual procedure. Portions of the seminiferous tubules set aside before fixation were stained with acetic=orcein and squashed between a microscope slide and a coverglass for rapid evaluation of spermatogenic cells. Samples of tubules from 4 patients were incubated in Basal Medium (Eagle) with Earle's balanced salt solution (2 mM L-glutamine added) containing 50 izCi/ml of aH-uridine (5,6-aHuridine, sp. act. 42.4 Ci/mmoi, New England Nuclear, Boston, Mass. USA). After 3 hours of incubation alb 34~C, the samples were rinsed several times with non-radioactive medium and either fixed immediately or incubated further in non-radioactive medium containing 100 ~g/mI of cold uridine (Sigma Chemical Co., St. Louis, Mo. USA) for another 30 minutes period and fixed thereafter. These postincubation procedures were designed to wash out unbound all. uridine from the samples. These procedures were tested in specimens from mouse testes and compared the results with previous autoradiographic data obtained by injecting ~H-uridine intratesticularly (Kierszenbaum and Tres, 1974a). Similar nucleolar and chromosomal labeling sites were obtained with both procedures. For electron microscope autoradiography, thin sections with gold interference colors were transferred to Parlodion-coated half microscope slides split lengthwise. Before coating with emulsion, sections were stained with uranyl acetate and lead citrate. A thin carbon layer was evaporated over the stained sections and the slides then dipped in a diluted Ilford L4 emulsion (Ilford, Ltd., Ilford, Essex, England). After exposure for 1 to 6 months, the preparations were developed with the gold latensification-Elon-ascorbic acid procedure (Salpeter and Bachmann, 1972; Wisse and Totes, 1968), fixed with Kodak Rapid Fixer (Eastman Kodak, Co., Rochester, N.Y., U.S.A.) and rinsed in water, the specimens were stripped off onto a water surface. Copper electron microscope grids were placed over the sections and picked up on a polyethylene film strip (Parafilm "M", American Can Co., Neenah, Wis., U.S.A.). Label distribution within nuclei was determined quantitatively using tissues incubated with all-uridine followed by a chase with cold uridine, as indicated above. Grain densities overlying 10 [tinu units of surface area were scored and the values obtained were corrected for background density. Micrographs were taken with Siemens Elmiskop I and JEM 100B electron microscopes operated at 80 kV. Each instrument was calibrated with carbon grating replicas.
Results
Nucleolar Organization at Di[ferent Stages of the Meiotic Prophase A t e m p o r a l correlation b e t w e e n chromosomal p a i r i n g a n d 8H-uridine labeling of distinctive nucleolar c o m p o n e n t s in various meiotic prophase stages of h u m a n spermatocytes (exposed for 3 hours to radioactive uridine) can be achieved i n electron micrographs of autoradiograms. The results suggest a real relationship b e t w e e n t h e two e v e n t s d u r i n g the first p a r t of t h e meiotic cycle. Thus, a t leptotene, w h e n chromosomal cores (Fig. 1) a n d c h r o m a t i n c o n d e n s a t i o n are r e a d i l y found, one can detect small nucleoli, each characterized b y a n a c c u m u l a t i o n of dense fibrils s u r r o u n d i n g a center of more e x t e n d e d fibrils (Fig. 1). A t early zygotene,
RNA Synthesis in the Human Testis
143
Fig. 1. Leptotene spermatoeyte displaying a chromosomal core (arrow) and a nucleolus formed by dense fibers (]d) surrounding a fihrillar center (re). The scale mark indicates 1 ~m Fig. 2. Middle-to-late zygotene spermatoeyte. The nucleolus is formed by a fibrillar center (fc) surrounded by dense fibrils (Jd) and a fibrillo-granular component (It). Note that a portion of the fibrillar center is apparently continuous with loosely extended chromatin fibrils. Arrows indicate chromosomal cores. The scale mark indicates 1 ~zm
chromosomal cores and short segments of synaptonemal complexes in autosomes indicate initiation of pairing of homologous chromosomes. At early zygotene, aH-uridine labeling of nucleoli is mainly associated with dense fibrillar and fibrillo-granular components (Figs. 3 and 4). At late zygotene synaptonemal complexes predominate and the condensed ehromatin mass of the sex pair can be recognized (Solari and Tres, 1970a, b). During this stage, band-shaped nucleoli located next to the nuclear envelope (Fig. 2) display a fibrillar center (presumably consisting of proteins ; l~eeher et al., 1970) directly related to dispersed ehromatin fibers and separated b y a dense fibrillar shell from fibrillo-granular components extending toward the inner nuclear region. As spermatoeytes approach the end of zygotene, nueleolar components start their natural segregation process and silver grains accumulate predominantly on the dense fibrillar areas (Fig. 5). Furthermore, homologous pairing and formation of synaptonemal complexes is completed at late zygotene. Chromatin condensation of autosomes fluctuates throughout male meiotic prophase. This serves as an additional feature for recognition of prophase stages
144
L.L. Tres
Fig. 3. Autoradiograph showing early zygotene spermatocytes, tteavily labeled Ser~oli cell nuclei (S) contrast with less labeled zygotene spermatocytes (arrows). The nucleolus (Nu) of a Sertoli cell is well labeled. Exposure time: 65 days. Incubated with 31I-uridine for 3 hours. Postincubated with cold uridine for 30 minutes. Developing procedure: Gold latensificationElon-ascorbic acid procedure. The scale mark indicates 2 ~zm
