extfieldsofstudy: Biology Medicine

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Developmental Regulation of Spliced Leader RNA Gene inLeishmania donovani Amastigotes Is Mediated by Specific Polyadenylation*

Leishmania cycles between the insect vector and its mammalian host undergoing several important changes mediated by the stage-specific expression of a number of genes. Using a genomic differential screening approach, we isolated differentially expressed cosmid clones carrying several copies of the mini-exon gene. We report that the spliced leader (SL) RNA, essential for the maturation of all pre-mRNAs by trans-splicing, is developmentally regulated in Leishmania donovani amastigotes and that this regulation is rapidly induced upon parasite growth under acidic conditions. Stage-specific regulation of the SL RNA is associated with the expression of a larger ∼170-nucleotide transcript that bears an additional 15-nucleotide sequence at its 3′-end and is polyadenylated in contrast to the mature SL RNA. The poly(A)+ SL RNA represents 12–16% of the total SL transcript synthesized in amastigotes and is 2.5–3-fold more stable than the poly(A)− transcript. The poly(A)+ SL transcript is synthesized specifically from one class of the genomic mini-exon copies. Polyadenylation of the SL RNA may control the levels of the SL mature transcript under amastigote growth and may represent an additional step in the gene regulation process during parasite differentiation.

Leishmania is a protozoan parasite that infects and replicates within mammalian macrophages, thereby causing a wide spectrum of diseases in humans. The parasite's life cycle comprises two stages involving two different hosts: the flagellated promastigote stage, which is present in the gut of the sand fly vector, and the aflagellated non-motile amastigote present in the phagolysosomes of mammalian macrophages. During its developmental cycle, Leishmania adapts to the different environments encountered by undergoing a series of morphological and biochemical changes mediated by the differential expression of a variety of genes. Until now, only few of these genes have been identified, and the mechanisms of their stage-specific regulation are still poorly understood.
In Leishmania, several studies have shown that post-tran-scriptional regulation of developmentally expressed transcripts involved sequences present mainly in the 3Ј-untranslated regions (1-3) but also in 5Ј-untranslated regions (1) and in intergenic regions between tandemly repeated genes (4,5). Similarly, in trypanosomes, elements in the 3Ј-untranslated and intergenic regions of many mRNAs were found to be involved in the post-transcriptional regulation of gene expression, RNA stability, or translation (6 -10). The predominance of post-transcriptional over transcriptional controls is probably because of the organization of the trypanosomatid genome in large polycistronic transcription units (11)(12)(13)(14). Maturation of all pre-mRNAs in these organisms involves two RNA processing reactions; trans-splicing (15,16) and 3Ј-end cleavage linked to polyadenylation (17,18). Polycistronic precursor RNAs are processed into monocistronic units by the excision of intergenic sequences and the addition of a 5Ј-capped 39-nucleotide spliced leader (SL 1 or mini-exon) to the 5Ј-end of each pre-mRNA by trans-splicing. The mini-exon sequence is donated from a small independent RNA, the SL RNA or mini-exon-derived RNA, which is required for RNA processing, transcript stability, and initiation of translation in a variety of trypanosomatid protozoa (16). Trans-splicing constitutes an essential step in transcript's maturation and, as such, may serve as an important mode for the control of gene expression in kinetoplastida. It has been previously shown that exposure of Trypanosoma brucei procyclics to severe heat shock inhibited trans-splicing and processing of the ␣and ␤-tubulin transcripts (19).
In this study we report that the SL RNA of Leishmania donovani is developmentally regulated in the amastigote stage. This regulation involves a specific polyadenylation of a longer SL transcript, which seems to be synthesized specifically from one class of the genomic mini-exon repeats. This is the first time where polyadenylation of a normally non-polyadenylated RNA is reported as part of a putative regulatory mechanism aiming at controlling gene expression in a specific stage of Leishmania's life cycle. Specific polyadenylation of the SL RNA may represent an additional step in the gene regulation process during Leishmania differentiation into the amastigote form.

EXPERIMENTAL PROCEDURES
Parasite Strains and Culture Media-L. donovani donovani Sudanese 1S2D and L. donovani infantum LEM1317 (from Dr. Bastien, Montpellier, France) strains were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated calf serum and 5 mg/ml hemin. Promastigotes were cultured in RPMI at pH 7.0 and 25°C. To induce axenic differentiation we used a slightly modified protocol published by Joshi et al. (20) where stationary phase parasites were transferred into RPMI medium supplemented with 20% calf serum and MES * This work was supported by Medical Research Council Grant gr-14 500 (to B. P.). 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  to maintain the pH at 5.0 and grown for 5 days at 25°C before being shifted to 37°C at a 5% CO 2 atmosphere for an additional 24 h. Amastigotes were also isolated from the spleens of gold Syrian hamsters infected intraperitoneally with 5 ϫ 10 7 L. donovani 1S2D amastigotes as described (21).
Nucleic Acid Preparations and Analyses-Total RNA of L. donovani promastigotes and amastigotes was isolated using the guanidinium isothiocyanate method with TRIzol (Life Technologies, Inc.). cDNAs corresponding to all poly(A) ϩ mRNA transcripts were synthesized using an oligo(dT) 18 primer and the Superscript II reverse transcriptase (Life Technologies, Inc.). Southern and Northern blot hybridizations were performed following standard procedures (22). The two probes used for these studies correspond to the intron sequence of the spliced leader RNA gene generated by PCR using P1 (5Ј-ATTGGTATGCGAAACT-TCCG-3Ј) and P2 (5Ј-CCGCTACGGAAGCCGCCGGC-3Ј) primers (see Fig. 8) and to the whole mini-exon repeat, as part of the 434-bp NcoI fragment. Neomycin phosphotransferase (neo) and hygromycin phosphotransferase (hyg) specific probes were made by PCR and used for determining the vector copy number in the transfectants. The L. donovani infantum LEM1317 cosmid library was prepared by ligating sizeselected Sau3AI partial digest products of genomic DNA with the shuttle vector cL-HYG (23), cleaved by BamHI, and packaged using the Gigapack Gold II kit (Stratagene). The library was gridded onto nylon membranes at the Toronto Genome Center (Hospital for Sick Children) to generate high density filters of 92 ϫ 4 ϫ 4. High density filters of the same series were hybridized independently to L. donovani amastigote and promastigote 32 P-labeled cDNA probes.
Recombinant DNA Constructs and Transfections-Constructs pSP␣neo␣-miniexA, pSL␣neo␣-miniexB, and pSL␣neo␣-miniexC bearing the three different mini-exon genomic copies of L. infantum LEM1317 were made as follows. The 424-bp NcoI fragments (copies B and C) from cosmid 3H4 (carrying ϳ20 kilobase pairs of the mini-exon locus) were subcloned into the NcoI site of pSL1180 (Amersham Pharmacia Biotech), and the SmaI-BamHI ␣neo␣ expression cassette (24) from vector pSP72␣neo␣ was introduced into EcoRV-BamHI sites of pSL1180 to generate pSL␣neo␣-miniexB and pSL␣neo␣-miniexC, respectively. The 434-bp NcoI fragment (copy A) was subcloned into the BamHI site of pSP72␣neo␣ expression vector following a Klenow polymerase treatment. Expression of the neo gene in pSP72␣neo␣ and pSL␣neo␣ is driven by the intergenic region of the ␣-tubulin gene (represented here by ␣) (25). The cDNAs of the poly(A) ϩ and poly(A) Ϫ SL transcripts (see below) were amplified by RT-PCR using P1 (see Fig.  8) and an oligo(dT) 18 as primers and subcloned into pCR TM 2.1 (Invitrogen) to generate pSL-RNA(A) ϩ and pSL-RNA(A) Ϫ vectors, respectively. The nucleotide sequence of the different genomic mini-exon repeats of L. donovani 1S2D and L. infantum LEM1317 and of the cDNAs was determined by an ABI Prism 377 DNA automated sequencer. 10 -20 g of plasmid or cosmid DNAs were used for transfections into L. donovani by electroporation as described previously (24).
Nuclear Run-on and RNA Stability Assays-Nuclear run-on assays were performed essentially as described by Quijada et al. (3) using nuclei freshly isolated from L. donovani exponential phase promastigote and amastigote cultures. To look for RNA stability, late-log phase promastigotes were submitted to growth conditions mimicking those found in the phagolysosomes at pH 5.0 for 5 days followed by 1-2 h of incubation at 37°C in the presence of 5% CO 2, prior to the addition of 10 g/ml actinomycin D (Sigma). Cells were harvested by centrifugation, and total RNA was extracted at different time points of actinomycin D treatment: 0, 30 min, 1, 2, 4, and 8 h. Total RNA samples were subjected to Northern blot analysis using the intron sequence of the mini-exon gene as a probe. Quantitation of the SL transcripts was done by densitometric analysis using a PhosphorImager with the Image-Quant 3.1 software.
Mapping of the 3Ј-End of Poly(A) Ϫ and Poly(A) ϩ SL RNAs-To map the 3Ј-end of the polyadenylated SL RNA, a double-stranded cDNA was synthesized from axenic amastigotes using P1 and an oligo(dT) 18 as primers. 3Ј-RACE studies were used to map the 3Ј-end of the nonpolyadenylated SL transcript. A poly(A) tail was added at the 3Ј-end of this transcript following incubation of 10 g of total L. donovani promastigote RNA with 5 units of poly(A) polymerase (Life Technologies, Inc.) according to the manufacturer's recommendations. RT-PCR reactions were carried out using the above set of primers. The PCR products were then gel-excised, further purified using phenol/chloroform extraction, subcloned into the pCR TM 2.1 vector, and sequenced.

