TISSUE AND CELL, 1992 24 (1) 75-&l @ 1992 Longman Group UK Ltd.
JUAN
C. CAVICCHIA
and ALFONSINA
MORALES
CHARACTERIZATION OF NUCLEAR PORE DISTRIBUTION IN FREEZE-FRACTURE REPLICAS OF SEMINIFEROUS TUBULES ISOLATED BY TRANSILLUMINATION Keywords: Spermatogenesis,
seminiferous epithelium, nuclear pores, testis, freeze-fracture
ABSTRACT. Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII-VIII, IX-Xl, XII-XIV, and V-VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX-XI and XIIXIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V-VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII-VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX-XI) and zygotene (XII-XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina,and by being rather numerous and found in all the stages explored. Diplotene spermatocytes hale the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores take on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphaseanaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores,
Introduction
which has been termed fibrous lamina (Fawcett, 1966) or dense lamella (Kalifat et al., 1967). There is strong evidence that this structure serves as a skeletal support for the nuclear envelope and as attachment points both for nuclear pore complexes and elements of the chromatin (Aaronson and Blobel, 1974, 1975; Scheer et al., 1976). Nuclear pore complexes are dynamic structures which are subjected to marked morphological variations under different conditions. Their number increases during interphase in synchronized Hela cells and doubles in 48 hrs after phytochemagglutinin stimulation in lymphocytes (Maul et al., 1972). These events are correlated with protein synthesis and DNA replication. Pores also vary in number during hormonal stimulation in rat seminal vesicles (Ortiz and Cav-
The nuclear envelope is a complex eukaryotic
membrane organelle whose biological behavior under diverse physiological requirements is largely undefined at present. Its main architectural features are an inner and an outer membrane joined at the pore complexes, which constitute the major passageways of molecules between nucleus and cytoplasm. Attached to the inner aspect of the nuclear envelope is a proteinaceous electron-dense layer, biochemically different from the nuclear matrix (Gerace et al., 1978), Instituto de Histologia y Embriologia, Fact&ad de Ciencias Medicas, Universidad National de Cuyo, Mendoza, Argentina. Received 7 December 1990. Revised 24 September 1991. 75
CAVICCHIA
16
icchia, 1990)) during different life cycle stages in a ciliated protozoan (Dallai and Luporini, 1982), in a slime mold (Aldrich and Pendland, 1981) and in the Chinese hamster cells (Scott et al., 1971). Germ cell differentiation is a complex process where numerous cell types mature synchronously; therefore, the seminiferous epithelium seems to be a suitable model for the study of nuclear pore changes which may occur under physiological influences. Many developmental features were reported to occur during spermatogenesis, e.g. disappearance of the fibrous lamina during pachytene stage (Stick and Schwartz, 1982), a change in distribution of chromatin condensation in different spermatogonia (Cavicchia and Dym, 1978), spermatocytes and spermatids (Russell and Frank, 1978; Kretser and Kerr, 1988), and variations in the chromosome-nuclear envelope attachments during meiosis (Solari, 1969). These events might be closely related to changes in the nuclear pore patterns of germ cells. Freeze-fracture replicas allow us to explore extensive membrane areas. However, due to the difficulty in recognizing different germ cell types present in the seminiferous epithelium, previous descriptions of nuclear pore patterns in freeze-fracture replicas have been limited only to Sertoli cells, and to the general characteristics of spermatogonia, spermatocytes and spermatids (Fawcett and Chemes, 1979; Liankai, 1987). The present investigation was undertaken combining the isolation of those particular segments of the seminiferous tubules which contain specific germ cell associations (Parvinen and Vanha Perttula, 1972), allowing their proper identification with the freeze-fracture procedure.
AND MORALES
With these combined methods, we were able to characterize the nuclear pore pattern of type A and Type B spermatogonia, as well as of preleptotene, leptotene, zygotene, pachytene, diplotene, and secondary spermatocytes. Material and Methods Adult Holtzman albino rats weighing approximately 350 g, fed on laboratory chow and water ad libitum, were housed in animal room at 20” C. The lighting regimen was 12112. They were anesthetized with intraperitoneal pentobarbital sodium and the testes exposed through a scrotal incision. The tunica albuginea was opened, the seminiferous tubules were placed in a Petri dish containing a Ringer-Krebs sucrose solution and isolated according to the technique described by Christensen and Mason (1965). Segments of seminiferous tubules were studied in transmitted light under a Nikon dissecting microscope (X40-100 magnification) and cut in suitable l-2 mm fragments with iridectomy scissors. Four main types of light absorption were selected: 1) Stages VII-VIII with a very dark homogeneous central absorption due to mature spermatozoa ready to be released into the tubular lumen. 2) Stages IX-XI, which follow, homogeneously transparent because spermiation is over and no spermatozoa are present. There is a sharp demarcation between these two segment types. 3) Stages V-VI showing a spot-like arrangement in the tubule center apparently caused by maturing spermatids attached to the Sertoli cells (Parvinen and Vanha Perttula,
Fig. 1. A Sertoli cell. The infolded nuclear envelope (N) exhibits randomly distributed pores. Arrows indicate the Sertoli cell lateral membranes. G, neighboring germ cells. BL, basal lamina. Stages XII-VIX of the seminiferous epithelium. x 11,CKtO. Fig. 2. A round nuclear profile of a type A spermatogonium with a slight clumping of nuclear pores. Stages VII-VIII. BL, basal lamina. The characteristic pits of pinocytotic vesicles in the interlamellar, peritubular cells (IC) are indicated by arrows. x 10,000. Fig. 3. Stages V-VI. A germ cell with extensive attachment to the basal lamina (BL) probably a typ B spermatogonium. Its nucleus (N) displays intense pore aggregation. IC, interlamellar cell. Arrow: inter-germ cell bridge. xlO,OLKl. Fig. 4. Stages VII-VIII. A group of preleptotene spermatocytes close to the basal lamina (BL). Their nuclei (N) exhibit marked aggregation of nuclear pores. x4ooO.
