EXPERIMENTAL
AND
hiOLECULAR
Effects
31, 56-69
PATHOLOGY
of a-Amanitin on RNA Rat Seminiferous KARL-OVE
Department Receiced
of Pathology, August
Unioersity
29, 1978,
and
( 19%)
Synthesis Tubules
in Cultured
S~DERSTR~M of Turku, in recked
SF-20520 form
Turku
Nocember
53, Finlantl 17, 1978
The effect of the specific RNA polymerase II inhibitor a-amanitin on the cell morphology and RNA synthesis during spermatogenesis was investigated. Light microscopic autoradiography showed that a-amanitin largely decreased the rate of RNA synthesis in the pachytene spermatocytes and in the spermatids. Degenerative changes occurred most rapidly in the mid pachytene spermatocytes and in the intermediate and type B spermatogonia. Already after 14 hr of culture with a-amanitin (10 ~g/ml) a part of these cells showed progressive degenerative changes beginning with the clumping together of the chromosomes. This indicates that at least a part of the HnRNA which is synthesized in the lampbrush loops of the pachytene chromosomes is needed to maintain the normal structure and function of the pachytene spermatocytes. Biochemical evidence suggested also that the formation of HnRNA was specifically inhibited in the pachytene spermatocytes by ol-amanitin.
INTRODUCTION Several autoradiographic studies about the RNA synthesis during spermatogenesis have been made (Monesi, 1964, 1965; Tres, 1975; Loir, 1972; Kierszenbaum and Tres, 1974a, b, 1975; SGderstrijm and Parvinen, 1976a). These studies have shown that during the mid pachytene stage of the meiotic prophase there is a rapid increase in the RNA synthesis of the primary spermatocytes. This RNA synthesis takes place at the same time as the lampbrush loops of the pachytene chromosomes are formed (Kierszenbaum and Tres, 1974b) and the synthesized RNA molecules remain in contact with the chromosomes for a long time (Monesi, 1965, 1971). Biochemical studies have shown that this RNA is mostly HnRNA (Muramatsu et al., 19%) which seems to have a long life time (Saderstrijm and Parvinen, 1976a; Siiderstriim, 1976). The activation of the RNA synthesis during meiotic prophase is a general phenomenon because also in oocytes a high rate of RNA synthesis has been reported to exist in the lampbrush loops during pachytene and diplotene stages (Hartung and Stahl, 1976). This RNA, as shown for some larger lampbrush loops, is stored in the amphibian oocytes for about 10 days (Gall and Callan, 1962) and in dipteran spermatocytes for 20 to 30 hours (Hennig, 1967). The polypeptid toxin a-amanitin isolated from the toadstool Amanita phalloides ( Wieland, 1968) selectively inhibits the nucleoplasmic RNA polymerase II of mammalian cells (Jacob et al., 197Oa, b). The RNA polymerase II is involved in the synthesis of HnRNA (Roeder and Rutter, 1970; Zylber and
0014-4800/79/040056-14$02.00/O
Copyright 0 1979 by Academic Press,Jnc. All rights of reproduction in any form reserved.
EFFECTS
OF ALPHA-AMANITIN
ON SPERMATOGENESIS
57
Penman, 1971) which seems to be the main transcriptional product of the pachytene spermatocytes (Sijderstrom and Parvinen, 1976a; Soderstrom, 1976). In order to obtain information about both the mode of action of a-amanitin and the function of HnRNA synthesized during spermatogenesis and particularly during the mid pachytene stage of the meiotic prophase the effects of a-amanitin on the RNA synthesis, histology and ultrastructure of the spermatogenic cells of the rat were studied. MATERIALS
AND METHODS
Isolation of Cells The seminiferous epithelium consists of the Sertoli cells, the spermatogonia, the primary spermatocytes, the secondary spermatocytes and the spermatids. The different cell types are not randomly located in the seminiferous epithelium of the tubules but are arranged in association with constant cell composition. In the rat 14 different cell associations have been described (Leblond and Clermont, 1952). They follow each other in a regular manner and form the wave of the seminiferous epithelium (Perey et al., 1961). In a preparation microscope the different stages absorb light in a different way (Parvinen and Vanha-Perttula, 1972) allowing the isolation of living segments of the tubules with known cell associations. Incubation and Labeling Conditions First a piece of the tubule containing a complete wave of the seminiferous epithelium was isolated. This piece was then transferred under sterile conditions to a Petri dish and placed on lens paper lying on a stainless steel grid at the level of the culture medium (Johansson, 1975). The culture medium used was Parker 199 with Earle’s salts containing sodmm-G-penicillin (50 U/ml, Leiras, Turku, and Finland) and streptomycin sulphate (50 pg/ml Laake, Oy, Turku, and Finland). When the effect of a-amanitin was studied the culture medium contained 5, 10, or 20 pg/ml of a-amanitin (Sigma, St. Louis, MO, USA). The tubules were cultured between 2 and 20 hr in an atmosphere of 95% O3 and 5% CO, at 31°C. After this the tubulus was incubated in the presence of [3H]uridinc (100 &i/ml) for 2 hours, cut into smaller about 2 mm long pieces and the stage of each piece was accurately identified using phase contrast microscopy (Soderstrom and Parvinen, 1976a). The small pieces were then processed for histology, electron microscopy, autoradiography or biochemical analyses. In the control experiments tubules were incubated and treated in the same way except that no a-amanitin was present in the incubation media. In order to control that the obtained results were not an artefact caused by the lack of hormones in the incubation media, tubules were also incubated in the presence of FSH (5 pg/ml, NIH-ovine FSH-S9) and testosterone (0.1 pg/ml). Histology and Electron Microscopy The pieces of the tubules were tixed in Bouin’s fluid, embedded in paraffin, cut in 5 ,.m-~sections and stained either with Harris’ hematoxylin and eosin or
5s
KARL-OVE
SdDERSTRijM
with the periodic acid shiff method, with hematoxylin as counterstain. For electron microscopy the accurately identified tubular segments were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 3 hours, postfixed in 1% osmium tetroxide in 0.1 M 2,4,6-collidine buffer at pH 7.4 for 2 hours, dehydrated, embedded in epon and 700 A thick sections were cut with an LKB Huxley ultramicrotome. The sections were stained with 4% uranyl acetate for 30 min at +6O”C and 0.2% lead citrate for 2 min at room temperature. The observations were made with a Jeol Jem-T8 electron microscope. Autoradiography For light microscopic autoradiography the 5 pm thick sections were placed on glass slides and dipped in Kodak NTB 3 emulsion. Due to the high level of radioactivity in the cells the exposure time was limited to 1 or 3 days. The autoradiograms were thereafter developed in Kodak D 19 developer for 4 min at 20°C and stained with Harris’ hematoxylin and eosin. For high resolution autoradiography the 700 -4 thick sections were placed on glass slides coated with a thin collodion membrane. The slides were dipped in Ilford L 4 emulsion so that a monolayer was formed. Exposure times from 1 to 4 months were used. The autoradiograms were developed with Microdol X (Kodak Limited, England) and fixed with solifix. After this the specimens were stripped off the slide in water, placed on copper grids (Rogers, 1967) and stained with uranyl acetate and lead citrate as described previously. Extraction
and Electrophoretic
Analyses
of RNA
Stages VII containing the actively HnRNA synthesizing mid pachytene spermatocytes were chosen for biochemical analyses. The RNA was extracted from the tubules with a phenol SDS extraction method (Soderstrom, 1976). The tubules were first homogenized in 1 ml of 0.1 M Tris-HCl buffer pH 7.4 containing 0.570 SDS, and 100 pg pronase treated E. coli reference and carrier RNA. Immediately after the homogenization was started, 1.5 ml of water saturated freshly distilled phenol was added. The homogenization was performed at 0°C for 5 min and at 20°C for 5 min. Thereafter the solution was shaken vigorously for 1 min. The water and the phenol phases were separated with a rapid centrifugation and the phenol was removed. To the buffer 0.25 PI of 4 M NaCl was added and the RNA was precipitated with 2.5 volumes of cold absolute ethanol. The RNA was stored overnight at -20°C and the ethanol was carefully removed. The RNA was dissolved in 50 ~1 of 0.02 M Tris-HCI buffer pH 7.4 with 0.5% SDS. To avoid aggregation of the RNA the dissolving solution contained urea (8 M). The dissolved RNA was fractionated with electrophoresis in an agarose gel (1% agarose, Marine Colloids Inc., Springfield, NJ, USA) in 0.02 M Tris-HCI buffer pH 8.0 with 0.002 AZ EDTA and 0.5y0 SDS, using an Ortec 4010 pulsed constant power supply with the settings at 100 V, 100 mA, and 500 pulses/s. The localization of the E. coZi reference RNA bands was determined by their UV light absorption. After the electrophoresis the gels were washed in 5% TCA for I hr and distilled water for 2 hours at +4”C with several changes of the water. The gel was sliced in I.1 mm long pieces and the radioactivity in each piece was determined by a low background
EFFECTS
F.._ _ %
.