Differential Expression of the SL RNA Gene in L. donovani
Amastigotes-To identify genes that are developmentally regulated, we developed a genomic differential screening approach where high density filters, each covering approximately one parasite genome, were used. A genomic cosmid library made from L. donovani infantum strain LEM1317 was gridded onto high density filters (1536 cosmid clones/filter), which were hybridized with 32 P-labeled cDNA probes derived from promastigote and amastigote RNAs. Comparative analysis of the hybridization signal intensity between the promastigote and amastigote cDNA probes in four series of high density filters allowed the identification of a number of cosmid clones that were much more expressed in amastigotes. Normalization of the amount of bacteria layered at each position was obtained by hybridization with a vector-specific probe (hyg). Among a large number of clones selected on the basis of their higher expression in amastigotes, 10 were related. Partial sequence analysis of these clones indicated that we were dealing with the spliced leader RNA gene locus. From those 10, cosmid 3H4 was chosen for further analysis (Fig. 1A). Southern blot differential hybridization of cosmid 3H4 using promastigote and amastigote cDNA probes revealed the presence of a 434-bp NcoI genomic fragment hybridizing much stronger to the amastigote cDNA (Fig. 1B). The nucleotide sequence of this fragment confirmed the presence of the mini-exon gene repeat.
Total RNA preparations from promastigotes and axenic amastigotes were hybridized with a probe corresponding to the intron sequence of the L. infantum mini-exon repeat that we have sequenced. Two transcripts of different sizes were detected in the amastigote RNA preparation, one corresponding to the expected ϳ86-nt SL RNA and a second larger transcript of ϳ170 nt ( Fig. 2A). In addition, a 2.5-fold increase in the 86-nt SL transcript was observed in the amastigote RNA preparation ( Fig. 2A). To determine whether this developmental regulation of the SL transcript was in fact a physiological event and was not because of an artifact due to the axenic conditions used for parasite differentiation, we tested amastigotes freshly isolated from infected hamsters. As shown in Fig. 2B, a larger size SL transcript was detected in the RNA preparation from hamsterderived amastigotes similar to what was seen when using the FIG. 1. Differential genomic screening for the isolation of amastigote-specific clones. A, differential hybridization of one set of high density filters of the L. donovani infantum LEM1317 cosmid library with promastigote (Pro) and amastigote (Am) cDNA probes. The arrow indicates cosmid 3H4 selected for these studies on the basis of its higher expression in amastigotes. B, Southern blot hybridization of cosmid 3H4 with promastigote and amastigote cDNA probes. The arrow pinpoints a ϳ434-bp NcoI fragment showing a differential hybridization signal. This fragment contained the mini-exon repeat as demonstrated by sequence analysis. axenic amastigotes. The larger SL transcript was estimated, by densitometric analysis using PhosphorImager scanning of several independent Northern blots, to represent 12-16% of the total SL RNA synthesized within the amastigote cell.
Stage-specific Expression of the SL RNA Is Induced upon Growth at Acidic Conditions-During their life cycle Leishmania are exposed to temperature and pH changes that trigger stage-specific differentiation (26). Elevated temperature or acidic pH has been associated with the induced expression of a number of genes (21,27,28). To assess the role of pH or of temperature in the differential regulation of the SL RNA, different culture conditions were tested. L. donovani promastigotes were subjected to a pH shift from pH 7.0 to 5.0 for 5 days at 25°C or to a temperature shift at 37°C in a 5% CO 2 environment, or to both. Total RNA was extracted at different time points and analyzed by Northern blot using the PCR-amplified spliced leader intron sequence as a probe. As shown in Fig. 3 acidic pH seems to be the main factor that induces the expression of the larger ϳ170-nt SL transcript but is also responsible for the increased levels of the ϳ86-nt SL RNA. This pH-dependent induction of the SL transcripts occurs rapidly within the first hours of incubation under acidic conditions ( Fig. 3 and not shown). Developmental regulation of the SL RNA is specific to the amastigote stage because no larger transcript or any significant increase in the SL RNA levels were observed in stationary or metacyclic L. donovani cultures (Fig. 3).
Differential Regulation of the SL RNA Is Because of a Specific Polyadenylation in Amastigotes-We hypothesized that the larger 170-nt SL RNA present in amastigotes might be the result of an alternative 3Ј-end formation or of a post-transcriptional modification of the transcript. Several previous studies have reported that the kinetoplastid SL RNA, as for most of the SL RNAs in nematodes (29), is not polyadenylated and that it is constitutively expressed throughout the developmental stages of these parasites (30,31). However, the SL transcript has been detected within the poly(A) ϩ RNA fraction of T. brucei bloodstream forms (32). To test whether polyadenylated SL RNA could also be found in Leishmania, we first synthesized cDNAs from total RNA of L. donovani promastigotes and axenic amastigotes using and oligo(dT) primer, allowing only the polyadenylated RNAs to be reverse transcribed, and hybridized them with the mini-exon repeat as a probe. Because of the presence of the 39-nt spliced leader at the 5Ј-ends of all pre-mRNAs, a hybridization smear corresponding to all polyadenylated transcripts was observed in both promastigote and amastigote cDNAs (Fig. 4A). A cDNA fragment corresponding to the SL RNA gene was found exclusively in the amastigote cDNA preparation, strongly suggesting that polyadenylated forms of the SL RNA could indeed be present in Leishmania amastigotes (Fig. 4A). To verify whether this poly(A) ϩ SL transcript was also found in L. donovani amastigotes isolated from infected hamsters, similar experiments were performed, and they gave rise to identical results (data not shown). The presence of the poly(A) ϩ SL transcript in amastigotes was confirmed by RT-PCR studies where a cDNA synthesized from a poly(A) ϩ fraction was subjected to PCR amplification using a combination of P1 (see Fig. 8) and oligo(dT) primers to amplify part of the SL transcript that lacks the common 39-nt spliced leader. A ϳ130-bp cDNA fragment amplified by PCR and corresponding to the poly(A) ϩ SL RNA was detected by hybridization only in the amastigote poly(A) ϩ fraction (Fig. 4B). PCR products hybridizing to the intron probe were extracted from the agarose gel and subcloned into vector pCR TM 2.1. Sequence analysis definitively confirmed the poly(A) ϩ addition in the SL transcript expressed specifically in the amastigote stage (Fig. 5A).
To compare the 3Ј-end of the poly(A) ϩ and poly(A) Ϫ SL transcripts, we also mapped the 3Ј-end of the normally non-polyadenylated transcript in our L. donovani 1S2D strain by a 3Ј-RACE assay. The L. donovani poly(A) Ϫ SL RNA ended at position 86 (Fig. 5A), slightly different from what has already been published for other Leishmania species where the 3Ј-end was mapped at nucleotide 89 (31) or nucleotide 96 (33). Sequence comparison between the cDNAs corresponding to the polyadenylated and non-polyadenylated SL transcripts revealed a significant difference at their 3Ј-ends. Indeed, the poly(A) ϩ SL RNA contained 15 additional nucleotides, ending by four Ts part of the 8T-track sequence (see Fig. 8) in comparison with the poly(A) Ϫ transcript (Fig. 5A). A long poly(A) tail of at least 46 As was added immediately after the four Ts. Considering that the poly(A) ϩ SL RNA is ϳ170 nt long, the poly(A) tail could include as much as 80 As. Comparison of the predicted secondary structures of the poly(A) Ϫ and poly(A) ϩ SL RNAs using the SQUIGGLES program did not suggest a sig-nificant overall change in the free energy required for structure formation (⌬G), but it revealed the presence of a third stem loop structure at the 3Ј-end of the SL poly(A) ϩ RNA (Fig. 5B), which could possibly be a substrate for RNA binding proteins.
Polyadenylation of SL RNA Increases Its Stability-The effect of pH on the stability and accumulation of the SL transcripts was addressed. We first evaluated, by run-on experiments, whether the increase in poly(A) Ϫ SL RNA accumulation or the presence of the poly(A) ϩ SL transcript in amastigotes (see Figs. 2 and 3) was because of higher SL RNA synthesis rates in this parasite stage. Nuclei were freshly isolated from promastigotes, and axenic amastigotes and radiolabeled nascent RNAs were extracted and used to probe the mini-exon and control DNA sequences. No differences were observed in the overall synthesis of SL RNA between promastigotes and amastigotes (Fig. 6), suggesting that the regulation occurs post-transcriptionally. Although secondary structure predictions did not suggest that the addition of the 15 nucleotides at the 3Ј-end of the larger SL RNA should increase its stability significantly (see Fig. 5B), it is, however, known that polyadenylation in general increases message stability (for review see Ref. 34). We therefore looked to see whether the poly(A) ϩ SL RNA was more stable compared with the poly(A) Ϫ transcript. Because we showed that the maximum accumulation of the polyadenylated SL RNA is observed in cells maintained at pH 5.0 (Fig. 3), we evaluated the stability of the SL transcripts under these growth conditions. In these assays, L. donovani promastigotes were transferred from pH 5.0 and 25°C to 37°C for 1 h to adapt in these conditions prior to the addition of 10 g/ml actinomycin D, an inhibitor of RNA synthesis. Total RNA samples were extracted at different time points following the addition of actinomycin D to the culture medium and ana- lyzed by Northern blot using the spliced leader intron as a probe. The relative amount of the poly(A) ϩ and poly(A) Ϫ SL transcripts present in the cell at each time point was compared with the RNA levels present at time 0. Quantitative evaluation of the SL RNA levels obtained by densitometric analysis of radiolabeled membranes using a PhosphorImager demonstrated that the poly(A) ϩ SL RNA was 2.5-3-fold more stable compared with the non-polyadenylated transcript (Fig. 7, A  and B). Similar results were obtained from two more independent experiments. The small shortening of the poly(A) ϩ SL RNA observed over time may correspond to differences in RNA migration in that part of the gel (not shown). In light of these results, the half-lives were established at 4 h for the poly(A) ϩ SL RNA and at 2 h for the poly(A) Ϫ transcript.
