28 s and 18 s Ribonucleic Acid from Mammalian Spermatozoa CHARLES J. BETLACH AND ROBERT P. ERICKSON2 Department of Pediatrics, University of California, S a n Francisco, Sun Francisco, California 94143
ABSTRACT The majority of RNA in spermatozoa from the epididymis and ductus deferens of mice was found to be 28 s and 18 s RNA. This RNA was also seen in human ejaculated spermatozoa. Our evidence indicates that little or none of the 28 s and 18 s RNA is transcribed in the maturing mouse spermatozoa and that the majority is synthesized in the primary spermatocyte. This RNA may be necessary to maintain post-meiotic translation during spermatogenesis.
Recent advances in nucleic acid biochemistry have provided new approaches to the question of the quantity and purpose of spermatozoa1 RNA. RNA polymerase has been shown to be present in early spermatids (Moore, '71); and autoradiographic studies (Monesi, '71; Kierszenbaum and Tres, '75) and cell separation studies (Meistrich, '72; Lee and Dixen, '72) on mouse testicles have demonstrated a small burst of postmeiotic nuclear RNA synthesis that declines upon spermatozoan maturation. Active mitochondrial RNA synthesis is ejaculated bovine sperm has been reported (F'remkumar and Bhargava, '72) and other workers have demonstrated the synthesis of mitochondrial RNA in hamster and rat epididymal spermatozoa (MacLaughlin and Terner, '73). Our initial investigations of RNA in spermatozoa from the epididymides and ductus deferentes of mice (Betlach and Erickson, '73) revealed quantities greater than were expected on the basis of others' results with ejaculated spermatozoa (Mann, '51; Bhargava et al., '59). A n attempt to identify this RNA with polyacrylamide electrophoresis revealed a 11 s (190,000 dalton) RNA (Betlach and Erickson, '73). The purpose of this paper is to explain the presence of this 11 s RNA and to further characterize it. METHODS AND MATERIALS
Sperm preparation Maturing mouse spermatozoa were obtained as previously described (Betlach and Erickson, '73) from the epididymides and ducti deferentes of mature random bred Swiss Webster mice. Spermatozoa were colJ. EXP. ZOOL.,198:
49-56
lected separately from the cauda epididymis, caput epididymis and the ductus deferens, were counted in a hemacytometer, and pooled. This total sperm pool was a mixture and represented 26% from the ductus deferens, 50% from the cauda epididymis, and 24% from the caput epididymis. The cytoplasmic droplet was rarely seen. Human ejaculated spermatozoa were washed twice in 0.9% NaCl within onehalf hour of collection. R N A extraction The spermatozoa pellets were suspended in 100 times their volume with ice cold 0.05 M sodium acetate, 0.01 M EDTA at pH 5.1 which also contained 1% bentonite as a RNase inhibitor. The RNA extraction was performed with phenol and choloroform as previously described (Betlach and Erikson, '73) except that the entire extraction procedure was performed on ice so as to minimize RNase activity.
Polyacrylamide gel electrophoresis The purified RNA was electrophoresed on polyacrylamide disc gels containing 2.4% acrylamide and 0.12 % bisacrjrlamide (Betlach and Erickson, '73). Disc gels were run at room temperature (22 "-25 C) in 0.04 M Tris 0.02 M sodium acetate, 0.001 M EDTA buffer at pH 7.8 for two hours at 5 mampslgel. Each gel was 0.6 cm in diamO
1 This work was supported by a grant from the National Institutes of Health, Bethesda, Maryland (HD05259). 1The recipient of a Research Career Development Award from the National Institutes of Child Health and Human Development.
49
50
CHARLES J . BETLACH AND ROBERT P. ERICKSON
eter and 10 cm long. Gels to be stained were fixed for 15 minutes in 1 M acetic acid and stained for one hour in 0.2% methylene blue. They were destained in distilled water. These disc gels were scanned with a Canalco (Model G) densitometer. Gels that contained a radioactive sample were frozen on dry ice and sliced into 1 mm slices on a gel slicer (Misco Scientific, Berkeley, California). Each slice was placed in a counting vial with 1 ml of 9 parts NCS (Nuclear Chicago Solubilizer): 1 part water and heated at 40°C for two hours. After cooling, 10 ml of tolueneOmnifluor (New England Nuclear) scintillant was added to each vial. They were counted in a Beckman LS-250 liquid scintillation counter.
coated with a 1:l mixture of NTB 3 emulsion (Kodak) and distilled water (Baserga and Malamid, '69). The slides were exposed for 45 days at 4°C and developed in D-19 developer at 20°C. The cells were not stained.
Labeling of R N A in vitro Spermatozoa were removed sterilely and placed in Tyrode's solution plus 100 units penicillin Glml. The cells were at a concentration of 4-5 X lOVm1, and were labelled at 30"C with shaking for 90 minutes. The RNA precursor used was 5-3H-uridine at 10 ~ C i l m l(specific , activity: 28 Cilmmole from Schwarz-Mann). After labeling, the spermatozoa were pelleted at 600 X g for 15 minutes and washed three times with Tyrode's solution with cold uridine added. RNA extractions were performed on these samples.
