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Cell Growth & Differentiation Vol. 10, 19-26, January 1999
© 1999 American Association for Cancer Research

Two Alternatively Spliced Meig1 Messenger RNA Species Are Differentially Expressed in the Somatic and in the Germ-Cell Compartments of the Testis1

Leah Ever, Rachel Steiner, Sarah Shalom and Jeremy Don2

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Previous studies regarding the transcriptional pattern of the murine Meig1 gene (formally designated meg1) suggested that its transcription is restricted to germ cells at the first meiotic prophase, in both primary spermatocytes and primary oocytes. However, protein analysis revealed that certain forms of the MEIG1 protein exist in testes of early postnatal pups at stages that have no germ cells in the testis, excluding very few primitive type A spermatogonia cells. This suggested that MEIG1 expression is not confined to germ cells. In this study, we show that testicular somatic cells do, indeed, express MEIG1. This is especially evident in Leydig cells, where this protein is highly abundant. We also demonstrate that alternatively spliced mRNAs of Meig1 are differentially transcribed in the germ cell and the somatic compartments of the testis. There is a very low level of somatic transcript, whether labile or transcriptionally regulated, in contrast to the abundant MEIG1 protein in the somatic cells. This implies that the somatic transcript is very efficiently translated and reconfirms that protein levels do not necessarily reflect transcript abundancy. Structural features of the Meig1 transcript that would be expected to inhibit translation are discussed in light of the efficient translation of this RNA species.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Spermatogenesis is a complex process in which mitotically dividing spermatogonial stem cells differentiate into mature spermatozoa. This hormonally regulated process takes place in the seminiferous tubules that occupy the testis. The only cells within these tubules are the Sertoli cells, somatic cells attached to the basal membrane of the tubules, and the germ cells that are nourished and supported by the Sertoli cells during their differentiation and migration from the basal compartment to the lumen (1) . Located between the tubules, in the interstitial regions, are the Leydig cells, somatic cells that are responsible for the testosterone production that is vital for the execution of normal spermatogenesis. The function of both Sertoli and Leydig cells is hormonally regulated by gonadotropins. Luteinizing hormone stimulates testosterone production in Leydig cells, whereas follicle-stimulating hormone binds to receptors on the basal surface of the Sertoli cells and, through this interaction, modulates germ cell development (2 , 3) . Local communication between both somatic and germ cells within the testis is vital to normal spermatogenesis. This communication is mediated by various growth factors such as the basic fibroblast growth factor, several members of the transforming growth factor ß superfamily, epidermal growth factors, and others (4, 5, 6, 7, 8) .

Spermatogenesis can be divided into three main stages: (a) mitotic proliferation of spermatogonial stem cells and premeiotic differentiation of spermatogonia cells to primary spermatocytes; (b) meiotic differentiation of primary spermatocytes to haploid early round spermatids; and (c) spermiogenesis, the cellular and nuclear reorganization process that turns spermatids into spermatozoa (9) . These stages are structurally and morphologically well defined. However, due to a lack of an efficient system for germ cell differentiation in vitro, the molecular basis of spermatogenesis is still poorly understood. Nonetheless, an increasing number of genes that are expressed during mammalian spermatogenesis have been identified in recent years. Some of these genes encode proteins thought to be involved with various regulatory mechanisms. These include, among others, protein kinases such as Mak (10 , 11) , Gek1 (12) , Nek2 (13 , 14) , Ayk1 (15) , and some forms of cyclin-dependent kinases (16 , 17) . It also includes zinc finger containing genes Zfp 35, Zfp-38, and Zfp 51 (18, 19, 20) and neuropeptide coding genes such as POMC and proenkephalin (21 , 22) . Nevertheless, most of these kinases have not been assigned specific substrates, and most of the other genes (only a few of which were mentioned) have not been assigned specific spermatogenic functions.