Stage-specific Polyadenylation of One Class of the Mini-exon Genomic Copies-To examine whether differences within the non-transcribed spacer of the mini-exon genomic copies could account for the formation of the new 3Ј-end of the poly(A) ϩ SL RNA, the nucleotide sequence of several copies was determined. Several ϳ434-bp NcoI copies derived from cosmid 3H4 were subcloned into pSP␣neo␣ and PSL␣neo␣ expression vectors, and their nucleotide sequence was compared. By compiling the sequencing data from a large number of clones (ϳ20), we classified the mini-exon gene repeats into three classes (A-C). The alignment of the nucleotide sequences of these three different copies along with a previously published copy derived from another L. infantum strain is presented in Fig. 8. As expected, the 39-nt spliced leader was highly conserved, whereas three nucleotide changes and one short deletion were observed in the intron sequence of the two L. infantum strains. However, considerable differences were noted within the nontranscribed spacers of the two L. infantum strains (see Fig. 8). Analysis of the three copies of the LEM1317 strain showed only one difference located between nucleotides 140 and 150. Copy A indeed contains a 10-bp GC-rich stretch, which is absent in copies B and C, where only a single base pair mismatch from a T to C at position 150 distinguishes these two copies (Fig. 8).
We tested whether one of these genomic copies was associated with the larger ϳ170-nt SL transcript. Accordingly, we made the appropriate constructs to overexpress each one of the three mini-exon gene repeats (A-C) in L. donovani (Fig. 9A). We also transfected cosmid 3H4, which contains the three different classes of the mini-exon repeats. Stable transfectants were subjected to differentiation from promastigotes to axenic amastigotes. RNA samples from wild type and transfectants were analyzed by Northern blot using a probe corresponding to the spliced leader intron sequence. A significant increase in the poly(A) ϩ SL transcript was detected only in the transfectants expressing copy A or cosmid 3H4, respectively (Fig. 9B). No differences between the transfectants carrying copy B or C and the wild-type strain were observed, suggesting a certain specificity of the polyadenylation process. To ascertain these results we compared the copy number of the expression vectors in the transfectants by hybridization studies using neo and hyg genes as probes. No significant differences in the copy number of the three mini-exon repeats were noted between transfectants (Fig.  9C), thus supporting further the implication of copy A in the formation of the longer SL transcript that becomes polyadenylated. By random sequencing of a large number of clones derived from cosmid 3H4 we roughly estimated that each class is equally represented in this cosmid. DISCUSSION Using a genomic differential screening approach based on the hybridization of high density filters with promastigote and amastigote cDNA probes, we have isolated a number of differentially expressed cosmid clones that contained several miniexon gene repeats. In kinetoplastida, the SL RNA is encoded by a cluster of ϳ150 -200 tandemly repeated copies per genome. Each repeat comprises a highly conserved 39-bp exon sequence, a moderately variable (55-101 bp) intron sequence, and a nontranscribed highly variable spacer between different genus and species (35)(36)(37)(38). The spliced leader, a common 39-nucleotide sequence, is transferred via trans-splicing from the precursor SL RNA to the 5Ј-end of all trypanosomatid pre-mRNAs.
We have shown that stage-specific regulation of the SL RNA in both L. donovani and L. infantum is mediated by a specific polyadenylation process. Upon growth of the parasite at acidic pH, 12-16% of the total SL RNA becomes polyadenylated, giving rise to a larger 170-nt transcript in addition to the 86-nt mature SL RNA (Figs. 2, 3, and 5A). A temperature shift alone (25-37°C) was not capable of inducing the expression of the polyadenylated SL RNA or of increasing the amount of the non-polyadenylated transcript (Fig. 3). Several reports have confirmed the constitutive expression of the SL RNA in all developmental stages of trypanosomes and Leishmania, and it is generally assumed that the SL RNA is not polyadenylated (30,31). Similarly, spliced leader RNAs in the nematode Caenorhabditis elegans are normally not polyadenylated although one case has been reported (29). Polyadenylated forms of the SL transcript have been identified in poly(A) ϩ RNA fractions from short stumpy bloodstream forms of T. brucei, however (32).
Mapping of the 3Ј-end of the poly(A) Ϫ and poly(A) ϩ SL RNAs revealed that the poly(A) ϩ SL RNA was longer by 15 nucleotides compared with the 86-nt non-polyadenylated SL transcript (Fig. 5A). Little is known about the process of trypanosomatid mRNA 3Ј-end formation. Recent data reported that transcription termination of the Leishmania tarentolae SL RNA is mediated by the downstream T-track at the base of stem-loop III (39). We have shown that an alternative maturation site was used by the poly(A) ϩ SL RNA and that this transcript was ended by four Ts as part of an 8Ts track sequence in the mini-exon gene repeats present in our strain (Figs. 5A and 8). We have also demonstrated that only one of the three different classes of the mini-exon gene in L. infantum, the mini-exon copy A, showed a significant increase in the levels of the poly(A) ϩ SL transcript under pH stress at relatively similar rates as those of cosmid 3H4 carrying the three classes of the mini-exon genomic copies (Fig. 9B). These results support the implication of this particular mini-exon copy as a template for the formation of the longer 170-nt SL transcript. It is not clear, however, how the 10-bp GC-rich motif present only in the non-transcribed spacer of copy A between nucleotides 140 and 150 could influence maturation of the SL RNA. It is possible that changes in the secondary structure could allow the polymerase to pause further downstream. Alternatively, it is possible that the mini-exon copy A is transcribed by a different polymerase. Reports on the type of polymerase that transcribes the mini-exon gene of kinetoplastid protozoa have yielded conflicting data. It has been reported that the miniexon repeats are transcribed by polymerase II (40,41), but other studies support that they are transcribed by polymerase III (42). It is assumed from several studies that the tandemly repeated mini-exon genes in trypanosomatids are independently transcribed (41,43), and this is in line with the possibility that some mini-exon copies could be transcribed by different polymerases.
It is assumed that the 3Ј-end of mature mRNAs is generated by a cleavage of the pre-mRNA at the polyadenylation site followed by the addition of the poly(A) tail (44). No conserved sequence motif like the AAUAAA of higher eukaryotes has been identified, however, upstream of the kinetoplastid mRNA polyadenylation sites, suggesting a mechanism different from the one used in other systems (15). One important factor determining the site of polyadenylation in Leishmania is the positioning of a functional splice acceptor site immediately downstream as polyadenylation and trans-splicing seem to be coupled in this organism (17). Accurate polyadenylation in trypanosomatids is often determined by pyrimidine-rich elements in the intergenic regions (45)(46)(47). Polypyrimidine tracts are recognized by both the trans-splicing and polyadenylation machineries either sequentially or simultaneously (18). The additional 15-nt sequence part of the very 3Ј-end of the poly(A) ϩ SL RNA is rich in pyrimidines (10 nucleotides of 15), and it contains an AG splice acceptor site immediately preceded by a pyrimidine tract (5Ј-AGGCGCTCTTTT-3Ј) (see Fig.  5A). This splice acceptor site may be recognized by splicing factors that could activate polyadenylation. This has been seen in higher eukaryotes (48).
Although polyadenylation has been reported in bacteria and some plastid mRNAs to accelerate transcript degradation (49,50) it is generally assumed that in most eukaryotic systems the poly(A) tract protects mRNAs from rapid degradation (51)(52)(53). In addition, the poly(A) tail influences nuclear processing of the FIG. 8. Sequence alignment of the three L. donovani infantum LEM1317 mini-exon gene copies. The ϳ434-bp NcoI fragments derived from cosmid 3H4 were sequenced, and copies A, B, and C, which can be found under the accession numbers AF097653, AF097654, and AF097655, respectively, were aligned together with the mini-exon repeat of another L. donovani infantum MHOM 80 strain. The 39-nt common spliced leader is underlined. Open boxes indicate the differences depicted between the nucleotide sequences of the mini-exon gene repeats. Primers P1 and P2 used for PCR and RT-PCR studies are also indicated.
pre-mRNA, nucleocytoplasmic transport, and translation (34,(53)(54)(55). Polyadenylation of the SL RNA in Leishmania increases its stability by a factor of 2.5-3 as estimated from the actinomycin D data (Fig. 7). The presence of the 15-nt additional sequence at the 3Ј-end of the poly(A) ϩ SL RNA does not seem to introduce any significant change in the ⌬G (from Ϫ21.2 to Ϫ18.8 kcal/mol) required for RNA structure formation, but it allows the formation of a stem-loop structure at the 3Ј-end of the transcript (Fig. 5B). Stem-loop structures at the 3Ј-ends of many transcripts function as binding sites for regulatory proteins that could affect RNA transport, translation, and half-life (34). It is possible that the poly(A) ϩ SL RNA is exported into the cytoplasm as part of a regulatory mechanism to decrease the overall levels of SL RNA in the nucleus of the amastigote cells. Amastigotes replicate more slowly than promastigotes, and it is likely that less SL RNA is required for mRNA processing by trans-splicing. This is in line with our observation that even the poly(A) Ϫ SL RNA accumulates in the amastigote stage. Indeed, a 2.5-fold increase in SL transcript levels was seen during differentiation of the parasite into the amastigote form (Figs. 2 and 3). The half-life of transcripts could change in response to nutrient levels, cell growth rates, and temperature shifts, among other things (56), and this could also be the case for the SL RNA under acidic stress. It is also possible that the polyadenylated SL transcript cannot be used as a donor of the 39-nt spliced leader sequence to the 5Ј-end of all pre-mRNAS. As a consequence, this should decrease trans-splicing reactions in amastigotes by ϳ15%. It has been shown that L. tarentolae SL transcripts that were malformed by more than 1 nucleotide at their 3Ј-ends were unable to participate in trans-splicing or acquiring a proper cap 4 methylation (39).
During their life cycle Leishmania protozoans alternate from the promastigote to the amastigote stage, undergoing several morphological and biochemical changes that are monitored by the differential expression of a variety of genes. Previous studies have reported amastigote-specific regulation of a number of genes: hsp83 (27), hsp100 (57), histone H1 (58), cpb cysteine proteinases (59), A2 gene family (21), and the LmcDNA16 gene family (60). Our studies demonstrated that part of the spliced leader RNA in L. donovani amastigotes is polyadenylated. This polyadenylation is stage-regulated and may correspond to a general post-transcriptional mechanism operating upon parasite differentiation to control gene expression.