RESULTS
Labeling of R N A in vivo Fifty mice were anesthetized with sodium pentobarbital and injected intratesticularly with 20 pCi 5-3H-uridine (specific activity: 28 Cilmmole from Schwarz-Mann) in 20 ~1 per testicle. A 30 gauge needle was used to penetrate the skin and enter the center of the testicle. Epididymal and ductus deferens spermatozoa were removed from ten mice at six to seven day intervals and the RNA was extracted as previously described. Autoradiography Approximately 105 in vivo 5-3H-uridine labelled spermatozoa were smeared on acid-cleaned microscope slides. The smears were air dried and fixed for ten minutes in 3:l methano1:acetic acid. They were then treated on ice for five minutes with cold 5 % trichloroacetic acid (TCA) to remove soluble material. The dried slides were
Quantification of R N A and DNA RNA was hydrolyzed for one hour at 37°C in 0.3 N NaOH. The free ribose was assayed colorimetrically by the orcinol method (Habel and Salzman, '69). DNA was hydrolyzed from the above pellet with 1.5% perchloric acid for 20 minutes at 90°C. The supernatant was tested for hydrolyzed DNA by the Burton modification of the diphenylamine colorimetric test for DNA (Burton, '56). What classe(s) of R N A are present in spermatozoa? Polyacrylamide electrophoresis of unlabeled RNA extracted from mouse epididymal and ductus deferens spermatozoa revealed molecular weight equivalents to 28 s and 18 s RNA when stained with 0.2% methylene blue (fig. 1). These molecular weights, expressed as Svedberg units, were obtained from a plot of the log of Svedberg units against the electrophoretic migration distance of control RNA (Weinberg and Penman, '70). Mouse liver and HeLa cell RNA were used as controls and were purified and electrophoresed in the same manner as the spermatozoa RNA. The faint bands after the 28 s and the broad shoulder after the 18 s (fig. 1) may be spermatozoa mitochondrial ribosomal RNA (23 s and 16 s). The broad band of SDS (sodium dodecyl sulfate) was from the RNA sample that was placed on the gel and unfortunately obscures the 4 s and 5 s RNA. The 11 s RNA, which we previously reported (Betlach and Erickson, '73), was not seen when a RNase inhibitor and cold phenol extraction were used (METHODS AND MATERIALS). We have observed that freezethawed spermatozoa caused mouse liver ribosomal RNA to break down to a 11 s RNA. Could the spermatozoa1 28 s and 18 s RNA be an artifact? The possibility that the spermatozoa
51
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
5-
43-
I II
-t 28s
+18s
-t 4s
Fig. 1 Scans of disc acrylamide gel electrophoresis of maturing mouse spermatozoa RNA and mouse liver RNA (--------). The gels were stained with 0.2% methylene blue and scanned on a Canalco Model G densitometer. (-),
RNA was actually from epithelial or other became very radioactive but the spermatocells minced in the preparation of the zoa harvested from such epididymides were mouse spermatozoa was considered but unlabeled in their 28 s and 18 s RNA. seemed unlikely. Microscopic counting of An experiment with fresh ejaculated the cells revealed that there was less than human spermatozoa was performed to see 1 % contamination of the final spermatozoa if contaminating RNA was responsible for preparation by other cells. The majority of the 28 s and 18 s RNA seen. In the normal these contaminating cells were RNA free human male, the ejaculate has been reerythrocytes. Nonetheless, it was possible ported (Levis and Whalen, '76) and has that small amounts of RNA had become been microscopically observed by us and adsorbed onto the surface of the spermato- other colleagues to be relatively free from zoa. Several experiments ruled this out: other cellular material. RNA extracted After intratesticular injection of 5-3H-uri- from human ejaculated spermatozoa condine, the epididymal cell ribosomal RNA tained a 28 s and a 18 s component (fig. 2),
CHARLES J. BETLACH AND ROBERT P. ERICKSON
52
Fig. 2 Disc acrylamide gel electrophoresis of ejaculated human spermatozoa RNA ( I ) and HeLa cell RNA control (2).
further suggesting that the 28 s and 18 s RNA extensively studied in mouse spermatozoa had not been released from diced epithelial cells and adsorbed onto the spermatozoa.
W h e n during spermatogenesis is the spermatozoa1 28 s and I 8 s R N A synthesized? In vitro labeling with 5-3H-uridine was used to determine whether this 28 s and 28 s RNA were actively being synthesized in the maturing mouse spermatozoa. Ninety minute incubation with 5-3H-uridine gave 8.02 X 1 0 5 DPM/109 spermatozoa. The
RNA extracted from these sperm contained 0.040 pic0 moles 3H-uridineper mg of RNA. Polyacrylamide gel electrophoresis of this labeled RNA disclosed no discernable radioactive peaks but gave a 28 s and 18 s peak with 0.2 % methylene blue stain. The time of the 28 s and 18 s spermatozoal RNA synthesis during the course of spermatogenesis was further studied with sperm labeled in vivo with 5-3H-uridine given intratesticularly. This was performed in order to determine if the synthesis of the RNA found in maturing mouse spermatozoa had occured pre- or post-meiotically. Sperm were collected at weekly intervals. The RNA was extracted and the amount of 3H incorporation into RNA was determined (table 1). The total radioactivity was initially high, dropped off, and rose again on day 35. The 3H in the RNA was initially high and has a peak at 28 days post injection. After day 35, the total cellular radioactivity dropped rapidly. This radioactive profile was reproducible. Hydrolysis and analysis (METHODS AND MATERIALS) of the RNA and DNA indicated that more than 60% of the radioactivity on day 35 was in the DNA. On day 28 or earlier less than 15% of the radioactivity was in the DNA. These data show that extensive isotope reutilization had occurred in the weeks following the injections, but the highest specific activity in spermatozoal RNA was found at 28 days and at 6 days. RNA was extracted (METHODS AND MATERIALS) from the cauda epididymis sperm 36 days after the injection of 5-3H-uridine. The RNA was electrophoresed on polyacrylamide gels and the radioactive profile was determined (fig. 3). A 28 s, 18 s and 4 s 3H labeled RNA was present.
