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Regular Article |
a Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
ABSTRACT
Cell division requires that cyclin-dependent kinases (Cdks) be activated by phosphorylation. In mitotic cells, this is accomplished by the Cdk-activating-kinase (CAK), which is a complex of cyclin H and Cdk7. There are currently no data on the role of CAK in meiotic cells. Previously, we have shown that cyclin A1 is meiosis-specific and forms an active kinase with Cdk2. Because cyclin A1 is required for meiosis, and its associated kinase must be phosphorylated (activated), we propose that cyclin H/Cdk7 function to activate cyclin A1/Cdk2 in meiotic cells. Here, we show that cyclin H and Cdk7 are present during meiosis. Using reverse transcription-polymerase chain reaction and in situ hybridization, we show that the mRNAs encoding cyclin H and Cdk7 are abundant in spermatocytes. Immunohistochemistry localized cyclin H and Cdk7 to the nucleus of spermatocytes in stages IV to XII of the spermatogenic cycle, overlapping the same stages that express cyclin A1-associated kinases. Finally, immunoprecipitation and histone H1-kinase assays of cyclin H and Cdk7 from testicular extracts show that these proteins interact to form an active kinase. We conclude that cyclin H/Cdk7 complexes are present and during meiosis, form active complexes in testicular cells and are strong candidates for the activating kinase for cyclin A1-associated kinase.
developmental biology, gametogenesis, meiosis, spermatogenesis, testes
INTRODUCTION
The cyclin-dependent kinases (Cdks) are key regulators of the cell cycle whose function is critical for normal cell division [1–3]. The activity of these proteins has been widely studied in model systems of the mitotic cell cycle [1, 4–6]. Biochemical regulation of the Cdks is accomplished by obligatory binding of a regulatory cyclin subunit, phosphorylation (that can either activate or inhibit), and interaction with both activating and inhibiting proteins [7, 8]. While much is known about the general biochemistry of Cdks, their cellular function is not limited to cell division; these kinase complexes, which can be highly regulated, control events as wide ranging as transcriptional activation and signal transduction [5, 9, 10]. For example, the cyclin H/Cdk7 complex controls cell division due to its function as a Cdk-activating kinase (CAK), but also acts in transcriptional regulation in association with transcription factor II H (TFIIH) [8, 11, 12].
A Cdk-activating kinase was first identified in Xenopus egg and mammalian cell extracts that would activate Cdk1 (reviewed in [13], see also [14]). Phosphorylation of Cdk1 at threonine-161 by CAK allows biochemical control and subsequent activation of kinase activity [15, 16]. Activating phosphorylation by CAK has now been shown to occur for other Cdks, such as Cdk2 and Cdk4 [16–18], suggesting a global role for CAK in activating many different cyclin/Cdk complexes. Purification of CAK activity showed that CAK is a complex of cyclin H and Cdk7 [19]. The cyclin H/Cdk7 complex has also been shown to associate with the TFIIH basal transcription machinery and phosphorylate RNA polymerase [12, 20, 21]. This suggests that there may be two pools of cyclin H/Cdk7 complexes, one complex that acts in concert with TFIIH to affect transcription and another that functions as a CAK to activate Cdks [13, 22, 23].
Much of our understanding of the CAK function of cyclin H/Cdk7 has come from biochemical studies performed in vitro [16, 23, 24]. There is evidence, however, pointing to an in vivo functional role for cyclin H/Cdk7 as a bona fide CAK. In the mitotic cell cycle, disruption of cyclin H/Cdk7 or its homologues in many different systems leads to loss of CAK activity and the loss of activation of the appropriate downstream Cdks [14, 16, 25]. Indeed, studies in Drosophila suggest that CAK activity by cyclin H/Cdk7 is essential for normal cell division. Mutations in Cdk7 lead to an arrest of cell division and other cellular defects [26]. Thus, it is clear that maintenance of CAK activity is important. This is accomplished in part by constitutive expression of cyclin H and Cdk7 throughout the cell cycle, except at M-phase [25, 27]. Following activation of cyclin B/Cdk1 in G2-phase, Cdk7 activity decreases to undetectable levels during M-phase of mitosis [27, 28].