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Additive and supra-additive cytotoxicity of cisplatin-taxane combinations in ovarian carcinoma cell lines

The purpose of this study was to compare the growth-inhibitory effect of cisplatin–paclitaxel with that obtained with a cisplatin–docetaxel combination and to assess the type of interaction. Concomitant use of taxanes and cisplatin was studied in seven human ovarian carcinoma cell lines, using the 96-well plate clonogenic assay. Chemosensitivity was expressed in terms of IC50 values, the drug concentration causing 50% inhibition of clonogenic survival. The type of interaction was studied using the area under the survival curve ratios (AUC ratios) obtained by numerical integration. Comparison of the AUC ratio and the surviving fraction (SF) value after taxane alone was made using Student's t-test. The influence of the drug concentration was tested by one-way analysis of variance (Anova). A supra-additive or additive effect was seen when seven ovarian carcinoma cell lines were exposed to paclitaxel or docetaxel concomitantly with cisplatin. A supra-additive effect was found in four cell lines (UT-OC-3, UT-OC-4, UT-OC-5 and SK-OV-3) after simultaneous use of cisplatin with all docetaxel concentrations tested, and in two cell lines (UT-OC-4 and SK-OV-3) when cisplatin was used concomitantly with paclitaxel. A more pronounced supra-additive effect was seen with the combination of cisplatin and docetaxel. The degree of supra-additivity was dose dependent, with increasing synergy after a higher taxane dose. The data obtained in this study suggest that a supra-additive or additive effect can be achieved in ovarian carcinoma with the concomitant use of cisplatin and a taxane. © 1999 Cancer Research Campaign

The efficacy of paclitaxel and cisplatin combinations has been shown in two recent phase III trials (McGuire et al, 1996;Piccart et al, 1997), and this combination is currently widely used as the primary regimen for ovarian carcinoma. Docetaxel is another actively studied taxane. In vitro studies have shown that compared with paclitaxel, it has higher intracellular accumulation and binding to microtubules, as well as lower e fflux and dissociation from microtubules (Riou et al, 1994;Lavelle et al, 1995). Phase I trials on docetaxel-cisplatin treatments are also ongoing. This combination appears promising in non-small-cell lung cancer and in a few other solid tumour types, including colorectal, head and neck, gastric and breast cancer (Burris et al, 1995). Preliminary results from phase II clinical trials on the use of docetaxel in advanced ovarian cancer have confirmed the data obtained from preclinical studies (Kaye et al, 1995). Cisplatin is the most e ffective single chemotherapeutic agent in the treatment of ovarian carcinoma (Thigpen et al, 1989;Advanced Ovarian Cancer Trialists Group, 1991). The role of docetaxel in the management of this disease will, therefore, depend on the cytotoxic e ffect achieved with docetaxel-cisplatin therap y.
We have recently studied the sensitivity of cisplatin, paclitaxel and docetaxel in seven epithelial ovarian carcinoma cell lines using a clonogenic assa y. The IC 50 values of these drugs varied between 0.3 and 1.5 pM, 0.4 and 3. 4 nM and 0.2 and 2. 3 nM respectively (Engblom et al, 1996(Engblom et al, , 1997. On a molar basis, docetaxel was more cytotoxic than paclitaxel in six out of seven cell lines. The purpose of this study was to make a comparison between combinations of cisplatin-paclitaxel and of cisplatin-docetaxel in ovarian carcinoma cell lines, and to assess the types of interaction obtained. To our knowledge, comparative in vitro studies have not been previously published.

Cell lines
Seven ovarian carcinoma cell lines were tested in this stud y. The cell lines used, their histological type, plating e fficiencies (PE) and passages used are listed in Table 1. The SK-O V-3 and the CAO V-3 cell lines (Fogh et al, 1977;Untch et al, 1994) were obtained from the American Type Culture Collection (Rocville, MD, USA), and five cell lines (UT-OC-1, U T-OC-2, U T-OC-3, U T-OC-4 and UT-OC-5) have been established recently at the University of Turku by the author for correspondence. The U T-OC-5 cell line was derived from a metastatic omental tumou r, whereas the other cell lines were established from primary tumours. The donor of the UT-OC-2 cell line had been treated with four courses of vincristine, doxorubicin and cyclophosphamide and radiotherapy for pulmonary metastases before the cell line was established from a primary tumour outside the radiation field. The donors of the UT-OC-4 and U T-OC-5 cell lines had received pelvic radiotherapy Additive and supra-additive cytotoxicity of cisplatinÐtaxane combinations in ovarian carcinoma cell lines for cervical cancer 30 and 5 years before the diagnosis of ovarian carcinoma. The donors of the UT-OC-1 and UT-OC-3 cell lines had not received any cytotoxic therapy before the establishment of the cell lines.

Cell culture
Before the experiments, the cells were kept in logarithmic growth in T25 culture flasks by passing weekly in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 2 mM L-glutamine, 1% non-essential amino acids, 100 U ml -1 streptomycin, 100 U ml -1 penicillin and 10% fetal bovine serum (FBS). Cells in mid-logarithmic growth (40-60% confluence) were used for the experiments and fed with fresh medium on the day before plating.

Drug preparation
Cisplatin (Platinol) 0.5 mg ml -1 was diluted with growth medium to get a stock solution of 100 µg ml -1 . Final cisplatin dilutions of 0.05-0.6 µg ml -1 were used, and new stock solutions were made for each experiment. Paclitaxel (Taxol, kindly provided by Bristol-Myers Squibb) was initially dissolved in 0.9% sodium chloride to get a solution of 0.1 mM. Stock solutions were prepared in Ham's F-12 medium containing 10% FBS to obtain a solution of 100 nM, and stored at -40°C. Final dilutions of 0.4-5 nM paclitaxel were used for the experiments. Docetaxel (Taxotere, 807.9 mg, kindly provided by Rhone-Poulenc Rorer) was diluted in 1 ml of ethanol to obtain a stock solution of 0.1 mM and stored at -40°C. These solutions were further diluted in sterile water to obtain a solution of 100 nM immediately before each experiment. Final dilutions of 0.3-4 nM docetaxel were used for the experiments. We have previously studied the sensitivity of cisplatin, paclitaxel and docetaxel in these cell lines (Engblom et al, 1996(Engblom et al, , 1997. The IC 50 values obtained in these experiments are given in Table 1 and were used as the basis of drug concentrations used in this study. The paclitaxel concentrations used in this study corresponded to 25-100% of the IC 50 values of the cell lines. The docetaxel concentration varied from 25% to 150% of the IC 50 value.

Clonogenic assay
The 96-well plate clonogenic assay based on limiting dilutions was used. The assay has been described earlier in detail (Grènman et al, 1989;Rantanen et al, 1994). The cells were harvested with trypsin-EDTA to obtain a single-cell suspension, counted and diluted in Ham's F-12 medium containing 15% FBS. The number of cells plated per well was adjusted according to the PE of the cell line. The desired concentrations of paclitaxel or docetaxel were added in a stock solution containing 4167 cells ml -1 , and diluted in 25 ml of growth medium. A concentration of two cells per well is achieved by applying 100 µl of this stock solution to each well of the 96-well plate. The desired cisplatin concentrations along with the same paclitaxel or docetaxel concentration as on the day before were added in 100 µl of growth medium after the plates had been incubated for 24 h. All the drugs were allowed to stay on the plates throughout the whole incubation period. The plates were incubated at 37°C with 5% carbon dioxide for 4 weeks, after which the number of wells containing coherent, living colonies, consisting of 32 cells or more, was counted using an inverted phase-contrast microscope.

Data analysis
PE was calculated by the formula PE = -ln (number of negative wells/total number of wells)/number of cells plated per well (Thilly et al, 1980). Fraction survival data were fitted to the linear quadratic model, F = exp [-(αD+βD 2 )] and a microcomputer program was used to obtain the area under the curve (AUC) by numerical integration. The simultaneous effects of cisplatin and paclitaxel or docetaxel were determined as the ratio between the AUC for cisplatin plus paclitaxel or docetaxel, divided by the AUC for cisplatin alone. This AUC ratio was compared with the surviving fraction (SF) after the indicated dose of paclitaxel or docetaxel alone. Comparison of the AUC ratio and the SF value was made using the Student's t-test. The influence of the taxane concentration on the amount of additive or supra-additive cytotoxic effect was tested by one-way analysis of variance (ANOVA). The schedule of the drug administration is important and the achieved growth inhibition can vary from a subadditive to a supraadditive effect. To describe the type of interaction, we have used the term additive of the sum of individual effects. The term supraadditive is used if the combined effect exceeds the sum of individual effects. Some authors use the term synergy, which we have interpreted here as supra-additivity.