wherein, after a progressive condensation during leptotene, zygotene, and early pachytene, chromatin decondenses at middle and late pachytene (see summary diagram in Fig. 9). As pachytene stage advances, autosomal ehromatin adopts a more even, extended pattern except at the ehromatin region associated with nucleoli (basal knobs) which become completely condensed at late pachytene (Fig. 7). This last feature contrasts with the dispersed chromatin region associated with fibrillar centers as observed at zygotene (Fig. 2). To an increased degree, condensation of the basal knob region of autosomal bivalents coincides with a distinctive rearrangement of nucleolar components [nucleolar segregation (Simard and Bernhard, 1966); Fig. 7] and a decline of aH-uridine labeling (Figs. 6 and 7 inset). Results of quantitative autoradiographic analyses of nucleolar and extranneteolar labeling sites throughout meiotic prophase stages (obtained from spermatocytes from different seminiferous tubules) are shown in Fig. 8. The radioactivity profiles indicate that nucleolar RNA synthesis precedes chromosomal I~NA synthesis. It was previously indicated in the mouse testis that chromo-
I~NA Synthesis in the Human Testis
145
l~'ig. 4. High magnification of zygotene nuclei illustrated in Fig. 3 indicating the distribution of nucleolar silver grains. The scale mark is 2 ~m
Fig. 5. Lute zygotene spermatocyte. The plain arrow indicates ~ nueleolar fibrillar center partially surrounded by dense fibrils (/d) conspicuously aH-uridine-labeled. (/g) fibrillo-granular region free of silver grains. Portions of chromosomal cores (crossed arrow) and of a synaptonemal complex (sc) are displayed. 3H-uridine Lbeling as Fig. 3. Exposure time: 65 days. Scale mark: 1 ~xm
146
L.L. Tres
Fig. 6. Autoradiograph of an early pachytene spermatocyte displaying considerable nuclear att-nridine labeling. No radioactivity is found over the sex chromosomal pair (X Y). Arrows indicate dense bodies projecting from the X Y chromosomal dense mass. Granular components of a spheric nucleolus (Nu) are weakly labeled. 3H-uridine labeling as Fig. 3. Exposure time: 65 days. Scale mark: 2 ~m
somal RNA synthesis corresponds, in a great extent, to heterogeneous nuclear RlqA-protein complexes transcribed at chromosomal loops (Kierszenbaum and Tres, 1974b).
Structural Features during Di//erentiation o/the X Y Chromosomal Pair Electron micrographs vf human X Y chromosomes have indicated (Solari and Tres, 1970a, b) that the paired structures can be identified during zygoteneearly pachytene as a condensed chromatin mass with two cores of different length. The cores share a short synaptonemal complex at their mutual ends. The transient appearance of dense bodies along the cores of both X and Y chromosomes at late zygotene-early pachy~ene (Figs. 6 and 9) followed by "ring-li]ce structures" at middle-late pachytene (Figs. 7 and 9) are correlated with events occurring during autosomal nucleolar organization and extranueleolar 3H-uridine labeling in human spermatocytes. The major features are: First, the condensed (heterochromatie) sex chromosomes display little or no RNA synthesis during meiotic prophase (Fig. 6) ; second, although a nucleolus is frequently observed next to the sex chromosomes (Solari and Tres, 1970a, b; Luciani, 1970a, b), the X and Y chromosomes presumably
RNA Synthesis in the Human Testis
147
Fig. 7. Middle-to-late pachytene spermatocyte. The sex chromosomes (X Y) show ring-like structures (arrow). A nucleolus, attached to an entirely condensed basal knob (BK) shows three regions: a fibrillar center (re), a librillar dense region ([d) 0.nd a large granular region (g). Scale mark: 1 Bm. Inset: Autoradiograloh of a nucleolus at the same meiotic stage. A few silver grains are seen over the spherical granular structure. Exposure time: 70 days. Pulse labeling as Fig. 3. Scale mark: 0.5 [zm do n o t organize a nucleolns as i n d i c a t e d b y i n situ ribosomal I~NA-DNA hybridization studies (Henderson et al., 1972); third, dense bodies along the axial cores of the sexual pair, formerly appearing in t h r e e - d i m e n s i o n a l reconstructions of
148
L . L . Tres
35
z~fch
romosomalRNA
25
%
nucleolar RNA
=1. o
s O W
m
mW
z
z
-z
I..,U I"-
W S
"'
N
0 8
n"W ,,~ I.-
w>-
13W ~.
--
i
z
.-II-
>-
W I--
0
Fig. 8. Silver grains scored ever nueleolar and nucleoplasmie surface areas in electron microscope autoradiographs of human spermatocytes after 3 hours of 3I-I-uridine labeling. Ten spermatocyte nuclei per meiotic prophase stage were counted. The surface area (10 tzm 2) was taken by superimposing over autoradiographs of fixed magnification (• 15000) a lattice with regularly spaced points. The grain density distribution over each nuclear area was scored and divided by the number of lattice points
Fig. 9. Diagram indicating relevant features of the sexual pair (X, Y) and autosomal nucleolar development and changes of autosomal chromatin condensation (Chr. Con&)throughout meiotic prophase stages of human sperm~toeytes. The basal knob (BK) increases its condensation as the spermatocyte moves to late paehytene. The segregation of nucleolar components (]c, fibrillar centers, ], fibrils, and g, granules) parallels the progressive condensation of the basal knob. The diagramatic representation of the sex chromosomal pair is based on threedimensional reconstructions obtained from serial sections (Solari and Tres, 1970a; Tres, 1970)
]~I~A Synthesis in the Human Testis
149
the sex pair (Solari and Tres, 1970a) and more recently in spreads of human spermatoeytes (Moses et al., 1975), characterize late zygotene-early pachytene stages (Fig. 9). After these stages nucleolar I~NA synthesis declines, and fourth, ring-like structures along cores of sex chromosomes, are observed during middlelate pachytene stages, when chromosomal I~NA synthesis rises to a maximal level in autosomal bivalents following a decrease of nucleolar RNA transcription (Fig. 8).