Where i s the spermatozoa1 R N A located? The location of the in vivo labeled RNA
TABLE I T h e appearance of 5-3H-uridine in m o u s e spermatozoa R N A after intratesticular injections of 5-3H-uridine Days post injection
6 14 21 28 35
DPM/testicle
64.69X 33.63X 16.28x 12.66X 5.89x
104
lo* 104 104 104
DPM/lOs sperm
13.70X 105 3.70X 105 3.35x 105 8.10X 105 12.40X 1 0 5
% of 3H i n sperm DNA
pmoles 3H-uridine mg sperm RNA
5
0.0164 0.0092 0.0086 0.0146 0.0049
10
15 15 60
53
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
+
28s
t
t
18s
4s
FRACTION NUMBER Fig. 3 Radioactive profile of a disc acrylamide nel electrophoresis of cauda epididymis mouse spermatozoa 3H-RNA 36 days after intratesticular injection of 5-3H-uridine.
Fig. 4 Autoradiography of maturing mouse spermatozoa labeled in vivo with intratesticular injections of 5-3H-uridine. Spermatozoa 21 days and 35 days after the injection. X 380.
in spermatozoa was looked at with autoradiography (fig. 4). Pretreatment of the cells, while on the slides, with cold 5 % trichloroacetic acid remove most of the unincorporated or acid soluble radioactivity. The grains seen in figure 4 represent labeled RNA and/or other macromolecules. At both 21 days and 35 days the grains were seen only in the sperm head, and there
were no grains in the tail, midpiece or acrosome. At day 21, the grains appear to be near the border of the cell rather than clustered in the center. The dense grains at day 35 probably represent the re-utilization of much of the label into DNA. DISCUSSION
The 11 s spermatozoa1 RNA previously
54
CHARLES J. BETLACH AND ROBERT P. ERICKSON
described by us (Betlach and Erikson, '73) appears to be a degradation product, one of the possible explanations suggested at that time. This fragment was apparently due to ribonuclease activity associated with spermatozoa, since an alternative extraction procedure utilizing bentonite and cold phenol did not produce the 11 s fragment. The scans of stained gels of spermatozoa1 RNA, prepared with extra precautions against ribonucleases, demonstrated the typical 28 s and 18 s pattern of mammalian ribosomal RNA. It is unlikely that this RNA was from contaminating cells, since all contaminating epithelial cells were removed from sperm preparations by a slow speed (10 x g) 30 seconds centrifugation. It was possible, however, that small amounts of R N A had become adsorbed onto the surface of the spermatozoa. This possibility seems ruled out by the studies on spermatozoa harvested after intratesticular injections with 5-3H-uridine. The epididyma1 cells were very radioactive after this injection, but the spermatozoa harvested from such epididymides did not have labeled 28 s and 18 s RNA. The presence of a 28 s and 18 s RNA in human ejaculated sperm confirms the results seen with mouse spermatozoa. The next logical question is whether this sperm R N A is being synthesized in the maturing spermatozoa. In vitro labeling with 5-3H-uridine produced a minimal amount of incorporation into R N A and gave no identifiable peaks with disc gel electrophoresis. It appears from this data that little or no 28 s and 18 s R N A is being actively synthesized in maturing mouse spermatozoa. If the 28 s and 18 s R N A was not being actively synthesized in sperm, at what stage in spermatogenesis was it produced? By correlating the time of appearance of a radioactive R N A peak and the time sequence of the mouse sperm maturation, it was possible to approximately determine at what maturational stage this particular macromolecule was synthesized. The in vivo 5-3H-uridine studies indicated that the R N A maximum was 28 days after the injection of the isotope and corresponded to premeiotic RNA synthesis in the primary spermatocyte. In mice, it takes 27 to 28 days for a germ cell to develop from a spermatogonia to a spermatozoa in the caput epididvmis (Monesi, '71: Ghosal and Mukhkrjee; '71) and 34 days to reach the
ductus deferens (Nelson, '71). The total radioactivity on day 35 (table 1) mainly represents tritium that has entered D N A in late spermatogonia or early spermatocytes. This accounts for the dense grains seen on the day 35 autoradiography (fig. 4). Apparently much of the tritium was converted in the spermatogonia to a form that could be incorporated into DNA. The initial high level of radioactivity at day 6 corresponds to the picture seen in the in vitro labeling and probably represents uptake andlor incorporation into epididymal sperm mitochondria. The presence of a radioactive 28 s, 18 s and 4 s R N A in sperm 36 days after the in vivo injection demonstrates that the radioactive R N A seen is the same as the 28 and 18 s R N A in figure 1. R N A extraction from day 28 cells was inefficient because of low sperm count. The presence of no grains (fig. 4) in the midpiece indicates that there is little or no label uptake by the mitochondria. It appears from the in vitro and in vivo studies that little or no 28 s and 18 s R N A is being synthesized in the maturing