OF
ALPHA-AMANITIN
ON
SPERMATOGENESIS
59
4
a!
*
FIG. 4. An autoradiogram of stage X of rat spermatogenesis with [3H]uridine. Only occasional grains are seen over the step X spermatids (arrow) appear normal. The exposure time
cultured for 14 hr with pachytene spermatocytes was 1 day. Xl2fiO.
a-amanitin ( 10 rg/ml) (ps). These cells are
and thereafter morphologically
labeled normal.
for 2 hr Also the
and thereafter labeled for 2 hr FIG. 3. An autoradiogram of stage V of rat spermatogenesis cultured for 14 hr with a-amanitin (10 pg/ml) with [‘Hluridine. No labeling is seen over the pachytene spermatocytes (ps) and only occasional grains are seen over the young round spermatids (St). However, only some pachytene spermatocytes degenerate whereas most of them show a normal morphology. The B-spermatogoniti The exposure time was 1 day. X1250. (sg) show pycnotic nuclei as a sign of degeneration.
b
0 0
(10 pg/ml) and thereafter are unlabeled as also in over them. No morphological
labeled for physiological degenera-
(10 pg/ml) and thereafter labeled for 2 hr for 14 hr with a-amanitin (sg) are unlabeled while occasional grains are seen over the young spermatocytes is normal whereas the intermediate type spermatogonia The exposure time was 1 day. X1250. (arrow).
a-amanitin FIG. 6. An autoradiogram of stage XIV of rat spermatogenesis cultured for 14 hr with as well as the spermatids 2 hr with [‘H]uridine. The cells going through the meiotic divisions (M) conditions. The zygotene spermocytes (z) are unlabeled while in control samples some grains are seen tive changes are seen in any cell type of this stage. The exposure time was 1 day. X1250.
FIG. 5. An autoradiogram of stage III of rat spermatogenesis cultured with [‘Hluridine. The pachytene spermatocytes and the spermatogonia spermatids (St). The morphology of the spermatids and the pachytene undergo degenerative changes. Some label is seen over the Sertoli cells
63
KARL-OVE
SOIlERSTROM
high sensitivity gas counter (Parvinen et al., 1973) owing to the low level of radioactivity in the slices. For control purposes the RNA synthesis in the separated epididymal tubules were analyzed in the same way. RESULTS The control experiments showed that the morphology of the spermatogenic cells did not change in any of the stages during 22 hours of culture under the described conditions. The addition of FSH and testosterone to the culture medium had no effect on the cellular morphology during the 22 hours of culture. When the tubules were incubated in vitro without a-amanitin or hormones in the presence of [ 3H]uridine a high level of RNA synthesis could be seen in the mid pachytene spermatocytes and in the spermatogonia whereas the young round spermatids were only slightly labeled (Fig 1). The addition of testosterone and FSH to the culture medium did not cause any change in the labeling of the cells during a 22 hour culture. Histological and phase contrast microscopic studies showed also that the cells most affected by a-amanitin were the pachytene spermatocytes. Particularly the mid pachytene spermatocytes in stages VI, VII, and VIII underwent rapid degeneration so that after 14 hours in the presence of 10 pg/ml of a-amanitin pachytene spermatocytes in various stages of cell degeneration could be observed (Fig. 2). After a 20 hour treatment with a-amanitin most of the mid pachytene spermatocytes showed marked degenerative changes. In the control tubules incubated without a-amanitin all pachytene spermatocytes appeared normal. During the early pachytene (stages I-V) the degeneration was more slow than during the mid pachytene stage and many cells seemed unaffected even after a 20 hour incubation with ol-amanitin (10 pg/ml) (Fig. 3). The late pachytene spermatocyes were also resistant and only a small number of these cells were seen degenerating during 20 hour culture with a-amanitin (10 pg/ml) (Fig. 4). Other sensitive cell types were the intermediate (In) and type B spermatogonia in stages III, IV, and V. These cells degenerated almost as rapidly as the mid pachytene spermatocytes (Fig. 5). The histology of the leptotene spermatocytes, zygotene spermatocytes and the spermatids appeared normal and the meiotic divisions were not affected during a 20 hr culture with cu-amanitin (10 pg/ml) (Fig. 6). Electron microscopic observations showed more in detail the cell degeneration process. The pachytene spermatocytes seemed normal during an 8 hour culture with a-amanitin (Fig. 7) but thereafter followed a condensation of the chromatin and the chromosomes clumped together forming large electron dense structures (Fig. 8). The autoradiographic observations showed that the labeling of the mid pachytene spermatocytes was greatly reduced already after 6 hour culture with a-amanitin. Although the labeling of the autosomes of the pachytene spermatocytes decreased the nucleolus remained labeled (Figs. 9 and 10). The labeling of the zygotene spermatocytes which have a low level of RNA synthesis did not seem to be affected by a-amanitin. Electron microscopy showed that the nuclear membrane of some spermatids was partly broken down and that the chromatoid body in some of the sperma-
EFFECTS
OF ALPHA-AMANITIN
ON SPERMATOGENESIS
63
FIG. 7. An electron microscopic picture of stage VII of the seminiferous epithelium of the rat after 8 hr culture with a-amanitin ( 10 pg/ml), The appearance of the spermatids (st) is normal. Some slight fragmentation can be detected in the nucleolus (n) of the pachytene spermatocyte (p) which otherwise are normal. a: acrosome of a spermatid. x3300. FIG. 8. An electron microscopic picture of stage VII of rat spermatogenesis cultured for 14 hr with a-amanitin (10 rg/mI). The mid pachytene spermatocytes (p) show changes of their chromatin which seems to aggregate into electron dense granular masses. The spermatids (st ) remain normal. Bl: basal Iamina. X3300.