A similar requirement and regulation of CAK activity in the mammalian meiotic cell cycle, however, is not yet known. During meiosis, unique cyclins and Cdk isoforms are expressed, including the meiosis-specific cyclin A1 and a potential Cdk2 isoform [29]. Cyclin A1 is required for meiosis; targeted deletion of cyclin A1 leads to meiotic and spermatogenic arrest and male infertility [30]. Cyclin A1 and its associated kinase are likely upstream of the final activation of the cyclin B1/Cdk1 kinase [31], and cyclin B1/Cdk1 is itself essential for meiosis [32]. Because these meiotic kinases likely require activation, we propose that cyclin H/Cdk7 phosphorylates them. For this hypothesis to be true, cyclin H and Cdk7 must be expressed and active in the same meiotic cell types as the cyclin A1-associated kinases. Here, we test this hypothesis by examining the expression of cyclin H and Cdk7 at both the mRNA and protein levels during meiosis and assaying testicular extracts for the ability of these proteins to interact directly and form an active kinase. Confirmation of this hypothesis would be markedly different from mitotic cells, in which cyclin H/Cdk7 activity is decreased during M-phase [27, 28]. Part of this work was presented at the 1998 Society for the Study of Reproduction Annual Meeting [33].
MATERIALS AND METHODS
Tissues and Cell Extracts
All animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility and all studies in this work were approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center. Normal tissues were obtained from CD-1 mice (Charles River Labs, Wilmington, DE). Prepubertal and peripubertal testes were obtained from mice at 7, 17, and 20 days postnatal, and adult tissues were from mice at least 35 days old unless noted. Mice mutant for the ckit/white spotted locus (Wv) and atrichosis locus (ATEB/Le a/a dat/deb) were purchased from the Jackson Labs (Bar Harbor, ME). Tissues dissected for RNA isolation or protein solubilization were immediately frozen in liquid nitrogen. Tissues used for in situ hybridization or immunohistochemistry were taken from animals perfused with PBS and 4% paraformaldehyde/PBS, then fixed overnight in 4% paraformaldehyde/PBS. For immunoprecipitation studies, testes from adult mice were dissected and immediately homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1.0% NP40, 0.5% deoxycholic acid, and 0.1% SDS) containing a protease inhibitor cocktail of aprotinin, leupeptin, and pepstatin (Sigma, St. Louis, MO) at 1 µg/ml each, trypsin inhibitor at 10 µg/ml, 0.1 mM PMSF, 10 mM NaF, and 10 mM NaVO4. All samples were centrifuged at 10 000 x g for 10 min and supernatants were collected. Protein quantitation was by the BCA assay (Pierce, Rockford, IL) and the lysates were either used immediately or frozen on dry ice for later analysis.
Cloning and Sequencing
An adult male mouse pachytene spermatocyte library in 
ZAPII (Stratagene, La Jolla, CA) was kindly provided by Dr. John McCarrey (Southwest Foundation for Biomedical Research, San Antonio TX) [34]. The library was screened with a 0.5-kilobase (kb) EcoRI fragment of mouse cyclin H (CycH; kindly provided by Dr. David Morgan, University of California, San Francisco) [19] and a 1.3-kb clone was isolated and characterized. DNA sequencing was performed using a Sequenase kit (USB, Cleveland, OH) and by the Biotechnology Core Facility of the Texas Tech University Center for Biotechnology and Genomics. Sequence identity was confirmed by BLAST search algorithms [35].