Meig1, formally designated meg1 (23 , 24) , is a murine gene that encodes a highly abundant 0.75-kb transcript reported to be expressed exclusively in the testis, in a germ cell-specific manner. Meig1 transcripts begin to accumulate in early stages of the first meiotic prophase, leptotene-zygotene, and are most abundant in pachytene spermatocytes. In females, transcripts were detected only in ovaries of embryos at days 16.5–17.5 of gestation, when the oocytes have reached the comparable meiotic stage (i.e., the pachytene stage of prophase I; Refs. 23 and 24 ), suggesting that Meig1 is involved in meiosis rather than in spermatogenesis-specific processes. Protein analysis revealed that the MEIG1 protein appears in multiple phosphorylated forms, including two dimeric forms of about Mr 31,000 and Mr 32,000. A developmentally regulated switch in the relative abundance of these two dimeric forms is apparent, where the Mr 31,000 form, which is tyrosine phosphorylated, becomes the dominant form once the cells enter meiosis (25) . MEIG1 is, therefore, likely to be involved in meiotic events, and dimerization and phosphorylation/dephosphorylation reactions seem to regulate its function. However, this latter study also revealed that equal amounts of the monomeric forms of the MEIG1 protein are present in testes of pups at all developmental stages tested, including pnd3 5. At this developmental stage, there are no germ cells in the testis, excluding very few primitive type A spermatogonia cells. This suggests that MEIG1 might be expressed in testicular somatic cells as well.

In this study, we show that MEIG1 is, indeed, expressed in testicular somatic cells and that the somatic and the germ cell proteins seem to be translated from distinct, alternatively spliced mRNAs.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
MEIG1 Is Expressed in Somatic Cells within the Testis.
We have previously shown that, in contrast to the expression pattern at the RNA level, where Meig1 transcripts were apparent in the testes of mice only from pnd 10 (24) , the MEIG1 protein, in its monomeric form, seems to be present at fairly constant levels at all of the testicular developmental stages that were examined (i.e., pnd 5–30; Ref. 25 ). At the early developmental stages (i.e., pnd 5–7), the vast majority of the cells are somatic cells (26 , 27) , suggesting that MEIG1 is expressed not only in germ cells but in somatic cells as well. To address this possibility, we analyzed by Western blotting (using highly specific anti-MEIG1 antibodies; Ref. 25 ) testicular protein extracts from germ cell-deficient mutant mice of the atricosis strain, as well as from heterozygous individuals for this mutation. The homozygous individuals for the mutant allele (at/at) are devoid of germ cells, whereas the somatic complement of the testis is phenotypically normal. Heterozygous individuals (at/+), are phenotypically and functionally indistinguishable from the normal wild type mice (28) . As can be seen in Fig. 1Citation , the monomeric forms as well as the Mr 32,000 dimeric form were readily detected in the at/at mutant testicular extract (with trace amounts only of the Mr 31,000 form), resembling the pattern obtained in pnd 5–7 testicular extract. However, the dominant Mr 31,000 dimeric form, together with the monomeric forms that appeared in the at/+ testicular extract, resembled the pattern obtained in normal mature mice. These results support our assumption that somatic cells within the testis do, indeed, express MEIG1. We next wished to determine which cell type within the mutant testis express the MEIG1 protein. The signal observed could be derived from the interstitial cells, Sertoli cells, or the very few undifferentiated spermatogonia cells, that might occasionally be found in the testis of these mutants. Immunohistochemical analysis of paraffin sections of at/at testes revealed that the interstitial somatic cells are the main source of MEIG1 in the mutant testis, whereas only very low levels could be attributed to Sertoli cells (Fig. 2a)Citation . Three types of control experiments where executed: (a) preimmune serum was used as a primary antibody; (b) anti-MEIG1 antibodies, which were preincubated overnight with recombinant MEIG1, were used as the primary antibody; and (c) only the second antibody was applied to the sections. All of these control experiments revealed no signal above background (data not shown). These results confirm the ability of MEIG1 to be expressed in testicular somatic cells.



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Fig. 1. Western blot analysis of testicular proteins ({approx}50 µg) extracted from testes of germ cell-deficient mutant atricosis mice, homozygous for the mutant allele (at/at), and from testes of the phenotypically normal heterozygous mice (at/+). Blots were probed with affinity purified anti-MEIG1 antibodies. Size markers (kDa) are indicated at the left.