tids acquired a ring like appearance in stages I to VII (Fig. 11). This change in the chromatoid body was first seen after 12 hour of culture with ar-amanitin. The nature of the RNA synthesis inhibited by a-amanitin was studied biochemically. The electrophoretic distribution of the RNA synthesized in stage VII of rat spermatogenesis which contains the most actively RNA synthesizing
64
KARL-OVE
SBDERSTROM
FIG. 9. A control high resolution autoracliogram of a pachytene spcrmatocyte from stage VII of rat spermatogenesis cultured for 6 hr without n-amanitin followed by a 2. hr pulse with [3H]uridine. The nucleus is heavily labeled but almost no grains can be seen in the cytoplasm. The exposure time was 2 months. n: nucleus. ~1365. FIG. 10. A high resolution autoradiogram of a pachytene spermatocyte from stage VII of rat spermatogenesis cultured for 6 hr with or-amanitin (IO ~g/ml) followed by a 2 hr pulse with [3H]uridine. The labeling of the autosomes has markedly decreased whereas the nucleolus (nu) is labeled suggesting a normal rate of rRN.4 formation. The exposure time was 2 months. m: mitochondria. ~1365.
EFFECTS
OF
ALPHA-AMANITIN
ON
SPERMATOGENESIS
65
FIG. 11. A high resolution autoradiogram of a spermatid from stage VI of rat spcrmatogenesis after 20 hr culture with ar-amanitin (10 pg/ml) followed by a 2 hr pulse with ‘H-uridine. The nucleus (n) and the cytoplasm of the spermatid are unlabelled. The chromatoid body (cb) has a peculiar ring shaped structure which is not normally seen. Magnification 30,000X.
pachytene primary spermatocytes shows that during a 2 hr pulse with [3H]uridine almost only HnRNA is formed. a-amanitin greatly reduced the rate of this HnRNA synthesis in 4 hours (Fig. 12a). The synthesis of the smaller molecular weight RNA (4s) did not seem to be sensitive to a-amanitin although after S-hour culture almost no HnRNA synthesis could be observed (Fig. 12b). In the control experiments with epididymal tubules the HnRNA synthesis also seemed to be inhibited by a-amanitin although the synthesis of rRNA was unaffected and clear 28s and 18s peaks could be seen in the electrophoresis. DISCUSSION The RNA synthesis during spermatogenesis shows many peculiar features not observed in other cells. There is a high level of RNA synthesis in the spermatogonia that gradually decreases with the progressive increase in the amount of heterochromatin in the cells. The RNA synthesis is at a low level during leptotene, zygotene, and early pachytene. In mid pachytene primary spermatocytes there is a rapid increase in the RNA synthetic rate which then decreases during late pachytene. A low level of RNA synthesis persists in the early spermatids up to step 8. No RNA synthesis exists in the late spermatids (Monesi, 1964, 1965, 1971; SGderstriim and Parvinen, 1976a). The high level of
KARL-OVE
66
StiDERSTRiiM
RNA synthesis in the mid pachytene spermatocytes occurs at the same time as the lampbrush loops of the chromosomes are formed (Kierszenbaum and Tres, 197413). Biochemical studies have shown that most of the RNA synthesized in the mid pachytene spermatocytes seems to be HnRNA (Soderstrom, 1976; Soderstrom and Parvinen, 1976a). The present study showed that the specific inhibitor of RNA polymerase II o-amanitin had a selective action on the different spermatogenic cell types. Particularly the pachytene spermatocytes in their maturation stages showed a variable sensitivity to this drug. The action of a-amanitin on the pachytene
5 100
50
IO k 100
50
10
IO
30
33 Slice
40
50
N$@
Fro 12. The electrophoretic distribution of RNA from stage VII of rat spermatogenesis after 2 hr culture followed by a 2 hr pulse with and after 6 hr culture [3H] uri d ine (a) followed by a 2 hr pulse with [*H]uridine (b). Solid circles indicate the distribution of RNA from tubules cultured without or-amanitin and open circle with or-amanitin ( 10 pg/ml). Abscissa: Migration of RNA in the gel (slice number). Ordinate: ‘H cpm per slice divided by tubule length in mm.
EFFECTS
OF ALPHA-AMANITIN
ON SPERMATOGENESIS
67
spermatocytes correlated with the rate of RNA synthesis in these cells. In the early pachytene spermatocytes with a low rate of RNA synthesis, a-amanitin had only a slight effect but in the mid pachytene stage with a high rate of RNA synthesis, a-amanitin caused degeneration in the majority of the cells during a 26 hour observation period. Late pachytene spermatocytes with a low level of RNA synthesis were also more resistant to the action of a-amanitin. This may be due to long lived RNA molecules synthesized during mid pachytene that ensure cellular viability during late pachytene despite the block of HnRNA synthesis caused by a-amanitin. These results also indicate that at least a part of the HnRNA synthesized in the mid pachytene spermatocytes is necessary for the normal development of these cells and is not primarily stored for later use. It has also been observed that a large portion of the RNA synthesized in mid pachytene spermatocytes has a poly( A) sequence characteristic of mRNA (Soderstrom, 1976) and that protein synthesis is more active in mid pachytene spermatocytes than at earlier or later developmental stages (Monesi, 1965; Davis and Fir-lit, 1970). However, the RNA synthesis during mid pachytene may have other functions also which is indicated by the rapid changes in the ultrastructure of the chromatin caused by a-amanitin before any degenerative changes can be seen in the cytoplasm. The level of RNA synthesis is low in the leptotene, zygotene, and early pachytene spermatocytes and these cells seem also to be resistant to the action of a-amanitin. An interesting finding is the degeneration of the In and B spermatogonia caused by a-amanitin although the level of RNA synthesis in these cells is relatively low. The HnRNA synthesis is probably necessary for the normal development of these cells. Another possible explanation is that the spermatogonia are located in the basal compartment of the seminiferous epithelium between the Sertoli cell junctions and the basal lamina (Dym and Fawcett, 1970) where they may be more susceptible to the toxic action of a-amanitin than in the adluminal compartment above the Sertoli cell junctions. This does not, however, explain why the A spermatogonia which are also in the same basal compartment are not affected by a-amanitin in the same way. The results of this study are in some respects comparable to the effects of actinomycin D on the pachytene spermatocytes (Barcellona and Brinkley, 1973). In a high concentration actinomycin D inhibits, however, all RNA synthesis and not specifically the HnRNA synthesis. That the results of this study were not caused by the lack of hormones in the incubation media was shown by the control experiments when the tubules were incubated in the presence of FSH and testosterone and no differences in the results could be seen. However, it is known that tubular cells are actively engaged in FSH-dependent mRNA synthesis (Reddy and Villee, 1975). The reason why FSH and testosterone did not have any effect on the RNA synthesis in this experiment might be that 22 hours of culture in the absence of hormones is too short a time for the depletion of intercellular hormonal complexes. This is also supported by the findings that the first morphological changes in the spermatogonic cells occur 3 days after hypophysectomy (Clermont and Morgenthaler, 1955). These changes occur in the step 17 to 19 spermatids which do not synthesize RNA. Spermatids seemed to be little affected by a-amanitin. This is probably due to the low level of RNA synthesis in the spermatids (Monesi, 1964, 1965; Soder-
68
KARL-OVE
SiiDERSTRiiM
striim and Parvinen, 1976a). However, the cytoplasmic cell organelle of the spermatids, the chromatoid body, showed a ring like appearance after 14 hours of culture in the presence of Lu-amanitin. This change in the morphology of the chromatoid body is similar to that which is seen when spermatids are treated with actinomycin D ( SGderstrbm, 1977). Previously it has been shown that the chromatoid body is labeled with [3H]uridine when the spermatids are pulse labeled for 2 hours and thereafter chased for 14 hours in the presence of unlabeled uridine (Saderstriim and Parvinen, 1976b). That a-amanitin causes structural changes in the chromatoid body indicates not only that it contains RNA but also that at least a part of this RNA might be mRNA. This leads to the interesting suggestion that the chromatoid body might contain stored RNA molecules which are used later during spermiogenesis when transcription in the spermatid nucleus has ceased but protein synthesis still persists at a high level. ACKNOWLEDGMENTS I would like to thank technical assktance. This
Mrs. work
Marita SaderstrGm and was supported by a grant
Mrs. from
Leena Simola for their excellent the Finnish hledical Foundation.