Discussion
Electron microscope autoradiographic evidence from the human testis indicates that during meiotic prophase autosomal bivalents are involved in both nuc]eolar and chromosomal t~NA synthesis. These findings agree with previous experimental results of transcription activities in the mouse testis (Kierszenbaum and Tres, 1974a, b). A quantitative analysis of autoradiographie data from the human testis indicates that at middle pachyiene the rate of chromosomal RNA synthesis is at a peak. Peak production of nucleolar RNA is earlier, during zygotene and takes place at well defined loci of some acrocentric autosomes. Solari and Tres (1970a, b) have previously described fine structural and histochemical features of meiotic prophase stages in the human testis. The present results extend this information by defining sites of atI-uridine labeling as well as the characteristic structural sequence during nucleolar organization. The relevant aspects of nucleolar formation during meiotic prophase in man are: (1) A progressive condensation of the autosomal ehromatin region closely associated with nueleo]i, leading to the formation of well defined basal knobs at late paehytene, and (2), a rearrangement or segregation of nuclcolar components coinciding with the formation of basal in acrocentric autosomes knobs and a decrease in nucleolar aH-uridine labeling. Polycistronic genes for precursors of ribosomal I~NA occur in eukaryotic genomes and are located in specific chromosomal nucleolar organizer regions (Wallace and Birnstiel, 1966). In situ ribosomal I%NA-DNA hybridization procedures in somatic cells (Henderson et al., 1972) indicate that, in man, the satellite region of acrocentric autosomes (chromosomes 13, 14, t5, 21 and 22) contain ribosomal DNA cistrons. The association of nucleoli with basal knobs of human autosomal bivalents was indicated by light microscopy (Ferguson-Smith, 1964; Solari and Tres, 1967; Luciani, 1970a, b) and electron microscopy (Solari and Tres, 1967, 1970a, b; Tres, 1970). Furthermore, the nueleolar organizers of mouse chromosomes are situated near the centromeric heterochr0matin (Levan et al., 1962) which contains satellite DNA (Purdue and Gall, 1970). Nevertheless, the specific region of the basal knob involved in the molecular organization of precursors of ribosomal I~NA has not yet been identified. Knowledge of these facts favours inferring that the progressive chromatin condensation of the basal knob region in acrocentrie autosomes might affect the efficiency of ribosomal DNA cistrons, prompting the cessation of precursors of ribosomal RNA synthesis. In fact, these changes are accompanied by a decrease in ~H-uridine labeling and by nucleolar changes referred to as nucleolar segregation observed from late zygotene on. Morphological and biochemical evidence from several tissues indicates that
150
L.L. Tres
nucleolar segregation can be induced by inhibitors of nucleolar I~NA synthesis (for a complete review, see Busch and Smetana, 1970). From the foregoing discussion, it seems apparent that regulation of ribosomal gene function depends to a certain extent on controlling chromatin organization of basal knob segments of acrocentric bivalents. The evolution of autosomal nucleoli varies in different species. For instance, in the mouse testis, nucleolar masses, once detached from their primary autosomal nucleolar organizer loci during middle paehytene, migrate toward the sex chromosomal mass, rearrange their nucleolar components and form a natural segregated nucleoli which do not incorporate aH-uridine (Kierszenbaum and Tres, 1974a). I n human spermatocytes, this migrating behavior is not observed and nucleoli segregate at their putative primary point. Since nucleolar masses have been observed next to the X rY chromosomal pair in human (Solari and Tres, 1970a, b; Lueiani, 1970a, b) and mouse spermatocytes (Solari, 1964; Monesi, 1965; Kierszenbaum and Tres, 1974a), one question that arise is the possibility of a nucleolar organizing activity of the sex chromosomes as postulated by Ohno et al. (1957). I n this respect, in situ ribosomal t{NA-DNA hybridization procedures in human (Henderson et al., 1972) and mouse (Henderson et al., 1974) somatic cells have shown that several acrocentric chromosomes (but not X and J(chromosomes) contain ribosomal DNA eistrons. Additional data from autoradiographic experiments in mouse spermatoeytes (Kierszenbaum and Tres, 1974a) indicate that the condensed sex chromosomes do not display a structural and aH-uridine-labeling sequence as described for nucleoli associated with autosomal bivalents or during the organization of somatic cell nucleoli (i.e., fibrillar nucleolar components serve as precursors for the granular material, Noel et al., 1971). Finally, it has been proposed on the basis of light microscopy (Luciani, 1970a, b) and electron microscopy (Solari and Tres, 1970a, b) that " m a i n " and "secondary" nueleoli may be present in human pachytene spermatocytes. Nevertheless, autoradiographic data together with a structural evaluation of nucleolar organizing features preceding late pachytene, indicate that all nucleoli evolve almost synchronoulsy during the appropiate meiotic stages. Therefore, the concept of main and secondary nucleoli should be discarded.
Acknowledgments. The study reported here was initiated in the Centro de Investigaciones sobre Reproduceion, Facultad de Medicina, Universidad de Buenos Aires, Argentina. I wish to thank Dr. tt. S. Bennett for his support and interest. I am also grateful to Dr. M. J. Moses for criticism of the manuscript. This work was made possible by a grant from The l~oekefeller Foundation to The Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill.