CAVICCHIA
78
1972), and located immediately previous to stages VII-VIII. 4) Stages XII-XIV, which follow along the tubule, stages IX-XI and present central light spots apparently due to spermatids with an increasing density of acrosomal material (Parvinen and Vanha Perttula, 1972). For histological identification, samples of these tubular segments were transferred into 5% glutaraldehyde in cacodylate buffer pH 7.4, postfixed in a solution containing 2 parts of 2% osmium tetroxide and 1 part of 1% potassium ferrocyanide, embedded in Medcast, sectioned in 1 pm sections and stained with 1% toluidine blue sodium borate. The classification of Perey et al. (1961) was utilized for identification of the stages (I-XIV) of the seminiferous epithelium in the rat. A useful examination of the paper by Perey et al. can be found in Steinberger and Steinberger (1975). The criteria for identification of rat spermatocytes in 1 pm sections after plastic embedding (Russell and Frank, 1978) were also followed. In this manner the transilluminated appearance of each tubular segment was compared with the histological picture of cross tubular sections in the same piece of tissue. We found good correlation between both methods. The selected sets of tubular fragments containing specific stages of the seminiferous epithelium were fixed by immersion in 5% glutaraldehyde-cacodylate buffer for 20 min and placed in 30% glycerol for 2 hr. Sets of 3-4 tubule fragments were longitudinally aligned on gold freeze-fracture holders, rapidly frozen in liquid Freon 22 and stored in liquid nitrogen. The material was fractured in a Balzers BAF 301 at -105” C and shadowed with platinum followed by
AND MORALES
carbon. Replicas were cleaned with sodium hypochlorite, mounted on copper grids and observed in either a Siemens Ehniskop IA or I. The criteria for identification of each cell are progressively indicated in order to be recognized in the freeze-fracture replicas, type by type, in the course of the ‘Results’ section. 30 seminiferous segments from each set of stages were fractured and approximately 20 micrographs for each cell type were taken. As we found outstanding consistency of morphological characteristics for each of the analyzed cells, we estimated that the sample was appropriate. Results
Figure 1 provides a view of the seminiferous epithelium in a freeze-fracture replica. Sertoli cell nuclei, constantly found along the seminiferous tubule, are characterized by morphological parameters already described (Fawcett and Chemes, 1979) e.g., a highly infolded nuclear envelope and a random distribution of nuclear pore complexes. Type A spermatogonia are the only basal compartment cells with extensive surface contact with the basal lamina in the isolated stages IX-XI and XII-XIV. They are oval shaped cells. Their freeze-fracture nuclear profile (Fig. 2) are also oval or less frequently spherical. The nuclear pore pattern is constantly characterized by either a random distribution of nuclear pores or by a slight degree of pore clumping. The apparent density of pores in the freeze-fractured nuclear membranes is much the same in all the stages studied. Germ cells in stages V-VI, which also display extensive contact with the basal lamina and con-
Fig. 5. Stages VII-VIII. Two preleptotene spermatocytes at higher magnification with spherical, centrally located nuclei (N) with intense pore clustering. Arrows indicate inter-Sertoli interdigitations. x lO,OC@. Fig. 6. Germ cell close to the basal lamina found in stages IX-XI, likely to be a leptotene spermatocyte. The nuclear pore aggregation is less evident than in preleptotene spermatocytes. x1o,oc@. Fig. 7. Germ cell with similar characteristics and location to the one in the previous figure but found in stages XII-XIV, likely to be a zygotene spermatocyte. The nuclear pore pattern also has similar characteristics. x 15,CW Fig. 8. Pachytene spermatocytes are large cells always detached from the basal lamina. Their round nucleus (N) shows a conspicuous clustering of pores separated by large pore free areas. Stages IX-XI. x 8000.
CAVICCHIA
80
sistently contain spherical nuclei, are likely to be type B spermatogonia (Perey et al., 1961). They possess, instead, tightly aggregated nuclear pores (Fig. 3). Preleptotene spermatocytes are only found in stages VIVIII (Perey et al., 1961). They are rather numerous forming groups of cells in close vicinity to the basal lamina; such vicinity in this case is an additional reference parameter. Although still anchored to the basal lamina, some of these cells are already detached from it (Fig. 4). Their nuclei (78pm in diameter) are smaller than those found in both type A or B spermatogonia, perfectly spherical and located in the central part of the cell. They exhibit an intense clustering of nuclear pores (Figs 4, 5) forming groups of 15-30, separated by pore-free membranes areas. Leptotene (stages IX-XI) (Fig. 6) and zygotene (stages XII-XIV) (Fig. 7) spermatocytes (nuclear diameter 9-10 ,nm) are also rounded cells, larger than preleptotene spermatocytes and positioned higher in the tubule, though still not far from the basal lamina. This position allows us to tell the difference between leptotene or zygotene and type A spermatogonia (in direct contact to the basal lamina), on the one hand, and the groups of pachytene or diplotene spermatocytes (located higher in the seminiferous epithelium), on the other hand. The two latter cell types exhibit, moreover, other distinguishing features, indicated below, mainly their large nuclear sizes, which permit their discrimination from leptotenes (found in stages IX-XI) or zygotenes (in tubular segments containing stages XII-
AND MORALES
XIV). Since careful examination of the freeze-fracture material from both sets of stages did not disclose any difference regarding nuclear pore pattern (compare Figs 6 and 7), the following description applies to both leptotene and zygotene spermatocytes. The nuclear pore arrangement changes to a more even distribution, although clearly maintaining some degree of pore aggregation. Identification of pachytene spermatocytes in freeze-fracture replicas is facilitated by: a) their large size (nuclear diameter 10-13 pm), b) their location in the seminiferous epithelium where groups of basal compartment cells (e.g., preleptotene spermatocytes in the segments containing stages VII-VIII) are interposed between them and the basal lamina, and c) by being rather numerous and found in all the segments studied. The fracture faces of the round pachytene nuclei show a conspicuous clustering of pores (in groups of N-70) separated by large porefree areas (Fig. 8). Diplotene spermatocytes, found exclusively in the seminiferous tubules containing stage XIII, are similar to pachytene in their location within the seminiferous epithelium, in their relationship with other more basal cells (zygotenes in this case), and in their large size and shape. Diplotene cells have round nuclei with the largest diameter (Russell and Frank, 1978), a characteristic which is also observed in the freeze-fracture preparations (diameter of the largest nuclear profiles, 14-15 pm). The nuclear pore complexes are numerous here and apparently randomly distributed (Fig. 9). In freeze-fracture preparations of tubular