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from different tissues using the Trizol Reagent according to the manufacturer's recommendations (Life Technologies, Rockville, MD). Following DNAse I treatment, 2.5 µg of RNA was incubated for 2 min at 42°C in 48 µl RT mix (1x RT buffer, 10 mM dithiothreitol [DTT], 0.125 mM each dNTPs, 1 µM oligo dT15), 2 µl of 200 U/µl Superscript II RT (Life Technologies) was added and the reaction incubated at 42°C for 1 h. Control cDNA production was the same except that RT was omitted. The cDNA reaction was stopped by incubation at 70°C for 15 min and then 3 µl of cDNA was used for PCR. All PCR reactions were carried out using a Rapidcycler air thermal cycler (Idaho Technology, Salt Lake City, UT) in a volume of 10 µl, containing 0.2 mM dNTPs, 0.5 µM primers, 0.05 µM Taq polymerase (Sigma Chemical Company, St. Louis, MO), and the optimum concentration of the Idaho Technology ATC Mg++ buffer. Also, all reactions were repeated using at least three different RNA preparations of tissues from different animals. Conditions for PCR were optimized empirically for each primer set and the number of cycles were confirmed to be within the linear range. The primers and PCR conditions for each gene are as follows: CycH, forward primer: 5'-CCTCGGATAATAATGCTTAC-3'; reverse primer: 5'-TCATAGCCTTTCCTCTTC-3'; expected product: 625 base pairs (bp); ATC buffer: 4 mM, Rapidcycler settings: denaturation at 94°C for 0 sec; annealing at 55°C for 0 sec; elongation at 72°C for 30 sec; first hold of 94°C for 30 sec; second hold of 72°C for 60 sec, 30 cycles. The cyclin H primers were designed against the full-length CycH cDNA obtained by library screening (accession number AF287135). Cdk7, forward primer: 5'-AACACCAACCAAATCGTC-3', reverse primer: 5'-CTACTCCCACACCATACATC-3', expected product: 500 bp, ATC buffer: 4 mM, Rapidcycler settings: denaturation at 94°C for 0 sec; annealing at 60°C for 0 sec; elongation at 72°C for 30 sec; first hold of 94°C for 30 sec; second hold of 72°C for 60 sec, 30 cycles. GAPDH, forward primer: 5'-AAGGTCGGAGTCAACGGATT', reverse primer: 5'-TTGATGACAAGCTTCCCGTT-3', expected product: 175 bp, ATC buffer: 2 mM, Rapidcycler settings: denaturation at 94°C for 0 sec; annealing at 55°C for 0 sec; elongation at 72°C for 30 sec; first hold of 94°C for 30 sec; second hold of 72°C for 60 sec, 30 cycles. Amplified DNA products were visualized by ethidium bromide staining of products run on 1.2% agarose gels.
In Situ Hybridization
Paraformaldehyde-fixed tissues were embedded in paraffin, cut into 6-µm sections, and analyzed by in situ hybridization using our standard procedures [36, 37]. Briefly, histological sections were deparaffinized in xylene, dehydrated through an alcohol series, deproteinated with 20 µg/ml proteinase K, and acetylated with 0.1 M acetic anhydride/0.25% triethanolamine prior to hybridization. Slides were hybridized overnight at 50°C with 35S-labeled UTP sense or antisense RNA probes transcribed from the T7 or T3 promoters of linearized plasmids using the appropriate RNA polymerase (Promega, Madison, WI), following the manufacturer's suggested protocols. Slides were washed at a final stringency of 0.1x saline-sodium citrate (SSC) at 65°C for 2 h. Autoradiography was performed for 21 days. Developed slides were counterstained with hematoxylin and eosin and viewed on an Olympus BX-60 photomicroscope under brightfield and epiluminescence optics. Photomicrographs were taken using Fujicolor SuperG100 film.
Immunoblot Analysis
Extracted proteins (10 µg/lane) in 1x Laemmli sample buffer were electrophoretically separated on 12% SDS polyacrylamide gels and blotted onto nitrocellulose filters using standard techniques (e.g., [29]). The filters were treated with 0.3% H2O2 in 1x T/S (0.01 M Tris, 0.15 M NaCl pH 7.4) [38], blocked in Blotto (4% nonfat dry milk, 0.2% Tween 20 in 1x T/S), and incubated with primary antibodies for 1 h. Primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA; CycH polyclonal, #sc-855; Cdk7 polyclonal, #sc-828; Cdk7 monoclonal, #sc-7344) were used at 0.5 µg/ml diluted in Blotto. Blots were washed and incubated for 1 h. Following washing three times for 10 min, the blots were exposed to ECL Western blotting detection reagents (Amersham Pharmacia, Piscataway, NJ), following the manufacturer's suggested protocols, and exposed to film. To determine equal loading, the blots were stripped by incubation in 2% SDS, 62.5 mM Tris pH 6.8, 100 mM ß-mercaptoethanol for 30 min at room temperature, and reprobed with an antibody to GAPDH (Research Diagnostics, Flanders, NJ; #RDI-TRK564-6C5) at 0.1 µg/ml for 1 h, and treated as above. All immunoblotting experiments were repeated at least three times with protein extracts from different animals.