mouse spermatozoa. The very nature of the compact nucleus in spermatozoa would preclude any R N A transcription. However, it appears that approximately 85% of the R N A in spermatozoa is 28 s and 18 s R N A and the majority is produced in the primary spermatocyte. This is in good agreement with recent electron microscopical observations suggesting ribosomal R N A transcription in spermatocytes and heterogeneous R N A transcription in spermatids (Kierszenbaum and Tres, '75). Amino acid analyses have shown that the amino acid composition of bovine spermatozoa changes with passage through the epididymis (Lavon et al., '71); and ejaculated rabbit spermatozoa sequester the twenty common amino acids into trichloroacetic acid insoluble material (Busby et al., '74). The phenylalanine and histidine incorporation was observed to be due to non-covalent sequestering (Busby et al., '74). There is incorporation of selenium into developing spermatozoa which suggests an increase in sulfur containing amino acids (Gunn et al., '67). These proteins are thought to be protamine-like histones (Calvin and Bedford, '71). Monesi suggests that some of the messenger R N A that is produced during meiosis is stable and directs protein synthesis during post-
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
meiotic spermatogenesis (Monesi, '74). Galdieri demonstrates a reduction of ribosomal RNA maturation in the mouse spermatocyte (Galdiere and Monesi, '74). They speculated that this may lead to the storage of nucleolus ribosomal RNA precursors used for protein synthesis during post-meiotic spermatogenesis. We propose here that the 28 s and 18 s RNA seen in spermatozoa is necessary for this post-meiotic translation, is produced in the primary spermatocyte and is utilized throughout spermatogenesis. LITERATURE CITED Baserga, R., and D. Malamid 1969 Autoradiography. Techniques and Application. Harper and Row, Publishers, New York. Betlach, C. J., and R. P. Erickson 1973 Aunique RNA species from maturing mouse spermatozoa. Nature, 242: 114-115. Bhargava, P. M., M. Bishop and T. S. Work 1959 The chemical composition of Bull semen with special reference to nucleic acids, free nucleotides and free amino acids. Biochemical J., 73: 242247. Burton, K. 1956 A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical J., 62: 315-323. Busby, W. F., Jr., P. Hele and M. C. Chag 1974 Apparent amino acid incorporation by ejaculated rabbit spermatozoa. Biochim. Biophys. Acta, 335: 246-259. Calvin, H. I., and J. M. Bedford 1971 Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fert., Suppl., 13: 65-75. Galdieri, M., and V. Monesi 1974 Ribosomal RNA in mouse spermatocytes. Expt. Cell Research, 85: 287-295. Ghosal, S. L., and B. B. Mukherjee 1971 The chronology of DNA synthesis, meiosis and spermatogenesis in the male mouse and golden hamster. Can. J. Genet. Cytol., 13: 672-682. Gunn, S. A,, T. C. Gould and W. A. D. Anderson 1967 Incorporation of selenium into spermato-
55
genic pathway in mice. Proc. Soc. Expt. Biol. and Med., 124: 1260-1263. Kierszenbaum, A. L., and L. Tres 1975 Structural and transcriptional features of the mouse spermatid genome. J. Cell Biology, 65: 258-270. Lavon, U., R. Volcani and D. Damon 1971 The proteins of Bovine spermatozoa from the caput and cauda epididymides. J. Reprod. Fert., 24: 2 19-232. Lee, I . P., and R. L. Dixen 1972 Antineoplastic drug effects on spermatogenesis studied by velocity sedimentation cell separation. Tox. and Appl. Pharmacol., 23: 2 0 4 1 . Levis, W. R., and J. J. Whalen 1976 Mixed cultures of sperm and leukocytes as a measure of histocompatibility in man. Science, 191: 302304. MacLaughlin, J., and C. Terner 1973 Ribonucleic acid synthesis by spermatozoa from the rat and hamster. Biochem. J., 133: 635-639. Mann, T. 1951 Mammalian semen: Composition, metabolism and survival. Biochem. Soc. Symp., 7: 11-23. Meistrich, M. L. 1972 Separation of mouse spermatogenic cells by velocity sedimentation. J. Cell Physiol., 80: 299-312. Monesi, V. 1971 Chromosome activities during meiosis and spermiogenesis. J . Reprod. Fert., SUppl., 13: 1-14. 1974 Nucleoprotein synthesis in spermatogenesis. In: Male Fertility and Sterility, Proceedings of the Seroro Symposia. R. E. Mancini and L. Martini, eds. Academic Press, London, pp. 59-87. Moore, G . P. M. 1971 DNA-dependent RNA synthesis in fixed cells during spermatogenesis in mouse. Exptl. Cell Res., 68: 4 6 2 4 6 5 . Nelson, L. 1971 Differentiation of the male germ cell. In: Developmental Aspects of Cell Cycle. I. L. Cameron, ed. Academic Press, New York, pp. 243-278. Premkumar, E., and P. M. Bhargava 1972 Transcription and translation i n Bovine spermatozoa. Nature (New Biology), 240: 139-143. Shatkin, A. J. 1969 Colorimetric reactions for DNA, RNA, and protein determinations. In: Fundamental Technique i n Virology. K. Habel and N. P. Salzman, eds. Academic Press, New York, pp. 231-237. Weinberg, R. A., and S . Penman 1970 Processing of 45 s nucleolar RNA. J. Mol. Biol., 47: 169178.