References Busch, H., Smetana, K.: The Nucleolus. New York: Academic Press 1970 Ferguson-Smith, M. A. : The sites of nucleolus formation in human pachytene chromosomes. Cytogenetics 8, 124-134 (1964) Henderson, A. S., Eicher, E.M., Yu, M. T., Atwood, K. C. : The chromosomal location of ribosomal DNA in the mouse. Chromosoma (Berl.) 49, 155-160 (1974)
RNA Synthesis in the Human Testis
151
Henderson, A. S., Warburton, D., Atwood, K. C. : Loc~tion of ribosomal DNA of the human chromosome complement. Prec. nat. Acad. Sci. (Wash.) 69, 3394-3398 (1972) Kierszenbaum, A.L., Tres, L.L.: Nucleolar and perichromosom~l RNA synthesis during meiotic proph~se in the mouse testis. J. Cell Biol. 60, 39-53 (1974a) Kierszenbaum, A. L., Tres, L. L. : Transcription sites in spread meiotic prophase chromosomes from mouse spermatocytes. J. Cell Biol. 68, 923-935 (1974b) Levan, A., Hsu, T. C., Stich, H. F. : The idiogram of the mouse. Hereditas (Lund) 48, 677-689 (1962) Luciani, J. M.: Les chromosomes melotiques de l'homme. I. La meiosis normale. Ann. Gtn@t. 13, 101-111 (1970a) Luciani, J.M.: Les chromosomes meiotiques de l'homme. II. Le nuclOole. Les chiasmas. III. La sterglit@ masculine. Ann. G@ntt. 18, 169-182 (1970b) Monesi, V.: Synthetic activities during spermatogenesis in the mouse. Exp. Cell Res. 89, 197-224 (1965) Moses, M. J., Counce, S. J., Paulson, D. F. : Synaptonem~l complex complement of man in spreads of spermatoeytes, with details of the sex chromosome pair. Science 187, 363-365 (1975) Noel, d. S., Dewey, W. C., Abel, J. H., Thompson, R. P.: Ultrastructure of the nucleolus during the Chinese hamster cell cycle. J. Cell Biol. 49, 830-847 (1971) Ohno, S., Kaplan, W., Kinosit~, R.: Heteroehromatic regions and nucleolus organizers in chromosomes of the mouse, Mus musculus. Exp. Cell Res. 18, 358-364 (1957) Purdue, M. L., Gall, J. G.: Chromosomal localization of mouse satellite DNA. Science 168, 1356-1358 (1970) Recher, L., Whitescarver, J., Briggs, L. : A cytochemic~l and radioautographic study of human tissue culture nueleoli. J. Cell Biol. 45, 479-492 (1970) Salpeter, M. M., Bachmann, L. : Autoradiography. In: Principles and techniques of electron microscopy. Biological applications (M. A. Hayat, ed.), vol. 2, p. 221-278. New York: Van Nostrand Reinhold Company 1972 Simard, R., Bernhard, W.: The phenomenon of nucleolar segregation. Specific action of certain ~ntimetabolites. Int. J. Cancer 1, 463-479 (1966) Solari, A. J. : The morphology and ultrastructure of the sex vesicle in the mouse. Exp. Cell Res. 86, 160-168 (1964) Solari, A. J., Tres, L. L.: The ultrastrueture of the human sex vesicle. Chromosoma (Berl.) ~2, 16-37 (1967) Solari, A.J., Tres, L.L.: The three-dimensional reconstruction of the XY chromosomal pair in human spermatocytes. J. Cell Biol. 45, 43-53 (1970a) Solari, A.J., Tres, L.L.: Ultrastructure and histochemistry of the nucleus during male meiotic prophase. In: The human testis (E. Rosenberg and A. Paulsen, eds.), p. 127-138. New York: Plenum Press 1970b Tres, L.L.: Estudio ultraestruetural e histoquimico de las etapas premeitticas y meittieas de la espermatogenesis humana. Thesis. Facultad de Yiedicina, Universidad de Buenos Aires 1970 Utakoji, T. : Chronology of nucleic ~cid synthesis in meiosis of the male Chinese hamster. Exp. Cell Res. 42, 585-596 (1966) Wallace, H., Birnstiel, M. L. : Ribosomal cistrons and nucleolar organizer. Biochim. biophys. Acta (Amst.) 114, 296-310 (1966) Wisse, E., Tates, A. D. : A gold latensification-Elon ascorbic acid developer for Ilford L4 emulsion. Prec. 4th. Europ. Reg. Conf. Electron Microscopy, Rome, p. 465-466 (1968) Wollam, D. H. M., Ford, E. H. R.: The fine structure of the mammalian chromosome in meiotic prophase with special reference to the synaptinemal complex. J. Anat. 98, 163-173 (1964) Received July 7-August 12, 1975 / Accepted duly 29, 1975 by J. G. Gall Ready for press August 14, 1975
Nucleolar RNA Synthesis of Meiotic Prophase Spermatocytes in the Human Testis Laura L. Tres The Laboratories for Reproductive Biology, Division of Health Affairs, 111 Swing Building, University of North Carolina at Chapel Hill, Chapel Hill, N. C. 27514, U.S.A. Abstract. Human meiotic prophase spermatocyte nuclei were studied by electron microscope autoradiography after a 3 hours aH-uridine labeling pulse, followed by postincubation in non-radioactive medium. In autosomes, 3tt-uridine nucleolar labeling reaches a peak during early-middle zygotene prior to the peak labeling of chromosomal RNA species at middle pachytene. Transcription activities of sex chromosomes are inconspicuous when compared with that of autosomes. An increasing condensation of nucleolar-associated chromatin in acrocentric bivalents contributes to the formation of basal knobs in human pachytene spermatocytes. Upon completion of knob formation, nucleolar components segregate and the uptake of aH-uridine decreases. These findings suggest that the template capability of ribosomal DI~A cistrons, located next to the basal knob region, is largely associated with a dispersed state of chromatin whereas increased chromatin condensation is correlated with a restriction of ribosomal RNA transcription.
