Haploinsufficiency of Cytochrome P450 17
-Hydroxylase/17,20 Lyase (CYP17) Causes Infertility in Male Mice
Ying Liu,
Zhi-Xing Yao,
Claude Bendavid,
Carol Borgmeyer,
Zeqiu Han,
Luciane R. Cavalli,
Wai-Yee Chan,
Janet Folmer,
Barry R. Zirkin,
Bassem R. Haddad,
G. Ian Gallicano and
Vassilios Papadopoulos
Department of Biochemistry and Molecular Biology (Y.L., Z.-X.Y., Z.H., W.-Y.C., V.P.), Oncology (C.Be., L.R.C., B.R.H.), Cell Biology (C.Bo., G.I.G.), and Pediatrics (W.-Y.C.), Georgetown University Medical Center, Washington, D.C., 20057; Laboratory of Clinical Genomics (W.-Y.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Department of Biochemistry and Molecular Biology (J.F., B.R.Z.), The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Vassilios Papadopoulos, Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20057. E-mail: papadopv{at}georgetown.edu.
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ABSTRACT
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Cytochrome P450 17
-hydroxylase/17,20-lyase (CYP17) is critical in determining cortisol and sex steroid biosynthesis. To investigate how CYP17 functions in vivo, we generated mice with a targeted deletion of CYP17. Although in chimeric mice Leydig cell CYP17 mRNA and intratesticular and circulating testosterone levels were dramatically reduced (80%), the remaining testosterone was sufficient to support spermatogenesis as evidenced by the generation of phenotypical black C57BL/6 mice. However, male chimeras consistently failed to generate heterozygous CYP17 mice and after five matings chimeric mice stopped mating indicating a change in sexual behavior. These results suggested that CYP17 deletion caused a primary phenotype (infertility), probably not due to the anticipated androgen imbalance and a secondary phenotype (change in sexual behavior) due to the androgen imbalance. Surprisingly, CYP17 mRNA was found in mature sperm, and serial analysis of gene expression identified CYP17 mRNA in other testicular germ cells. CYP17 mRNA levels were directly related to percent chimerism. Moreover, more than 50% of the sperm from high-percentage chimeric mice were morphologically abnormal, and half of them failed the swim test. Furthermore, 60% of swimming abnormal sperm was devoid of CYP17. These results suggest that CYP17, in addition to its role in steroidogenesis and androgen formation, is present in germ cells where it is essential for sperm function, and deletion of one allele prevents genetic transmission of mutant and wild-type alleles causing infertility followed by change in sexual behavior due to androgen imbalance.
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INTRODUCTION
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CYTOCHROME P450 17
-hydroxylase/17,20-lyase (CYP17, EC 1.14.99.9) is a microsomal monoxygenase that mediates the 17
-hydroxylation of pregnenolone or progesterone to yield 17
-OH pregnenolone or 17
-OH progesterone, and the cleavage of the c17,20 bond of these steroids, leading into biosynthesis of cortisol and sex steroids (Refs.1, 2, 3 ; Fig. 1
). Thus, CYP17 plays a key role in determining the balance between corticosteroids and steroid sex hormones, although various intracellular factors may influence the catalytic activity of the enzyme (2, 4). CYP17 deficiency in humans, due to complete or partial combined deficiencies of 17
-hydroxylase/17,20-lyase activities, results in impaired production of cortisol, androgens and estrogens, and overproduction of mineralocorticoids (5) leading to hypertension, pseudohermaphroditism, and delay in sexual maturation (5, 6, 7).
To investigate how CYP17 functions in vivo, we generated mice with a targeted deletion of CYP17. In this article, we report our surprising findings that CYP17, in addition to its role in steroidogenesis, is present in germ cells where it is required for fertility.