REFERENCES BARCELLONA, W. J., and BHIKKLEY, B. R. ( 1973). Eff ec t.s 0 f actinomycin D on spermatogenesis in the Chinese hamster. Biol. Reprod. 8, 335-349. Q uantitative CLERMONT, Y., and MORGENTHALER, H. (1955). study of spermatogenesis in the hypophysectomized rat. Endocrinology 57, 369-382. DAVIS, J. R., and FIRLIT, F. C. (1970). In The Testis III (A. D. Johnson, W. R. Comes, and N. L. Wandermark, eds.), pp. 259-306. Academic Press, Inc., New York. DYM, M., and FAWCETT, D. W. ( 1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308-326. GALL, J. G., and CALLAN, H. G. ( 1962). [3H]uridine incorporation in lampbrush chromosomes. Proc. Natl. Acad. Sci. USA 48, 562-570. HARTUNG, M., and STAHL, A. (1976). Incorporation of tritiated uridine during pachytene and diplotene stages in the oocytes of the Japanese quail (Coturnix coturnix japonica). Experientia 32, 96-98. HENNIG, W. ( 1967). Untersuchungen zur Struktur und Funktion des Lampbursten-Y-Chromosoms in der Spermatogenese von Drosphila. Chromosoma 22, 294-357. JACOB, S. T., SAJDEL, E. M., and MUNRO, H. N. (1970a). Different responses of soluble whole nuclear RNA polymerase and soluble nucleolar RNA polymerase to divalent cations and to inhibition by a-amanitin. Biochern. Biophys. Res. Comm. 38, 765-770. JACOB, S. T., SAJDEL, E. M., MUECKE, W., and MUNRO, H. N. (1970b). Soluble RNA polymerase of rat liver nuclei: properties, template specificity, and amanitin responses in vitro and in vivo. Cold Spring Harbor Symp. @ant. Biol. 35, 681-691. JOHANSSON, R. (1975). RNA, protein, and DNA synthesis stimulated by testosterone, insulin and prolactin in the rat ventral prostate cultured in chemically defined medium. Acta Endocr. 80, 761-774. KIERSZENBAUM, A. L., and TRES, L. L. (1974a). Nuclear and perichromosomal RNA synthesis during meiotic prophase in the mouse testis. J. Cell Biol. 60, 39-53. T ranscription KIERSZENBAUM, A. L., and TRES, L. L. (197413). sites in spread meiotic prophase chromosomes from mouse spermatocytes. J. Cell Biol. 63, 923-925. KIERSZENBAUM, A. L., and TRES, L. L. ( 1975). Structural and transcriptional features of the mouse spermatid genome. 1. Cell Biol. 65, 258-270. LEBLOND, C. P., and CLERMONT, Y. ( 1952). Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci. 55, 548-573. LOIR, M. (1972). MGtabolisme de l’acide ribom&%qnc rt dcs protbinrs danh lrzs spczrtnato-
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OF ALPHA-AMANITIN
ON SPERMATOGENESIS
69
cytes et les spermatides du belier (Ovis aries). 1. Incorporation et devenir de 1-[3H]uridine. Ann. Biol. Anim. Bioch. Biophys. 12, 203-219. MONESI, V. ( 1964). Ribonucleic acid synthesis during mitosis and meiosis in the mouse testis. J. Cell MONESI,
Biol.
22,
521632.
V. (1965).
Synthetic activities during spermatogenesis in the mouse RNA and protein. Exp. Cell Res. 39, 197-224. MONESI, V. ( 1971). Chromosome activities during meiosis and spermiogenesis. J. Reprod. Fert. Suppl. MURAMATSU,
13,
1-14.
M., UTAKOJI, T., and SUCANO, H. ( 1968). Rapidly-labelled nuclear RNA in Chinese hamster cells. Exp. Cell Res. 53, 278-283. PARVINEN, M., SOINI, E., and TUOHIMAA, P. ( 1973). An automatic gas counter for quantitative microdeterminations of tritium in biological material. Anal. Biochem. 55, 193-200. PARVINEN, M., and VANKA-PERTTULA, T. (1972). Identification and enzyme quantitation of the stages of the seminiferous epithelial wave in the rat. Anat. Rec. 174, 435450. PEREY, B., CLERMONT, Y., and LEBLOND, C. P. (1961). The wave of the seminiferous epithelium in the rat. Am. J. Anat. 108, 47-77. REDDY, P. R. K., and VILLEE, C. A., ( 1975). Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP. Biochem. Biophys. Res. Comm. 63, 1063-1069. ROEDER, R. G., and RUTTER, W. J. ( 1970). Specific nucleolar and nucleoplasmic RNA polymerases. Proc. Natl. Acad. Sci. USA 65, 675-689. ROGERS, A. W. (1967). Techniques of autoradiography. EIsevier Publishing Co., Amsterdam, 289-308. S~DERSTR~M, K.-O. ( 1976). Characterization of RNA synthesis in mid pachytene spermatocytes of the rat. Exp. Cell Res. 102, 237-245. S~DERSTR~M, K.-O. (1977). Effect of actinomycin D on the chromatoid body of rat spermatids. Cell Tiss. Res. 184, 411421. S~DERSTR~M, K.-O., and PARVINEN, M. (1976a). RNA synthesis in different stages of rat seminiferous epithelial cycle. Mol. Cell EndocrinoI. 5, 181-199. S~DERSTR~M, K.-O., and PARVINEN, M. (197613). Incorporation of [3H]uridine by the chromatoid body during rat spermatogenesis. J. Cell Biol. 70, 239-246. TRES, L. L. (1975). Nucleolar RNA synthesis of meiotic prophase spermatocytes in the human testis. Chromosoma 53, 141-151. WIELAND, T. ( 1968). Poisonous principles of mushrooms of the genus Amanita. Science 159, 946-952. ZYLBER, E., and PENMAN, S. (1971). Products of RNA polymerases in HeLa cell nuclei. PTOC. Natl. Acad. Sci. USA 68, 2861-2865.