Introduction Previous studies have indicated that uucleolar and chromosomal R N A synthesis takes place in mouse spermatocytes during meiotic prophase (Kierszenbaum and Tres, 1974a, b). The studies indicated further that nucleolar organizers may be located next to paracentromeric heterochromatic regions or basal knobs (Woollam and Ford, 1964) of some autosomal bivalents, whereas ribonucleoprotein complexes (presumably heterogeneous nuclear RNA) are transcribed at the ehromatin loops of all autosomes (Kierszenbaum and Tres, 1974b). These findings are based on light and electron microscope autoradiography and on visualization of structures displaying transcription relationships. Besides providing original evidences for a nucleolar RNA synthesis during meiotic prophase in mouse spermatocytes, we advanced an interpretation (Kierszenbaum and Tres, 1974a) of a puzzling fact described by Monesi (1965) and Utakoji (1966), namely that nucleoli, especially those associated with the sex chromosomes, do not incorporate labeled precursors for RNA. The association of nucleoli with specific terminally-placed chromosomal regions ("nucleolar chromomeres") of certain bivalents in human spermatocytes was described by Ferguson-Smith (1964)on the basis of light microscopy. Solari and Tres (1967, 1970a, b) have pointed out some fine structural aspects of nueleoli associated with autosomal bivalents in human pachytene spermatocytes. In this study, the nucleolar and chromosomal SH-uridine-labeling patterns seen at leptotene, zygotene, and pachytene stages in human spermatocytes are described using an electron microscope autoradiographie technique. Structural features of the sex chromosomes as related to nucleolar formation by autosomal
142
L.L. Tres
b i v a l e n t s are also reported since t h e y facilitates t h e i d e n t i f i c a t i o n of meiotic prophase stages i n the man.
Material and Hethods Biopsy samples from testes of 8 normal, untreated patients attending infertility clinics were examined. Selection of subjects was based on clinical and laboratory tests. Upon removal, a few seminiferous tubules were separated from the biopsies and the remaining portions were immediately fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 6.9) for 2 hours, washed in buffer, and postfixed in 1% buffered osmium tetroxide for 1 hour. Tissues were embedded in Maraglas (Polyseiences, Inc., Rydal, Pa. USA) according to the usual procedure. Portions of the seminiferous tubules set aside before fixation were stained with acetic=orcein and squashed between a microscope slide and a coverglass for rapid evaluation of spermatogenic cells. Samples of tubules from 4 patients were incubated in Basal Medium (Eagle) with Earle's balanced salt solution (2 mM L-glutamine added) containing 50 izCi/ml of aH-uridine (5,6-aHuridine, sp. act. 42.4 Ci/mmoi, New England Nuclear, Boston, Mass. USA). After 3 hours of incubation alb 34~C, the samples were rinsed several times with non-radioactive medium and either fixed immediately or incubated further in non-radioactive medium containing 100 ~g/mI of cold uridine (Sigma Chemical Co., St. Louis, Mo. USA) for another 30 minutes period and fixed thereafter. These postincubation procedures were designed to wash out unbound all. uridine from the samples. These procedures were tested in specimens from mouse testes and compared the results with previous autoradiographic data obtained by injecting ~H-uridine intratesticularly (Kierszenbaum and Tres, 1974a). Similar nucleolar and chromosomal labeling sites were obtained with both procedures. For electron microscope autoradiography, thin sections with gold interference colors were transferred to Parlodion-coated half microscope slides split lengthwise. Before coating with emulsion, sections were stained with uranyl acetate and lead citrate. A thin carbon layer was evaporated over the stained sections and the slides then dipped in a diluted Ilford L4 emulsion (Ilford, Ltd., Ilford, Essex, England). After exposure for 1 to 6 months, the preparations were developed with the gold latensification-Elon-ascorbic acid procedure (Salpeter and Bachmann, 1972; Wisse and Totes, 1968), fixed with Kodak Rapid Fixer (Eastman Kodak, Co., Rochester, N.Y., U.S.A.) and rinsed in water, the specimens were stripped off onto a water surface. Copper electron microscope grids were placed over the sections and picked up on a polyethylene film strip (Parafilm "M", American Can Co., Neenah, Wis., U.S.A.). Label distribution within nuclei was determined quantitatively using tissues incubated with all-uridine followed by a chase with cold uridine, as indicated above. Grain densities overlying 10 [tinu units of surface area were scored and the values obtained were corrected for background density. Micrographs were taken with Siemens Elmiskop I and JEM 100B electron microscopes operated at 80 kV. Each instrument was calibrated with carbon grating replicas.
Results
Nucleolar Organization at Di[ferent Stages of the Meiotic Prophase A t e m p o r a l correlation b e t w e e n chromosomal p a i r i n g a n d 8H-uridine labeling of distinctive nucleolar c o m p o n e n t s in various meiotic prophase stages of h u m a n spermatocytes (exposed for 3 hours to radioactive uridine) can be achieved i n electron micrographs of autoradiograms. The results suggest a real relationship b e t w e e n t h e two e v e n t s d u r i n g the first p a r t of t h e meiotic cycle. Thus, a t leptotene, w h e n chromosomal cores (Fig. 1) a n d c h r o m a t i n c o n d e n s a t i o n are r e a d i l y found, one can detect small nucleoli, each characterized b y a n a c c u m u l a t i o n of dense fibrils s u r r o u n d i n g a center of more e x t e n d e d fibrils (Fig. 1). A t early zygotene,
RNA Synthesis in the Human Testis
143
Fig. 1. Leptotene spermatoeyte displaying a chromosomal core (arrow) and a nucleolus formed by dense fibers (]d) surrounding a fihrillar center (re). The scale mark indicates 1 ~m Fig. 2. Middle-to-late zygotene spermatoeyte. The nucleolus is formed by a fibrillar center (fc) surrounded by dense fibrils (Jd) and a fibrillo-granular component (It). Note that a portion of the fibrillar center is apparently continuous with loosely extended chromatin fibrils. Arrows indicate chromosomal cores. The scale mark indicates 1 ~zm