Fig. 9. Stages XII-XIV. A large germ cell located far from the basal lamina, likely to be a diplotene spermatocyte exhibits randomly distributed nuclear pores. x8000. Fig. 10. Freeze-cross section of small vesicle profiles forming rows (dotted lines) alternated with smooth areas. These structures might well correspond to the spindle vesicles described in thin sections of metaphase-anaphase spindles (Pleshkewych and Levine, 1975). Stages XIIXIV. XlO,cKlO Fig. 11. A large. round nuclear profile found far from ihe basal lamina in stages XII-XIV and intermingled with the spindles profiles described in the previous figure. This germ cell might be a secondary spermatocyte. Its nucleus exhibits a paucity of nuclear pores randomly distributed. x 10,000. Fig. 12. Lateral view of a round spermatid with a developing acrosome cap (A). Its nuclear envelope shows a few pores (arrows) in the post-acrosomal region. No nuclear pores arc observed below the acrosome. Stages V-VI. ~15,000. Fig. 13. A caudal view of a similar spermatid. The nuclear pores are gathered around the implantation fossa (F) of the flagellum. x 10.000.
CAVICCHIA AND MORALES
82
stages XII-XIV, fragments containing groups of numerous round cells, centrally located within the seminiferous epithelium, with a nuclear diameter about 9-10 pm are frequently found. For these characteristics and also because they are found intermingled with metaphase-anaphase profiles (Fig. lo), a feature also present in stage XIV, they can be secondary spermatocytes (Russell and Frank, 1978; Kretser and Kerr, 1988). Their nuclear membranes in freeze-fracture are characterized by a paucity of pores in an apparently random distribution (Fig. 11). Round spermatids, with a developing acrosome cap and an axonemal profile, are spermatocytes smaller than secondary (nuclear diameter 8-9 pm) and found in the tubular segments containing stages V-VI. Nuclear pores have disappeared below the expanding acrosome (Fig. 12), and pore migration proceeds towards the caudal pole of the nucleus (Fig. 13). Since the process of spermiogenesis in the rat was extensively studied with freeze-fracture (Fawcett and Chemes, 1979) no further description is needed. Other cell types such as intermediate spermatogonia which could not be isolated within specific transilluminated seminiferous tubules are beyond the scope of the present study. Discussion Electron micrographs of thin sections provide little information about the distribution pattern and density of nuclear pores. In freeze-fracture preparations, instead, the cleavage plane tends to follow the hydrophobic region of the membranes affording extensive en face views of the nuclear envelope and its nuclear pore complexes. Freezefracture studies of the seminiferous epithelium have allowed, therefore, other authors (Fawcett and Chemes, 1979; Liankai, 1987) to identify several nuclear pore patterns belonging to specific cell types. However, since spermatogenesis is a complex process involving many germ cells in different degrees of development, it is quite difficult, if not impossible, by freeze-fracture alone to characterize them further. The isolation by transillumination of seminiferous tubule segments containing specific stages of the seminiferous cycle proved a valuable procedure for identification. Combining both methods
and utilizing secondary parameters for cell identification in freeze-fracture, such as nuclear size and shape, cell location within the seminiferous epithelium or vicinity to other structures, we were able to go further in the identification procedure characterizing other germ cell nuclei. The most prominent feature which seems to emerge from the present report is the close correspondence between chromatin arrangements and nuclear pore patterns exhibited by any of the identified cells. The two main categories of spermatogonia, type A and B, with chromatin arrangement respectively described as ‘dust like’ (Regaud, 1901) due to its homogeneous, finely granular distribution, also observed in electron micrographs (Cavicchia and Dym, 1978), and ‘crust like’, which show, in conventional electron microscopy, coarse clumps of chromatin attached to the nuclear envelope, display quite different nuclear pore patterns: the former, homogeneous distribution, and the latter, evident pore clustering. Similarly, preleptotene spermatocytes with numerous dense masses of chromatin mainly associated with the nuclear envelope (Solari, 1969; Cavicchia and Dym, 1978) exhibit in freeze-fracture preparations a striking clustering of pores separated by pore-free areas in freezefracture preparations. Nuclear pore complexes change again to a more homogeneous distribution in leptotene spermatocytes which are recognized in thin sections by disappearance of the characteristic preleptotene chromatin clumps associated with the nuclear membrane. Chromatin here becomes rather homogeneous forming diffuse thin threads (Solari, 1969). In pachytene spermatocytes, a progressive chromosome condensation takes place. Chromosomes adopt the so-called ‘bouquet’ arrangement forming long loops with their ends attached to the nuclear envelope and the condensed X-Y pair (‘sex vesicle’), which also displays increasing nuclear membrane contact (Solari, 1969; Fawcett, 1981). Coincidently with these intranuclear events, nuclear pore distribution changes to a pattern of marked clustering, which reverses again in diplotene, showing an even distribution of nuclear pores. Here, decondensation of chromosomes reaches a maximum (Solari, 1969). Due to the increased size of the nucleus many areas are
NUCLEAR PORE DISTRIBUTION
IN ISOLATED
TUBULES
free from condensed chromatin (Russell and Frank, 1978). In secondary spermatocytes, nuclear pores, now evenly distributed, also correspond to poor chromatin condensation and a nucleoplasm with very low stain affinity (Russell and Frank, 1978). All these features coincide in indicating a close correlation between chromatin condensation and nuclear pore clustering. In agreement with our results, during interphase, in cockroach blood cell and Malpighian tubule nuclei which contain large amounts of heterochromatin, an intense clustering of nuclear pores was also reported (Teigler and Baerwald, 1972). Maul et al. (1971) have suggested that the heterochromatin lying beneath the surface of the nuclear membrane may influence the pattern seen in nuclear pores. Dallai and Luporini (1982) also observed that in the macronuclei of a ciliated protozoan, either in thin sections and in freeze-fracture replicas, pore clusters are confined to areas free of underlying heterochromatin masses. Co-incidentally, other consistent structures which can make close apposition to the nuclear envelope from either the inside, e.g., the nucleoli, or the outside, e.g., vacuoles (Severs et al., 1976) or the acrosome during spermiogenesis (Fawcett and Chemes, 1979) may influence pore arrangement.