 


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Fig. 2. a, immunohistochemical localization of the MEIG1 protein in testis sections of a germ cell-deficient mutant atricosis mouse (at/at). After incubation with affinity purified rabbit anti-MEIG1 antibodies, staining was visualized with FITC-conjugated goat antirabbit. A, double staining of nuclei with propidium iodide (red fluorescence) and MEIG1 localization (green fluorescence). The two images are superimposed. B, interstitial localization of MEIG1. Bar indicates size in µm. b, immunohistochemical localization of the MEIG1 protein in testis sections of a normal adult mouse. A, double staining of nuclei with propidium iodide (red fluorescence) and MEIG1 localization (green fluorescence). The two images are superimposed. B, localization of MEIG1 to Leydig cells in the interstitial regions. C, higher magnification of a double-stained tubule. D, MEIG1 localization to germ cells within it. Bars indicate sizes in µm. c, negative control for immunohistochemical analysis. A, double staining of nuclei with propidium iodide (red fluorescence) and anti-MEIG1 antibodies that were preincubated overnight with recombinant MEIG1 (as primary antibody), followed by treatment with FITC-conjugated goat antirabbit (green fluorescence). The two images are superimposed. B, green fluorescence only shows no signal above background over tissue.

 
Testicular somatic expression of MEIG1 has been shown in mutant testes and in early postnatal developmental stages (data not shown). In both cases, however, differentiated germ cells are absent. We, therefore, wished to determine whether somatic cells express MEIG1 in normal testis containing differentiated germ cells. Immunohistochemical analysis performed on paraffin-embedded testis sections from normal mature mice showed that very high levels of MEIG are expressed in Leydig cells, which are located in the interstitial regions between tubules (Fig. 2b)Citation . When higher magnification was used, it became clear that although germ cells do express MEIG1, Leydig cells express the highest levels of MEIG1, per cell. Control experiments performed as described above did not reveal any signal above background (Fig. 2c)Citation . We conclude that MEIG1 is normally expressed in both the germ cell and the somatic compartments of the testis.

Alternatively Spliced mRNAs of Meig1 Are Differentially Expressed in Somatic and in Germ-Cells.
In recent studies, Meig1 transcripts could not be detected in the testis of either at/at mutant mice or pups at early developmental stages (23 , 24) . We, therefore, performed a more detailed analysis of RNA expression in these tissues. As previously described by Don and Wolgemuth (23) , Meig1 encodes two alternatively spliced transcripts, represented by clones 11a2 and 2a2, both of which are about 0.75 kb long. These two transcripts share the same ORF and 3'UTR, but differ in their 5' UTR (Fig. 3A)Citation . It is possible that the two transcripts are differentially expressed in the somatic and the germ cell compartments of the testis and that the level of the somatic transcript in the testis is under the detection threshold by Northern analysis of total RNA. If that were the case, one would expect the somatic transcript to be enriched for in testis of early postnatal pups, so that it can be detected in poly(A)+ RNA using the 5' UTR of the somatic transcript as a probe. For this experiment, an EcoRI-SacI fragment and an EcoRI-NaeI fragment from the 5' UTR of clones 11a2 and 2a2, respectively (Fig. 3A)Citation , were used as probes for Northern analysis of 8 µg of poly(A)+ RNA from testes of pups at different postnatal ages (pnd 5–30). When the 2a2-specific probe was used, signal could first be detected in d12 RNA and it increased along with testicular development, in accordance with the known pattern of Meig1 transcripts. However, when the 11a2-specific probe was used on the same blot, after stripping off the 2a2 probe, signal could clearly be detected in the early postnatal stages (pnd 5, 7, and 10), and it decreased along with testicular development (Fig. 4)Citation . Considering that testicular development entails a dramatic increase in the proportion of germ cells coupled with a dramatic decrease in the proportion of the somatic cells (26) , these results support the notion that the 2a2 clone represents a germ cell-specific transcript, whereas the 11a2 clone represents a somatic-specific transcript. To further examine this possibility, in situ hybridization experiments were performed on paraffin-embedded testis sections from normal mature mice, as well as from at/at mutants. Antisense S35{alpha}UTP-labeled riboprobes corresponding to the two transcript-specific fragments were used for the assay, whereas sense probes were used as controls. Using the 2a2-specific antisense probe, a very intense signal could be obtained over primary spermatocytes, which persisted developmentally through to round spermatids, within all tubules, independent of the stage of their seminiferous epithelium cycle (Fig. 5, A–D)Citation . No signal above background was detected in the interstitial regions of the adult testis using this 2a2-specific antisense probe (Fig. 5, A–D)Citation , nor over the whole section when sense 2a2 probe was used (data not shown). These results are similar to those obtained in previous studies (23) . However, an apparent, though weak, signal was consistently seen over interstitial regions when hybridization took place with the 11a2-specific antisense probe (Fig. 5, E–H)Citation . This signal was limited to the interstitial regions because no signal above background could be observed over the germ cells within the tubules. Sense-oriented 11a2 probes did not reveal any signal above background (data not shown). Furthermore, when the 11a2 transcript-specific antisense probe was applied to testis sections from at/at mutant mice, an apparent signal was noted over the enlarged interstitial regions, but not within the tubules (Fig. 6, A and B)Citation , suggesting expression by somatic cells. When the 2a2-specific antisense probe was used for hybridization on these sections, no signal could be obtained (Fig. 6, C and D)Citation , as in the case of the sense control hybridizations (data not shown), suggesting expression specificity to germ cells. We conclude that the two alternatively spliced mRNAs of Meig1, represented by clones 11a2 and 2a2, are differentially expressed in the somatic and in the germ cell compartments of the testis. The 2a2 transcript is germ cell specific, whereas the 11a2 is somatic specific. Interestingly, although the germ cell-specific transcript (2a2) could not be detected in ovaries of mature females (in accordance with our previous results; Ref. 24 ), reverse transcription-PCR analysis, using primers specific to the 11a2 5' UTR, revealed that low levels of the somatic transcript are present in the ovary (data not shown). However, the identity of the cells within the ovary that express the somatic transcript and the MEIG1 protein are presently under study.