chromosomal cores and short segments of synaptonemal complexes in autosomes indicate initiation of pairing of homologous chromosomes. At early zygotene, aH-uridine labeling of nucleoli is mainly associated with dense fibrillar and fibrillo-granular components (Figs. 3 and 4). At late zygotene synaptonemal complexes predominate and the condensed ehromatin mass of the sex pair can be recognized (Solari and Tres, 1970a, b). During this stage, band-shaped nucleoli located next to the nuclear envelope (Fig. 2) display a fibrillar center (presumably consisting of proteins ; l~eeher et al., 1970) directly related to dispersed ehromatin fibers and separated b y a dense fibrillar shell from fibrillo-granular components extending toward the inner nuclear region. As spermatoeytes approach the end of zygotene, nueleolar components start their natural segregation process and silver grains accumulate predominantly on the dense fibrillar areas (Fig. 5). Furthermore, homologous pairing and formation of synaptonemal complexes is completed at late zygotene. Chromatin condensation of autosomes fluctuates throughout male meiotic prophase. This serves as an additional feature for recognition of prophase stages
144
L.L. Tres
Fig. 3. Autoradiograph showing early zygotene spermatocytes, tteavily labeled Ser~oli cell nuclei (S) contrast with less labeled zygotene spermatocytes (arrows). The nucleolus (Nu) of a Sertoli cell is well labeled. Exposure time: 65 days. Incubated with 31I-uridine for 3 hours. Postincubated with cold uridine for 30 minutes. Developing procedure: Gold latensificationElon-ascorbic acid procedure. The scale mark indicates 2 ~zm
wherein, after a progressive condensation during leptotene, zygotene, and early pachytene, chromatin decondenses at middle and late pachytene (see summary diagram in Fig. 9). As pachytene stage advances, autosomal ehromatin adopts a more even, extended pattern except at the ehromatin region associated with nucleoli (basal knobs) which become completely condensed at late pachytene (Fig. 7). This last feature contrasts with the dispersed chromatin region associated with fibrillar centers as observed at zygotene (Fig. 2). To an increased degree, condensation of the basal knob region of autosomal bivalents coincides with a distinctive rearrangement of nucleolar components [nucleolar segregation (Simard and Bernhard, 1966); Fig. 7] and a decline of aH-uridine labeling (Figs. 6 and 7 inset). Results of quantitative autoradiographic analyses of nucleolar and extranneteolar labeling sites throughout meiotic prophase stages (obtained from spermatocytes from different seminiferous tubules) are shown in Fig. 8. The radioactivity profiles indicate that nucleolar RNA synthesis precedes chromosomal I~NA synthesis. It was previously indicated in the mouse testis that chromo-
I~NA Synthesis in the Human Testis
145
l~'ig. 4. High magnification of zygotene nuclei illustrated in Fig. 3 indicating the distribution of nucleolar silver grains. The scale mark is 2 ~m
Fig. 5. Lute zygotene spermatocyte. The plain arrow indicates ~ nueleolar fibrillar center partially surrounded by dense fibrils (/d) conspicuously aH-uridine-labeled. (/g) fibrillo-granular region free of silver grains. Portions of chromosomal cores (crossed arrow) and of a synaptonemal complex (sc) are displayed. 3H-uridine Lbeling as Fig. 3. Exposure time: 65 days. Scale mark: 1 ~xm
146
L.L. Tres
Fig. 6. Autoradiograph of an early pachytene spermatocyte displaying considerable nuclear att-nridine labeling. No radioactivity is found over the sex chromosomal pair (X Y). Arrows indicate dense bodies projecting from the X Y chromosomal dense mass. Granular components of a spheric nucleolus (Nu) are weakly labeled. 3H-uridine labeling as Fig. 3. Exposure time: 65 days. Scale mark: 2 ~m
somal RNA synthesis corresponds, in a great extent, to heterogeneous nuclear RlqA-protein complexes transcribed at chromosomal loops (Kierszenbaum and Tres, 1974b).
Structural Features during Di//erentiation o/the X Y Chromosomal Pair Electron micrographs vf human X Y chromosomes have indicated (Solari and Tres, 1970a, b) that the paired structures can be identified during zygoteneearly pachytene as a condensed chromatin mass with two cores of different length. The cores share a short synaptonemal complex at their mutual ends. The transient appearance of dense bodies along the cores of both X and Y chromosomes at late zygotene-early pachy~ene (Figs. 6 and 9) followed by "ring-li]ce structures" at middle-late pachytene (Figs. 7 and 9) are correlated with events occurring during autosomal nucleolar organization and extranueleolar 3H-uridine labeling in human spermatocytes. The major features are: First, the condensed (heterochromatie) sex chromosomes display little or no RNA synthesis during meiotic prophase (Fig. 6) ; second, although a nucleolus is frequently observed next to the sex chromosomes (Solari and Tres, 1970a, b; Luciani, 1970a, b), the X and Y chromosomes presumably
RNA Synthesis in the Human Testis
147
Fig. 7. Middle-to-late pachytene spermatocyte. The sex chromosomes (X Y) show ring-like structures (arrow). A nucleolus, attached to an entirely condensed basal knob (BK) shows three regions: a fibrillar center (re), a librillar dense region ([d) 0.nd a large granular region (g). Scale mark: 1 Bm. Inset: Autoradiograloh of a nucleolus at the same meiotic stage. A few silver grains are seen over the spherical granular structure. Exposure time: 70 days. Pulse labeling as Fig. 3. Scale mark: 0.5 [zm do n o t organize a nucleolns as i n d i c a t e d b y i n situ ribosomal I~NA-DNA hybridization studies (Henderson et al., 1972); third, dense bodies along the axial cores of the sexual pair, formerly appearing in t h r e e - d i m e n s i o n a l reconstructions of
148
L . L . Tres
35
z~fch
romosomalRNA
25
%
nucleolar RNA
=1. o
s O W
m
mW
z
z
-z
I..,U I"-
W S
"'
N
0 8
n"W ,,~ I.-
w>-
13W ~.