ABSTRACT The majority of RNA in spermatozoa from the epididymis and ductus deferens of mice was found to be 28 s and 18 s RNA. This RNA was also seen in human ejaculated spermatozoa. Our evidence indicates that little or none of the 28 s and 18 s RNA is transcribed in the maturing mouse spermatozoa and that the majority is synthesized in the primary spermatocyte. This RNA may be necessary to maintain post-meiotic translation during spermatogenesis.
Recent advances in nucleic acid biochemistry have provided new approaches to the question of the quantity and purpose of spermatozoa1 RNA. RNA polymerase has been shown to be present in early spermatids (Moore, '71); and autoradiographic studies (Monesi, '71; Kierszenbaum and Tres, '75) and cell separation studies (Meistrich, '72; Lee and Dixen, '72) on mouse testicles have demonstrated a small burst of postmeiotic nuclear RNA synthesis that declines upon spermatozoan maturation. Active mitochondrial RNA synthesis is ejaculated bovine sperm has been reported (F'remkumar and Bhargava, '72) and other workers have demonstrated the synthesis of mitochondrial RNA in hamster and rat epididymal spermatozoa (MacLaughlin and Terner, '73). Our initial investigations of RNA in spermatozoa from the epididymides and ductus deferentes of mice (Betlach and Erickson, '73) revealed quantities greater than were expected on the basis of others' results with ejaculated spermatozoa (Mann, '51; Bhargava et al., '59). A n attempt to identify this RNA with polyacrylamide electrophoresis revealed a 11 s (190,000 dalton) RNA (Betlach and Erickson, '73). The purpose of this paper is to explain the presence of this 11 s RNA and to further characterize it. METHODS AND MATERIALS
Sperm preparation Maturing mouse spermatozoa were obtained as previously described (Betlach and Erickson, '73) from the epididymides and ducti deferentes of mature random bred Swiss Webster mice. Spermatozoa were colJ. EXP. ZOOL.,198:
49-56
lected separately from the cauda epididymis, caput epididymis and the ductus deferens, were counted in a hemacytometer, and pooled. This total sperm pool was a mixture and represented 26% from the ductus deferens, 50% from the cauda epididymis, and 24% from the caput epididymis. The cytoplasmic droplet was rarely seen. Human ejaculated spermatozoa were washed twice in 0.9% NaCl within onehalf hour of collection. R N A extraction The spermatozoa pellets were suspended in 100 times their volume with ice cold 0.05 M sodium acetate, 0.01 M EDTA at pH 5.1 which also contained 1% bentonite as a RNase inhibitor. The RNA extraction was performed with phenol and choloroform as previously described (Betlach and Erikson, '73) except that the entire extraction procedure was performed on ice so as to minimize RNase activity.
Polyacrylamide gel electrophoresis The purified RNA was electrophoresed on polyacrylamide disc gels containing 2.4% acrylamide and 0.12 % bisacrjrlamide (Betlach and Erickson, '73). Disc gels were run at room temperature (22 "-25 C) in 0.04 M Tris 0.02 M sodium acetate, 0.001 M EDTA buffer at pH 7.8 for two hours at 5 mampslgel. Each gel was 0.6 cm in diamO
1 This work was supported by a grant from the National Institutes of Health, Bethesda, Maryland (HD05259). 1The recipient of a Research Career Development Award from the National Institutes of Child Health and Human Development.
49
50
CHARLES J . BETLACH AND ROBERT P. ERICKSON
eter and 10 cm long. Gels to be stained were fixed for 15 minutes in 1 M acetic acid and stained for one hour in 0.2% methylene blue. They were destained in distilled water. These disc gels were scanned with a Canalco (Model G) densitometer. Gels that contained a radioactive sample were frozen on dry ice and sliced into 1 mm slices on a gel slicer (Misco Scientific, Berkeley, California). Each slice was placed in a counting vial with 1 ml of 9 parts NCS (Nuclear Chicago Solubilizer): 1 part water and heated at 40°C for two hours. After cooling, 10 ml of tolueneOmnifluor (New England Nuclear) scintillant was added to each vial. They were counted in a Beckman LS-250 liquid scintillation counter.
coated with a 1:l mixture of NTB 3 emulsion (Kodak) and distilled water (Baserga and Malamid, '69). The slides were exposed for 45 days at 4°C and developed in D-19 developer at 20°C. The cells were not stained.
Labeling of R N A in vitro Spermatozoa were removed sterilely and placed in Tyrode's solution plus 100 units penicillin Glml. The cells were at a concentration of 4-5 X lOVm1, and were labelled at 30"C with shaking for 90 minutes. The RNA precursor used was 5-3H-uridine at 10 ~ C i l m l(specific , activity: 28 Cilmmole from Schwarz-Mann). After labeling, the spermatozoa were pelleted at 600 X g for 15 minutes and washed three times with Tyrode's solution with cold uridine added. RNA extractions were performed on these samples.