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Fig. 3. A, schematic presentation of the two alternatively spliced Meig1 transcripts represented by the cDNA clones 11a2 and 2a2. The two transcripts share the same Meig1 ORF and 3' UTR, but differ in their 5' leader sequences. The 5' leader of the 11a2 transcript contains a short upstream ORF. The restriction sites used to generate transcript-specific probes are indicated. This diagram is not drawn to the exact scale. B, a potential stable secondary structure ({Delta}G = -62.7 Kcal/mol) that can be formed by the 5' leader of the 11a2 transcript. The 5' end of this structure consists of the EcoRI site and the GCC nucleotides at its 3' end are followed by the AUG of the major Meig1 ORF.

 


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Fig. 4. Northern blot analysis of poly(A)+ RNA (8 µg) from testes of pups at different postnatal ages (pnd 5–30). An EcoRI-Nae1 fragment and an EcoRI-SacI fragment from the 5' UTR of clones 2a2 and 11a2, respectively, were used as transcript-specific probes. The same blot was used for both hybridizations so that the 2a2 probe was stripped off the membrane before the 11a2-specific probe was applied. Exposure time to the phosphor screen was10 h. After 11a2 hybridization, the blot was stripped and rehybridized with a ß-actin probe to verify RNA integrity (data not shown).

 


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Fig. 5. In situ hybridization analysis of testis sections from normal adult animals after hybridization with antisense {alpha}35S-UTP-labeled RNA probe specific to clone 2a2, bright field (A) and dark field (B); exposure time, 10 days. C, bright field of higher magnification of some of the tubules shown in A. D, dark field view after hybridization with antisense probe. Bright field (E) and dark field (F) after hybridization with antisense {alpha}35S-UTP-labeled RNA probe specific to clone 11a2; exposure time, 10 days. G, bright field of higher magnification of some of the tubules shown in E. H, dark field view after hybridization with antisense probe. Arrows point to interstitial regions with mild expression of the 11a2 transcript (F and H). Sense {alpha}35S-UTP-labeled RNA probe, specific for both the 2a2 and the 11a2 transcripts, were used for control hybridizations. No signal above background was detected with the sense controls (data not shown). Bar, 50 µm.