AND
hiOLECULAR
Effects
31, 56-69
PATHOLOGY
of a-Amanitin on RNA Rat Seminiferous KARL-OVE
Department Receiced
of Pathology, August
Unioersity
29, 1978,
and
( 19%)
Synthesis Tubules
in Cultured
S~DERSTR~M of Turku, in recked
SF-20520 form
Turku
Nocember
53, Finlantl 17, 1978
The effect of the specific RNA polymerase II inhibitor a-amanitin on the cell morphology and RNA synthesis during spermatogenesis was investigated. Light microscopic autoradiography showed that a-amanitin largely decreased the rate of RNA synthesis in the pachytene spermatocytes and in the spermatids. Degenerative changes occurred most rapidly in the mid pachytene spermatocytes and in the intermediate and type B spermatogonia. Already after 14 hr of culture with a-amanitin (10 ~g/ml) a part of these cells showed progressive degenerative changes beginning with the clumping together of the chromosomes. This indicates that at least a part of the HnRNA which is synthesized in the lampbrush loops of the pachytene chromosomes is needed to maintain the normal structure and function of the pachytene spermatocytes. Biochemical evidence suggested also that the formation of HnRNA was specifically inhibited in the pachytene spermatocytes by ol-amanitin.
INTRODUCTION Several autoradiographic studies about the RNA synthesis during spermatogenesis have been made (Monesi, 1964, 1965; Tres, 1975; Loir, 1972; Kierszenbaum and Tres, 1974a, b, 1975; SGderstrijm and Parvinen, 1976a). These studies have shown that during the mid pachytene stage of the meiotic prophase there is a rapid increase in the RNA synthesis of the primary spermatocytes. This RNA synthesis takes place at the same time as the lampbrush loops of the pachytene chromosomes are formed (Kierszenbaum and Tres, 1974b) and the synthesized RNA molecules remain in contact with the chromosomes for a long time (Monesi, 1965, 1971). Biochemical studies have shown that this RNA is mostly HnRNA (Muramatsu et al., 19%) which seems to have a long life time (Saderstrijm and Parvinen, 1976a; Siiderstriim, 1976). The activation of the RNA synthesis during meiotic prophase is a general phenomenon because also in oocytes a high rate of RNA synthesis has been reported to exist in the lampbrush loops during pachytene and diplotene stages (Hartung and Stahl, 1976). This RNA, as shown for some larger lampbrush loops, is stored in the amphibian oocytes for about 10 days (Gall and Callan, 1962) and in dipteran spermatocytes for 20 to 30 hours (Hennig, 1967). The polypeptid toxin a-amanitin isolated from the toadstool Amanita phalloides ( Wieland, 1968) selectively inhibits the nucleoplasmic RNA polymerase II of mammalian cells (Jacob et al., 197Oa, b). The RNA polymerase II is involved in the synthesis of HnRNA (Roeder and Rutter, 1970; Zylber and
0014-4800/79/040056-14$02.00/O
Copyright 0 1979 by Academic Press,Jnc. All rights of reproduction in any form reserved.
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Penman, 1971) which seems to be the main transcriptional product of the pachytene spermatocytes (Sijderstrom and Parvinen, 1976a; Soderstrom, 1976). In order to obtain information about both the mode of action of a-amanitin and the function of HnRNA synthesized during spermatogenesis and particularly during the mid pachytene stage of the meiotic prophase the effects of a-amanitin on the RNA synthesis, histology and ultrastructure of the spermatogenic cells of the rat were studied. MATERIALS
AND METHODS
Isolation of Cells The seminiferous epithelium consists of the Sertoli cells, the spermatogonia, the primary spermatocytes, the secondary spermatocytes and the spermatids. The different cell types are not randomly located in the seminiferous epithelium of the tubules but are arranged in association with constant cell composition. In the rat 14 different cell associations have been described (Leblond and Clermont, 1952). They follow each other in a regular manner and form the wave of the seminiferous epithelium (Perey et al., 1961). In a preparation microscope the different stages absorb light in a different way (Parvinen and Vanha-Perttula, 1972) allowing the isolation of living segments of the tubules with known cell associations. Incubation and Labeling Conditions First a piece of the tubule containing a complete wave of the seminiferous epithelium was isolated. This piece was then transferred under sterile conditions to a Petri dish and placed on lens paper lying on a stainless steel grid at the level of the culture medium (Johansson, 1975). The culture medium used was Parker 199 with Earle’s salts containing sodmm-G-penicillin (50 U/ml, Leiras, Turku, and Finland) and streptomycin sulphate (50 pg/ml Laake, Oy, Turku, and Finland). When the effect of a-amanitin was studied the culture medium contained 5, 10, or 20 pg/ml of a-amanitin (Sigma, St. Louis, MO, USA). The tubules were cultured between 2 and 20 hr in an atmosphere of 95% O3 and 5% CO, at 31°C. After this the tubulus was incubated in the presence of [3H]uridinc (100 &i/ml) for 2 hours, cut into smaller about 2 mm long pieces and the stage of each piece was accurately identified using phase contrast microscopy (Soderstrom and Parvinen, 1976a). The small pieces were then processed for histology, electron microscopy, autoradiography or biochemical analyses. In the control experiments tubules were incubated and treated in the same way except that no a-amanitin was present in the incubation media. In order to control that the obtained results were not an artefact caused by the lack of hormones in the incubation media, tubules were also incubated in the presence of FSH (5 pg/ml, NIH-ovine FSH-S9) and testosterone (0.1 pg/ml). Histology and Electron Microscopy The pieces of the tubules were tixed in Bouin’s fluid, embedded in paraffin, cut in 5 ,.m-~sections and stained either with Harris’ hematoxylin and eosin or
5s
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with the periodic acid shiff method, with hematoxylin as counterstain. For electron microscopy the accurately identified tubular segments were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 3 hours, postfixed in 1% osmium tetroxide in 0.1 M 2,4,6-collidine buffer at pH 7.4 for 2 hours, dehydrated, embedded in epon and 700 A thick sections were cut with an LKB Huxley ultramicrotome. The sections were stained with 4% uranyl acetate for 30 min at +6O”C and 0.2% lead citrate for 2 min at room temperature. The observations were made with a Jeol Jem-T8 electron microscope. Autoradiography For light microscopic autoradiography the 5 pm thick sections were placed on glass slides and dipped in Kodak NTB 3 emulsion. Due to the high level of radioactivity in the cells the exposure time was limited to 1 or 3 days. The autoradiograms were thereafter developed in Kodak D 19 developer for 4 min at 20°C and stained with Harris’ hematoxylin and eosin. For high resolution autoradiography the 700 -4 thick sections were placed on glass slides coated with a thin collodion membrane. The slides were dipped in Ilford L 4 emulsion so that a monolayer was formed. Exposure times from 1 to 4 months were used. The autoradiograms were developed with Microdol X (Kodak Limited, England) and fixed with solifix. After this the specimens were stripped off the slide in water, placed on copper grids (Rogers, 1967) and stained with uranyl acetate and lead citrate as described previously. Extraction
and Electrophoretic
Analyses
of RNA
Stages VII containing the actively HnRNA synthesizing mid pachytene spermatocytes were chosen for biochemical analyses. The RNA was extracted from the tubules with a phenol SDS extraction method (Soderstrom, 1976). The tubules were first homogenized in 1 ml of 0.1 M Tris-HCl buffer pH 7.4 containing 0.570 SDS, and 100 pg pronase treated E. coli reference and carrier RNA. Immediately after the homogenization was started, 1.5 ml of water saturated freshly distilled phenol was added. The homogenization was performed at 0°C for 5 min and at 20°C for 5 min. Thereafter the solution was shaken vigorously for 1 min. The water and the phenol phases were separated with a rapid centrifugation and the phenol was removed. To the buffer 0.25 PI of 4 M NaCl was added and the RNA was precipitated with 2.5 volumes of cold absolute ethanol. The RNA was stored overnight at -20°C and the ethanol was carefully removed. The RNA was dissolved in 50 ~1 of 0.02 M Tris-HCI buffer pH 7.4 with 0.5% SDS. To avoid aggregation of the RNA the dissolving solution contained urea (8 M). The dissolved RNA was fractionated with electrophoresis in an agarose gel (1% agarose, Marine Colloids Inc., Springfield, NJ, USA) in 0.02 M Tris-HCI buffer pH 8.0 with 0.002 AZ EDTA and 0.5y0 SDS, using an Ortec 4010 pulsed constant power supply with the settings at 100 V, 100 mA, and 500 pulses/s. The localization of the E. coZi reference RNA bands was determined by their UV light absorption. After the electrophoresis the gels were washed in 5% TCA for I hr and distilled water for 2 hours at +4”C with several changes of the water. The gel was sliced in I.1 mm long pieces and the radioactivity in each piece was determined by a low background
EFFECTS
F.._ _ %
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4
a!