RESULTS
Labeling of R N A in vivo Fifty mice were anesthetized with sodium pentobarbital and injected intratesticularly with 20 pCi 5-3H-uridine (specific activity: 28 Cilmmole from Schwarz-Mann) in 20 ~1 per testicle. A 30 gauge needle was used to penetrate the skin and enter the center of the testicle. Epididymal and ductus deferens spermatozoa were removed from ten mice at six to seven day intervals and the RNA was extracted as previously described. Autoradiography Approximately 105 in vivo 5-3H-uridine labelled spermatozoa were smeared on acid-cleaned microscope slides. The smears were air dried and fixed for ten minutes in 3:l methano1:acetic acid. They were then treated on ice for five minutes with cold 5 % trichloroacetic acid (TCA) to remove soluble material. The dried slides were
Quantification of R N A and DNA RNA was hydrolyzed for one hour at 37°C in 0.3 N NaOH. The free ribose was assayed colorimetrically by the orcinol method (Habel and Salzman, '69). DNA was hydrolyzed from the above pellet with 1.5% perchloric acid for 20 minutes at 90°C. The supernatant was tested for hydrolyzed DNA by the Burton modification of the diphenylamine colorimetric test for DNA (Burton, '56). What classe(s) of R N A are present in spermatozoa? Polyacrylamide electrophoresis of unlabeled RNA extracted from mouse epididymal and ductus deferens spermatozoa revealed molecular weight equivalents to 28 s and 18 s RNA when stained with 0.2% methylene blue (fig. 1). These molecular weights, expressed as Svedberg units, were obtained from a plot of the log of Svedberg units against the electrophoretic migration distance of control RNA (Weinberg and Penman, '70). Mouse liver and HeLa cell RNA were used as controls and were purified and electrophoresed in the same manner as the spermatozoa RNA. The faint bands after the 28 s and the broad shoulder after the 18 s (fig. 1) may be spermatozoa mitochondrial ribosomal RNA (23 s and 16 s). The broad band of SDS (sodium dodecyl sulfate) was from the RNA sample that was placed on the gel and unfortunately obscures the 4 s and 5 s RNA. The 11 s RNA, which we previously reported (Betlach and Erickson, '73), was not seen when a RNase inhibitor and cold phenol extraction were used (METHODS AND MATERIALS). We have observed that freezethawed spermatozoa caused mouse liver ribosomal RNA to break down to a 11 s RNA. Could the spermatozoa1 28 s and 18 s RNA be an artifact? The possibility that the spermatozoa
51
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
5-
43-
I II
-t 28s
+18s
-t 4s
Fig. 1 Scans of disc acrylamide gel electrophoresis of maturing mouse spermatozoa RNA and mouse liver RNA (--------). The gels were stained with 0.2% methylene blue and scanned on a Canalco Model G densitometer. (-),
RNA was actually from epithelial or other became very radioactive but the spermatocells minced in the preparation of the zoa harvested from such epididymides were mouse spermatozoa was considered but unlabeled in their 28 s and 18 s RNA. seemed unlikely. Microscopic counting of An experiment with fresh ejaculated the cells revealed that there was less than human spermatozoa was performed to see 1 % contamination of the final spermatozoa if contaminating RNA was responsible for preparation by other cells. The majority of the 28 s and 18 s RNA seen. In the normal these contaminating cells were RNA free human male, the ejaculate has been reerythrocytes. Nonetheless, it was possible ported (Levis and Whalen, '76) and has that small amounts of RNA had become been microscopically observed by us and adsorbed onto the surface of the spermato- other colleagues to be relatively free from zoa. Several experiments ruled this out: other cellular material. RNA extracted After intratesticular injection of 5-3H-uri- from human ejaculated spermatozoa condine, the epididymal cell ribosomal RNA tained a 28 s and a 18 s component (fig. 2),
CHARLES J. BETLACH AND ROBERT P. ERICKSON
52
Fig. 2 Disc acrylamide gel electrophoresis of ejaculated human spermatozoa RNA ( I ) and HeLa cell RNA control (2).
further suggesting that the 28 s and 18 s RNA extensively studied in mouse spermatozoa had not been released from diced epithelial cells and adsorbed onto the spermatozoa.
W h e n during spermatogenesis is the spermatozoa1 28 s and I 8 s R N A synthesized? In vitro labeling with 5-3H-uridine was used to determine whether this 28 s and 28 s RNA were actively being synthesized in the maturing mouse spermatozoa. Ninety minute incubation with 5-3H-uridine gave 8.02 X 1 0 5 DPM/109 spermatozoa. The
RNA extracted from these sperm contained 0.040 pic0 moles 3H-uridineper mg of RNA. Polyacrylamide gel electrophoresis of this labeled RNA disclosed no discernable radioactive peaks but gave a 28 s and 18 s peak with 0.2 % methylene blue stain. The time of the 28 s and 18 s spermatozoal RNA synthesis during the course of spermatogenesis was further studied with sperm labeled in vivo with 5-3H-uridine given intratesticularly. This was performed in order to determine if the synthesis of the RNA found in maturing mouse spermatozoa had occured pre- or post-meiotically. Sperm were collected at weekly intervals. The RNA was extracted and the amount of 3H incorporation into RNA was determined (table 1). The total radioactivity was initially high, dropped off, and rose again on day 35. The 3H in the RNA was initially high and has a peak at 28 days post injection. After day 35, the total cellular radioactivity dropped rapidly. This radioactive profile was reproducible. Hydrolysis and analysis (METHODS AND MATERIALS) of the RNA and DNA indicated that more than 60% of the radioactivity on day 35 was in the DNA. On day 28 or earlier less than 15% of the radioactivity was in the DNA. These data show that extensive isotope reutilization had occurred in the weeks following the injections, but the highest specific activity in spermatozoal RNA was found at 28 days and at 6 days. RNA was extracted (METHODS AND MATERIALS) from the cauda epididymis sperm 36 days after the injection of 5-3H-uridine. The RNA was electrophoresed on polyacrylamide gels and the radioactive profile was determined (fig. 3). A 28 s, 18 s and 4 s 3H labeled RNA was present.