RESULTS
Cisplatin and taxanes had either an additive or supra-additive growth inhibitory effect in all cell lines studied. The type and magnitude of growth inhibition varied between individual cell lines. In most of the cell lines, higher taxane concentrations  Engblom et al (1996); b Engblom et al (1997).
increased the extent of supra-additive effect. In some cell lines, lower drug concentrations caused an additive effect, whereas higher concentrations were supra-additive. Furthermore, on a molar basis, docetaxel-cisplatin combinations had more pronounced cytotoxic effects than paclitaxel-cisplatin combinations. A supra-additive effect was seen more frequently with a cisplatin-docetaxel combination than with a cisplatin-paclitaxel combination (Tables 2 and 3). The type of interaction after paclitaxel and cisplatin and the statistical significance of supra-additivity is presented in Table 2. Dose dependency of the magnitude of the interaction is presented in Table 4. All paclitaxel concentrations used concomitantly with   In CAO V-3 cells, the combined effect was additive ( P-values 0.11 and 0.25) when cisplatin was added to 0.1 or 0. 2 nM paclitaxel, which corresponds to 50% or 100% of the previously determined IC 50 concentration. In contrast, 0. 3 n M paclitaxel caused a clear supra-additive (P = 0.0065) e ffect. In U T-OC-1 cells, an additive growth inhibitory e ffect (P-values 0.45 and 0.30) was noticed with 0.6 and 0. 8 nM paclitaxel, corresponding to 43% and 57% of the IC 50 dose ( Table 2). The U T-OC-1 cells showed a clear supra-additive e ffect when 1.0 nM of paclitaxel was combined with cisplatin. In cell lines showing supra-additivity with lower paclitaxel doses, the increasing paclitaxel dose resulted in increased supra-additivit y. In the U T-OC-1, U T-OC-4 and SK-O V-3 cell lines, the degree of supra-additivity was found to be directly correlated to the dose of paclitaxel and this correlation was statistically significant (Table 4). The fitted survival curves of the seven ovarian carcinoma cell lines with three various paclitaxel doses combined with cisplatin are shown in Figure 1.
The type of interaction after docetaxel and cisplatin and the statistical significance of supra-additivity is shown in Table 3, and the dose dependency of interaction is presented in Table 4. In four cell lines (SK-OV-3, UT-OC-3, U T-OC-4 and U T-OC-5), a supraadditive effect was found after simultaneous use of cisplatin with all tested docetaxel concentrations. The lowest docetaxel dose used in the CAO V-3 and U T-OC-1 cells, corresponding to 50% and 25%, respectivel y, of the IC 50 doses of the cell lines, caused a pure additive effect (P-values 0.10 and 0.15), whereas with a higher docetaxel dose supra-additivity was found. The U T-OC-2 cell line was an exception because the combined e ffect was supraadditive with the lowest docetaxel dose, and additive with the two higher doses (Table 3). The degree of supra-additivity was dose dependent in SK-O V-3, UT-OC-3 and U T-OC-4 cell lines.
Increasing the docetaxel dose resulted in a clearer supra-additive effect. The same phenomenon was noticed also in CAO V-3 and UT-OC-1 cells, though in these cell lines the lowest docetaxel dose caused a purely additive e ffect (Table 3). The fitted survival curves of the seven cell lines after concomitant exposure to docetaxel and cisplatin are shown Figure 1.

DISCUSSION
In this stud y, we demonstrated a supra-additive or additive growth-inhibitory e ffect when human ovarian carcinoma cells were exposed to paclitaxel or docetaxel concomitantly with cisplatin. This e ffect was found to be dose dependent with the combination of paclitaxel and cisplatin in three cell lines and with the combination of docetaxel and cisplatin in four out of seven cell lines (Table 4). The U T-OC-2 cell line was an exception; simultaneous docetaxel and cisplatin caused a clear supra-additive e ffect with the lowest docetaxel dose and an additive e ffect with the two higher doses. In our previous stud y, we have shown that clonogenic cell survival after paclitaxel or docetaxel exposure clearly correlated in six out of seven ovarian carcinoma cell lines (Engblom et al, 1997). The only exception was the U T-OC-2 cell line. This result was consistent in repeated experiments. On a molar basis, all seven ovarian cell lines showed more pronounced supra-additivity with the combination of docetaxel and cisplatin compared with paclitaxel and cisplatin.
The effects of paclitaxel in combination with cisplatin were initially reported by Citardi and colleagues in 1990 in mouse leukaemia L1210 cells. They demonstrated the superiority of paclitaxel given before cisplatin compared with other regimens (Citardi et al, 1990). In ovarian cancer cell lines, the decrease of cell viability was significantly greater with the combination of paclitaxel and cisplatin compared with exposure to a single drug (Untch et al, 1994). With human ovarian carcinoma cells, additive or supra-additive e ffect was found when the cells were exposed to paclitaxel before cisplatin. Conversel y, if cisplatin was given first, antagonism was observed (Parker et al, 1993;Jekunen et al, 1994;Kiyozuka et al, 1995). In the current experiments, an additive or supra-additive inhibitory e ffect was seen in all cell lines when the taxane were administered concomitantly with cisplatin. This is in line with previously published reports showing an additive (Saunders et al, 1992;Jekunen et al, 1994) or supra-additive (Parker et al, 1993) e ffect with the cisplatin-paclitaxel combination in ovarian cell lines. The growth inhibitory e ffect of docetaxel combined with cytotoxic agents has not been studied as widely as that of paclitaxel. In a study with human breast carcinoma cells, an additive or supra-additive e ffect was noticed after cells pretreated with edatrexate were treated with docetaxel. Howeve r, antagonism was evident when the schedule was reversed (Chou et al, 1996). In the present stud y, we demonstrated a supra-additive or additive growth inhibitory e ffect when docetaxel was given concomitantly with cisplatin. Moreove r, on a molar basis, this combination was more effective than the combination of paclitaxel and cisplatin.
The type and degree of the growth inhibitory e ffect varied with different doses of the taxanes. Increasing paclitaxel doses resulted in increasing supra-additivity in three out of seven cell lines. The same kind of dose-dependent interaction was found in the breast cancer cell lines (Koechli et al, 1993). The dose dependency of the cisplatin-docetaxel combination was even more pronounced because the degree of supra-additivity was dose dependent in three cell lines. In an additional two cell lines, the lowest docetaxel dose had an additive e ffect and higher doses had a supra-additive growth inhibitory e ffect.
It has been demonstrated in several studies that on a molar basis docetaxel is more potent than paclitaxel as a single drug (Kelland et al, 1992;Riou et al, 1992;Hill et al, 1994;Engblom et al, 1997). In the present stud y, a greater supra-additive e ffect was achieved with the combination of docetaxel and cisplatin compared with the Table 4 The dose dependence of additive and supra-additive cytotoxic effect. The influence of the concentration was tested by one-way analysis of variance (Anova) and the P-values after cisplatin plus paclitaxel and cisplatin plus docetaxel are listed below. A statistically significant direct correlation between increasing the taxane dose and the amount of synergy is found when P is < 0.05

Cell line Cisplatin plus paclitaxel Cisplatin plus docetaxel
UT combination of paclitaxel and cisplatin. Studies evaluating the mechanism of action of these two taxanes have shown that, in comparison with paclitaxel, docetaxel is slightly more active as a tubulin assembly promoter and microtubule stabilizer, and approximately twofold more potent as an inhibitor of microtubule depolymerization (Gueritte-Voegelein et al, 1991). Furthermore, the effective affinity of docetaxel for the microtubule binding site is 1.9-fold greater than that of paclitaxel (Diaz et al, 1993). These differences in mechanism of action may explain the differences in the cytotoxic effect achieved with concomitant use of cisplatin and these two taxanes.
Peak plasma concentrations achieved with a 24-h paclitaxel infusion have ranged from 0.23 to 0.43 µM, and with a 3-h infusion from 2.5 to 4.3 µM (Huizing et al, 1993). After a 1-h infusion, the peak plasma concentration for docetaxel has been 4.46 µM (Hino et al, 1995), and for cisplatin 2.5 µg ml -1 (Gullo et al, 1980). The current experiments were performed using paclitaxel concentrations of 0.1-3 nM, docetaxel doses of 0.1-1.5 nM and cisplatin doses of 0.01-0.6 µg ml -1 , which were clearly below the peak plasma concentrations achieved for these drugs. In vitro, the duration of both paclitaxel (Rowinsky et al, 1988;Arbuck et al, 1993;Lopes et al, 1993;Georgiadis et al, 1994) and docetaxel (Hill et al, 1994) exposure has a great impact on the growth-inhibitory effect of the drug.
In fact, in studies combining taxanes and radiation, increasing the time of exposure has been reported to be more important than increasing the drug concentration (Schiff et al, 1995). In the present study, the time of exposure was long and was kept constant, and the interaction of cisplatin and taxanes was studied as the function of drug concentrations.
The efficacy of the cisplatin-paclitaxel combination has been demonstrated in clinical use. Incorporating paclitaxel into first-line therapy has improved the survival in stage III and stage IV ovarian carcinoma (McGuire et al, 1996;Piccart et al, 1997). The therapeutic effect of docetaxel-cisplatin combination is under investigation. The results of the present study indicate that on a molar basis the combination of docetaxel-cisplatin is more cytotoxic than the combination of paclitaxel-cisplatin. If the toxicity profile of the docetaxel-cisplatin combination is acceptable, a randomized trial comparing the two taxane-cisplatin combinations is warranted.