Immunohistochemistry
All immunohistochemical procedures followed our previously published protocols [29, 39]. Briefly, perfused, fixed tissues were embedded in paraffin and sectioned at 6 µm. Immunohistochemical analyses were performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Histological sections were deparaffinized in xylene, hydrated through an alcohol series, and washed with H2O. Antigen recapture was performed by boiling slides in a microwave for 10 min in 0.01 M citrate buffer pH 6.0 [40]. Following treatment with 0.03% H2O2 in methanol for 20 min, sections were washed with 1x PBS containing 0.1% Triton X-100 (PBSTr). Slides were blocked for 1 h at room temperature with 2.5% goat serum in PBSTr. All primary antibody incubations were carried out overnight at 4°C in a humidified chamber using the following concentrations in PBS: anti-CycH, 0.5 µg/ml; anti-Cdk7 monoclonal, 0.5 µg/ml. Appropriate control antibodies were diluted at the same concentration as their respective primary antibody. After incubation, slides were washed three times for 10 min each with PBSTr and processed using the Vectastain ABC kit following the manufacturer's suggestions. Diaminobenzidine-stained slides were counterstained with hematoxylin and viewed on an Olympus BX-60 photomicroscope under brightfield optics. Photomicrographs were taken using Fujicolor 100 film.
Immunoprecipitation and Kinase Assays
Testicular lysates (described above) were preabsorbed for 1 h at 4°C with 30 µl of a 20% slurry of protein A-sepharose beads (PAS beads; blocked with 3% nonfat dry milk in RIPA and then equilibrated with RIPA). Antibodies were preconjugated to PAS beads and incubated on a rocking platform overnight at 4°C at a rate of 0.5 µg antibody/ml lysate. Western blot analyses of immunoprecipitated proteins were performed according to our published methods [37]. Histone H1 kinase assays were performed based on the method of Buchkovich [41] with our modifications [29]. Briefly, following two washes in RIPA, immunoprecipitates were washed two times in kinase buffer (50 mM HEPES pH 7, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT). Washed PAS beads were resuspended in 50 µl kinase buffer containing 100 µM ATP, 50 µg/ml Histone H1 (Sigma), 5 µCi 
-32P-labeled ATP (NEN-Perkin Elmer, Boston, MA). Reactions were incubated at 30°C for 10 min and then stopped by immediately adding 50 µl of 2x Laemmli sample buffer. Samples were vortexed, boiled for 5 min, and then run on 12% SDS-PAGE gels. Gels were dried and autoradiography was performed for 18 h. The data presented are representative of at least three different experiments using extracts from different animals.
RESULTS
As mentioned above, because the activity of the cyclin A1 kinase is required for meiosis in males [30], in the present study we have examined the expression of CycH and Cdk7 to test the hypothesis that a CycH/Cdk7 complex is present in order to activate the cyclin A1 kinase during meiosis in males. Furthermore, in humans, both CycH and Cdk7 mRNAs are abundantly expressed in the testis [42], providing further impetus for our focused efforts on mouse spermatogenesis.
Cloning of Murine Cyclin H
Prior to carrying out a detailed analysis of the expression pattern of CycH and Cdk7 during spermatogenesis, we cloned a full-length mouse CycH cDNA. Using a partial, uncharacterized cDNA clone of mouse CycH [19] (kindly provided by Dr. David Morgan, University of California, San Francisco), a 1.2-kb cDNA containing the coding region of mouse CycH (CcnH) was cloned from an adult mouse pachytene spermatocyte cDNA library [34] (a kind gift of John McCarrey, Southwest Foundation for Biomedical Research, San Antonio TX). This sequence has been placed in the GenBank database (accession number AF287135) and is identical to a number of overlapping mouse expressed sequence tags (numbers AA276851, AW538719, and AA672454) and shows 95% similarity at the amino acid level to human CycH.
Developmental and Lineage Expression of CycH/Cdk7 mRNA
To ascertain whether CycH and Cdk7 are expressed in the same cell types as cyclin A1, the expression pattern of CycH and Cdk7 during spermatogenesis was determined using an RT-PCR approach. We used the above spermatocyte CycH cDNA and a full-length mouse Cdk7 cDNA [18] (kindly provided by Dr. Charles Sherr, St. Jude's Children's Hospital) to design gene-specific PCR primers. The primers are from the coding regions of both CycH and Cdk7, which amplify 625-bp and 560-bp fragments, respectively. The identity of these fragments was confirmed by TA-cloning and sequencing. As shown in Figure 1A, both genes are present throughout testicular development with an increase in expression levels being apparent as spermatogenesis proceeds from the mitotic stages (Day 7) to the meiotic stages (Days 17 and 20). Because spermatogenesis advances in a characteristic developmental fashion [43, 44], it is possible to examine whether genes are more likely to be expressed in meiotic germ cells based on their relative abundance at different ages. Our data show similar expression patterns throughout development, suggesting expression of CycH and Cdk7 mRNAs in both mitotic and meiotic cell types.