--
i
z
.-II-
>-
W I--
0
Fig. 8. Silver grains scored ever nueleolar and nucleoplasmie surface areas in electron microscope autoradiographs of human spermatocytes after 3 hours of 3I-I-uridine labeling. Ten spermatocyte nuclei per meiotic prophase stage were counted. The surface area (10 tzm 2) was taken by superimposing over autoradiographs of fixed magnification (• 15000) a lattice with regularly spaced points. The grain density distribution over each nuclear area was scored and divided by the number of lattice points
Fig. 9. Diagram indicating relevant features of the sexual pair (X, Y) and autosomal nucleolar development and changes of autosomal chromatin condensation (Chr. Con&)throughout meiotic prophase stages of human sperm~toeytes. The basal knob (BK) increases its condensation as the spermatocyte moves to late paehytene. The segregation of nucleolar components (]c, fibrillar centers, ], fibrils, and g, granules) parallels the progressive condensation of the basal knob. The diagramatic representation of the sex chromosomal pair is based on threedimensional reconstructions obtained from serial sections (Solari and Tres, 1970a; Tres, 1970)
]~I~A Synthesis in the Human Testis
149
the sex pair (Solari and Tres, 1970a) and more recently in spreads of human spermatoeytes (Moses et al., 1975), characterize late zygotene-early pachytene stages (Fig. 9). After these stages nucleolar I~NA synthesis declines, and fourth, ring-like structures along cores of sex chromosomes, are observed during middlelate pachytene stages, when chromosomal I~NA synthesis rises to a maximal level in autosomal bivalents following a decrease of nucleolar RNA transcription (Fig. 8).
Discussion
Electron microscope autoradiographic evidence from the human testis indicates that during meiotic prophase autosomal bivalents are involved in both nuc]eolar and chromosomal t~NA synthesis. These findings agree with previous experimental results of transcription activities in the mouse testis (Kierszenbaum and Tres, 1974a, b). A quantitative analysis of autoradiographie data from the human testis indicates that at middle pachyiene the rate of chromosomal RNA synthesis is at a peak. Peak production of nucleolar RNA is earlier, during zygotene and takes place at well defined loci of some acrocentric autosomes. Solari and Tres (1970a, b) have previously described fine structural and histochemical features of meiotic prophase stages in the human testis. The present results extend this information by defining sites of atI-uridine labeling as well as the characteristic structural sequence during nucleolar organization. The relevant aspects of nucleolar formation during meiotic prophase in man are: (1) A progressive condensation of the autosomal ehromatin region closely associated with nueleo]i, leading to the formation of well defined basal knobs at late paehytene, and (2), a rearrangement or segregation of nuclcolar components coinciding with the formation of basal in acrocentric autosomes knobs and a decrease in nucleolar aH-uridine labeling. Polycistronic genes for precursors of ribosomal I~NA occur in eukaryotic genomes and are located in specific chromosomal nucleolar organizer regions (Wallace and Birnstiel, 1966). In situ ribosomal I%NA-DNA hybridization procedures in somatic cells (Henderson et al., 1972) indicate that, in man, the satellite region of acrocentric autosomes (chromosomes 13, 14, t5, 21 and 22) contain ribosomal DNA cistrons. The association of nucleoli with basal knobs of human autosomal bivalents was indicated by light microscopy (Ferguson-Smith, 1964; Solari and Tres, 1967; Luciani, 1970a, b) and electron microscopy (Solari and Tres, 1967, 1970a, b; Tres, 1970). Furthermore, the nueleolar organizers of mouse chromosomes are situated near the centromeric heterochr0matin (Levan et al., 1962) which contains satellite DNA (Purdue and Gall, 1970). Nevertheless, the specific region of the basal knob involved in the molecular organization of precursors of ribosomal I~NA has not yet been identified. Knowledge of these facts favours inferring that the progressive chromatin condensation of the basal knob region in acrocentrie autosomes might affect the efficiency of ribosomal DNA cistrons, prompting the cessation of precursors of ribosomal RNA synthesis. In fact, these changes are accompanied by a decrease in ~H-uridine labeling and by nucleolar changes referred to as nucleolar segregation observed from late zygotene on. Morphological and biochemical evidence from several tissues indicates that
150
L.L. Tres
nucleolar segregation can be induced by inhibitors of nucleolar I~NA synthesis (for a complete review, see Busch and Smetana, 1970). From the foregoing discussion, it seems apparent that regulation of ribosomal gene function depends to a certain extent on controlling chromatin organization of basal knob segments of acrocentric bivalents. The evolution of autosomal nucleoli varies in different species. For instance, in the mouse testis, nucleolar masses, once detached from their primary autosomal nucleolar organizer loci during middle paehytene, migrate toward the sex chromosomal mass, rearrange their nucleolar components and form a natural segregated nucleoli which do not incorporate aH-uridine (Kierszenbaum and Tres, 1974a). I n human spermatocytes, this migrating behavior is not observed and nucleoli segregate at their putative primary point. Since nucleolar masses have been observed next to the X rY chromosomal pair in human (Solari and Tres, 1970a, b; Lueiani, 1970a, b) and mouse spermatocytes (Solari, 1964; Monesi, 1965; Kierszenbaum and Tres, 1974a), one question that arise is the possibility of a nucleolar organizing activity of the sex chromosomes as postulated by Ohno et al. (1957). I n this respect, in situ ribosomal t{NA-DNA hybridization procedures in human (Henderson et al., 1972) and mouse (Henderson et al., 1974) somatic cells have shown that several acrocentric chromosomes (but not X and J(chromosomes) contain ribosomal DNA eistrons. Additional data from autoradiographic experiments in mouse spermatoeytes (Kierszenbaum and Tres, 1974a) indicate that the condensed sex chromosomes do not display a structural and aH-uridine-labeling sequence as described for nucleoli associated with autosomal bivalents or during the organization of somatic cell nucleoli (i.e., fibrillar nucleolar components serve as precursors for the granular material, Noel et al., 1971). Finally, it has been proposed on the basis of light microscopy (Luciani, 1970a, b) and electron microscopy (Solari and Tres, 1970a, b) that " m a i n " and "secondary" nueleoli may be present in human pachytene spermatocytes. Nevertheless, autoradiographic data together with a structural evaluation of nucleolar organizing features preceding late pachytene, indicate that all nucleoli evolve almost synchronoulsy during the appropiate meiotic stages. Therefore, the concept of main and secondary nucleoli should be discarded.
