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Testis |
INSERM U418/INRA UMR 1245,3 Hôpital Debrousse, 69322 Lyon, cedex 05, France
Laboratoire de Biologie Moléculaire de la Cellule,4 CNRS UMR 5161, Ecole Normale Supérieure de Lyon, 69364 Lyon cedex 07, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, kinases, meiosis, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and Coculture of Rat Sertoli Cells and PS
These experiments were conducted according to the Guide for Care and Use of Laboratory Animals. Sertoli cells and PS were isolated and cultured as previously described, and cell viability was assessed by trypan blue exclusion [6]. The purity of the PS fraction was assessed by flow cytometry (see below; 94% ± 3% of cells were 4C cells, 3% ± 2% were 2C cells, and 2% ± 0.5% were 1C cells, n = 5). It was found that 15% of elutriated PS were early PS (stages XIV to IV of the rat seminiferous epithelium [9]), 60% were middle PS (stages V to IX), and 25% were late PS (stages X to XIII; M.H. Perrard, personal communication).
Immunocytochemical Studies on Cultured Cells
Cells were rinsed with phosphate buffer saline (PBS) and fixed directly in the well with Bouin fixative for 20 min at room temperature. After washing with PBS, fixed cells were permeabilized with 0.25% Triton X-100 in PBS containing 1% bovine serum albumin (BSA) for 20 min at room temperature. The immunocytochemical reaction was performed using an antiphosphorylated serine 10 (ser10) of histone H3 (Upstate Biotechnology, Euromedex, Mundolsheim, France) as a primary antibody used at a dilution of 1:300 in blocking buffer: 2% BSA, 10% fetal calf serum (Gibco BRL Life Technologie, Cergy-Pontoise, France) in PBS for 3 h at 4°C and revealed with an LSAB2 kit (DAKO, Trappes, France) using avidin-biotin-peroxidase complex as a staining reaction and amino-3-ethyl-9-carbazole as a chromogen. Phosphorylation of histone H3 on ser10 is correlated with chromosome condensation at G2/M in spermatocytes [10, 11], and this antibody staining provides a marker for cells in the division phases.
Immunolabeling of Cells for Flow Cytometry
At selected days of culture, cells were detached from culture wells by trypsinization, washed, then fixed with ice-cold 70% ethanol. Immunolabeling of cultured germ cells and flow cytometric analyses were performed essentially as described by Godet et al. [3]. After washing with PBS, fixed cells were resuspended in 150 µl of permeabilizing buffer: 0.25% Triton X-100 and 1% BSA in PBS for 20 min on ice. The cells were exposed to anti-vimentin antibody (clone V9; DAKO) used at a dilution of 1:500 in blocking buffer for 3 h at 4°C. After three washes in PBS/BSA 1%, the cells were exposed to 70 µl of fluorescein (FITC)-conjugated rabbit anti-mouse immunoglobulins (IgG; DAKO) used at a dilution of 1:60 in blocking buffer for 1 h at 4°C. After washing, the cells were incubated with 100 µl of anti-CDK1 antibody (clone 17; Santa-Cruz, Tébu, Le Perray-en-Yvelines, France) or anti-cycB1 antibody (clone GNS1; Becton Dickinson, Le pont-de-Claix, France), both used at a dilution of 1:200 in PBS/BSA 1% and left overnight at 4°C. After washing, CDK1- or cycB1-labeled cells were incubated with 70 µl of phycoerythrin (PE)-conjugated rabbit anti-mouse IgG (DAKO) used at a dilution of 1:60 in PBS/BSA 1% for 1 h at 4°C. Before analysis, Hoechst 33342 (Sigma, La Verpillière, France) was added to the suspensions of labeled cells at a final concentration of 0.12 µg/ml. Two negative controls were included: cells incubated with mouse IgG1 negative control (DAKO) and FITC- or PE-conjugated secondary antibody.
Flow Cytometric and Computer Analyses
After immunolabeling, cells were analyzed using a FacsStar Plus Cytometer (Becton Dickinson) equipped with a 50-mW argon laser tuned to 448 nm and an INNOVA 300 ion multilined/UV laser tuned to UV. Emission fluorescence was measured with a DF 530/30 filter for FITC, a DF 575/26 filter for PE and DF 424/44 filter for Hoechst. Data acquisition and analysis were performed with "CellQuest" software (Becton Dickinson). Six data parameters were acquired and stored in list mode files: linear forward light scatter (FSC) and linear side angle light scatter (SSC), which roughly represent cell size and cellular granularity, respectively; logarithmic (log) PE (CDK1), log FITC (vimentin), and linear PE (cycB1) to detect the immunolabeling; and linear Hoechst to measure the DNA content of the different population of cells. Contaminating events such as debris and clumped cells were eliminated from the analysis. Each acquisition was performed on 50 000 cells negative for vimentin. Flow cytometry using three different parameters (FSC, SSC, and ploidy) allowed the identification of middle to late PS and RS in pure fractions, whereas secondary spermatocytes (SII) were contaminated by doublets of round spermatids. Then the SII were separated from doublets of RS on the basis of their approximately threefold higher content of CDK1. Each sorted population was further characterized by microscopic examination [3]. Hence the percentage of each category of germ cells was multiplied by the total number of cells per well in order to obtain the absolute number of PS, SII, and RS.