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In the next experiment, we examined whether CycH and Cdk7 expression is limited to the germ lineage or not. We obtained cDNAs from testes of animals mutant at either the ckit or atrichosis loci. Mice homozygous for these genes have no germ cells, due to agenesis of the germ lineage [45, 46], but do contain the normal complement of the testicular somatic cells, specifically Leydig and Sertoli cells. Results of RT-PCR from the mutant animals are shown in Figure 1B. This analysis reveals a slight decrease in the amount of CycH or Cdk7 expression in the homozygous mutants compared with their normal, heterozygous littermates. These data reveal that mRNAs for both CycH and Cdk7 are abundantly expressed in both the germinal and somatic lineages of the testis, but do not indicate in which cell types these genes are expressed.
Localization of CycH and Cdk7 Transcripts in the Testis
To localize the transcripts for CycH and Cdk7 in the testis, we performed in situ hybridization using 35S-labeled antisense probes to CycH and Cdk7. As shown in Figure 2, hybridization signal of both CycH and Cdk7 was readily apparent over most seminiferous tubules, which is consistent with the data from RT-PCR. Rather than a homogenous pattern of expression in all tubules, however, we observed that some tubule cross-sections show increased levels of hybridization than others. Cross-sections of seminiferous tubules show characteristic patterns of germ cell associations, which have been divided into 12 stages of the seminiferous cycle [47]. Different stages contain germ cells (especially meiotic cells) at different stages of the cell cycle. Our data show that the most abundant signal for CycH is over mid-pachytene spermatocytes in tubule stages V to IX (Fig. 2A), whereas the most abundant for Cdk7 is over more mature pachytene and diplotene spermatocytes in tubule stages IX to XII (Fig. 2C). This suggests that the mRNAs for these genes may be in spermatocytes of different meiotic stages.
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Based on the data shown in Figure 1B showing easily detectable levels of RNA in the testicular somatic cells, we expected that both Leydig cells and Sertoli cells would exhibit abundant hybridization signal in the in situ hybridization experiments. This was not the case. We detected levels of signal significantly above background (see sense probe labeled sections, Fig. 2, B and D) over spermatogenic tubules, which have both Sertoli and germ cells, but little if any signal above background was detected over cells in the interstitial regions, including that of Leydig cells (Fig. 2, A and C).
CycH and Cdk7 Protein Expression During Testicular Development
The differences in the abundance of CycH and Cdk7 genes in differently staged tubules suggested the possibility that expression of the two proteins was uncoupled and that they may be expressed in different cell types. To determine the expression pattern of the CycH and Cdk7 proteins during spermatogenesis, we used Western blot and immunohistochemical analysis of different testicular samples. First, samples of testicular protein of animals of different ages were examined by immunoblot using antibodies specific for CycH and Cdk7. Similar to the mRNA expression, both the 37-kDa CycH and the 39-kDa Cdk7 proteins were readily detectable in each of the different testicular samples (Fig. 3). In samples from animals of different ages, we observed an apparent slight increase in the amount of both CycH and Cdk7 in testes of Day 7 animals through Day 17 and Day 20 until adulthood (Fig. 3A).
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To examine the lineage specificity of CycH and Cdk7 protein expression, similar immunoblot experiments were performed with testicular extracts of animals mutant for either the atrichosis or ckit loci. As shown in Figure 3B, the heterozygous mutant animals showed similar levels of both proteins as normal adult mice (Fig. 3A), whereas there was a decrease in the amount of CycH and Cdk7 protein detected in animals that were homozygous for either mutation. This suggests that although a majority of the protein is in germ cells, there is still a substantial amount of protein present in testicular somatic cell types.