83
One aspect that remains unresolved is how nuclear pore complexes in germ cells change in density and distribution during spermatogenesis. It is known that some turnover of nuclear pores takes place in somatic cells during different physiological activities (Maul et al., 1973; Ortiz and Cavicchia, 1990) although a genera1 pattern is probably maintained by attachment of the pore complexes to the underlying fibrous lamina through connecting fibrillar elements @cheer et al., 1976; Allen and Douglas, 1989). The fact that the fibrous lamina is absent in male germ cells during meiosis (Stick and Schwartz, 1982) can explain in part the apparent freedom that may be necessary for the process by which the nuclear pore pattern changes so dynamically during meiosis, as shown in the present paper. Acknowledgements
This investigation was supported by grants from CONICET (Consejo National de Investigaciones Cientificas y Tecnicas and CIUNC (Consejo de Investigaciones de la Universidad National de Cuyo), Argentina. The technical assistance of Mr. Osvaldo Arango is highly appreciated. We thank Dr. Fabio L. Sacerdote for linguistic assistance with the manuscript.
References
Aaronson, R. P. and Blob& G. 1974. On the attachment of the nuclear pore complex. J. Cell Biol., 62, 76754. Aaronson, R. P. and Blobel, G. 1975. Isolation of nuclear pore complexes in association with a lamina. Proc. Narl. Acad. Sci. USA, 72, 1007-1011. Aldrich, H. C. and Pendland, J. C. 1981. Nuclear pores during the cell cycle in a slime mold. Physarum polycephalum. Ti.wue Cell, 13,431-439.
Allen, .I. L., Douglas, M. G. 1989. Organization of the nuclear pore complex in Saccharomyces cereoisiae. J. Ubrmt. Mol. Res., 102, 95-108. Cavicchia, J. C. and Dym, M. 1978. Ultrastructural characteristics of monkey spermatogonia and preleptotene spermatocytes. Biol. Reprod., 18, 219-228. Christensen, A. K. and Mason, N. R. 1965. Comparative ability of seminiferous tubules and interstitial tissue of rat testes to synthesize androgens from progesterone-4-14C in oifro. Endocrinology, 76, 646-656. Dallai, R. and Luporini, P. 1982. Variation in the nuclear pore distribution through different life cycle of the ciliated protozoan Euplotes crassu.x .I. Submicrosc. Cytol., 14, 107-121. Fawcett, D. W. 1966. On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. Am. J. Amt., 119, 129-146. Fawcett, D. W. 1981. Nuclear Envelope. In The Cell, pp. 266-280. W. B. Saunders Company. Philadelphia, London, Toronto. Fawcett, D. W. and Chemes, H. E. 1979. Changes in distribution of nuclear pores during differentiation of the male germ cells. Tissue Cell, 11, 147-162. Gerace, L., Blum, A. and Blobel, G. 1978. Immunochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. J. Cell Biol., 79, 546-566.