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RESULTS AND DISCUSSION
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Mouse CYP17 is encoded by a single gene located on chromosome 19 (8). Using CYP17 cDNA [Ref.8 ; National Center for Biotechnology Information (NCBI) GenBank accession no. NM_007809] as a probe, we screened a mouse BAC (bacterial artificial chromosome) library and isolated a clone containing CYP17 genomic DNA (120 kb). The CYP17 gene was sequenced and shown to include eight exons (Fig. 2a
). The sequence of the entire CYP17 gene sequence was deposited in the NCBI GenBank (accession no. AY594330). A targeting vector was constructed with the neomycin resistance (neo) gene (2.0 kb) used to replace the entire (exons and introns, 8.2 kb) CYP17 gene as well as 5' and 3' DNA fragments (> 3 kb) (Fig. 2b
). The rationale for replacing the entire gene instead of interrupting one of the exons was to avoid potential recombination with sequences homologous to the remaining genomic sequences of the CYP17 gene. The targeted allele was generated by homologous recombination (Fig. 2c
). The KpnI linearized targeting vector was transfected into 129/SvJ agouti embryonic stem (ES) cells derived from 129/SvJ-strain agouti mice and G418-resistant positive clones were selected. The integration of the CYP17 deletion in the ES cell-selected clones was evaluated by Southern blot (Fig. 2d
): hybridization of the genomic DNA digested with XbaI and ClaI with the designed 3' probe resulted in the identification of a 7.0-kb fragment in the ES wild-type genomic DNA and 7.0- and 3.7-kb fragments in the ES cells containing a CYP17 deleted allele (Fig. 2d-A
); hybridization of the genomic DNA digested with XhoI and AflII with the designed 5' probe resulted in the identification of a 5.8-kb fragment in the ES wild-type genomic DNA and 5.8- and 4.3-kb fragments in the ES cells containing a CYP17-deleted allele (Fig. 2d-B
). Three ES clones with CYP17 deleted allele were selected. To assess whether the insertion of the neo gene alters the expression of other genes, we monitored the expression of As3mt (Mus musculus arsenic methyltransferase; NM_020577) and sfxn2 (Mus musculus sideroflexin 2; NM_053196) genes present upstream and downstream of CYP17 in chromosome 19, respectively. RT-PCR analysis of the expression of these two genes indicated that the insertion of neo gene did not affect gene expression (Fig. 2e
). The three selected positive clones were injected into blastocysts from C57BL/6-strain mice.

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Fig. 2. Strategy for CYP17 Gene Knockout in ES Cells Using Gene Targeting
Schematic representations: a, the genomic CYP17 wild-type locus; b, the targeting vector; c, the targeted locus showing exons (E1E8), restriction sites of KpnI (used to linearize the targeting vector), ClaI and XbaI (resulting in the 7.0-kb fragment in wild-type and 3.7-kb fragment in the targeted gene by hybridization with the 3' probe), XhoI and AflIII (resulting in the 5.8-kb fragment in wild-type and 4.3-kb fragment in the targeted gene by hybridization with the 5' probe), and the respective positions of the 3' and 5' probes. d, Southern blot analysis of wild-type (+/+) and generated CYP17 (+/) ES cell clones demonstrating the successful homologous recombination of CYP17: A, digestion with by XbaI and ClaI followed by hybridization with the 3' probe; B, digestion with XhoI and AflII followed by hybridization with the 5' probe. e, RT-PCR analysis of As3mt and sfxn2 gene expression in wild-type (+/+) and CYP17 deleted (+/) ES cell clones.
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Twelve chimeric male mice were generated (from 93 newborn pups, 81 mice were with black coat; 13% chimeric). From those chimeric mice, eight showed 7580% chimerism, two showed 50% chimerism, and two showed 2530% chimerism (Fig. 3a
). To generate CYP17 heterozygous (+/) mice, all of the chimeric males were mated with wild-type C57Bl/6 females. Unfortunately, no agouti offspring were produced. All pups had black coats. However, during these studies we noted that all of the chimeric males were only capable of four to five matings, generating four to five litters of black pups. After that, male chimeric mice consistently failed to mate even in the continuous presence of female mice and/or in the presence of new female mice partners, thus indicating a change in sexual behavior, probably due to the reduced testosterone levels (see data below; Ref.9). It should be noted that, in contrast to CYP17 chimeric mice, healthy male mice kept mating through their adulthood.

