ABSTRACT
As components of the 42S storage particles (thesauri-somes), thesaurin a and thesaurin b are involved in the long-term storage of tRNA and 5S RNA in previtello-genic oocytes of Xenopus laevis. Thesaurin a and thesaurin b are among the most abundant proteins in previtellogenic oocytes. We show here that the mRNAs encoding thesaurin a and thesaurin b are present not only in previtellogenic oocytes but also in pre-meiotic germ cells (oogonia). These mRNAs can also be detected in spermatogonia and early spermatocytes, and are translated into protein in testis, as they are in ovary. We conclude that male germ cells mimic female germ cells in several aspects of gene activity related to RNA accumu-lation and metabolism.
Introduction
Ribosomal and transfer RNA (tRNA) accumulation in amphibian and teleost oocytes is a long-term process involving the storage of 5S RNA and tRNA in early diplotene cells (Ford, 1971; Denis and Mairy, 1972; Picard and Wegnez, 1979; Denis and le Maire, 1983). In Xenopus laevis these cells contain two major kinds of nucleoprotein particles (7S and 42S) which are thought to protect 5S RNA and tRNA against degradation (Denis and le Maire, 1983).
The 7S particles contain one molecule of 5S RNA and one molecule of a 38 ×X103Mr protein which is identical to transcription factor IILA (TFIIIA; Picard and Wegnez, 1979; Pelham and Brown, 1980; Honda and Roeder, 1980). The 42S particles are composed of four subunits, each of which contains one molecule of 5S RNA, three molecules of tRNA, two molecules of a 50×103Mr protein known as thesaurin a, and one molecule of a 40×l03Mr protein known as thesaurin b (Picard et al. 1980). Thesaurin a (also designated 42Sp50 or 42Sp48) binds aminoacyl tRNA within the 42S particle (Picard et al. 1980) and is homologous to elongation factor la (EF-lα, Viel et al. 1987; Djé et al. 1990). It also delivers aminoacyl tRNA to the A site of ribosomes, a function normally served by EF-lα (Mattaj et al. 1987; Viel et al. 1991). Thesaurin b (also designated 42Sp43) binds 5S RNA within the 42S particle (Picard et al. 1980; Joho et al. 1990), and is homologous to TFIIIA (Joho et al. 1990). However, thesaurin b does not act as a transcription factor (Joho et al. 1990).
The 7S and 42S particle proteins (TFIIIA, thesaurin a and thesaurin b) are by far the most abundant proteins in previtellogenic oocytes (stage I) of X. laevis (Denis and le Maire, 1983; Viel et al. 1990). TFIIIA has a somatic counterpart (Pelham et al. 1981; Shastry et al. 1984; Kim et al. 1990), and can therefore be considered as a ubiquitous gene product (Kim et al. 1990). In contrast, thesaurins a and b have no equivalent in somatic cells (Mattaj et al. 1983; Joho et al. 1990).
The mRNAs encoding TFIIIA and both thesaurins can each be detected as a single band by northern blot analysis of ovary and oocyte RNA (Ginsberg et al. 1984; Djé et al. 1990; Joho et al. 1990). The amount of thesaurin mRNA per cell is highest in previtellogenic oocytes and drops sharply in larger oocytes, so as to become undetectable or hardly detectable in eggs and early embryos (Djé et al. 1990; Joho et al. 1990). The drop in TFIIIA mRNA content in growing oocytes is less steep, so that eggs and early embryos still contain substantial amounts of this mRNA (Ginsberg et al. 1984).
An important point that could not be settled by the northern blot analyses concerns the stage of the female germ cell differentiation at which the TFIIIA and thesaurin genes start being transcribed. We used in situ hybridization to detect TFIIIA and thesaurin tran-scripts in sections of immature ovaries, which contain germ cells of various stages. We found that all transcripts are already present in oogonia, i.e. before the germ cells begin to increase in size. All three mRNAs accumulate up to the end of the previtellogenic period (stage I). During later oogenesis TFIIIA and thesaurin b mRNAs disappear more slowly than does thesaurin a mRNA. Thesaurin b mRNA is still abundant in early vitellogenic oocytes (stage II). TFIIIA mRNA can be detected up to mid-vitellogen-esis.