extfieldsofstudy: Medicine Biology

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Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

D URING cell division, a bipolar spindle is assembled to allow the segregation of two sets of chromosomes. The view of spindle assembly and its influence on M phase completion is based mostly on experiments performed on mitotic cells.
In mitosis, spindle assembly proceeds in two major steps. First, the two centrosomes, which have nucleated microtubules, migrate to opposite sides of the nucleus. As a consequence, spindle bipolarity is established before nuclear envelope breakdown. Second, after nuclear envelope breakdown, dynamic microtubules are randomly captured and stabilized by the kinetochores that are located at each chromosome's centromere. Sister kinetochores rarely at-tach simultaneously, leading to a poleward motion of the chromosomes. The chromosomes move to the equatorial plane of the spindle when both sister kinetochores are connected to opposite poles by kinetochore fibers. This random and continuous process ends with the sharp alignment of all the chromosomes on the metaphase plate (for review see Rieder and Salmon, 1998). This allows the transition to anaphase and the segregation of the two sets of chromatids.
During meiosis, two successive M phases occur without an intermediate S phase, so that haploid gametes are produced. The second meiotic division is equational and is effectively a mitotic division. The first meiotic division is unique in that it is reductional: homologous chromosomes that just underwent meiotic recombination are segregated while the cohesion between sister chromatids is maintained.
In the present work, we studied the assembly of the first meiotic spindle and its involvement in the first meiotic M phase completion using the mouse oocyte as a model. In the mouse oocyte, the first meiotic M phase is very long, lasting from 6 to 11 h, depending on the genetic back-ground (Polanski, 1986(Polanski, , 1997. The activity of the major M phase kinase (p34cdc2/cyclin) that directly or indirectly promotes spindle assembly increases slowly and reaches a plateau that is maintained for hours (Choi et al., 1992). One peculiarity of the mouse oocyte is that the oocytes lack centrioles (Szöllösi et al., 1972). The progressive aggregation of numerous microtubule organizing centers (MTOCs) 1 form the spindle poles (Maro et al., 1985). Thus, the bipolarity of the spindle is not predefined (reviewed in  in contrast to mitosis. Finally, a stable bipolar spindle is present in the oocyte during most of the first meiotic M phase (Verlhac et al., 1994).
We show here that the formation of a bipolar spindle and the congression of chromosomes near the equatorial plane of the spindle is performed in the absence of kinetochore fibers. This late prometaphase spindle is then maintained for 4 h. The ability of kinetochores to interact with microtubule ends (to form kinetochore fibers) is acquired at the end of the first meiotic M phase and this event allows the metaphase I to anaphase I transition. Moreover, continuous presence of the prometaphase spindle is not required for progression through the first meiotic M phase.

Collection and Culture of Oocytes
To obtain immature oocytes arrested at prophase I of meiosis, the ovaries were removed from 5-6-wk-old Swiss female mice (Animalerie Spécialisée de Villejuif, Center National de la Recherche Scientifique, France) and transferred to prewarmed (37 Њ C) M2 medium supplemented with 4 mg/ml BSA (Whittingham, 1971) and 50 g/ml dibutyryl cyclic AMP (db-cAMP), which prevents immature oocytes from undergoing germinal vesicle breakdown (GVBD). The ovarian follicles were punctured to release the enclosed oocytes, and immature oocytes displaying a germinal vesicle (GV) were collected. Oocytes undergoing meiotic maturation were synchronized by scoring GVBD. Synchronized groups of oocytes were cultured in M2 medium under liquid paraffin oil at 37 Њ C.
Metaphase II-arrested oocytes were recovered from mice superovulated by intraperitoneal injections of pregnant mare gonadotrophin (PMSG; Intervet) and human chorionic gonadotrophin (hCG; Intervet) 48 h apart. Ovulated oocytes were released from the ampullae of oviducts 12.5 or 17 h after hCG. The cumulus cells were dispersed by brief exposure to 0.1 M hyaluronidase (Sigma Chemical Co.) and after careful washing, all oocytes were put in culture.

Immunofluorescence
The fixation and labeling of oocytes were performed as described in Kubiak et al. (1992). As the first layer, we used either the rat mAb YL1/2 specific for tyrosinated ␣ -tubulin (Kilmartin et al., 1982) or a rabbit polyclonal antibody directed against cytoplasmic linker protein 170 (CLIP-170;Dujardin et al., 1998). As the second layer, we used either a fluorescein-conjugated anti-rat antibody or a fluorescein-conjugated anti-rabbit antibody (Miles). The chromatin was visualized using propidium iodide (Molecular Probes; 5 mg/ml in PBS). Samples were observed with a Leica TCS4D confocal microscope.

Video Microscopy
Time-lapse studies on living mouse oocytes were performed with a computer-assisted image analysis system (Oncor Imaging), coupled to a cooled CCD camera (CH 2400; Photometrics) and an epiillumination inverted microscope (Zeiss Axiovert 135). Oocytes were cultured at 37 Њ C in an adaptable cell culture chamber (Life Science Resources) in a 2-ng/ml vital bis benzimide (Hoechst 33258, Sigma Chemical Co.) M2 medium. Chromosome images were acquired with a 20 ϫ objective (total optical magnification was 200) and the following filter set: bandpass filter BP 365/ 12 nm, chromatic beam splitter FT 395 nm, longwave pass filter LP 397 nm. A log 2 neutral filter was placed in the bulb light path to minimize photo damage of oocytes by UV light. Under these conditions, images were acquired every 180 s with a 1 to 2 s exposure time. Images were digitized in 512 ϫ 512 array (binning mode) and fluorescence intensity levels were coded on 16 bits. Images were then converted in 8 bits TIFF format. Image processing was realized with NIH Image 1.62 and Adobe Photoshop 4.0. The printed outputs were done on a sublimation color printer (ColorEase, Kodak).

Transmission EM
For transmission EM, the oocytes were prefixed with 3% paraformaldehyde in PHEM (25 mM Hepes, 60 mM Pipes, 10 mM EGTA, 2 mM MgCl 2 , pH 7.4) containing 0.5% Triton X-100 for 30 min. Then the oocytes were fixed with 2% glutaraldehyde and 0.2% tannic acid in a phosphate buffer, postfixed on ice with 0.5% OsO 4 for 10 min, and stained en bloc with uranyl acetate. Samples were then embedded in epon in Beam's capsule and sectioned on a Reichert ultramicrotome before observations under a Philips EM410 at 80 kV. Thick sections were used to locate the spindles.

Kinetics of the First Meiotic Spindle Formation
1 h after GVBD, short microtubules are radiating around the chromosomes (Fig. 1 A). 2 h after GVBD, the microtubules have formed a bipolar spindle and the chromosomes are dispersed within the structure (Fig. 1 B). 4 h after GVBD, the spindle has acquired a barrel shape morphology, typical of meiosis ( Fig. 1 C), and the chromosomes are located near the equatorial plane of the spindle. From 4 to 8 h after GVBD, the spindle morphology remains unchanged with unaligned chromosomes (Fig. 1, D and E). 8 h after GVBD, metaphase plates could be observed (Fig. 1  F) with the chromosomes perfectly aligned and a symmetrical arrangement of their arms. Anaphase (Fig. 1 G) and telophase ( Fig. 1 H) figures were also present. Finally, we observed that the spindle length decreases 2-8 h after GVBD (reaching ف 50% of its initial length) before elongation at anaphase.

Chromosome Dynamics during the First Meiotic M Phase
The immunofluorescence study of fixed material did not allow us to observe changes in dynamics of spindle morphology and chromosome position from 4 to 8 h after GVBD. The chromosomes are not sharply aligned dur-ing this period, suggesting that it corresponds to a long prometaphase. To address this point, chromosomes were stained with a vital dye and observed in vivo using a cooled CCD camera.
During the metaphase II arrest, the chromosomes are aligned on the metaphase plate and do not move (Fig. 2  A). In contrast, during the last 4 h of the first meiotic M phase, the chromosomes oscillate with a low amplitude around the equatorial plane of the spindle (Fig. 2 B). For example, the top right chromosome protruding from the main mass in Fig. 2 B, at 27 min (arrow) has moved back into it at 87 min, and out again at 105 min; the center right chromosome at 60 min (arrowhead) is in the main mass, out at 105 min, and in again at 117 min. They became aligned in metaphase for only a short period (12.2 min Ϯ 3.9; n ϭ 16) immediately before their segregation in anaphase ( Fig. 2 B, at 195 min). Thus, during virtually all of the second half of the first meiotic M phase, the spindle appears to be maintained in a late prometaphase state.