84
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Kalifat, S. R.. Bouteille, M. and D&me, .I. 1967. Etude ultrastructurale de la lamelle dense observe au contact de la membrane nuclaire interne. J. de Microscopic, 6, 101%1026. Kretser, D. M. and Kerr, J. B. 1988. The cytology of the testis. In The Physiology of Reproduction (eds. E. Knobil and J. Neil1 et al.), pp. 551-649. Academic Press, New York. Liankai, D. 1987. A morphological study on the membrane structure and the distribution of nuclear pore during spermiogenesis. Kexue Tongbao. 32, 1642-1646. Maul. H. M., Hsu, B. Y. L., Borun, T. M. and Maul. G. G. 1973. Effect of metabolic inhibitors on nuclear pore formation during the Hela S3 cell cycle. J. CeN Biol.. 59, 669-676. Maul, G. G., Maul, H. M., Scogna. J. E.. Lieberman, M. W.. Stein, G. S., Hsu. B. Y. and Borun, T. W. 1972. Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in Hela cells during the cell cycle. J. Cell Bio/. , 55,43%447. Maul, G. G., Price, J. W. and Lieberman, M. W. 1971. Formation and distribution of nuclear pore complexes in interphase. J. Cell Biol.. 51, 405-418. Ortiz. H. E. and Cavicchia, J. C. 1990. Androgen-induced changes in nuclear pore number and in tight junctions in rat seminal vesicle epithelium. Amt. Rec.. 226, 129-134. Parvinen, M. and Vanha-Perttula, T. 1972. Identification and enzyme quantification of the stages of the seminiferous epithelial wave in the rat. Amt. Rec.. 174, 435-450. Percy, B., Clermont, Y. and Leblond. C. P. 1961. The wave of the seminiferous epithelium in the rat. Am. J. Anat., 108, 47-77. Pleshkewych. A. and Levine. L. 1975. Phase contrast and electron microscopic observations on a membranous complex in primary spermatocytes of the mouse. J. CeN Sci., 18, 1-17. Regaud, C. 1901. Etudes sur la structure des tubes seminiferes et sur la spermatogentse chez les mammiferes. Arch. Amt. Microskop., 4, 101-156, 231-380. Russell. L. and Frank, B. 1978. Characterization of rat spermatocytes after plastic embeddmg. Archiues ofAndrology. 1, 5-18. Scheer, U.. Kartenbeck. J.. Trendelenburg, F., Stadler. J. and Franke, W. W. 1976. Experimental disintegration of the nuclear envelope. J. Cell Biol.. 69, 1-18. Scott, R. E., Carter. R. L. and Kidwell, W. R. 1971. Structural changes in membranes of synchronized cells demonstrated by freeze-cleavage. Nat.NewBiol., 233, 219229. Severs, N. J.. Jordan. E. G. and Williamson, D. W. 1976. Nuclear pore absence from areas of close association between nucleus and vacuoles in synchronous yeast cultures. J. Ultrastruct. Res.. 54, 374387. Solari. A. J. 1969. The evolution of the ultrastructure of the sex chromosomes (sex vesicle) during meiotic prophase in mouse spermatocytes. J. Ultrartruct. Res., 27, 289-305. Steinberger, E. and Steinberger. A. 1975. Spermatogenic function of the testis. In Male Reproductive System (eds. R. 0. Greep and D. W. Hamilton). pp. 1-19. Am. Physiol. Sot.. Bethesda, Maryland. Stick, R. and Schwartz. H. 1982. The disappearance of the nuclear lamina during spermatogenesis. CPNDiffrentration. II, 235. Teigler, D. J. and Baerwald, R. J. 1972. A freeze-etch study of clustered nuclear pores. r&sue Cell, 4, 447-456.
JUAN
C. CAVICCHIA
and ALFONSINA
MORALES
CHARACTERIZATION OF NUCLEAR PORE DISTRIBUTION IN FREEZE-FRACTURE REPLICAS OF SEMINIFEROUS TUBULES ISOLATED BY TRANSILLUMINATION Keywords: Spermatogenesis,
seminiferous epithelium, nuclear pores, testis, freeze-fracture
ABSTRACT. Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII-VIII, IX-Xl, XII-XIV, and V-VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX-XI and XIIXIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V-VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII-VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX-XI) and zygotene (XII-XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina,and by being rather numerous and found in all the stages explored. Diplotene spermatocytes hale the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores take on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphaseanaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores,
Introduction
which has been termed fibrous lamina (Fawcett, 1966) or dense lamella (Kalifat et al., 1967). There is strong evidence that this structure serves as a skeletal support for the nuclear envelope and as attachment points both for nuclear pore complexes and elements of the chromatin (Aaronson and Blobel, 1974, 1975; Scheer et al., 1976). Nuclear pore complexes are dynamic structures which are subjected to marked morphological variations under different conditions. Their number increases during interphase in synchronized Hela cells and doubles in 48 hrs after phytochemagglutinin stimulation in lymphocytes (Maul et al., 1972). These events are correlated with protein synthesis and DNA replication. Pores also vary in number during hormonal stimulation in rat seminal vesicles (Ortiz and Cav-
The nuclear envelope is a complex eukaryotic
membrane organelle whose biological behavior under diverse physiological requirements is largely undefined at present. Its main architectural features are an inner and an outer membrane joined at the pore complexes, which constitute the major passageways of molecules between nucleus and cytoplasm. Attached to the inner aspect of the nuclear envelope is a proteinaceous electron-dense layer, biochemically different from the nuclear matrix (Gerace et al., 1978), Instituto de Histologia y Embriologia, Fact&ad de Ciencias Medicas, Universidad National de Cuyo, Mendoza, Argentina. Received 7 December 1990. Revised 24 September 1991. 75
CAVICCHIA
16
icchia, 1990)) during different life cycle stages in a ciliated protozoan (Dallai and Luporini, 1982), in a slime mold (Aldrich and Pendland, 1981) and in the Chinese hamster cells (Scott et al., 1971). Germ cell differentiation is a complex process where numerous cell types mature synchronously; therefore, the seminiferous epithelium seems to be a suitable model for the study of nuclear pore changes which may occur under physiological influences. Many developmental features were reported to occur during spermatogenesis, e.g. disappearance of the fibrous lamina during pachytene stage (Stick and Schwartz, 1982), a change in distribution of chromatin condensation in different spermatogonia (Cavicchia and Dym, 1978), spermatocytes and spermatids (Russell and Frank, 1978; Kretser and Kerr, 1988), and variations in the chromosome-nuclear envelope attachments during meiosis (Solari, 1969). These events might be closely related to changes in the nuclear pore patterns of germ cells. Freeze-fracture replicas allow us to explore extensive membrane areas. However, due to the difficulty in recognizing different germ cell types present in the seminiferous epithelium, previous descriptions of nuclear pore patterns in freeze-fracture replicas have been limited only to Sertoli cells, and to the general characteristics of spermatogonia, spermatocytes and spermatids (Fawcett and Chemes, 1979; Liankai, 1987). The present investigation was undertaken combining the isolation of those particular segments of the seminiferous tubules which contain specific germ cell associations (Parvinen and Vanha Perttula, 1972), allowing their proper identification with the freeze-fracture procedure.