Male germ cells also contain thesaurin a and thesaurin b transcripts. Both mRNAs are present in pre-meiotic germ cells (spermatogonia). Primary sper-matocytes, like early vitellogenic oocytes, no longer contain thesaurin a mRNA, but still contain thesaurin b mRNA. Both mRNAs are translated into protein in testis. However, thesaurins a and b are far less abundant in male gonads than in female ones. In contrast to thesaurin a and b mRNAs, TFIIIA mRNA cannot be detected in male germ cells by in situ hybridization.
The distribution of thesaurin a and thesaurin b transcripts in male and female germ cells is similar to that of the mRNA encoding the oocyte form of elongation factor 1α (EF-lαO; Abdallah et al. 1991). Therefore, a pool of genes that are permanently repressed in somatic cells become specifically activated in immature germ cells of both sexes. The products of these genes can be defined as markers of germ cell differentiation.
Materials and methods
In situ hybridization was carried out as described previously (Hourdry et al. 1988), with 35S-labeled antisense and sense RNA probes transcribed from TFHIA, thesaurin a and thesaurin b cDNAs (Ginsberg et al. 1984; Djé et al. 1990; Joho et al. 1990). Sections (7.5μm) of ovaries and testes from juvenile adults (3–5 cm from mouth to anus) were incubated with the labeled probes, washed and autoradiographed for 7 to 11 days. The number of silver grains on the autoradiograms was measured with an automatic microdensitometer. Stages of oogenesis were identified by measuring the diameter of equatorially sectioned cells (Dumont, 1972). Stages of spermatogenesis were identified by microscopic observation of Feulgen-stained testis sections.
SI nuclease protection assay was carried out with antisense single-stranded cDNA probes corresponding to the 3’-parts of thesaurin a cDNA (Djé et al. 1990) and thesaurin b cDNA (Joho et al. 1990). The probes were generated in Blues-cript(+) KS (Stratagene) by primer extension, using seque-nase (USB), zr-[32P]dATP, and either the reverse primer (in the case of thesaurin a cDNA), or the T3 primer (in the case of thesaurin b cDNA; Davis et al. 1986). The thesaurin a and thesaurin b probes were purified with phenol and digested with EcoRI and Mstl, respectively. This generates a 330bp-fragment (240 pb from thesaurin a cDNA linked to 90 bp from the vector), and a 410bp-fragment (330 bp from thesaurin b cDNA linked to 80bp form the vector). Both fragments were purified by electrophoresis in a polyacrylamide denaturing gel (Maniatis et al. 1982). The bands of expected size were eluted from the gel. Aliquots of each probe (IfPctsmin−1) were hybridized for 3h at 45°C with 1/zg, 50μg and 50μg, respectively of total RNA from ovary, testis and liver of young adults (Djé et al. 1990). The hybrids were then digested with 2.5 units of SI nuclease (Promega). Control samples were simply incubated with 10μg of tRNA, or incubated with 10μg of tRNA and digested with SI nuclease. Protected and non-protected fragments were separated by electrophoresis in a 8.3 M urea, 6% sequencing gel for 75 min.
Post mitochondrial extracts from adult testes and ovaries were fractionated by sucrose density centrifugation, and analyzed for thesaurin a, thesaurin b, tRNA and 5S RNA content by polyacrylamide gel electrophoresis followed by immunoblotting or silver staining (Viel et al. 1990, 1991).