Cell-Type Specific Localization of CycH and Cdk7 in the Testis
To identify which testicular cell types in both the somatic and germinal lineages express CycH and Cdk7, histological sections of mouse testis were examined by immunohistochemistry using the same antibodies as those for the immunoblot analyses. As shown in Figure 4, both CycH and Cdk7 are readily detected in the nuclei of many germ cells throughout development. Surprisingly, not all germ cell subtypes express CycH and Cdk7 to the same degree. In sections from Day 7 postnatal animals, most of the early undifferentiated spermatogonia have abundant CycH and Cdk7 in their nuclei, whereas many of the smaller, differentiated spermatogonia show much lower levels (Fig. 4, A and B). As development proceeds, the expression levels in germ cells further decline from late B-type spermatogonia to preleptotene spermatocytes (Fig. 4, C and D) until there is little if any expression in early prophase leptotene and zygotene spermatocytes (Fig. 4, C–F). As spermatocytes enter the pachytene stage, expression levels increase again and remain at easily detectable levels in the nuclei of these later prophase spermatocytes throughout the remainder of the meiotic cell cycle and into round spermatids (Fig. 4, E and F). Thus, at the protein level, both CycH and Cdk7 are expressed in the same cell types, indicating that there may be a slight delay in the translation of CycH because the CycH mRNA was more abundant at slightly earlier stages than was Cdk7 (see Fig. 2). These data are in general agreement with the mRNA expression patterns, and further pinpoint the cellular localization of CycH and Cdk7 in germ cells of the different meiotic stages.
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In addition to the germ cells, these immunohistochemical experiments show that CycH and Cdk7 are easily detected in Sertoli cells, with increasing abundance during development as Sertoli cells differentiate. The abundant Sertoli cell expression of both CycH and Cdk7 is also seen by examining tubules from the ckit (Fig. 4, G and H) and atrichosis (not shown) mutant mice. This result was not possible to determine from the developmental analyses shown in either Figures 1B or 3B. On the other hand, both CycH and Cdk7 were greatly diminished or not detectable in Leydig cells of mature animals (see Fig. 4, E–H), confirming the results suggested by the in situ hybridization experiments.
Assembly of Active CycH/Cdk7 in the Testis
The expression data presented above indicate that CycH and Cdk7 are both present in later-stage spermatocytes, the same germ cell types that express cyclin A1 [29]. We next used immunoprecipitation and Western blot analysis to determine whether CycH and Cdk7 interact directly in testicular extracts. Testicular extracts were prepared and subjected to immunoprecipitation using either CycH or Cdk7 antibodies. The immunoprecipitates were then immunoblotted and the cognate-bound protein was detected using the same antibodies. Figure 5 shows that the 37-kDa CycH protein is present in extracts immunoprecipitated with Cdk7 and that the 39-kDa Cdk7 protein is present in extracts immunoprecipitated with CycH. These data show that CycH and Cdk7 do interact directly in extracts from testicular cells. Furthermore, it appears that a slightly larger pool of Cdk7 is available for immunoprecipitation by polyclonal antibodies to Cdk7 as opposed to a monoclonal antibody. In addition to immunoblots of the precipitated proteins, we also performed histone H1 kinase assays to confirm that these complexes in the testis are active (Fig. 5, lower panel). Together, these data show that CycH and Cdk7 form an active complex.
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DISCUSSION
We have presented evidence showing that CycH and Cdk7, the major components of the Cdk activating kinase, CAK, are present and active in the testis. Furthermore, these proteins are present in the same cell types as cyclin A1 [29] and cyclin B1 [48]. We and others have shown previously that the meiotic divisions are likely controlled, in part, by the action of the cyclin A1- and cyclin B1-associated kinases [29–32]. In fact, gene ablation studies in the mouse have shown that cyclin A1 is required for meiotic cell cycle progression [30] and strongly implicate cyclin B1 for a similar role [32]. Thus, because these meiotic cyclin kinases are important, it is our hypothesis that they are activated by a CAK.
Our data show that the most abundant expression of CycH and Cdk7 is in late prophase spermatocytes and spermatocytes undergoing the meiotic divisions (see Fig. 4). A summary of the CycH and Cdk7 expression patterns compared to that of the cyclin A1 kinases is shown in Figure 6 (see also Fig. 4). Therefore, we propose that the CycH and Cdk7 proteins present in meiotic cells as described here act as a CAK to activate the meiotic Cdks. Although it is not technically feasible to isolate enough purified cyclin A1- and cyclin B1-associated kinases from late-stage meiotic cells to directly show that these kinases are phosphorylated by CAK, studies are ongoing to determine the meiotic substrates of the CycH/Cdk7 complexes present in male germ cells. As there is currently no evidence that the single subunit Cdk activating kinase, CAK1, which is responsible for CAK activity in budding yeast, is expressed in mammals [49, 50], our data are consistent with the likelihood that Cdk-activating kinase activity in male germ cells is provided by the CycH/Cdk7 complex.