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Fig. 3. Leydig Cell CYP17 mRNA and Androgen Levels in Wild-Type and CYP17 Chimeric Mice
a, Black and chimeric pups produced with CYP17+/ ES cells. b, CYP17 mRNA levels in interstitial Leydig cells captured from wild-type and high-percentage chimeric mice. c, Intratesticular testosterone levels in wild-type and high-percentage chimeric mouse testes. d, Serum testosterone level in wild-type and high-percentage chimeric mice. Data shown are means ± SEM; *, P < 0.05; **, P < 0.01 compared with wild type.
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The fact that only offspring with black coats were generated from male chimeric mice suggested that the CYP17 gene deletion could have caused a primary phenotype other than an androgen imbalance that was expected. To begin determining the function of CYP17, it was imperative that we investigate whether an androgen imbalance was evident in animals lacking one CYP17 allele in chimeric tissues. For that, laser capture microdissection was employed to capture androgen producing interstitial Leydig cells from wild-type and chimeric mice. Interestingly, a population of randomly picked interstitial Leydig cells from high-percentage (over 75%) chimeric mice contained reduced amounts of CYP17 mRNA when compared with an equal number of Leydig cells captured from control mice (Fig. 3b
). Reduction of Leydig cell CYP17 levels resulted in 80% decrease in intratesticular (Fig. 3c
) and circulating (Fig. 3d
) testosterone levels, in agreement with the critical role of this enzyme in androgen biosynthesis (10). Although high levels of testosterone are critical for spermatogenesis and sperm function in the rat (11), primate (12), and human (13), this might not be the case for the mouse where normal spermatogenesis is maintained in the presence of very low intratesticular testosterone levels (2% of control) (14). In this later study, although spermatogenesis was arrested at round-spermatid stages 78 in the 2-month-old LH receptor knockout mouse, spermatogenesis was completed up to elongated spermatids at late stages 1316, when spermatids are ready for spermiation, indicating complete spermatogenesis (14). In our studies, where 2- to 6-month-old animals were used, despite the low levels of testosterone found in chimeric mice, spermatogenesis was not arrested, but high levels of abnormal sperm were formed (see below). Taken together, these data, while demonstrating the critical role of CYP17 in androgen formation, may not account for the inability of the male CYP17 chimeric mice to generate heterozygous offspring.
In search of the reason behind this infertility, we found that although little difference in the number of sperm in chimeric vs. wild-type mice was observed, a direct loss of function was attributed to the loss of the allele in mature sperm. Checking the morphology of the generated sperm, we observed that higher percentage of abnormal sperm was present in the chimeric male mouse sperm, accounting for 25% and 50% abnormal sperm in mice with lower (2530%) and higher (7580%) chimerism, respectively (Fig. 4a
). It should be noted that the presence of 10% abnormal sperm in wild-type healthy male mice was common (Fig. 4a
). Figure 4b
shows abnormal chimeric male mouse sperm (indicated with arrows) and normal sperm (indicated with arrowheads) present in sperm from high-percentage chimeric mice (images 1 and 2, low magnification view). Two main abnormalities were observed: sperm were bent at a point between the middle and end piece and sperm tails formed distinct loops (indicated by yellow arrows in images 3 and 4; higher magnification view). These morphological defects of the sperm seen in vitro were also seen in situ in epididymes collected from wild-type (image 5) and high-percentage chimeric male mice (images 6 and 7). Swim-up test demonstrated that 50% of abnormal sperms from chimeric mice failed to swim-up in contrast to the wild-type sperm (Fig. 4c
). To determine whether the high percentage of abnormal sperm found in chimeric mice was due to CYP17 deletion, we performed fluorescence in situ hybridization (FISH) using a CYP17 DNA probe and a control probe (PAP7 in BAC library; Ref.15). Figure 4d
shows that morphologically normal sperm were labeled with both the CYP17 (red) and control (green) probes (Fig. 4d
, A and C), whereas most of the abnormal sperm hybridized only with the control probe (Fig. 4d
, B and D). At least 300 sperm were scored for morphology and signals. The percentage of morphologically abnormal swimming sperm without a signal for CYP17 was over 60% (Fig. 4e
), suggesting that the CYP17 deletion affected both sperm morphology and motility, leading to sperm lacking the ability to fertilize oocytes and infertility.