Chromatin-Microtubule Interactions during the First Meiotic M Phase
In mitosis, the accurate alignment of chromosomes on the metaphase plate is due to kinetochore-microtubule end interactions. To understand the peculiar congression of homologous chromosomes during the first meiotic M Figure 2. Chromosome movements during the metaphase II arrest (A) and the first meiotic M phase (B). Pictures were taken every 3 min. Because the chromosomes did not move in metaphase II, one picture out of two is shown in A (6 min interval).
The Journal of Cell Biology, Volume 146, 1999 4 was not detected (Fig. 3 B, arrows). However, the kinetochores and adjacent chromatin appeared to be pulled out from the main chromatin mass. This likely is due to lateral interactions between some microtubules and the kinetochores, in the absence of stable kinetochore bundles. 8 h after GVBD, end interactions were not detected at some kinetochores ( Fig. 3 C, arrows), but they were observed in others ( Fig. 3 D, arrows), as in metaphase II-arrested oocytes (not shown). When a systematic analysis of microtubule end interactions with kinetochores was performed at 6 and 7 h after GVBD (Table I), none were observed at phase, we studied kinetochore-microtubule end interactions using EM.
2 h after GVBD, no kinetochore-microtubule end interactions were observed (Fig. 3 A). Numerous microtubules interacted with the chromosome surface or penetrated within the chromosome (Fig. 3 A, arrows). A similar situation was observed 4 h after GVBD (not shown). 6 h after GVBD, microtubules interacted with the chromosome surface, but no longer penetrated chromosomes (Fig. 3 B). Some microtubules were present near the kinetochores, but insertion of microtubule ends in the kinetochore plate 5 6 h, the spindle areas facing the kinetochores being deprived of microtubules (Fig. 4, A and B). A few microtubule ends were observed interacting with kinetochores at 7 h (Fig. 4 C, arrows), however, they were less numerous than at 8 h (compare Figs. 3 and 4, C). All kinetochore regions observed were stretched towards the poles, suggesting that unstable lateral interactions with microtubules are able to position the kinetochores towards the poles.
These data suggest that the long late prometaphase state is correlated with the lack of kinetochore fibers. During most of the first meiotic M phase, the microtubules interact directly with the surface of the chromosomes, and even penetrate within the chromatin, from 2 to 4 h after GVBD. These interactions could provide the forces leading to chromosome congression near the equatorial plane.

CLIP-170 Is Present on Kinetochores Until the End of Late Prometaphase
CLIP-170 is associated with kinetochores during prometaphase in somatic cells (Dujardin et al., 1998) until the formation of kinetochore fibers (Dujardin, D., and J. de Mey, manuscript in preparation). We first checked whether this was the case in mouse oocytes. In metaphase II-arrested oocytes, kinetochore fibers are present and no CLIP-170 staining was observed on the chromosomes (Fig.  5 A). After total depolymerization of microtubules by 10 g/ml nocodazole, CLIP-170 staining was observed on kinetochores (Fig. 5 B). This staining disappeared rapidly when microtubules polymerized after nocodazole removal (Fig. 5, C and D). Thus, in mouse oocytes, CLIP-170 behaves as it does in somatic cells: it is associated with kinetochores in the absence of kinetochore fibers.
During the first meiotic M phase, CLIP-170 stains kinetochores already at GVBD and persists until 7 h after GVBD (Fig. 6, A-C). At this time, some kinetochores are no longer stained (Fig. 6 C). By 8 h after GVBD, the staining has disappeared from most kinetochores when chromosomes were still not aligned ( Fig. 6 D), and completely  *Using the Fisher's exact test, the difference in the number of kinetochores observed between the two groups is not significant ( P ϭ 0.1871). ‡ Using the Fisher's exact test, the difference in the number of kinetochores with attached microtubules between the two groups is significant ( P Ͻ 0.0001).
The Journal of Cell Biology, Volume 146, 1999 6 absent at metaphase (Fig. 6 E). Thus, we can conclude that CLIP-170 remains on kinetochores until ف 7-8 h after GVBD and disappears just before the final alignment of the chromosomes. It is noteworthy that bivalents are correctly oriented (kinetochores of homologous chromosomes facing opposite poles) 5 h after GVBD, which suggests that lateral interactions between some microtubules and the kinetochores take place in the absence of stable kinetochore bundles. These data confirm our EM observations and show that the long late prometaphase state is correlated with the lack of kinetochore fibers.

The Continuous Presence of the Prometaphase Spindle Is Not Required for Completion of the First Meiotic M Phase
Our previous data show that, during most of the first meiotic M phase (6 out of 8 h), the chromosomes oscillate near the equatorial plane of a bipolar spindle. This observation led us to ask whether the persistence of this prometaphase structure was required for the assembly of a functional spindle and completion of the first meiotic M phase.
In mitosis, the duration of M phase is strongly correlated with the time devoted to spindle assembly and chromo-some congression (Rieder et al., 1994). This dependence was confirmed in one-cell mouse embryos incubated after nuclear envelope breakdown for 1 ( n ϭ 56) or 2 h ( n ϭ 50) in 10 g/ml nocodazole. The drug was then removed to allow spindle reformation and the time of cell division was measured. A 1 h incubation in nocodazole delays cell division by 1 h (160 min vs. 100 min after nuclear envelope breakdown in control oocytes) and a 2 h incubation leads to a 2 h delay (210 min vs. 100 min after nuclear envelope breakdown).
A similar experiment was performed on oocytes during the first meiotic M phase. Starting 2 h after GVBD (when a bipolar spindle is already present), oocytes were incubated in nocodazole for increasing periods, varying from 2 to 6 h. Oocytes were then removed from the drug and the time of polar body extrusion (PBE) was measured. In all cases, the resulting metaphase II-arrested oocytes contained only monovalent chromosomes, as checked by pro-  pidium iodide staining. That the spindles reformed after nocodazole treatments led to the correct segregation of homologous chromosomes. Incubations in nocodazole for 2 or 3 h did not induce a delay in PBE. However, longer incubations (4, 5, and 6 h) resulted in significant delays (Table II).
To determine whether these delays were due to the duration of the incubation in nocodazole or to the time of drug removal, we measured the kinetics of PBE in groups of oocytes incubated for 1 h in nocodazole and removed from the drug 5, 6, 7, and 8 h after GVBD (Table II). The comparison of the results of the two experiments shows that the time of PBE depends only on the time of nocodazole removal, whatever the duration of nocodazole treatment.
Spindle destruction before 5 h after GVBD does not induce any significant delay in PBE; it continues to occur at ف 8 h after GVBD. Thus, the continuous presence of a prometaphase spindle is not required during the first meiotic M phase. This implies that the length of the first meiotic M phase is not strictly controlled by the microtubule network, but by some other unknown factor.
In contrast, nocodazole removal after 6 h after GVBD led to a linear increase in the time of PBE. However, polar bodies were extruded invariably 2.6 h after removal from the drug, probably corresponding to the minimum time required for spindle reformation and PBE. This observation suggests that the event controlling the length of the first meiotic M phase takes place between 6 and 8 h after GVBD.

Setting Up of Stable Kinetochore-Microtubule End Interactions Determines the Duration of the First Meiotic M Phase
To determine the nature of the event controlling the length of the first meiotic M phase, we took advantage of previous observations. We studied spindle reformation after a 2 h incubation in nocodazole and drug removal 8 h after GVBD (NZ/6h-8h; when this event is supposed to have occurred) or 5 h after GVBD (NZ/3h-5h; when the spindle would have been present for a few hours until this event takes place).
In both experiments, 20 min after drug removal a bipolar structure formed, although it was larger in the NZ/3h-5h group (Fig. 7, A and E). 50 and 120 min after drug removal, the chromosomes were localized near the equatorial plane (Fig. 7, B, C, F, and G). However, the spindle length did not change until metaphase in the NZ/6h-8h group (Fig. 7, E-G), while it decreased progressively until 120 min in the NZ/3h-5h group (Fig. 7 C). This late prometaphase spindle was maintained until 7.5 h after GVBD (Fig. 7 D), just before PBE. In the NZ/6h-8h group, the chromosomes seemed to congress progressively to the equatorial plane of the spindle (Fig. 7, E-G). PBE occurred 2.5 h after removal of the drug (Fig. 7 H). In the NZ/6h-8h group, nocodazole induced chromosome dispersal in some oocytes ( ف 1/3). After drug removal, a spindle reformed around each group of chromosomes and a polar body was extruded for each spindle. However, the only difference between the two groups was that the prometaphase spindle persisted for 3 h in the NZ/3h-5h group.
This result was reinforced by live observation of chromosome movements after nocodazole treatment. In the NZ/3h-5h group, chromosomes kept oscillating near the equatorial plane until metaphase, as in untreated oocytes (not shown). In the NZ/6h-8h group, some individual chromosomes were observed moving towards the poles (Fig. 8, 0 to 6 min) and then congressing to the equatorial plane (Fig. 8, 6 to 30 min). Anaphase occurred after the alignment of the last chromosome (Fig. 8, 48 min).
Thus, the process of spindle reformation differs in the two groups. In the NZ/3h-5h, the process is very similar to the one observed in control oocytes during meiosis I, with the chromosomes oscillating during a long late prometaphase. In the NZ/6h-8h group, chromosomes behave as they do in mitosis. We observed that the kinetochore fibers (stable kinetochore-microtubule end interactions) are established only at the end of the first meiotic M phase, in contrast to mitosis, where they can form as soon as nuclear envelope breakdown. This led us to suspect that the setting up of these stable interactions between microtubule ends and kinetochore plates controls the duration of the first meiotic M phase. We studied the kinetochoremicrotubule end interactions after nocodazole removal using CLIP-170 staining. In the NZ/3h-5h group, CLIP-170 was still observed on kinetochores 7 h after GVBD (Fig. 9  A) and disappeared ف 8 h after GVBD, as in nontreated oocytes (not shown). In the NZ/6h-8h oocytes, CLIP-170 disappeared from many kinetochores as soon as 15 min after removal of the drug (Fig. 9 B). The staining was completely abolished after 30 min (Fig. 9 C). These results show that the setting up of stable kinetochore-microtubule end interactions occurs invariably ف 8 h after GVBD, suggesting that this event controls the exit from meiosis I.