 


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Fig. 6. In situ hybridization analysis of testis sections from a germ cell-deficient mutant atricosis mouse (at/at). Bright field (A) and dark field (B) after hybridization with antisense {alpha}35S-UTP-labeled RNA probe specific to clone 11a2; :Exposure time, 10 days. Bright field (C) and dark field (D) after hybridization with antisense {alpha}35S-UTP-labeled RNA probe specific to clone 2a2; exposure time, 10 days. Sense {alpha}35S-UTP-labeled RNA probe, specific for both the 2a2 and the 11a2 transcripts, were used for control hybridizations. No signal above background was detected with the sense controls (data not shown). Bar, 50 µm.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Traditionally, genes that are expressed in the testis and are assumed to play some role during spermatogenesis are divided into three categories: (a) those that are expressed in all somatic tissues, but have a quantitatively distinct expression in the testis; (b) those that are expressed in other somatic tissues, but have a testis-specific transcript; and (c) those that are expressed exclusively in the testis. Inasmuch as no Meig1 transcripts were detected, in previous studies, in any male tissue except the testis, Meig1 was classified as a member of the third group (i.e., testis-specific) and assumed to be expressed in a germ cell-specific manner (23) . However, immunoblotting analysis of the MEIG1 protein profile during testicular development revealed steady state levels of the monomeric forms of MEIG1 independent of the developmental stage that was examined, including pnd 5 (25) . These results suggested that MEIG1 expression in the testis is not confined to germ cells because the testes of animals at early postnatal developmental stages do not contain germ cells, except for very few mitotically dividing spermatogonia cells (26 , 29) . This assumption has borne out during the present study, given that the MEIG1 protein was observed in testes of germ cell-deficient mutant mice and in the interstitial somatic cells of normal adult males, using immunoblotting and immunohistochemistry. Furthermore, MEIG1 levels in Leydig cells were higher than in any other cell type within the normal testis, suggesting that MEIG1 might play an important role in these somatic cells. Considering the switch in the phosphorylation state of the dimeric form of MEIG1, from a Mr 32,000 serine threonine phosphorylated form to a Mr 31,000 tyrosine phosphorylated form, as germ cells enter meiosis (25) , together with the fact that the Mr 32,000 form seems to be the predominant form expressed in somatic cells, as evident in early postnatal stages (25) and in germ cell-deficient mice (this study), it is reasonable to conclude that this protein plays differential roles in germ cells and in somatic cells. This conclusion is further supported by our recent findings that the Mr 31,000, but not the Mr 32,000 form, enters the nucleus of primary spermatocytes that have initiated meiosis and binds to the meiotic chromosomes, whereas no MEIG1 protein could be detected either in nuclear fractions or bound to mitotic chromosomes of somatic cells.4 This suggests that in germ cells MEIG1 plays a role during the meiotic differentiation. However, as of yet we do not have a clue as to the potential somatic function of this protein.

In this study, we demonstrated that the two alternatively spliced Meig1 transcripts are differentially expressed in somatic cells and in germ cells. The different 5' ends of the two RNA species suggest that the cell-type specificity might be elicited by the use of alternative promoters, as in the case of the proenkephalin gene (30) , the farnesyl PPi synthetase gene FPP (31) , and the Cu/Zn superoxide dismutase gene SOD1 (32) , all of which express somatic and germ cell-specific transcripts.

One practical conclusion that can be drawn from our results is that characterization of the expression pattern of a gene based on RNA analysis should be treated with caution. Although not common, our results demonstrate that RNA expressed at undetectable levels, or very transiently can, nevertheless, support rather high steady-state levels of a protein in a specific tissue. Other examples of such uncoupling of RNA and protein levels include cdc2 transcripts in mammalian cells, in which the level of the p34cdc2 protein remains constant all along the cell cycle while the mRNA level oscillates in a cell cycle-dependent manner (33) , mammalian testis Cu/Zn superoxide dismutase, SOD-1 (32) , and Saccharomyces cerevisiae RAD50 transcripts (34) .