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Fig. 4. Morphological and Functional Characterization of Sperm from Wild-Type and Chimeric Mice
a, Percentage of abnormal sperm in wild-type, 30%, and 80% chimeric mice. b, Microscopic view of sperm obtained from the epididymis of chimeric mice. Image 1 is a low-magnification view: arrows point to abnormal sperm, whereas arrowheads point to relatively normal looking sperm; image 2 shows a higher magnification view; images 3 and 4 show morphologically abnormal sperm. Yellow arrows point to the connection between the middle and end piece of sperm. Images 57 were obtained from in vivo perfused epididymes and shows normal sperm from a wild-type animal (image 5) and abnormal sperm from a chimeric mouse (images 6 and 7). c, Mobility of sperm from wild-type and chimeric mice. d, FISH for CYP17 detection in normal and abnormal sperm. Sperm is counterstained with DAPI (blue) to assess the morphology. Spectrum green-labeled control probe is shown in green and Cy3-labeled CYP17 probe in red. A, Normal sperm showing both the control and the CYP17 probes, green and red, respectively, in lower magnification view; B, abnormal sperm showing labeling with only the control probe (green) in lower magnification. C and D, High-magnification views of FISH images from normal (C) and abnormal (D) sperm. Yellow arrows point to the connection between the middle and end piece of sperm. e, Percentage of CYP17 gene deletion in chimeric swimming sperm of normal and abnormal morphology.
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These data were complemented with Southern blot analysis of sperm from chimeric mice, indicating that one copy of CYP17 gene was deleted (Fig. 5a
). To determine whether the disruption of one allele of CYP17 disturbs the production of 129/SvJ-strain sperm or interferes with transmission of the 129/SvJ sperm during reproduction, we performed a genotyping assay by PCR amplification of the polymorphic locus D2Mit94 (16) (Fig. 5b
). Our result indicates that over 50% sperm from chimeras were originated from ES cells (129/SvJ strain). Further PCR experiments using CYP17 and neo-specific primers (pairs 1 and 2, respectively) demonstrated that, as expected, all samples contained CYP17 (Fig. 5c
; lanes 16), the neo gene was present only in sperm and testes from chimeric mice and the percent of neo gene expression related to the percentage of chimerism (Fig. 5c
, lanes 7 and 10). In addition, CYP17 mRNA, measured by quantitative real-time PCR, was found in sperm from wild-type mice and sperm from high-percentage (7580%) chimeric mice contained 50% less CYP17 mRNA (Fig. 5d
). At last, immunoblot analysis of sperm protein extracts indicated that CYP17 protein levels were dramatically reduced in sperm from chimeric (high and low percentage) compared with wild-type mice (Fig. 5e
). These findings suggest that the absence of germ-line transmission of the mutant allele is not caused by the absence of ES cell-derived germ cells. Deletion of one allele of CYP17 results in a haploinsufficient phenotype of male infertility, as previously reported for protamines 1 and 2 (16) and the klhl10 gene (17).