Where i s the spermatozoa1 R N A located? The location of the in vivo labeled RNA
TABLE I T h e appearance of 5-3H-uridine in m o u s e spermatozoa R N A after intratesticular injections of 5-3H-uridine Days post injection
6 14 21 28 35
DPM/testicle
64.69X 33.63X 16.28x 12.66X 5.89x
104
lo* 104 104 104
DPM/lOs sperm
13.70X 105 3.70X 105 3.35x 105 8.10X 105 12.40X 1 0 5
% of 3H i n sperm DNA
pmoles 3H-uridine mg sperm RNA
5
0.0164 0.0092 0.0086 0.0146 0.0049
10
15 15 60
53
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
+
28s
t
t
18s
4s
FRACTION NUMBER Fig. 3 Radioactive profile of a disc acrylamide nel electrophoresis of cauda epididymis mouse spermatozoa 3H-RNA 36 days after intratesticular injection of 5-3H-uridine.
Fig. 4 Autoradiography of maturing mouse spermatozoa labeled in vivo with intratesticular injections of 5-3H-uridine. Spermatozoa 21 days and 35 days after the injection. X 380.
in spermatozoa was looked at with autoradiography (fig. 4). Pretreatment of the cells, while on the slides, with cold 5 % trichloroacetic acid remove most of the unincorporated or acid soluble radioactivity. The grains seen in figure 4 represent labeled RNA and/or other macromolecules. At both 21 days and 35 days the grains were seen only in the sperm head, and there
were no grains in the tail, midpiece or acrosome. At day 21, the grains appear to be near the border of the cell rather than clustered in the center. The dense grains at day 35 probably represent the re-utilization of much of the label into DNA. DISCUSSION
The 11 s spermatozoa1 RNA previously
54
CHARLES J. BETLACH AND ROBERT P. ERICKSON
described by us (Betlach and Erikson, '73) appears to be a degradation product, one of the possible explanations suggested at that time. This fragment was apparently due to ribonuclease activity associated with spermatozoa, since an alternative extraction procedure utilizing bentonite and cold phenol did not produce the 11 s fragment. The scans of stained gels of spermatozoa1 RNA, prepared with extra precautions against ribonucleases, demonstrated the typical 28 s and 18 s pattern of mammalian ribosomal RNA. It is unlikely that this RNA was from contaminating cells, since all contaminating epithelial cells were removed from sperm preparations by a slow speed (10 x g) 30 seconds centrifugation. It was possible, however, that small amounts of R N A had become adsorbed onto the surface of the spermatozoa. This possibility seems ruled out by the studies on spermatozoa harvested after intratesticular injections with 5-3H-uridine. The epididyma1 cells were very radioactive after this injection, but the spermatozoa harvested from such epididymides did not have labeled 28 s and 18 s RNA. The presence of a 28 s and 18 s RNA in human ejaculated sperm confirms the results seen with mouse spermatozoa. The next logical question is whether this sperm R N A is being synthesized in the maturing spermatozoa. In vitro labeling with 5-3H-uridine produced a minimal amount of incorporation into R N A and gave no identifiable peaks with disc gel electrophoresis. It appears from this data that little or no 28 s and 18 s R N A is being actively synthesized in maturing mouse spermatozoa. If the 28 s and 18 s R N A was not being actively synthesized in sperm, at what stage in spermatogenesis was it produced? By correlating the time of appearance of a radioactive R N A peak and the time sequence of the mouse sperm maturation, it was possible to approximately determine at what maturational stage this particular macromolecule was synthesized. The in vivo 5-3H-uridine studies indicated that the R N A maximum was 28 days after the injection of the isotope and corresponded to premeiotic RNA synthesis in the primary spermatocyte. In mice, it takes 27 to 28 days for a germ cell to develop from a spermatogonia to a spermatozoa in the caput epididvmis (Monesi, '71: Ghosal and Mukhkrjee; '71) and 34 days to reach the
ductus deferens (Nelson, '71). The total radioactivity on day 35 (table 1) mainly represents tritium that has entered D N A in late spermatogonia or early spermatocytes. This accounts for the dense grains seen on the day 35 autoradiography (fig. 4). Apparently much of the tritium was converted in the spermatogonia to a form that could be incorporated into DNA. The initial high level of radioactivity at day 6 corresponds to the picture seen in the in vitro labeling and probably represents uptake andlor incorporation into epididymal sperm mitochondria. The presence of a radioactive 28 s, 18 s and 4 s R N A in sperm 36 days after the in vivo injection demonstrates that the radioactive R N A seen is the same as the 28 and 18 s R N A in figure 1. R N A extraction from day 28 cells was inefficient because of low sperm count. The presence of no grains (fig. 4) in the midpiece indicates that there is little or no label uptake by the mitochondria. It appears from the in vitro and in vivo studies that little or no 28 s and 18 s R N A is being synthesized in the