Acknowledgments. The study reported here was initiated in the Centro de Investigaciones sobre Reproduceion, Facultad de Medicina, Universidad de Buenos Aires, Argentina. I wish to thank Dr. tt. S. Bennett for his support and interest. I am also grateful to Dr. M. J. Moses for criticism of the manuscript. This work was made possible by a grant from The l~oekefeller Foundation to The Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill.
References Busch, H., Smetana, K.: The Nucleolus. New York: Academic Press 1970 Ferguson-Smith, M. A. : The sites of nucleolus formation in human pachytene chromosomes. Cytogenetics 8, 124-134 (1964) Henderson, A. S., Eicher, E.M., Yu, M. T., Atwood, K. C. : The chromosomal location of ribosomal DNA in the mouse. Chromosoma (Berl.) 49, 155-160 (1974)
RNA Synthesis in the Human Testis
151
Henderson, A. S., Warburton, D., Atwood, K. C. : Loc~tion of ribosomal DNA of the human chromosome complement. Prec. nat. Acad. Sci. (Wash.) 69, 3394-3398 (1972) Kierszenbaum, A.L., Tres, L.L.: Nucleolar and perichromosom~l RNA synthesis during meiotic proph~se in the mouse testis. J. Cell Biol. 60, 39-53 (1974a) Kierszenbaum, A. L., Tres, L. L. : Transcription sites in spread meiotic prophase chromosomes from mouse spermatocytes. J. Cell Biol. 68, 923-935 (1974b) Levan, A., Hsu, T. C., Stich, H. F. : The idiogram of the mouse. Hereditas (Lund) 48, 677-689 (1962) Luciani, J. M.: Les chromosomes melotiques de l'homme. I. La meiosis normale. Ann. Gtn@t. 13, 101-111 (1970a) Luciani, J.M.: Les chromosomes meiotiques de l'homme. II. Le nuclOole. Les chiasmas. III. La sterglit@ masculine. Ann. G@ntt. 18, 169-182 (1970b) Monesi, V.: Synthetic activities during spermatogenesis in the mouse. Exp. Cell Res. 89, 197-224 (1965) Moses, M. J., Counce, S. J., Paulson, D. F. : Synaptonem~l complex complement of man in spreads of spermatoeytes, with details of the sex chromosome pair. Science 187, 363-365 (1975) Noel, d. S., Dewey, W. C., Abel, J. H., Thompson, R. P.: Ultrastructure of the nucleolus during the Chinese hamster cell cycle. J. Cell Biol. 49, 830-847 (1971) Ohno, S., Kaplan, W., Kinosit~, R.: Heteroehromatic regions and nucleolus organizers in chromosomes of the mouse, Mus musculus. Exp. Cell Res. 18, 358-364 (1957) Purdue, M. L., Gall, J. G.: Chromosomal localization of mouse satellite DNA. Science 168, 1356-1358 (1970) Recher, L., Whitescarver, J., Briggs, L. : A cytochemic~l and radioautographic study of human tissue culture nueleoli. J. Cell Biol. 45, 479-492 (1970) Salpeter, M. M., Bachmann, L. : Autoradiography. In: Principles and techniques of electron microscopy. Biological applications (M. A. Hayat, ed.), vol. 2, p. 221-278. New York: Van Nostrand Reinhold Company 1972 Simard, R., Bernhard, W.: The phenomenon of nucleolar segregation. Specific action of certain ~ntimetabolites. Int. J. Cancer 1, 463-479 (1966) Solari, A. J. : The morphology and ultrastructure of the sex vesicle in the mouse. Exp. Cell Res. 86, 160-168 (1964) Solari, A. J., Tres, L. L.: The ultrastrueture of the human sex vesicle. Chromosoma (Berl.) ~2, 16-37 (1967) Solari, A.J., Tres, L.L.: The three-dimensional reconstruction of the XY chromosomal pair in human spermatocytes. J. Cell Biol. 45, 43-53 (1970a) Solari, A.J., Tres, L.L.: Ultrastructure and histochemistry of the nucleus during male meiotic prophase. In: The human testis (E. Rosenberg and A. Paulsen, eds.), p. 127-138. New York: Plenum Press 1970b Tres, L.L.: Estudio ultraestruetural e histoquimico de las etapas premeitticas y meittieas de la espermatogenesis humana. Thesis. Facultad de Yiedicina, Universidad de Buenos Aires 1970 Utakoji, T. : Chronology of nucleic ~cid synthesis in meiosis of the male Chinese hamster. Exp. Cell Res. 42, 585-596 (1966) Wallace, H., Birnstiel, M. L. : Ribosomal cistrons and nucleolar organizer. Biochim. biophys. Acta (Amst.) 114, 296-310 (1966) Wisse, E., Tates, A. D. : A gold latensification-Elon ascorbic acid developer for Ilford L4 emulsion. Prec. 4th. Europ. Reg. Conf. Electron Microscopy, Rome, p. 465-466 (1968) Wollam, D. H. M., Ford, E. H. R.: The fine structure of the mammalian chromosome in meiotic prophase with special reference to the synaptinemal complex. J. Anat. 98, 163-173 (1964) Received July 7-August 12, 1975 / Accepted duly 29, 1975 by J. G. Gall Ready for press August 14, 1975