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Fig. 5. CYP17 Gene Targeting and Expression in Sperm
a, Southern blot analysis of genomic DNA from wild-type and chimeric mouse sperm. b, Genotypic analysis of sperm from wild-type and chimeric mice and the ES cell clone performed by PCR to detect the polymorphic locus D2Mit94, which displays a 194bp band for the 129 strain and 160 bp for the C57 strain. c, Lanes 16 are PCR products (1.8 kb) generated using primers Pair 1 for CYP17 recognizing the wild-type allele; Lanes 712 are PCR products (1.5 kb) generated using primers Pair 2 recognizing the targeted allele. Wt, Wild type; Ch, chimera; S, sperm; T, testis; L, low-percentage (30%) chimeric mouse; H, high-percentage (80%) chimeric mouse. d, Real-time quantitative RT-PCR used to compare the content of CYP17 transcripts in sperm from wild-type and chimeric mice. The relative fold change was defined as the transcript level of CYP17 in chimeric mice divided by that found in wild-type mice set as 1. Data shown is means ± SEM from a representative experiment performed in triplicates (P < 0.05 compared with wild type). Similar results were obtained in five independent experiments (each experiment performed in triplicates). e, Immunoblot analysis of sperm extracts from wild-type, low-percentage, and high-percentage chimeric mice was performed using either an anti-CYP17 antiserum (panel A) or anti-CYP17 antiserum preabsorbed with recombinant CYP17 protein (C). Striped membranes were blotted with an anti-GAPDH antiserum to examine protein loading (panels B and C).
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These studies make a strong case that a previously defined steroidogenic enzyme, which is supposed to be confined to the interstitial Leydig cells of the testis, is present in the nonsteroidogenic sperm, where it fulfills a function in spermatogenesis not yet identified. Interestingly, a search of a mouse germ cell serial analysis of gene expression (SAGE) database, using the tag (CCTTGACACC) at the 3' end of CYP17 mRNA with a Unigene Cluster ID 1262 identified three copies of CYP17 in spermatogonia (0.0027% of transcriptome) and seven copies (0.0062% of transcriptome) in pachytene spermatocytes, further supporting the finding that CYP17 is expressed in germ cells of the testis. In support of this finding, a recent report indicated that CYP17 was present in Leydig cells and spermatids in male raccoon dogs during the mating season, but only in Leydig cells during the nonmating season (18, 19). Thus, although the underlying mechanism by which CYP17 controls sperm structure and function and thus fertility is unknown, the results presented herein could explain why no agouti mice were generated.
During the preparation of this manuscript, Bair and Mellon (20) reported that deletion of mouse CYP17 gene cause early embryonic lethality. These authors were able to generate chimeric mice and heterozygotes that were phenotypically and reproductively normal, but CYP17/ zygotes died by embryonic d 7 (20). In contrast to our findings, no effect on androgen formation (the hallmark of CYP17 function) in the chimeric or heterozygote mice was observed, suggesting that in these mice CYP17 expression in steroidogenic organs might not have been knocked down. Moreover, no information was provided about the CYP17 protein levels either in heterozygotes or homozygote embryos. It is likely that the observed differences between the two studies might be due to the different approaches in gene targeting technology used. In the present study, we deleted the entire CYP17 gene and replaced it with the neo gene that may have resulted in the inactivation of neighboring genes. In contrast, Bair and Mellon interrupted the CYP17 gene at the level of exon 5, which may have allowed for the generation of a partial protein. The finding that the expression of As3mt and sfxn2 gene products, As3mt and sfxn2 genes, are located upstream and downstream of CYP17 in chromosome 19 was not altered by the insertion of the neo gene rules out the possibility that neo insertion may be at the origin of the observed phenotype. Thus, at present we have no explanation for the discrepancy between the phenotypes reported herein and by Bair and Mellon. Nevertheless, the results obtained in both studies clearly suggest that CYP17 exerts functions distinct to the well established 17
-hydroxylase/17,20-lyase activity.