*
FIG. 4. An autoradiogram of stage X of rat spermatogenesis with [3H]uridine. Only occasional grains are seen over the step X spermatids (arrow) appear normal. The exposure time
cultured for 14 hr with pachytene spermatocytes was 1 day. Xl2fiO.
a-amanitin ( 10 rg/ml) (ps). These cells are
and thereafter morphologically
labeled normal.
for 2 hr Also the
and thereafter labeled for 2 hr FIG. 3. An autoradiogram of stage V of rat spermatogenesis cultured for 14 hr with a-amanitin (10 pg/ml) with [‘Hluridine. No labeling is seen over the pachytene spermatocytes (ps) and only occasional grains are seen over the young round spermatids (St). However, only some pachytene spermatocytes degenerate whereas most of them show a normal morphology. The B-spermatogoniti The exposure time was 1 day. X1250. (sg) show pycnotic nuclei as a sign of degeneration.
b
0 0
(10 pg/ml) and thereafter are unlabeled as also in over them. No morphological
labeled for physiological degenera-
(10 pg/ml) and thereafter labeled for 2 hr for 14 hr with a-amanitin (sg) are unlabeled while occasional grains are seen over the young spermatocytes is normal whereas the intermediate type spermatogonia The exposure time was 1 day. X1250. (arrow).
a-amanitin FIG. 6. An autoradiogram of stage XIV of rat spermatogenesis cultured for 14 hr with as well as the spermatids 2 hr with [‘H]uridine. The cells going through the meiotic divisions (M) conditions. The zygotene spermocytes (z) are unlabeled while in control samples some grains are seen tive changes are seen in any cell type of this stage. The exposure time was 1 day. X1250.
FIG. 5. An autoradiogram of stage III of rat spermatogenesis cultured with [‘Hluridine. The pachytene spermatocytes and the spermatogonia spermatids (St). The morphology of the spermatids and the pachytene undergo degenerative changes. Some label is seen over the Sertoli cells
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high sensitivity gas counter (Parvinen et al., 1973) owing to the low level of radioactivity in the slices. For control purposes the RNA synthesis in the separated epididymal tubules were analyzed in the same way. RESULTS The control experiments showed that the morphology of the spermatogenic cells did not change in any of the stages during 22 hours of culture under the described conditions. The addition of FSH and testosterone to the culture medium had no effect on the cellular morphology during the 22 hours of culture. When the tubules were incubated in vitro without a-amanitin or hormones in the presence of [ 3H]uridine a high level of RNA synthesis could be seen in the mid pachytene spermatocytes and in the spermatogonia whereas the young round spermatids were only slightly labeled (Fig 1). The addition of testosterone and FSH to the culture medium did not cause any change in the labeling of the cells during a 22 hour culture. Histological and phase contrast microscopic studies showed also that the cells most affected by a-amanitin were the pachytene spermatocytes. Particularly the mid pachytene spermatocytes in stages VI, VII, and VIII underwent rapid degeneration so that after 14 hours in the presence of 10 pg/ml of a-amanitin pachytene spermatocytes in various stages of cell degeneration could be observed (Fig. 2). After a 20 hour treatment with a-amanitin most of the mid pachytene spermatocytes showed marked degenerative changes. In the control tubules incubated without a-amanitin all pachytene spermatocytes appeared normal. During the early pachytene (stages I-V) the degeneration was more slow than during the mid pachytene stage and many cells seemed unaffected even after a 20 hour incubation with ol-amanitin (10 pg/ml) (Fig. 3). The late pachytene spermatocyes were also resistant and only a small number of these cells were seen degenerating during 20 hour culture with a-amanitin (10 pg/ml) (Fig. 4). Other sensitive cell types were the intermediate (In) and type B spermatogonia in stages III, IV, and V. These cells degenerated almost as rapidly as the mid pachytene spermatocytes (Fig. 5). The histology of the leptotene spermatocytes, zygotene spermatocytes and the spermatids appeared normal and the meiotic divisions were not affected during a 20 hr culture with cu-amanitin (10 pg/ml) (Fig. 6). Electron microscopic observations showed more in detail the cell degeneration process. The pachytene spermatocytes seemed normal during an 8 hour culture with a-amanitin (Fig. 7) but thereafter followed a condensation of the chromatin and the chromosomes clumped together forming large electron dense structures (Fig. 8). The autoradiographic observations showed that the labeling of the mid pachytene spermatocytes was greatly reduced already after 6 hour culture with a-amanitin. Although the labeling of the autosomes of the pachytene spermatocytes decreased the nucleolus remained labeled (Figs. 9 and 10). The labeling of the zygotene spermatocytes which have a low level of RNA synthesis did not seem to be affected by a-amanitin. Electron microscopy showed that the nuclear membrane of some spermatids was partly broken down and that the chromatoid body in some of the sperma-
EFFECTS
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FIG. 7. An electron microscopic picture of stage VII of the seminiferous epithelium of the rat after 8 hr culture with a-amanitin ( 10 pg/ml), The appearance of the spermatids (st) is normal. Some slight fragmentation can be detected in the nucleolus (n) of the pachytene spermatocyte (p) which otherwise are normal. a: acrosome of a spermatid. x3300. FIG. 8. An electron microscopic picture of stage VII of rat spermatogenesis cultured for 14 hr with a-amanitin (10 rg/mI). The mid pachytene spermatocytes (p) show changes of their chromatin which seems to aggregate into electron dense granular masses. The spermatids (st ) remain normal. Bl: basal Iamina. X3300.