Two structural features characterize the 5' leader of the somatic Meig1 transcript: (a) it contains a short uORF that precedes the major ORF (Fig. 3A)Citation ; and (b) it can form a stable ({Delta}G = -62.7 Kcal/mol) secondary structure (Fig. 3B)Citation . According to the generally excepted model for selection of translation initiation site, the 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG codon that lies within a favorable sequence context (reviewed in Ref. 35 ). This implies that uORF would preclude access of the translation machinery to the second, downstream AUG, especially if the first one is within a preferred sequence context (35, 36, 37, 38) . Indeed, Oliveira and McCarthy (39) demonstrated by combining different 5'-UTRs to the chloramphenicol transacetylase ORF that an uORF is translated efficiently, but at the same time translation of the chloramphenicol transacetylase ORF is inhibited. Furthermore, it has been shown that secondary structures in the leader sequence, whether long or short, are major barriers to translation (35 , 40 , 41) . Because the somatic Meig1 transcript contains both translational inhibitory elements, one would expect it to be translated very inefficiently. However, the fact that the MEIG1 protein is fairly abundant in the testicular somatic cells, especially in Leydig cells, despite the low levels of somatic transcript, suggests highly efficient translation. Furthermore, in vitro translation experiments with the somatic and the germ cell transcripts revealed efficient translation of both transcripts without any notable differences (data not shown). These results, together with other examples of mRNAs that are efficiently translated despite an uORF or a structured 5' UTR, including the growth/differentiation factor 1 (42) , the retinoic acid receptor-ß2 (43) and the cauliflower mosaic virus 35 S RNA (44) , indicate that additional factors must regulate translational efficiency of mRNAs containing structured 5' UTR and uORF. An idea of what the nature of these factors maybe can be obtained from the yeast GCN4 mRNA, which has four uORFs. It has been shown that the translational control of this gene is mediated by: (a) phosphorylation, and hence reduced activity, of the eukaryotic initiation factor 2; (b) intercistronic distance; (c) sequences surrounding the termination codon of the uORF; and (d) sequences upstream to the uORF (38 , 45, 46, 47) . Yet other studies have shown that the actual short peptide encoded by the uORF might also play a regulatory role on the translation efficiency of the major downstream ORF (reviewed in Ref. 38 ). This suggests that various structural elements in the somatic Meig1 transcript might be involved in its efficient translation. The potential effect of the uORF, its surrounding sequences, and the peptide encoded by it on the efficiency of translation and stability of the somatic Meig1 transcript are presently under study.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Source of Tissues.
Mature BALB/c mice (>60 days of age) were used as a source of normal mouse tissues for all experiments, unless otherwise stated. Tissues for RNA isolation were frozen in liquid nitrogen immediately on dissection and stored at -70°C until used. All animals were sacrificed by cervical dislocation.

RNA Isolation and Northern Blot Hybridization Analysis.
RNA was isolated using the Tri-Reagent solution (Sigma Chemical Co.), according to the manufacturer’s protocol. Poly(A)+ RNA was selected using Stratagene’s mRNA isolation kit. RNA samples were electrophoresed on denaturing 1% agarose- 2.2 M formaldehyde gels, blotted onto Nytran membranes (Schleicher & Schuell), according to standard procedure (48) , and UV cross-linked to the membrane. Prehybridization and hybridization were performed at 42°C for 3–4 h and 15–20 h, respectively, as described in Don et al. (24) . Labeled probe (multiprime labeling system; Amersham Corp.) was added to the hybridization solution to a final concentration of 1–2 x 106 cpm/ml. Final washes, 20 min each, consisted of 0.1 x SSC, 0.1% SDS at 65°C, and 0.1 x SSC at 65°C, consecutively. Filters were then exposed to BAS-MP 2040S phosphor screen for the times indicated in the text, and the images were analyzed by BAS-1500 phosphor imager (Fujifilm).

Immunoblotting (Western) Analysis of Proteins.
Protein extracts were separated on a 15% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose filter (using the MiniProtein II cell and the MiniProtein II transfer cell, respectively; Bio-Rad), according to standard protocols. After electrotransfer, filters were washed in water for 3 min, monitored by staining with ponceau stain (Sigma Chemical Co.), and then blocked by 1% BSA in TBST [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 1 h at room temperature. Affinity purified rabbit anti-MEIG1 polyclonal antibodies (25) were used as a primary antibody at a dilution of 1:20,000. Unbound antibodies were removed by three TBST washes. Antirabbit IgG- alkaline phosphatase-conjugated antibodies (Promega) were used as secondary antibodies at a dilution of 1:7,500. Bands were detected by incubation with alkaline phosphatase substrate (Promega) for 15–30 min, followed by washing with water.