In conclusion, our results indicate that although the critical role of CYP17 in steroid synthesis is unquestionable and supported by the data presented herein, the presence of CYP17 in germ cell lineage of the testis and its link to fertility shown in the present studies raise the possibility that either germ cells and sperm have the ability to form androgens from pregnenolone or progesterone or this enzyme may have alternative functions affecting sperm formation. Indeed, there is evidence that germ cells express the cytochrome P450 aromatase (CYP19) and metabolize androgens to estrogens (21), and it was recently suggested that, in addition to its 17
-hydroxylase/17,20-lyase activity, CYP17 may exert additional catalytic roles (22), including squalene monoxygenase activity critical in cholesterol biosynthesis (23). One of these catalytic roles may involve a step critical in sperm formation, function, and fertility. Indeed, the presence of appropriate amounts of cholesterol in sperm has been shown to be critical in sperm structure and function (24).
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MATERIALS AND METHODS
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Gene Targeting and Chimeric Mice Generation
Using P450c17 cDNA (Ref.8 ; NCBI GenBank accession no. NM_007809) as a probe, a mouse BAC library was screened and a clone containing P450c17 genomic DNA (120 kb) was isolated (GenomeSystems Inc., St Louis, MO). Nucleotide sequencing was determined following the protocol of ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA). Gel electrophoresis and computer analyses were performed at Lombardi Comprehensive Cancer Center (Georgetown University). Several subclones were constructed in vectors, such as pCR2.1 (Invitrogen Life Technologies, Carlsbad, CA), pBlueScript (KS+) (Stratagene, La Jolla, CA) and pGT-N29 (New England Biolabs, Beverly, MA) vectors. The 3.3-kb 5' P450c17 DNA fragment and 3.5-kb 3' DNA fragment were constructed together with the neomycin resistance (neo) gene as targeting vectors. KpnI linearized targeting vector was transfected into ES cells derived from 129/SvJ-strain mice and G418-resistant clones were selected. The integration of the transfected gene in the selected ES cell clones was confirmed by Southern blot. ES clones with CYP17 deletion were microinjected into blastocysts from C57BL/6-strain mice with black coat phenotype and implanted into pseudopregnant foster mothers to generate chimeras. Approximately 2 months later, male chimeras were mated with C57BL/6 female mice to generate offspring (25).
Sperm Count and Swim-Up Test
Epididymes were dissected out at the vas deferens, and sperm from the cauda epididymis was transferred into PBS or in vitro fertilization medium and kept at 37 C for 20 min. Sperm was counted and either used for microscopy, light and FISH, or examined for its ability to swim using the swim-up test as described (26).
In Situ Sperm Morphology
Mice were perfused transcardially with 5% glutaraldehyde in 0.1 M sodium cacodylate buffer. Epididymes were removed and reimmersed in 5% glutaraldehyde overnight. Tissues were postfixed in cacodylate-buffered 1% osmium tetroxide, washed, dehydrated, and embedded in Epon 812. Semithin sections (1 µm) were mounted on glass slides and stained with toluidine blue. Bright-field images were captured with a Nikon Eclipse 800 microscope system (Konagawa, Japan) equipped with a Princeton Instruments charge-coupled device camera and digitized with IPLab software (Scanalytics, Fairfax, VA).
SAGE
Purified total RNA isolated from type A spermatogonia of 6-d-old mice and pachytene spermatocytes of 60-d-old mice were used to generate SAGE libraries using the I-SAGE kit (Invitrogen Life Technologies). 110,872 and 111,384 tags of the spermatogonial and the spermatocyte libraries, respectively, were sequenced (27). Genes encoded by the tag sequences were identified using the SAGEmap database from NCBI.