maturing mouse spermatozoa. The very nature of the compact nucleus in spermatozoa would preclude any R N A transcription. However, it appears that approximately 85% of the R N A in spermatozoa is 28 s and 18 s R N A and the majority is produced in the primary spermatocyte. This is in good agreement with recent electron microscopical observations suggesting ribosomal R N A transcription in spermatocytes and heterogeneous R N A transcription in spermatids (Kierszenbaum and Tres, '75). Amino acid analyses have shown that the amino acid composition of bovine spermatozoa changes with passage through the epididymis (Lavon et al., '71); and ejaculated rabbit spermatozoa sequester the twenty common amino acids into trichloroacetic acid insoluble material (Busby et al., '74). The phenylalanine and histidine incorporation was observed to be due to non-covalent sequestering (Busby et al., '74). There is incorporation of selenium into developing spermatozoa which suggests an increase in sulfur containing amino acids (Gunn et al., '67). These proteins are thought to be protamine-like histones (Calvin and Bedford, '71). Monesi suggests that some of the messenger R N A that is produced during meiosis is stable and directs protein synthesis during post-
28 s AND 18 s RNA FROM MAMMALIAN SPERMATOZOA
meiotic spermatogenesis (Monesi, '74). Galdieri demonstrates a reduction of ribosomal RNA maturation in the mouse spermatocyte (Galdiere and Monesi, '74). They speculated that this may lead to the storage of nucleolus ribosomal RNA precursors used for protein synthesis during post-meiotic spermatogenesis. We propose here that the 28 s and 18 s RNA seen in spermatozoa is necessary for this post-meiotic translation, is produced in the primary spermatocyte and is utilized throughout spermatogenesis. LITERATURE CITED Baserga, R., and D. Malamid 1969 Autoradiography. Techniques and Application. Harper and Row, Publishers, New York. Betlach, C. J., and R. P. Erickson 1973 Aunique RNA species from maturing mouse spermatozoa. Nature, 242: 114-115. Bhargava, P. M., M. Bishop and T. S. Work 1959 The chemical composition of Bull semen with special reference to nucleic acids, free nucleotides and free amino acids. Biochemical J., 73: 242247. Burton, K. 1956 A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical J., 62: 315-323. Busby, W. F., Jr., P. Hele and M. C. Chag 1974 Apparent amino acid incorporation by ejaculated rabbit spermatozoa. Biochim. Biophys. Acta, 335: 246-259. Calvin, H. I., and J. M. Bedford 1971 Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fert., Suppl., 13: 65-75. Galdieri, M., and V. Monesi 1974 Ribosomal RNA in mouse spermatocytes. Expt. Cell Research, 85: 287-295. Ghosal, S. L., and B. B. Mukherjee 1971 The chronology of DNA synthesis, meiosis and spermatogenesis in the male mouse and golden hamster. Can. J. Genet. Cytol., 13: 672-682. Gunn, S. A,, T. C. Gould and W. A. D. Anderson 1967 Incorporation of selenium into spermato-
55
genic pathway in mice. Proc. Soc. Expt. Biol. and Med., 124: 1260-1263. Kierszenbaum, A. L., and L. Tres 1975 Structural and transcriptional features of the mouse spermatid genome. J. Cell Biology, 65: 258-270. Lavon, U., R. Volcani and D. Damon 1971 The proteins of Bovine spermatozoa from the caput and cauda epididymides. J. Reprod. Fert., 24: 2 19-232. Lee, I . P., and R. L. Dixen 1972 Antineoplastic drug effects on spermatogenesis studied by velocity sedimentation cell separation. Tox. and Appl. Pharmacol., 23: 2 0 4 1 . Levis, W. R., and J. J. Whalen 1976 Mixed cultures of sperm and leukocytes as a measure of histocompatibility in man. Science, 191: 302304. MacLaughlin, J., and C. Terner 1973 Ribonucleic acid synthesis by spermatozoa from the rat and hamster. Biochem. J., 133: 635-639. Mann, T. 1951 Mammalian semen: Composition, metabolism and survival. Biochem. Soc. Symp., 7: 11-23. Meistrich, M. L. 1972 Separation of mouse spermatogenic cells by velocity sedimentation. J. Cell Physiol., 80: 299-312. Monesi, V. 1971 Chromosome activities during meiosis and spermiogenesis. J . Reprod. Fert., SUppl., 13: 1-14. 1974 Nucleoprotein synthesis in spermatogenesis. In: Male Fertility and Sterility, Proceedings of the Seroro Symposia. R. E. Mancini and L. Martini, eds. Academic Press, London, pp. 59-87. Moore, G . P. M. 1971 DNA-dependent RNA synthesis in fixed cells during spermatogenesis in mouse. Exptl. Cell Res., 68: 4 6 2 4 6 5 . Nelson, L. 1971 Differentiation of the male germ cell. In: Developmental Aspects of Cell Cycle. I. L. Cameron, ed. Academic Press, New York, pp. 243-278. Premkumar, E., and P. M. Bhargava 1972 Transcription and translation i n Bovine spermatozoa. Nature (New Biology), 240: 139-143. Shatkin, A. J. 1969 Colorimetric reactions for DNA, RNA, and protein determinations. In: Fundamental Technique i n Virology. K. Habel and N. P. Salzman, eds. Academic Press, New York, pp. 231-237. Weinberg, R. A., and S . Penman 1970 Processing of 45 s nucleolar RNA. J. Mol. Biol., 47: 169178.