Histone H1 Kinase Assay
Equal amounts of proteins (500 µg) from elutriated PS were precipitated with either anti-CDK1 (clone 17) or anti-CDK2 (clone D-12; Santa Cruz, Tébu); monoclonal antibodies; and 40 µl of a 50% (v/v) suspension of protein-A Sepharose per assay. After 3 h of incubation at 4°C, the beads were collected by centrifugation and washed four times with lysis buffer. Histone H1 kinase activities were performed in the absence or presence of 1 µM roscovitine according to the manufacturer's instructions (SignaTECT cdc2 Kinase Assay System, Promega, Charbonnières-Les-Bains, France).
Statistical Analysis
Analysis of variance followed by Fisher test was used throughout.
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RESULTS |
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Very few SII were present on Day 0 (2200 ± 600) of culture, but their number increased dramatically up to 28 900 ± 3300 on Day 1; thereafter, their number decreased until the end of the culture, but remained always higher than on Day 0 (P < 0.05; Fig. 1B). Likewise, only few RS contaminated elutriated PS (5600 ± 1200) at the time of seeding. However, the number of RS increased rapidly, reaching its maximal value on Day 2 (82 000 ± 6900; P < 0.001 vs. Day 0; i.e., 1 day later than for SII, which matches the duration of the stage XIV of the seminiferous epithelium of the rat) [9]. Thereafter, it decreased slightly between Day 3 (85 000 ± 4600) and Day 5 (53 500 ± 12 200; P < 0.02; Fig. 1C).
When 12.5 µM roscovitine was present in culture media from Day 0 onward, cell viability was identical to that of control cultures on Day 5 (74% ± 2%). Likewise, no effect of roscovitine on the number of PS was ever observed (Fig. 1A). By contrast, the number of SII in roscovitine-treated cultures was only 30%–60% of that of control cultures from Day 1 onward (P < 0.001; Fig. 1B), indicating that roscovitine inhibited the first meiotic division of PS. Moreover, very few RS were observed in the presence of the inhibitor (20%–25% of controls; Fig. 1C), which suggests that roscovitine also inhibited the second meiotic division.
In an attempt to confirm this assumption, in the second set of experiments roscovitine was added to culture media only from Day 1 onward (i.e., at the time when the number of SII was maximal). Under such conditions, the number of RS in roscovitine-treated cultures was decreased, when compared with controls, already from Day 2 (P < 0.02; Fig. 1D). It should be noted that when roscovitine was added on Day 1, the number of RS on Day 2 was higher than when roscovitine was put on Day 0. This results from the half-life of RS, which is 1.5 days under our culture conditions (M. Vigier, personal communication; see discussion below). Taken together, these results indicate that roscovitine is inhibiting both the first and the second meiotic divisions of spermatocytes.
In order to determine whether the inhibitory effect of roscovitine was reversible, in the next series of experiments cocultures of PS and Sertoli cells were treated with 12.5 µM roscovitine for 3 h, either on Day 0 or on Day 1, and then the cells were washed to remove the drug and the cultures were prolonged up to Day 3. As expected, roscovitine treatment did not modify the number of PS at any time (not shown). When roscovitine was added to culture media on Day 0, the number of SII on Day 1 was only 78% ± 2% of that of control (n = 3, P < 0.01), but thereafter the number of SII in roscovitine-treated wells was similar to that in control wells (Fig. 3A). Likewise, when roscovitine was added on Day 1, the number of RS was lower (65% ± 6%) than in controls (P < 0.02) on Day 2, but no longer different on Day 3 (Fig. 3B). These results demonstrate the reversibility of a short treatment with roscovitine on both the first and the second meiotic divisions.
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In order to define whether the effect of roscovitine on the meiotic divisions of germ cells involved the Sertoli cells, in the next experiments PS were preincubated for 3 h with 12.5 µM roscovitine, washed, and then seeded on Sertoli cell monolayers. Under such conditions, the number of SII on Day 1 of coculture was 79% ± 4% of that of control (n = 4; P < 0.02), which is identical to the percentage observed when the inhibitor was added to PS/Sertoli cell cocultures (see above). Likewise, the number of RS on Day 1 was very close when either PS together with Sertoli cells were treated with roscovitine for 3 h on Day 0 of coculture (13 900 ± 1100) or preincubated for 3 h with roscovitine before seeding on Sertoli cells (12 000 ± 1400). These data indicate that the effect of roscovitine on the meiotic divisions is exerted essentially, if not solely, through the germ cells.
This effect of roscovitine was dependent on the concentration of the inhibitor with an apparent half-maximum effect close to 15 µM, both for the first and the second meiotic divisions, as shown in Fig. 4.