FISH
Sperm samples were processed for FISH analysis as described earlier (28). Briefly, sperm samples were collected from dissected chimeric mouse epididymis and resuspended in in vitro fertilization media. After a swim up procedure, slides for FISH were prepared from the swimming fraction and dried at room temperature. The slides were fixed in absolute methanol for 2 min. Salt was removed with a single 2x standard saline citrate wash at room temperature for 2 min and dehydration was immediately performed in an ethanol series (70%, 95%, 100%). For the FISH analysis, two probes were used: a gene-specific probe using a CYP17 construct in pBluescript vector and a control probe obtained from a BAC clone containing the PAP7 gene that maps to a different chromosome (chromosome 1; 15). FISH was performed as described (29). Briefly, the probes were labeled by nick translation with two different fluorochromes: Cy3 (red) (Amersham Pharmacia Biotech, Piscataway, NJ) for the CYP17 probe and Spectrum Green (green) (Vysis, Dowers Grove, IL) for the control probe. Slides were denatured at 90 C for 10 min and dehydrated using an ethanol series (70%, 95%, and 100%). After an overnight hybridization at 42 C, scoring of cells and digital image acquisition were performed using a x100 objective mounted on a Leica DMRBE microscope (Leica, Wetzlar, Germany) equipped with optical filters for 4',6-diamidino-2-phenylindole (DAPI), Spectrum Green, Cy3, a triple bandpass (Chroma Technologies, Rockingham, VT) and a cooled charge-coupled device camera (Photometrics, Huntington Beach, CA). DAPI staining was used for sperm localization and evaluation of sperm morphology. At least 300 sperms were scored. Spectrum Green, Cy3, and DAPI digital images were acquired separately as gray-scale images then merged using the software IPLab 3.6 for Windows.
Laser Capture Microdissection
Frozen testis sections (67 µm) were prepared, fixed, and stained for hematoxylin and eosin. Laser capture microdissection was performed as described (30) using a PixCell II apparatus (Arcturus, Mountain View, CA). One hundred Leydig cells were pooled, and total RNA was extracted using the PicoPure RNA isolation kit (Arcturus).
Real-Time Quantitative RT-PCR
Sperm was collected from the epididymes of wild-type and chimeric mice using a micromanipulator (Eppendorf, Westbury, NY). Total sperm RNA was isolated using the RNAzol B reagent (Tel-Test, Inc., Friendswood, TX). Reverse transcription and real-time PCR were performed using the TaqMan reagent (PE Applied Biosystems). An Applied Biosystems Prism 7700 Sequence Detection System (PE Applied Biosystems) was used with the default thermal cycling program (95 C for 10 min followed by 40 cycles of 95 C, 15 sec, 60 C, 1 min). 18S rRNA was used as endogenous reference. The primers (in exon 5) used for real-time PCR amplification of CYP17 were designed by Primer Express (PE Applied Biosystems): sense, 5'-AAGGCCAGGACCCAAGTGT-3'; antisense, 5'-CCACCGTGACAAGGATATGCT-3'.
Testosterone Measurement
Blood was drown from wild-type and chimeric male mice into Serum Separation Blood Collection Tubes (BD Bioscience, San Jose, CA). The tubes were converted several times then sit on ice for a half hour. After a 1-h low-speed centrifugation, the serum was separated from blood cells. Wild-type and chimeric (high) mouse testes were homogenized, and organic extracts were prepared. Testosterone levels in serum and testes were measured by RIA as described (31).