Immunohistochemical Analysis.
An indirect immunofluorescent localization of the MEIG1 protein was applied. Sections of paraffin-embedded tissue ({approx}5 µm thick) were applied onto 3-aminopropyltriethoxy-silane (Sigma Chemical Co.)-treated slides, incubated overnight in 37°C, and kept in a dust-free box at room temperature until used. Upon usage, slides were heated to 58°C for 20 min and then deparaffinized in xylene for 20 min, rehydrated in decreasing ethanol concentrations (100%, 95%, 70%, and 50% for 2 min each), and washed in H2O for 10 min and in PBS for 20 min. Slides were blocked overnight (4°C) with PBS containing 20% normal goat serum and 0.1% Triton x-100. Primary antibody (rabbit anti-MEIG1), diluted 1:100 in blocking solution, was applied to the sections at room temperature for 1 h (alternatively, overnight at 4°C) and then removed by three washes in PBS-Tween (0.05% Tween 20) for 15 min each. FITC-conjugated goat antirabbit IgG antibody (Zymed), diluted 1:50 in PBS containing 20% goat serum, was used as a second antibody (1 h in the dark at room temperature), followed by two washes in PBS-Tween (10 min each). Nuclei were stained for 3 min with PI solution [10 mM Tris (pH 8.0), 1 mM NaCl, 0.1% NP40, 0.7 mg/ml RNase A, and 0.05 mg/ml propidium iodide), and the sections were washed twice in PBS-Tween (15 min each). One drop of antibleaching retardant (Bio-Rad) was added to the sample before sealing with a coverslip. Sections were viewed with the MRC 1024 confocal microscope (Bio-Rad), using an argon/krypton laser. Excitation was at 488 nm for FITC and at 568 nm for PI.

In Situ Hybridization.
For in situ hybridization analysis of testicular tissues, paraffin-embedded sections were used. Sections ({approx}5 µm) were treated as follows: deparaffination with xylene (10 min, twice), rehydration with decreasing ethanol concentrations (100%, 95%, 85%, 70%, 50%, and 30% for 2 min each), 0.85% saline and PBS washes (5 min each), postfixation in 4% paraformaldehyde (20 min), PBS washes (5 min, twice), proteinase K treatment for 8 min [20 µg/ml in 5 mM EDTA and 50 mM Tris-HCl (pH 7.5)], PBS wash (5 min), refixation in 4% paraformaldehyde (5 min), incubation in 0.1 M triethanolamine (pH 8.0) with acetic anhydride added to 0.25%, PBS and 0.85% saline washes (5 min each), and dehydration in increasing ethanol concentrations (50–95%, 2 min each and 2 min in 100%, twice). Prehybridization and hybridization were performed using the procedure described by Jaffe et al. (49) . Slides were viewed on a AH3-RFCA Olympus photomicroscope using bright and dark field optics.


    Acknowledgments
 
We are grateful to Dr. Ron Goldstin for helpful advice, Drs. Ron Wides and Shelley Schwarzbaum and to Mira Malkov for reviewing the manuscript and for critical comments.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by The Israel Science Foundation, administered by The Israel Academy of Sciences and Humanities; the Maria Rossi Ascoli Fund, administered by the Israeli Ministry of Health-The Chief Scientist’s Office (Grant 970404); a grant from the Ihel Foundation; and a grant from the Bar-Ilan committee for the promotion of research. Back

2 To whom requests for reprints should be addressed, at Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Phone: 972-3-5318963; Fax: 972-3-5351824; E-mail: don{at}mail.biu.ac.il Back

3 The abbreviations used are: pnd, postnatal day; UTR, untranslated region; ORF, open reading frame; uORF, upstream ORF. Back

4 R. Steiner, L. Ever, and J. Don. A 31 kDa dimeric form of MEIG1 localizes to the nucleus of spermatocytes as they initiate meiosis and binds to meiotic chromosomes, manuscript in preparation. Back

Received for publication 9/10/98. Revision received 11/19/98. Accepted for publication 11/24/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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