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In the next series of experiments, we researched as to whether the effect of roscovitine on male meiotic divisions was due to an inhibition of the histone H1 kinase activity of MPF as in the oocyte [1]. A 3-h treatment with 12.5 µM roscovitine either on Day 0 or on Day 1 did not modify the levels of either cycB1 or CDK1 in PS, SII, or RS at any day of a 3-day culture (Table 1 and data not shown). Since roscovitine acts as a competitive and reversible inhibitor of ATP binding and is removed after washing the cells (see above), histone H1 kinase activity of PS incubated with roscovitine could not be assessed directly. However, roscovitine inhibited the CDK1- and CDK2-associated histone H1 kinase activities of PS (Fig. 5). Taken together, these results suggest strongly that at least part of the inhibitory effect of roscovitine on the male meiosis is exerted through an inhibition of MPF activity of PS.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present results have shown that roscovitine, a potent inhibitor of MPF, CDK2-cyclin A, CDK2-cyclin E, CDK5-p25, and, to a lesser extent, MAP kinase activities [7, 8] is able to inhibit in a dose-dependent manner both the first and the second meiotic divisions of spermatocytes. At the concentration of 12.5 µM, which is close to that giving half of the apparent maximum effect (see below) on either the first or the second meiotic division, this effect was not due to cell toxicity. This is ascertained by similar cell viability in both control and roscovitine-treated cultures and the resumption of the meiotic process after removal of the inhibitor. Hence it appears that both the first and the second meiotic divisions of PS are regulated by some cyclin-dependent kinase(s), the activity of which can be inhibited by roscovitine.
Previous studies indicate that CDK1 and CDK2 are present in both PS and SII [2, 3, 18]. It has been reported recently that disruption of the CDK2 gene in the mouse leads to an arrest of spermatogenesis in late prophase of the first meiotic division [19]. Moreover, cyclin A1, which binds both CDK1 and CDK2, is needed for completion of the first meiotic prophase of spermatocytes [20]. However, CDK2 kinase activity is required for entry into mitosis as a positive regulator of MPF activity [19], and cyclin A1 has an essential role in initiating the activation of MPF at the meiotic G2/MI transition of male germ cells [21, 22]. Those data, together with the present results, including those showing similar levels of cycB1 and CDK1 in cultured PS and SII (Table 1), suggest strongly that both CDK1 and CDK2 are operating during the first and second meiotic divisions of spermatocytes. In addition, a possible involvement of CDK5 in this process cannot be excluded, since CDK5 was localized to the microtubules of metaphase spermatocytes [23]. The present data also fit rather well with recent results showing that a knockout of Cks2, a protein that interacts with CDK1, leads to defects in meiosis in males (and females) [24]. Specifically, germ cells do not progress past the first meiotic division as the result of a failure to enter anaphase. Since the defect involves the lack of interaction between Cks2 and a CDK, it gives further support to the requirement of cyclin-dependent kinase activity in male meiotic divisions.
It could seem surprising that a concentration of 12.5 µM roscovitine induced only a partial inhibition of the formation of RS, since it has been reported that the IC50 for roscovitine of the in vitro kinase activities of MPF and CDK2 cyclin A and E are 0.45 and 0.7 µM, respectively [7]. However, numerous parameters can influence the effects of an inhibitor on intact cells, such as cell membrane permeability, compartmentalization, and degradation. Indeed, in a variety of cultured cancer cells, IC50 values for roscovitine range from 7.9 to 30.2 µM [25]. Moreover, it must be recalled that the number of RS counted, on either day of culture, is the sum of the number of RS formed between this day and the day before and the number of the RS surviving from, but formed during, the preceding days. Since the half-life of RS in culture is 1.5 days (P. Durand, unpublished results), the number of RS formed every day can be easily calculated. Thus, it can be concluded that 12.5 µM roscovitine actually resulted in a complete inhibition of RS formation beyond 24 h of treatment. Finally, the IC50 values of 0.45 and 0.7 µM for roscovitine of purified CDK1 and CDK2, respectively, compare quite well with the concentration of 1 µM, which gave more than 80% of inhibition of the CDK1- or CDK2-associated histone H1 kinase activity of PS lysates (Fig. 5). Taken together, these data make it unlikely that the observed effects of roscovitine might be mediated through an inhibition of MAPK activity. Indeed, the IC50 of roscovitine on purified ERK1 and ERK2 was reported to be 34 and 14 µM, respectively [8]. This is 30- to 60-fold higher than the value needed for half inhibition of purified MPF, and even higher than the concentration of roscovitine that induced a complete inhibition of RS formation from cultured PS.
Thus a functional relationship has been identified and characterized between roscovitine-sensitive cyclin-dependent kinase activities and the spontaneous processing not only of the first meiotic division, but also, for the first time, of the second meiotic division of spermatocytes. Hence, these results support the importance of MPF for progression of male germ cells to meiotic metaphase I and metaphase II, as found in the oocyte [1].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Murielle Godet, INSERM U418/INRA UMR 1245, Hôpital Debrousse, 29, Rue S
ur Bouvier, 69322 Lyon cedex 05, France. FAX: 33 4 78256168; godet{at}lyon.inserm.fr
Received: 29 September 2003.
First decision: 10 October 2003.
Accepted: 25 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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