Results
In situ hybridization on sections of immature ovaries shows that thesaurin a and thesaurin b mRNAs are highly concentrated in oogonia and in early previtello-genic oocytes (stage I; Fig. 1A and B). This is also true for TFIIIA mRNA (data not shown). Larger previtello-genic oocytes give a fainter hybridization signal (Fig. 1A and B). This decrease in concentration is a dilution effect due to cytoplasmic expansion, since the total amount of thesaurin mRNA per cell augments until the end of stage I (Table 1 and Fig. 2). In vitellogenic oocytes the concentration and total amount of thesaurin a mRNA drop more rapidly (Figs 1A, 2 and Table 1) than those of thesaurin b mRNA (Fig. 1B, 2 and Table 1). TFIIIA mRNA accumulates up to stage III (Table 1 and Fig. 2). The concentration of all three mRNAs falls below the level of detection in late vitellogenic oocytes (stages V–VI), in cleaving eggs and in embryos up to the tadpole stage. These results agree with those of the northern blot analyses, and confirm the absence of thesaurin mRNAs in somatic cells (Ginsberg et al. 1984; Djé et al. 1990; Joho et al. 1990).
Amount of mRNA in germ cells of various stages as measured by quantitative in situ hybridization

In situ hybridization of thesaurin a and thesaurin b probes to sections of ovary and testis. (A) Section of immature ovary hybridized with thesaurin a probe. (B) Section of immature ovary hybridized with thesaurin b probe. (C and D) Sections of mature testis hybridized with thesaurin a probe. (E and F) Sections of mature testis hybridized with thesaurin b probe. Both probes label oogonia (og) and previtellogenic (stage I) oocytes (panels A and B), and the periphery (arrows) of the seminiferous cysts (sc) which contains mostly spermatogonia (panels C-F). The thesaurin b probe also labels early vitellogenic (stage II) oocytes (panel B), and a sub-peripheral region of the seminiferous cysts which contains primary spermatocytes (panels E and F). Intercystic spaces (is) and oocyte nuclei (n) show a moderate to very low signal. A–C and E, dark-field illumination; D and F, bright-field illumination. Scale bars correspond to 50μm.
In situ hybridization of thesaurin a and thesaurin b probes to sections of ovary and testis. (A) Section of immature ovary hybridized with thesaurin a probe. (B) Section of immature ovary hybridized with thesaurin b probe. (C and D) Sections of mature testis hybridized with thesaurin a probe. (E and F) Sections of mature testis hybridized with thesaurin b probe. Both probes label oogonia (og) and previtellogenic (stage I) oocytes (panels A and B), and the periphery (arrows) of the seminiferous cysts (sc) which contains mostly spermatogonia (panels C-F). The thesaurin b probe also labels early vitellogenic (stage II) oocytes (panel B), and a sub-peripheral region of the seminiferous cysts which contains primary spermatocytes (panels E and F). Intercystic spaces (is) and oocyte nuclei (n) show a moderate to very low signal. A–C and E, dark-field illumination; D and F, bright-field illumination. Scale bars correspond to 50μm.
Histogram showing the accumulation of thesaurin a (Tha) mRNA, thesaurin b (Thb) mRNA and TFIIIA mRNA in growing oocytes. Thesaurin a mRNA disappears more rapidly than thesaurin b mRNA in early vitellogenic oocytes (375 pm in diameter). In contrast, TFIIIA mRNA continues to accumulate in early vitellogenic oocytes.
Histogram showing the accumulation of thesaurin a (Tha) mRNA, thesaurin b (Thb) mRNA and TFIIIA mRNA in growing oocytes. Thesaurin a mRNA disappears more rapidly than thesaurin b mRNA in early vitellogenic oocytes (375 pm in diameter). In contrast, TFIIIA mRNA continues to accumulate in early vitellogenic oocytes.
Thesaurin a and thesaurin b mRNAs can also be detected in mature testis by in situ hybridization (Fig. 1C–F and Table 1). Thesaurin a mRNA is concentrated in a very thin layer of cells at the periphery of the seminiferous cysts (Fig. 1C and D). We identified these cells as spermatogonia. Thesaurin b mRNA is also present in a more internal region of the seminiferous cysts, which contains primary spermato-cytes (leptotene through pachytene-diplotene cells; Fig. 1E and F). Round spermatids and spermatozoa which fill the lumen of the cysts have little thesaurin mRNA (Table 1). The concentration of TFIIIA mRNA in male germ cells is below the level of detection by in situ hybridization.