Sperm Genotyping
Sperm was obtained from epididymis and DNA was extracted by Pico Pure DNA Extraction Kit (Arcturus). DNA extraction from the ES cell clone used was performed as described (25). Genotyping for a microsatellite in the D2Mit94 locus of mouse chromosome 2 was performed as described (16, 17) using the PCR Advantage Kit (CLONTECH, Palo Alto, CA)
Southern Blot and PCR Analysis
Genomic DNA was extracted from targeted ES cell, sperm, and testis. For Southern blot analysis (32), DNA was either digested with restrictive endonucleases ClaI and XbaI, and hybridized with 32P-labeled 3' probe, or digested with XhaI and AflII, and hybridized by 32P-labeled 5' probe. The PCR was performed by PCR advantage kit (CLONTECH) using a thermal cycling program (95 C for 2 min followed by 40 cycles of 95 C, 30 sec, 68 C, 6 min, then 1 cycle of 68 C, 12 min). CYP17 primers for PCR amplification were designed as: Pair1 (sense, 5'-gggttagagtcaacgagacaagccg-3'; antisense, 5'-ctagaggccgaatctaacgtcc-3') in CYP17 wild-type allele (1.8 kb); Pair2 (sense, 5'-cctcccctacccggtagaattga-3'; antisense, 5'-tggtggaccagtcagagtctgtga-3') in targeted allele (1.5 kb).
For the analysis of As3mt (Mus musculus arsenic methyltransferase; NM_020577) and sfxn2 (Mus musculus sideroflexin 2; NM_053196) gene expression, total RNA was isolated from wild-type ES cells and CYP17 deletion clones and reverse transcribed using the TaqMan reagent. The PCR was performed by PCR advantage kit using a thermal cycling program (95 C for 2 min followed by 35 cycles of 95 C, 30 sec, 68 C, 3 min, then 1 cycle of 68 C, 6 min). For As3mt sense primer 5'-tcatggctgcttcccgagacgctga-3' and antisense primer 5'-tgcactggcgaagagcaggctaa-3' were used giving a PCR product of approximately 1.2 kb in length. For sfxn2, sense primer 5'-agtgcactttcctggggcgcgtgaa-3' and antisense primer 5'-tgtgaatgccaggcaaacgctccca-3' were used giving a PCR product of approximately 1.2 kb in length. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous reference.
Immuno (Western) Blot Analysis
Sperm from cauda epididymis was used for Western blot analysis performed as previously described (33). Anti-CYP17 antiserum was a gift from Drs. D. B. Hales (University of Illinois, Chicago, IL) and A. Payne (Stanford University, Stanford, CA) and anti-GAPDH (Trevigen, Gaithersburg, MD) antisera were used a loading control. Protein standards for Western blot (MagicMark) were from Invitrogen Life Technologies. Immunoreactive proteins were detected using an enhanced chemiluminescence Western blot detection kit (Amersham Pharmacia Biotech). To determine the specificity of the immunoreactive bands seen, anti-CYP17 antiserum was preincubated for 2 h at room temperature with recombinant mouse CYP17 protein (protein ratio antiserum/CYP17 = 1/5) generated as previously described (23). Blots were striped and incubated with the preabsorbed antiserum.
Miscellaneous
Cell protein content was determined according to the method of Bradford (34) using BSA as standard. Statistical analysis of the data, expressed as mean ± SEM, was performed by ANOVA followed by the Student-Newman-Keuls test using the Instat (version 3.0) package from GraphPad (San Diego, CA).
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ACKNOWLEDGMENTS
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We thank Dr. Jun Liu (Georgetown University Medical Center, Washington, DC) for providing the FISH control probe (BAC containing mouse PAP7 DNA), Drs. D. B. Hales (University of Illinois, Chicago, IL) and A. Payne (Stanford University, Stanford, CA) for the P450c17 antiserum, and the Transgenic Shared Resource at the Lombardi Comprehensive Cancer Center.
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FOOTNOTES
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This work was supported by Grant IBN-0110711 from the National Science Foundation. The authors are grateful to the Transgenic Core Facility, funded by Grant P30 CA51008-13 from the National Cancer Institute, for their support.
First Published Online May 12, 2005
Abbreviations: BAC, Bacterial artificial chromosome; CYP17, cytochrome P450 17
-hydroxylase/17,20 lyase; DAPI, 4',6-diamidino-2-phenylindole; ES, embryonic stem; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SAGE, serial analysis of gene expression.
Received for publication October 18, 2004.
Accepted for publication May 6, 2005.
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