AND MORALES
With these combined methods, we were able to characterize the nuclear pore pattern of type A and Type B spermatogonia, as well as of preleptotene, leptotene, zygotene, pachytene, diplotene, and secondary spermatocytes. Material and Methods Adult Holtzman albino rats weighing approximately 350 g, fed on laboratory chow and water ad libitum, were housed in animal room at 20” C. The lighting regimen was 12112. They were anesthetized with intraperitoneal pentobarbital sodium and the testes exposed through a scrotal incision. The tunica albuginea was opened, the seminiferous tubules were placed in a Petri dish containing a Ringer-Krebs sucrose solution and isolated according to the technique described by Christensen and Mason (1965). Segments of seminiferous tubules were studied in transmitted light under a Nikon dissecting microscope (X40-100 magnification) and cut in suitable l-2 mm fragments with iridectomy scissors. Four main types of light absorption were selected: 1) Stages VII-VIII with a very dark homogeneous central absorption due to mature spermatozoa ready to be released into the tubular lumen. 2) Stages IX-XI, which follow, homogeneously transparent because spermiation is over and no spermatozoa are present. There is a sharp demarcation between these two segment types. 3) Stages V-VI showing a spot-like arrangement in the tubule center apparently caused by maturing spermatids attached to the Sertoli cells (Parvinen and Vanha Perttula,
Fig. 1. A Sertoli cell. The infolded nuclear envelope (N) exhibits randomly distributed pores. Arrows indicate the Sertoli cell lateral membranes. G, neighboring germ cells. BL, basal lamina. Stages XII-VIX of the seminiferous epithelium. x 11,CKtO. Fig. 2. A round nuclear profile of a type A spermatogonium with a slight clumping of nuclear pores. Stages VII-VIII. BL, basal lamina. The characteristic pits of pinocytotic vesicles in the interlamellar, peritubular cells (IC) are indicated by arrows. x 10,000. Fig. 3. Stages V-VI. A germ cell with extensive attachment to the basal lamina (BL) probably a typ B spermatogonium. Its nucleus (N) displays intense pore aggregation. IC, interlamellar cell. Arrow: inter-germ cell bridge. xlO,OLKl. Fig. 4. Stages VII-VIII. A group of preleptotene spermatocytes close to the basal lamina (BL). Their nuclei (N) exhibit marked aggregation of nuclear pores. x4ooO.
CAVICCHIA
78
1972), and located immediately previous to stages VII-VIII. 4) Stages XII-XIV, which follow along the tubule, stages IX-XI and present central light spots apparently due to spermatids with an increasing density of acrosomal material (Parvinen and Vanha Perttula, 1972). For histological identification, samples of these tubular segments were transferred into 5% glutaraldehyde in cacodylate buffer pH 7.4, postfixed in a solution containing 2 parts of 2% osmium tetroxide and 1 part of 1% potassium ferrocyanide, embedded in Medcast, sectioned in 1 pm sections and stained with 1% toluidine blue sodium borate. The classification of Perey et al. (1961) was utilized for identification of the stages (I-XIV) of the seminiferous epithelium in the rat. A useful examination of the paper by Perey et al. can be found in Steinberger and Steinberger (1975). The criteria for identification of rat spermatocytes in 1 pm sections after plastic embedding (Russell and Frank, 1978) were also followed. In this manner the transilluminated appearance of each tubular segment was compared with the histological picture of cross tubular sections in the same piece of tissue. We found good correlation between both methods. The selected sets of tubular fragments containing specific stages of the seminiferous epithelium were fixed by immersion in 5% glutaraldehyde-cacodylate buffer for 20 min and placed in 30% glycerol for 2 hr. Sets of 3-4 tubule fragments were longitudinally aligned on gold freeze-fracture holders, rapidly frozen in liquid Freon 22 and stored in liquid nitrogen. The material was fractured in a Balzers BAF 301 at -105” C and shadowed with platinum followed by
AND MORALES
carbon. Replicas were cleaned with sodium hypochlorite, mounted on copper grids and observed in either a Siemens Ehniskop IA or I. The criteria for identification of each cell are progressively indicated in order to be recognized in the freeze-fracture replicas, type by type, in the course of the ‘Results’ section. 30 seminiferous segments from each set of stages were fractured and approximately 20 micrographs for each cell type were taken. As we found outstanding consistency of morphological characteristics for each of the analyzed cells, we estimated that the sample was appropriate. Results
Figure 1 provides a view of the seminiferous epithelium in a freeze-fracture replica. Sertoli cell nuclei, constantly found along the seminiferous tubule, are characterized by morphological parameters already described (Fawcett and Chemes, 1979) e.g., a highly infolded nuclear envelope and a random distribution of nuclear pore complexes. Type A spermatogonia are the only basal compartment cells with extensive surface contact with the basal lamina in the isolated stages IX-XI and XII-XIV. They are oval shaped cells. Their freeze-fracture nuclear profile (Fig. 2) are also oval or less frequently spherical. The nuclear pore pattern is constantly characterized by either a random distribution of nuclear pores or by a slight degree of pore clumping. The apparent density of pores in the freeze-fractured nuclear membranes is much the same in all the stages studied. Germ cells in stages V-VI, which also display extensive contact with the basal lamina and con-
Fig. 5. Stages VII-VIII. Two preleptotene spermatocytes at higher magnification with spherical, centrally located nuclei (N) with intense pore clustering. Arrows indicate inter-Sertoli interdigitations. x lO,OC@. Fig. 6. Germ cell close to the basal lamina found in stages IX-XI, likely to be a leptotene spermatocyte. The nuclear pore aggregation is less evident than in preleptotene spermatocytes. x1o,oc@. Fig. 7. Germ cell with similar characteristics and location to the one in the previous figure but found in stages XII-XIV, likely to be a zygotene spermatocyte. The nuclear pore pattern also has similar characteristics. x 15,CW Fig. 8. Pachytene spermatocytes are large cells always detached from the basal lamina. Their round nucleus (N) shows a conspicuous clustering of pores separated by large pore free areas. Stages IX-XI. x 8000.