tids acquired a ring like appearance in stages I to VII (Fig. 11). This change in the chromatoid body was first seen after 12 hour of culture with ar-amanitin. The nature of the RNA synthesis inhibited by a-amanitin was studied biochemically. The electrophoretic distribution of the RNA synthesized in stage VII of rat spermatogenesis which contains the most actively RNA synthesizing
64
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FIG. 9. A control high resolution autoracliogram of a pachytene spcrmatocyte from stage VII of rat spermatogenesis cultured for 6 hr without n-amanitin followed by a 2. hr pulse with [3H]uridine. The nucleus is heavily labeled but almost no grains can be seen in the cytoplasm. The exposure time was 2 months. n: nucleus. ~1365. FIG. 10. A high resolution autoradiogram of a pachytene spermatocyte from stage VII of rat spermatogenesis cultured for 6 hr with or-amanitin (IO ~g/ml) followed by a 2 hr pulse with [3H]uridine. The labeling of the autosomes has markedly decreased whereas the nucleolus (nu) is labeled suggesting a normal rate of rRN.4 formation. The exposure time was 2 months. m: mitochondria. ~1365.
EFFECTS
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FIG. 11. A high resolution autoradiogram of a spermatid from stage VI of rat spcrmatogenesis after 20 hr culture with ar-amanitin (10 pg/ml) followed by a 2 hr pulse with ‘H-uridine. The nucleus (n) and the cytoplasm of the spermatid are unlabelled. The chromatoid body (cb) has a peculiar ring shaped structure which is not normally seen. Magnification 30,000X.
pachytene primary spermatocytes shows that during a 2 hr pulse with [3H]uridine almost only HnRNA is formed. a-amanitin greatly reduced the rate of this HnRNA synthesis in 4 hours (Fig. 12a). The synthesis of the smaller molecular weight RNA (4s) did not seem to be sensitive to a-amanitin although after S-hour culture almost no HnRNA synthesis could be observed (Fig. 12b). In the control experiments with epididymal tubules the HnRNA synthesis also seemed to be inhibited by a-amanitin although the synthesis of rRNA was unaffected and clear 28s and 18s peaks could be seen in the electrophoresis. DISCUSSION The RNA synthesis during spermatogenesis shows many peculiar features not observed in other cells. There is a high level of RNA synthesis in the spermatogonia that gradually decreases with the progressive increase in the amount of heterochromatin in the cells. The RNA synthesis is at a low level during leptotene, zygotene, and early pachytene. In mid pachytene primary spermatocytes there is a rapid increase in the RNA synthetic rate which then decreases during late pachytene. A low level of RNA synthesis persists in the early spermatids up to step 8. No RNA synthesis exists in the late spermatids (Monesi, 1964, 1965, 1971; SGderstriim and Parvinen, 1976a). The high level of
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RNA synthesis in the mid pachytene spermatocytes occurs at the same time as the lampbrush loops of the chromosomes are formed (Kierszenbaum and Tres, 197413). Biochemical studies have shown that most of the RNA synthesized in the mid pachytene spermatocytes seems to be HnRNA (Soderstrom, 1976; Soderstrom and Parvinen, 1976a). The present study showed that the specific inhibitor of RNA polymerase II o-amanitin had a selective action on the different spermatogenic cell types. Particularly the pachytene spermatocytes in their maturation stages showed a variable sensitivity to this drug. The action of a-amanitin on the pachytene
5 100
50
IO k 100
50
10
IO
30
33 Slice
40
50
N$@
Fro 12. The electrophoretic distribution of RNA from stage VII of rat spermatogenesis after 2 hr culture followed by a 2 hr pulse with and after 6 hr culture [3H] uri d ine (a) followed by a 2 hr pulse with [*H]uridine (b). Solid circles indicate the distribution of RNA from tubules cultured without or-amanitin and open circle with or-amanitin ( 10 pg/ml). Abscissa: Migration of RNA in the gel (slice number). Ordinate: ‘H cpm per slice divided by tubule length in mm.
EFFECTS
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spermatocytes correlated with the rate of RNA synthesis in these cells. In the early pachytene spermatocytes with a low rate of RNA synthesis, a-amanitin had only a slight effect but in the mid pachytene stage with a high rate of RNA synthesis, a-amanitin caused degeneration in the majority of the cells during a 26 hour observation period. Late pachytene spermatocytes with a low level of RNA synthesis were also more resistant to the action of a-amanitin. This may be due to long lived RNA molecules synthesized during mid pachytene that ensure cellular viability during late pachytene despite the block of HnRNA synthesis caused by a-amanitin. These results also indicate that at least a part of the HnRNA synthesized in the mid pachytene spermatocytes is necessary for the normal development of these cells and is not primarily stored for later use. It has also been observed that a large portion of the RNA synthesized in mid pachytene spermatocytes has a poly( A) sequence characteristic of mRNA (Soderstrom, 1976) and that protein synthesis is more active in mid pachytene spermatocytes than at earlier or later developmental stages (Monesi, 1965; Davis and Fir-lit, 1970). However, the RNA synthesis during mid pachytene may have other functions also which is indicated by the rapid changes in the ultrastructure of the chromatin caused by a-amanitin before any degenerative changes can be seen in the cytoplasm. The level of RNA synthesis is low in the leptotene, zygotene, and early pachytene spermatocytes and these cells seem also to be resistant to the action of a-amanitin. An interesting finding is the degeneration of the In and B spermatogonia caused by a-amanitin although the level of RNA synthesis in these cells is relatively low. The HnRNA synthesis is probably necessary for the normal development of these cells. Another possible explanation is that the spermatogonia are located in the basal compartment of the seminiferous epithelium between the Sertoli cell junctions and the basal lamina (Dym and Fawcett, 1970) where they may be more susceptible to the toxic action of a-amanitin than in the adluminal compartment above the Sertoli cell junctions. This does not, however, explain why the A spermatogonia which are also in the same basal compartment are not affected by a-amanitin in the same way. The results of this study are in some respects comparable to the effects of actinomycin D on the pachytene spermatocytes (Barcellona and Brinkley, 1973). In a high concentration actinomycin D inhibits, however, all RNA synthesis and not specifically the HnRNA synthesis. That the results of this study were not caused by the lack of hormones in the incubation media was shown by the control experiments when the tubules were incubated in the presence of FSH and testosterone and no differences in the results could be seen. However, it is known that tubular cells are actively engaged in FSH-dependent mRNA synthesis (Reddy and Villee, 1975). The reason why FSH and testosterone did not have any effect on the RNA synthesis in this experiment might be that 22 hours of culture in the absence of hormones is too short a time for the depletion of intercellular hormonal complexes. This is also supported by the findings that the first morphological changes in the spermatogonic cells occur 3 days after hypophysectomy (Clermont and Morgenthaler, 1955). These changes occur in the step 17 to 19 spermatids which do not synthesize RNA. Spermatids seemed to be little affected by a-amanitin. This is probably due to the low level of RNA synthesis in the spermatids (Monesi, 1964, 1965; Soder-
68
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striim and Parvinen, 1976a). However, the cytoplasmic cell organelle of the spermatids, the chromatoid body, showed a ring like appearance after 14 hours of culture in the presence of Lu-amanitin. This change in the morphology of the chromatoid body is similar to that which is seen when spermatids are treated with actinomycin D ( SGderstrbm, 1977). Previously it has been shown that the chromatoid body is labeled with [3H]uridine when the spermatids are pulse labeled for 2 hours and thereafter chased for 14 hours in the presence of unlabeled uridine (Saderstriim and Parvinen, 1976b). That a-amanitin causes structural changes in the chromatoid body indicates not only that it contains RNA but also that at least a part of this RNA might be mRNA. This leads to the interesting suggestion that the chromatoid body might contain stored RNA molecules which are used later during spermiogenesis when transcription in the spermatid nucleus has ceased but protein synthesis still persists at a high level. ACKNOWLEDGMENTS I would like to thank technical assktance. This
Mrs. work
Marita SaderstrGm and was supported by a grant
Mrs. from
Leena Simola for their excellent the Finnish hledical Foundation.