Protein Synthesis Is Not Required for the Setting Up of the Stable Kinetochore-Microtubule End Interactions
We tested whether the ability of kinetochores to interact with microtubules was dependent upon protein synthesis. Oocytes incubated in 10 g/ml of the protein synthesis in- hibitor, puromycin, for 5 h after GVBD, extruded their polar bodies at the same time as the control oocytes (481 Ϯ 33 min after GVBD, n ϭ 87 for puromycin; and 491 Ϯ 34 min after GVBD, n ϭ 81 for control; three experiments). Thus, protein synthesis during the last 3 h of the first meiotic M phase is not required for the setting up of stable kinetochore-microtubule end interactions.

Discussion
Our results support the following mechanism for first meiotic spindle assembly (Fig. 10). After entry into the first meiotic M phase, 2 h are required for the formation of a bipolar structure. For the next 2 h, the homologous chromosomes congress near the equatorial plane of the spindle. At that time, microtubules interact directly with the chromosomes' arms, but do not form kinetochore fibers. During the next 3 to 4 h, the chromosomes gather near the equatorial plane of the spindle. Unstable lateral interactions between microtubules and kinetochores may maintain a correct orientation of the sister kinetochores towards both poles. At 8 h after GVBD, the stable kinetochore-microtubule end interactions are set up, allowing the final alignment of the chromosomes on the metaphase plate. Metaphase lasts for a short time ( ف 12 min) and the metaphase-anaphase transition leads to the extrusion of the first polar body.

Formation of a Bipolar Spindle
In mitosis, the migration of the two centrosomes determines spindle bipolarity, even before nuclear envelope breakdown. Another component in establishing bipolarity is the presence of a kinetochore on each of the sister chromatids. They are located on opposite sides of the chromosome and stabilize microtubules nucleated by opposite poles, as soon as the cell enters prometaphase. In contrast,   . CLIP-170 staining after nocodazole treatment. CLIP-170 appears in green and chromosomes in red. 10-15 optical sections were merged for the final images. Oocytes were cultured in the presence of 10 g/ml nocodazole from 3 to 5 h after GVBD and fixed 2 h after removal of the drug (A). Oocytes were cultured in the presence of 10 g/ml nocodazole from 6 to 8 h after GVBD and fixed 15 (B) and 30 min (C) after removal of the drug. during the first meiotic M phase in mouse oocytes (and in other species, reviewed by , bipolarity is not predefined. Oocytes are lacking centrioles and poles assemble progressively by the aggregation of numerous MTOCs. Here, we show that a bipolar spindle is also formed without stable kinetochore-microtubule end interactions. First, we were not able to detect by EM any interaction between microtubule ends and kinetochore plates before 8 h after GVBD. Second, this observation was reinforced by the study of CLIP-170 in oocytes during the first meiotic M phase. Recently, Dujardin et al. (1998) reported that CLIP-170 associates with kinetochores during mitotic prometaphase until the formation of kinetochore fibers (Dujardin, D., and J. de Mey, manuscript in preparation). We first showed that this property of CLIP-170 was conserved during meiosis. In metaphase II-arrested oocytes, CLIP-170 is absent from kinetochores ( Fig. 4 A). After a pulse of nocodazole and a transient depolymerization of the microtubule network, the CLIP-170 staining reappears and fades away when microtubules reform (Fig. 4, B and C). During the first meiotic M phase, CLIP-170 is present on kinetochores when the bipolar spindle forms, showing that this process does not require any stable kinetochoremicrotubule interaction.
Chromatin, per se, is involved in spindle formation, since it has been shown to have a microtubule stabilizing activity in vivo (Karsenti et al., 1984;Maro et al., 1986) and in vitro (Dogterom et al., 1996). This effect constrains microtubule polymerization near the chromosomes (Zhang and Nicklas, 1995). However, the formation of a bipolar structure seems to be due to the properties of microtubules and associated factors only. We recently showed in mouse oocytes that normal bipolar spindles can form in cytoplasts deprived of chromosomes . In this case, the formation of bipolar spindles is due to the setting up and stabilization of antiparallel interactions between microtubules nucleated from distinct MTOCs and the capacity of these MTOCs to aggregate. In Xenopus egg extracts containing centrioles, the formation of a bipo-lar spindle around nonspecific DNA (Heald et al., 1996) was shown to require cytoplasmic dynein for microtubule organization into poles (Heald et al., 1997). Other motors could be required for the organization of microtubules into antiparallel arrays.

Chromosome Congression during the First Meiotic M Phase Is a Two-step Process
Stable kinetochore-microtubule end interactions are essential for the congression of chromosomes to the equatorial plane of the spindle during mitotic prometaphase. In contrast, during the first meiotic M phase in mouse oocytes, chromosomes congress to the vicinity of the equatorial plane without such stable interactions. Using EM, we showed that end interactions between microtubules and the kinetochore plate, as in metaphase II-arrested oocytes, are only observed ف 8 h after GVBD. The fact that the bivalents are correctly oriented (kinetochores of homologous chromosomes facing opposite poles) during all the first meiotic M phase and the stretching of the kinetochores towards the pole suggests lateral interactions between some microtubules and kinetochores, in the absence of stable kinetochore bundles. The CLIP-170 staining disappears from the kinetochores between 7 and 8 h after GVBD, reinforcing the fact that stable kinetochoremicrotubule end interactions are only established at the end of the first meiotic M phase, before the final alignment of chromosomes on the metaphase plate.
Previous studies have shown that direct interactions between microtubules and chromosomes are also involved in chromosome congression. First, a wind of microtubules exert some pushing forces on the chromosomes (Khodjakov et al., 1996). Second, similar forces are produced by several members of the kinesin family of microtubule motor proteins associated with chromatin, like Xklp1 during meiosis and mitosis  or NOD during meiosis I in Drosophila oocytes (Theurkauf and Hawley, 1992). During the first hours of the first meiotic M phase in mouse oocytes, numerous microtubules interact with the chromosomes arms and, more surprisingly, penetrate within them (Fig. 3 A).
Taken together, these observations suggest that during the long prometaphase of meiosis I, pushing forces, likely mediated by motor proteins, are exerted on the chromosomes and drive them to the vicinity of the equatorial plane of the spindle. Then, when kinetochore-microtubule end interactions are set up ف 7-8 h after GVBD, the chromosomes are sharply aligned on the metaphase plate by pushing and pulling forces exerted through the kinetochores.
The microinjection of an antikinetochore autoimmune serum in immature mouse oocytes impaired the formation of the metaphase I plate, but the microinjection of the same serum in cold-treated metaphase II oocytes did not interfere with the reformation of a metaphase II plate after warming (Simerly et al., 1990). However, the serum recognized the CENP-B protein that binds to centromeric sequences and seems to organize centromere satellite DNA into a higher order structure, which then directs centromere formation and kinetochore assembly (Goldberg et al., 1996;Iwahara et al., 1998). Such a role of CENP-B in kinetochore assembly, before or at the very beginning of M phase, may explain why microinjection of the serum did not interfere with metaphase plate formation when kinetochore-microtubule end interactions are possible because kinetochores are already formed. Moreover, we observed that the kinetochores are assembled already at the beginning of the first meiotic M phase, but are able to interact with microtubule ends only at the end of the first meiotic M phase.

Kinetochore Maturation Controls the Duration of the First Meiotic M Phase
In contrast to mitosis, spindle assembly does not determine the duration of the first meiotic M phase. Indeed, the continuous presence of the prometaphase spindle is not required for completion of the first meiotic metaphase. After spindle destruction by nocodazole, the minimum time needed for the formation of a functional spindle and PBE is 2.5 h. The study of the kinetochore-microtubule end interactions after nocodazole treatments leading to various delays in PBE shows that they are set up invariably ف 7-8 h after GVBD. Once they can be set up, the final alignment of the chromosomes occurs, followed by the metaphase/anaphase transition and extrusion of the polar body. These late events are likely under the control of the spindle assembly checkpoint, because after nocodazoleinduced spindle disruption and drug removal PBE was delayed (Table II), anaphase did not occur before the alignment of the last chromosome on the metaphase plate ( Fig.  7) and, when chromosomes were dispersed by the drug, spindles formed around each group of chromosomes, and polar bodies were synchronously extruded (Fig. 6 H).
The ability of the kinetochores to interact with microtubule ends is acquired at the end of the first meiotic M phase. This was confirmed in oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the timing of PBE, ف 7 and 11 h after GVBD, respectively . In both strains, kinetochore fibers are observed just before PBE (not shown). This suggests that a slow and progressive maturation of the kinetochore controls the duration of the first meiotic M phase. Protein synthesis during the last 3 h of the first meiotic M phase is not required for this process, suggesting that it requires some post translational modification of kinetochore component(s), only achieved at the end of the first meiotic M phase.
During the first meiotic M phase, the activity of MPF (M phase promoting factor) increases slowly and reaches a plateau that is maintained for hours (Choi et al., 1992). We recently showed that cyclin B synthesis controls the duration of the first meiotic M phase through the raise in MPF activity, the plateau of maximum activity being constant . The post-translational modification of kinetochores could be directly or indirectly under the control of this plateau of maximum MPF activity.
It is possible that some event(s) need(s) to be completed before the setting up of kinetochore fibers. Interestingly, chromatin appears not to be fully condensed during most of the first meiotic M phase, probably because the activation of MPF is slow and progressive. The lack of kinetochore fibers could prevent the chromosomes from being pulled apart by high intensity forces until condensation is sufficient to protect them from such tractions exerted on kinetochores.