CAVICCHIA
80
sistently contain spherical nuclei, are likely to be type B spermatogonia (Perey et al., 1961). They possess, instead, tightly aggregated nuclear pores (Fig. 3). Preleptotene spermatocytes are only found in stages VIVIII (Perey et al., 1961). They are rather numerous forming groups of cells in close vicinity to the basal lamina; such vicinity in this case is an additional reference parameter. Although still anchored to the basal lamina, some of these cells are already detached from it (Fig. 4). Their nuclei (78pm in diameter) are smaller than those found in both type A or B spermatogonia, perfectly spherical and located in the central part of the cell. They exhibit an intense clustering of nuclear pores (Figs 4, 5) forming groups of 15-30, separated by pore-free membranes areas. Leptotene (stages IX-XI) (Fig. 6) and zygotene (stages XII-XIV) (Fig. 7) spermatocytes (nuclear diameter 9-10 ,nm) are also rounded cells, larger than preleptotene spermatocytes and positioned higher in the tubule, though still not far from the basal lamina. This position allows us to tell the difference between leptotene or zygotene and type A spermatogonia (in direct contact to the basal lamina), on the one hand, and the groups of pachytene or diplotene spermatocytes (located higher in the seminiferous epithelium), on the other hand. The two latter cell types exhibit, moreover, other distinguishing features, indicated below, mainly their large nuclear sizes, which permit their discrimination from leptotenes (found in stages IX-XI) or zygotenes (in tubular segments containing stages XII-
AND MORALES
XIV). Since careful examination of the freeze-fracture material from both sets of stages did not disclose any difference regarding nuclear pore pattern (compare Figs 6 and 7), the following description applies to both leptotene and zygotene spermatocytes. The nuclear pore arrangement changes to a more even distribution, although clearly maintaining some degree of pore aggregation. Identification of pachytene spermatocytes in freeze-fracture replicas is facilitated by: a) their large size (nuclear diameter 10-13 pm), b) their location in the seminiferous epithelium where groups of basal compartment cells (e.g., preleptotene spermatocytes in the segments containing stages VII-VIII) are interposed between them and the basal lamina, and c) by being rather numerous and found in all the segments studied. The fracture faces of the round pachytene nuclei show a conspicuous clustering of pores (in groups of N-70) separated by large porefree areas (Fig. 8). Diplotene spermatocytes, found exclusively in the seminiferous tubules containing stage XIII, are similar to pachytene in their location within the seminiferous epithelium, in their relationship with other more basal cells (zygotenes in this case), and in their large size and shape. Diplotene cells have round nuclei with the largest diameter (Russell and Frank, 1978), a characteristic which is also observed in the freeze-fracture preparations (diameter of the largest nuclear profiles, 14-15 pm). The nuclear pore complexes are numerous here and apparently randomly distributed (Fig. 9). In freeze-fracture preparations of tubular
Fig. 9. Stages XII-XIV. A large germ cell located far from the basal lamina, likely to be a diplotene spermatocyte exhibits randomly distributed nuclear pores. x8000. Fig. 10. Freeze-cross section of small vesicle profiles forming rows (dotted lines) alternated with smooth areas. These structures might well correspond to the spindle vesicles described in thin sections of metaphase-anaphase spindles (Pleshkewych and Levine, 1975). Stages XIIXIV. XlO,cKlO Fig. 11. A large. round nuclear profile found far from ihe basal lamina in stages XII-XIV and intermingled with the spindles profiles described in the previous figure. This germ cell might be a secondary spermatocyte. Its nucleus exhibits a paucity of nuclear pores randomly distributed. x 10,000. Fig. 12. Lateral view of a round spermatid with a developing acrosome cap (A). Its nuclear envelope shows a few pores (arrows) in the post-acrosomal region. No nuclear pores arc observed below the acrosome. Stages V-VI. ~15,000. Fig. 13. A caudal view of a similar spermatid. The nuclear pores are gathered around the implantation fossa (F) of the flagellum. x 10.000.