REFERENCES BARCELLONA, W. J., and BHIKKLEY, B. R. ( 1973). Eff ec t.s 0 f actinomycin D on spermatogenesis in the Chinese hamster. Biol. Reprod. 8, 335-349. Q uantitative CLERMONT, Y., and MORGENTHALER, H. (1955). study of spermatogenesis in the hypophysectomized rat. Endocrinology 57, 369-382. DAVIS, J. R., and FIRLIT, F. C. (1970). In The Testis III (A. D. Johnson, W. R. Comes, and N. L. Wandermark, eds.), pp. 259-306. Academic Press, Inc., New York. DYM, M., and FAWCETT, D. W. ( 1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308-326. GALL, J. G., and CALLAN, H. G. ( 1962). [3H]uridine incorporation in lampbrush chromosomes. Proc. Natl. Acad. Sci. USA 48, 562-570. HARTUNG, M., and STAHL, A. (1976). Incorporation of tritiated uridine during pachytene and diplotene stages in the oocytes of the Japanese quail (Coturnix coturnix japonica). Experientia 32, 96-98. HENNIG, W. ( 1967). Untersuchungen zur Struktur und Funktion des Lampbursten-Y-Chromosoms in der Spermatogenese von Drosphila. Chromosoma 22, 294-357. JACOB, S. T., SAJDEL, E. M., and MUNRO, H. N. (1970a). Different responses of soluble whole nuclear RNA polymerase and soluble nucleolar RNA polymerase to divalent cations and to inhibition by a-amanitin. Biochern. Biophys. Res. Comm. 38, 765-770. JACOB, S. T., SAJDEL, E. M., MUECKE, W., and MUNRO, H. N. (1970b). Soluble RNA polymerase of rat liver nuclei: properties, template specificity, and amanitin responses in vitro and in vivo. Cold Spring Harbor Symp. @ant. Biol. 35, 681-691. JOHANSSON, R. (1975). RNA, protein, and DNA synthesis stimulated by testosterone, insulin and prolactin in the rat ventral prostate cultured in chemically defined medium. Acta Endocr. 80, 761-774. KIERSZENBAUM, A. L., and TRES, L. L. (1974a). Nuclear and perichromosomal RNA synthesis during meiotic prophase in the mouse testis. J. Cell Biol. 60, 39-53. T ranscription KIERSZENBAUM, A. L., and TRES, L. L. (197413). sites in spread meiotic prophase chromosomes from mouse spermatocytes. J. Cell Biol. 63, 923-925. KIERSZENBAUM, A. L., and TRES, L. L. ( 1975). Structural and transcriptional features of the mouse spermatid genome. 1. Cell Biol. 65, 258-270. LEBLOND, C. P., and CLERMONT, Y. ( 1952). Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci. 55, 548-573. LOIR, M. (1972). MGtabolisme de l’acide ribom&%qnc rt dcs protbinrs danh lrzs spczrtnato-
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cytes et les spermatides du belier (Ovis aries). 1. Incorporation et devenir de 1-[3H]uridine. Ann. Biol. Anim. Bioch. Biophys. 12, 203-219. MONESI, V. ( 1964). Ribonucleic acid synthesis during mitosis and meiosis in the mouse testis. J. Cell MONESI,
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Synthetic activities during spermatogenesis in the mouse RNA and protein. Exp. Cell Res. 39, 197-224. MONESI, V. ( 1971). Chromosome activities during meiosis and spermiogenesis. J. Reprod. Fert. Suppl. MURAMATSU,
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M., UTAKOJI, T., and SUCANO, H. ( 1968). Rapidly-labelled nuclear RNA in Chinese hamster cells. Exp. Cell Res. 53, 278-283. PARVINEN, M., SOINI, E., and TUOHIMAA, P. ( 1973). An automatic gas counter for quantitative microdeterminations of tritium in biological material. Anal. Biochem. 55, 193-200. PARVINEN, M., and VANKA-PERTTULA, T. (1972). Identification and enzyme quantitation of the stages of the seminiferous epithelial wave in the rat. Anat. Rec. 174, 435450. PEREY, B., CLERMONT, Y., and LEBLOND, C. P. (1961). The wave of the seminiferous epithelium in the rat. Am. J. Anat. 108, 47-77. REDDY, P. R. K., and VILLEE, C. A., ( 1975). Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP. Biochem. Biophys. Res. Comm. 63, 1063-1069. ROEDER, R. G., and RUTTER, W. J. ( 1970). Specific nucleolar and nucleoplasmic RNA polymerases. Proc. Natl. Acad. Sci. USA 65, 675-689. ROGERS, A. W. (1967). Techniques of autoradiography. EIsevier Publishing Co., Amsterdam, 289-308. S~DERSTR~M, K.-O. ( 1976). Characterization of RNA synthesis in mid pachytene spermatocytes of the rat. Exp. Cell Res. 102, 237-245. S~DERSTR~M, K.-O. (1977). Effect of actinomycin D on the chromatoid body of rat spermatids. Cell Tiss. Res. 184, 411421. S~DERSTR~M, K.-O., and PARVINEN, M. (1976a). RNA synthesis in different stages of rat seminiferous epithelial cycle. Mol. Cell EndocrinoI. 5, 181-199. S~DERSTR~M, K.-O., and PARVINEN, M. (197613). Incorporation of [3H]uridine by the chromatoid body during rat spermatogenesis. J. Cell Biol. 70, 239-246. TRES, L. L. (1975). Nucleolar RNA synthesis of meiotic prophase spermatocytes in the human testis. Chromosoma 53, 141-151. WIELAND, T. ( 1968). Poisonous principles of mushrooms of the genus Amanita. Science 159, 946-952. ZYLBER, E., and PENMAN, S. (1971). Products of RNA polymerases in HeLa cell nuclei. PTOC. Natl. Acad. Sci. USA 68, 2861-2865.