We wished to confirm the presence of thesaurin mRNAs in testis by a non-cytological method. North-ern blot analysis of total RNA from adult testis gave no significant hybridization signal with either cDNA. A more sensitive test resorting to protection of mRNA-cDNA hybrids against SI nuclease indicates that both thesaurin mRNAs are present in extracts of ovary and testis, but absent in those of liver (Fig. 3).
Nuclease protection assay of thesaurin a (A) and thesaurin b (B) cDNAs by RNA from liver (lanes L), ovary (lanes O) and testis (lanes T). Lanes C1 and C2 contain non-digested and non-protected probes, respectively. Lanes M contain DNA fragments of known sizes. Each non-digested DNA probe (lanes C1) includes the 3’ end of thesaurin cDNA linked to a small fragment of the cloning vector (Bluescript). This fragment is not protected against digestion, so that the nuclease-treated probes (lanes O and T) are shorter. The ovary tissue used in this experiment contained only previtellogenic oocytes. The testis tissue contained male germ cells at all stages of maturation.
Nuclease protection assay of thesaurin a (A) and thesaurin b (B) cDNAs by RNA from liver (lanes L), ovary (lanes O) and testis (lanes T). Lanes C1 and C2 contain non-digested and non-protected probes, respectively. Lanes M contain DNA fragments of known sizes. Each non-digested DNA probe (lanes C1) includes the 3’ end of thesaurin cDNA linked to a small fragment of the cloning vector (Bluescript). This fragment is not protected against digestion, so that the nuclease-treated probes (lanes O and T) are shorter. The ovary tissue used in this experiment contained only previtellogenic oocytes. The testis tissue contained male germ cells at all stages of maturation.
We also wanted to know if the germ cell-specific mRNAs are translated into protein in testis. No trace of 42S particles can be observed in testis extracts fractionated by sucrose density centrifugation (Fig. 4), and no significant amount of 5S RNA and tRNA can be detected in this region of the gradient. However, a 42S particle antiserum reveals the presence of both thesaur-ins in the 4–7S fraction (Fig. 4). Thesaurin a is also present in all fractions sedimenting between 4S and 80S (Fig. 4). This indicates that in male germ cells the amount of thesaurin a vastly exceeds the amount of thesaurin b. Furthermore, thesaurins a and b are apparently not associated in large, monodisperse nucleoprotein complexes, as they are in oocytes (Fig.4).
Fractionation of testis and ovary extracts by sucrose density centrifugation, followed by immunodetection of thesaurins a and b (insert). Thirty-two testes and six ovaries from young adults (3 cm from mouth to anus) were homogenized in 5 volumes of 50 mM Tris-HCl, 25 mM KC1, 5 mM MgC12. The clarified extracts were fractionated by centrifugation through two 15-30% sucrose density gradients (3h at 45 000 revs min−1 in a SW50.1 rotor). Aliquots (200 MI) from testis fractions 10 (42S) and 15 (7S) were concentrated by ethanol precipitation, fractionated by polyacrylamide gel electrophoresis in the presence of SDS, blotted onto nitrocellulose and probed with a 42S particle antiserum (lanes 1 and 2 of insert). Aliquots (8;d) from ovary fractions 10 and 15 were analyzed in the same way (lanes 3 and 4 of insert).
Fractionation of testis and ovary extracts by sucrose density centrifugation, followed by immunodetection of thesaurins a and b (insert). Thirty-two testes and six ovaries from young adults (3 cm from mouth to anus) were homogenized in 5 volumes of 50 mM Tris-HCl, 25 mM KC1, 5 mM MgC12. The clarified extracts were fractionated by centrifugation through two 15-30% sucrose density gradients (3h at 45 000 revs min−1 in a SW50.1 rotor). Aliquots (200 MI) from testis fractions 10 (42S) and 15 (7S) were concentrated by ethanol precipitation, fractionated by polyacrylamide gel electrophoresis in the presence of SDS, blotted onto nitrocellulose and probed with a 42S particle antiserum (lanes 1 and 2 of insert). Aliquots (8;d) from ovary fractions 10 and 15 were analyzed in the same way (lanes 3 and 4 of insert).