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Previously, we showed that earlier stages of male germ cell development were regulated, in part, by another A-type cyclin kinase, cyclin A2/Cdk2 [37]. This complex has been used as the prototype substrate for action by CAK [16, 24, 51–53]. Our data show that CycH and Cdk7 are expressed in an overlapping pattern with CycA2/Cdk2 in spermatogonia (see Figs. 4 and 6) but not to the same degree in preleptotene spermatocytes. It is possible that these cells represent the best candidates for expressing alternative CycH or Cdk7 isoforms, but it is also possible that the expression levels detected by immunohistochemistry are too low. Although we have not directly tested whether CycH and Cdk7 mRNAs are present in these cells, because there is little change in the expression levels detected by RT-PCR as development proceeds, it is possible that small amounts of the RNAs are present.
Another possible interpretation of our data comes from the function of CycH/Cdk7 not as a CAK, but instead as a component of the TFIIH basal transcription factor. It has been reported that the kinase component of TFIIH that phosphorylates the C-terminal domain of RNA polymerase II is a form of the CycH/Cdk7 complex that contains an additional factor, MAT-1 [12, 20, 21, 54]. Some of the components of the basal transcription machinery are known to be abundant in testis and are present in meiotic prophase cells, including RNA polymerase II [55–57]. We also know that the MAT-1 assembly factor is at least present in the mouse testis (E.A. Whitmire and S.E. Ravnik, unpublished observations). Thus, it is possible that a CycH/Cdk7 complex functions during meiosis to act on RNA polymerase II as a substrate. Studies in our laboratory are currently under way to determine in which cell types MAT-1 is expressed and whether a CycH/Cdk7/MAT-1 complex is an active kinase for the C-terminal domain of RNA polymerase II. It is interesting that a CycH/Cdk7/MAT-1 complex in HeLa cells has been shown to phosphorylate p53 [58] and it is known that p53 is abundant in spermatocytes [59] and is important for accurate monitoring of germ cell apoptosis [60, 61]. Clearly, CAK function during spermatogenesis is likely to be very complex and will take future experiments to fully dissect.
One puzzling aspect of the data presented here is the difference in expression of CycH and Cdk7 between adult Sertoli and Leydig cells. By immunohistochemistry, both CycH and Cdk7 are abundant in Sertoli cells, but not in Leydig cells. If CycH and Cdk7 functioned only as a CAK, this result might simply be related to minor differences in gene expression in two terminally differentiated, nondividing cell types. The possibility that CycH and Cdk7 functions during basal transcription, however, raises an interesting possibility of alternative mechanisms of TFIIH function in Leydig cells. We are currently performing a direct examination of expression of CycH and Cdk7, along with other TFIIH components, in Leydig cells to begin to answer that question.
In summary, we have presented evidence that CycH and Cdk7, at least, of the CAK/TFIIH components are abundantly expressed during the meiotic cell cycle, but there are intriguing differences in the cell type specificity of expression in other testicular cell types that remain to be studied.
ACKNOWLEDGMENTS
We wish to thank Dr. Charles Sherr (St. Jude's Children's Hospital, Nashville, TN) for the kind gift of the Cdk7 plasmid; and Dr. David Morgan University of California, San Francisco, for the partial CycH plasmid. Dr. John McCarrey is thanked for the use of the pachytene spermatocyte cDNA library. Editorial comments were thoughtfully provided by Drs. Clint MacDonald and Elmus Beale. The authors thank Ms. Callenda Hacker for assistance in cloning the full-length mouse CycH and the Biotechnology Core Facility of the Texas Tech University Center for Biotechnology and Genomics for sequencing efforts. The able technical assistance of Mr. Anthony B. LeGrow and Mrs. Patricia L. Frisbie is also gratefully acknowledged.
FOOTNOTES
First decision: 12 October 2000.
1 These studies were supported by grants from the South Plains Foundation, TTUHSC Seed Research Grant program, and National Institutes of Health HD36285 to S.E.R. Additional support to J.M.K., J.T.M., and R.K.B. was provided, in part, by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program at Texas Tech University. 
2 Correspondence. FAX: 806 743 2990; cbbser{at}ttuhsc.edu
Accepted: December 13, 2000.
Received: August 18, 2000.
REFERENCES
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