CAVICCHIA AND MORALES
82
stages XII-XIV, fragments containing groups of numerous round cells, centrally located within the seminiferous epithelium, with a nuclear diameter about 9-10 pm are frequently found. For these characteristics and also because they are found intermingled with metaphase-anaphase profiles (Fig. lo), a feature also present in stage XIV, they can be secondary spermatocytes (Russell and Frank, 1978; Kretser and Kerr, 1988). Their nuclear membranes in freeze-fracture are characterized by a paucity of pores in an apparently random distribution (Fig. 11). Round spermatids, with a developing acrosome cap and an axonemal profile, are spermatocytes smaller than secondary (nuclear diameter 8-9 pm) and found in the tubular segments containing stages V-VI. Nuclear pores have disappeared below the expanding acrosome (Fig. 12), and pore migration proceeds towards the caudal pole of the nucleus (Fig. 13). Since the process of spermiogenesis in the rat was extensively studied with freeze-fracture (Fawcett and Chemes, 1979) no further description is needed. Other cell types such as intermediate spermatogonia which could not be isolated within specific transilluminated seminiferous tubules are beyond the scope of the present study. Discussion Electron micrographs of thin sections provide little information about the distribution pattern and density of nuclear pores. In freeze-fracture preparations, instead, the cleavage plane tends to follow the hydrophobic region of the membranes affording extensive en face views of the nuclear envelope and its nuclear pore complexes. Freezefracture studies of the seminiferous epithelium have allowed, therefore, other authors (Fawcett and Chemes, 1979; Liankai, 1987) to identify several nuclear pore patterns belonging to specific cell types. However, since spermatogenesis is a complex process involving many germ cells in different degrees of development, it is quite difficult, if not impossible, by freeze-fracture alone to characterize them further. The isolation by transillumination of seminiferous tubule segments containing specific stages of the seminiferous cycle proved a valuable procedure for identification. Combining both methods
and utilizing secondary parameters for cell identification in freeze-fracture, such as nuclear size and shape, cell location within the seminiferous epithelium or vicinity to other structures, we were able to go further in the identification procedure characterizing other germ cell nuclei. The most prominent feature which seems to emerge from the present report is the close correspondence between chromatin arrangements and nuclear pore patterns exhibited by any of the identified cells. The two main categories of spermatogonia, type A and B, with chromatin arrangement respectively described as ‘dust like’ (Regaud, 1901) due to its homogeneous, finely granular distribution, also observed in electron micrographs (Cavicchia and Dym, 1978), and ‘crust like’, which show, in conventional electron microscopy, coarse clumps of chromatin attached to the nuclear envelope, display quite different nuclear pore patterns: the former, homogeneous distribution, and the latter, evident pore clustering. Similarly, preleptotene spermatocytes with numerous dense masses of chromatin mainly associated with the nuclear envelope (Solari, 1969; Cavicchia and Dym, 1978) exhibit in freeze-fracture preparations a striking clustering of pores separated by pore-free areas in freezefracture preparations. Nuclear pore complexes change again to a more homogeneous distribution in leptotene spermatocytes which are recognized in thin sections by disappearance of the characteristic preleptotene chromatin clumps associated with the nuclear membrane. Chromatin here becomes rather homogeneous forming diffuse thin threads (Solari, 1969). In pachytene spermatocytes, a progressive chromosome condensation takes place. Chromosomes adopt the so-called ‘bouquet’ arrangement forming long loops with their ends attached to the nuclear envelope and the condensed X-Y pair (‘sex vesicle’), which also displays increasing nuclear membrane contact (Solari, 1969; Fawcett, 1981). Coincidently with these intranuclear events, nuclear pore distribution changes to a pattern of marked clustering, which reverses again in diplotene, showing an even distribution of nuclear pores. Here, decondensation of chromosomes reaches a maximum (Solari, 1969). Due to the increased size of the nucleus many areas are
NUCLEAR PORE DISTRIBUTION
IN ISOLATED
TUBULES
free from condensed chromatin (Russell and Frank, 1978). In secondary spermatocytes, nuclear pores, now evenly distributed, also correspond to poor chromatin condensation and a nucleoplasm with very low stain affinity (Russell and Frank, 1978). All these features coincide in indicating a close correlation between chromatin condensation and nuclear pore clustering. In agreement with our results, during interphase, in cockroach blood cell and Malpighian tubule nuclei which contain large amounts of heterochromatin, an intense clustering of nuclear pores was also reported (Teigler and Baerwald, 1972). Maul et al. (1971) have suggested that the heterochromatin lying beneath the surface of the nuclear membrane may influence the pattern seen in nuclear pores. Dallai and Luporini (1982) also observed that in the macronuclei of a ciliated protozoan, either in thin sections and in freeze-fracture replicas, pore clusters are confined to areas free of underlying heterochromatin masses. Co-incidentally, other consistent structures which can make close apposition to the nuclear envelope from either the inside, e.g., the nucleoli, or the outside, e.g., vacuoles (Severs et al., 1976) or the acrosome during spermiogenesis (Fawcett and Chemes, 1979) may influence pore arrangement.
83
One aspect that remains unresolved is how nuclear pore complexes in germ cells change in density and distribution during spermatogenesis. It is known that some turnover of nuclear pores takes place in somatic cells during different physiological activities (Maul et al., 1973; Ortiz and Cavicchia, 1990) although a genera1 pattern is probably maintained by attachment of the pore complexes to the underlying fibrous lamina through connecting fibrillar elements @cheer et al., 1976; Allen and Douglas, 1989). The fact that the fibrous lamina is absent in male germ cells during meiosis (Stick and Schwartz, 1982) can explain in part the apparent freedom that may be necessary for the process by which the nuclear pore pattern changes so dynamically during meiosis, as shown in the present paper. Acknowledgements
This investigation was supported by grants from CONICET (Consejo National de Investigaciones Cientificas y Tecnicas and CIUNC (Consejo de Investigaciones de la Universidad National de Cuyo), Argentina. The technical assistance of Mr. Osvaldo Arango is highly appreciated. We thank Dr. Fabio L. Sacerdote for linguistic assistance with the manuscript.
References
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Allen, .I. L., Douglas, M. G. 1989. Organization of the nuclear pore complex in Saccharomyces cereoisiae. J. Ubrmt. Mol. Res., 102, 95-108. Cavicchia, J. C. and Dym, M. 1978. Ultrastructural characteristics of monkey spermatogonia and preleptotene spermatocytes. Biol. Reprod., 18, 219-228. Christensen, A. K. and Mason, N. R. 1965. Comparative ability of seminiferous tubules and interstitial tissue of rat testes to synthesize androgens from progesterone-4-14C in oifro. Endocrinology, 76, 646-656. Dallai, R. and Luporini, P. 1982. Variation in the nuclear pore distribution through different life cycle of the ciliated protozoan Euplotes crassu.x .I. Submicrosc. Cytol., 14, 107-121. Fawcett, D. W. 1966. On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. Am. J. Amt., 119, 129-146. Fawcett, D. W. 1981. Nuclear Envelope. In The Cell, pp. 266-280. W. B. Saunders Company. Philadelphia, London, Toronto. Fawcett, D. W. and Chemes, H. E. 1979. Changes in distribution of nuclear pores during differentiation of the male germ cells. Tissue Cell, 11, 147-162. Gerace, L., Blum, A. and Blobel, G. 1978. Immunochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. J. Cell Biol., 79, 546-566.
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