Discussion
We have shown that pre-meiotic germ cells of both sexes contain both thesaurin mRNAs. Thesaurin a mRNA disappears when the male germ cell enters meiosis, but accumulates in the female germ cell after the beginning of meiosis, up to mid-diplotene stage (end of stage I; Table 1). Thesaurin b mRNA is present during a longer period of male and female gametogen-esis, extending to prophase I of meiosis (leptotene-diplotene) for male germ cells, and to diplotene stage II for female germ cells (Table 1).
The pattern of thesaurin mRNA expression is similar, but not identical to that of EF-lαO mRNA. All three mRNAs are present in pre-meiotic germ cells of both sexes (Table 1; Abdallah et al. 1991). EF-lαO mRNA is also present in primary spermatocytes and oocytes, up to the end of prophase I (Abdallah et al. 1991). Thesaurin mRNAs disappear much earlier than EF-lαO mRNA during male and female gametogen-esis. An exception is thesaurin b mRNA in male germ cells, whose expression apparently covers the same period as that of EF-1αO mRNA, ie pre-meiosis and prophase I of meiosis (Table 1).
The EF-lαO and thesaurin genes belong to a small category of genes whose expression is restricted to male and female germ cells. Well characterized members of this category are the Drosophila cyclin B and vasa genes (Whitfield et al. 1989; Hay et al. 1988; Lasko and Ashburner, 1988, 1990). Both of these genes encode abundant maternal mRNAs, and are also expressed in male germ cells. Cyclin B is presumably involved in the control of germ cell division in the gonads (Whitfield et al. 1988). The function of the vasa gene product in gametogenesis is less clear. The vasa protein is homologous to eIF-4A (an ATP-dependent translation factor that binds to mRNA), and to other helicases (Hay et al. 1988; Lasko and Ashburner, 1988). This protein bears some functional similarity with the thesaurins, especially thesaurin a, which in oocytes acts as a GTP-dependent elongation factor (Mattaj et al. 1987; Viel et al. 1991).
Both thesaurins play a crucial role in RNA metab-olism of oocytes. These proteins protect tRNA and 5S RNA against degradation (Denis and le Maire, 1983), and contribute substantially to accumulation of these molecules in previtellogenic oocytes. The function of thesaurins a and b in spermatogonia and in spermato-cytes is unclear because RNA metabolism in these cells seems very different from that in oocytes. In particular, male germ cells do not accumulate large amounts of tRNA and 5S RNA. In spite of this, spermatogonia amplify to a small extent their ribosomal genes (Kalt and Gall, 1974), a process known to considerably increase the production capacity of oocytes for 28S and 18S RNA (Gall, 1968; Brown and Dawid, 1968). Similarly, male and female germ cells contain a specific form of elongation factor 1er (EF-lαO; Abdallah et al. 1991). One is tempted to conclude that male germ cells mimic female germ cells in all aspects of gene activity related to RNA accumulation and metabolism. This conclusion does not apply to derepression of the oocyte-type 5S RNA genes. Like amplification, this process enables oocytes to accumulate many RNA molecules in a relatively short time (Wegnez et al. 1972; Ford and Southern, 1973). Unlike oocytes, male germ cells do apparently not express more 5S RNA genes than somatic cells (Wegnez, 1973).
These observations make the presence of thesaurins a and b in male germ cells very intriguing. Whatever the function of their products in spermatogenesis, the thesaurin genes will hopefully allow identification and isolation of the factor(s) responsible for their activation in the germ cell line. A Xenopus germ line-specific transcription factor has recently been characterized (Tafuri and Wolffe, 1990). It will be interesting to determine if this factor can activate the EF-lαO and thesaurin genes.
ACKNOWLEDGEMENTS
We thank Dr K. E. Joho for the gift of thesaurin b cDNA and Dr D. Schoevaert for quantitative analysis of the autoradiograms. This work was supported by grants from ‘Fondation pour la Recherche médicale’ and ‘Ligue nationale contre le Cancer’.