| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Departments of Pathology and Molecular Medicine (G.M., M.P., H.L.), and Biochemistry (M.P.), Division of Cancer Biology and Genetics (G.M., H.L., M.P.), Cancer Research Institute (G.M., M.P.), Queen’s University, Kingston, Ontario, Canada K7L 3N6; and Institut de Génétique et de Biologie Moléculaire et Cellulaire (D.M., P.C.), Collège de France, Illkirch 67404, CU de Strasbourg, France
Address all correspondence and requests for reprints to: Dr. Martin Petkovich, Ph.D., Cancer Research Institute, Division of Cancer Biology and Genetics, Botterell Hall, Room 354, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: petkovic{at}post.queensu.ca.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RA signaling is mediated through two families of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which form functional heterodimers. Each family consists of three separate genes 
, ß, and 
, with several isoforms that differ in tissue distribution (2). In the absence of RA, RAR/RXR heterodimers bind to RA response elements and are complexed with transcriptional corepressors associated with histone deacetylase activity, resulting in chromatin condensation, and transcriptional silencing. In the presence of RA, corepressors are released, permitting liganded RAR/RXR to recruit coactivator complexes possessing histone transacetylase activity, which induces chromatin decondensation and subsequent activation of target gene transcription.
RA is synthesized by a family of retinaldehyde dehydrogenases (ALDH1A1, ALDH1A2, and ALDH1A3) that irreversibly oxidize retinal to form RA (3). Conversely, RA is catabolized by a family of cytochrome P450 (CYP) enzymes, CYP26A1, CYP26B1, and CYP26C1, which convert RA to more polar metabolites for excretion (4, 5, 6). Regions where Cyp26 genes are expressed are devoid of RA, and the complementary expression of CYP26 and ALDH is responsible for defining tissue distribution of RA. Perturbations of this endogenous distribution have severe consequences for the embryo because mice lacking either Cyp26a1 or Cyp26b1 die in utero, or shortly after birth, and exhibit abnormalities consistent with those seen in RA teratogenesis (7, 8, 9).
Although the role of CYP26B1 in regulating limb development has been documented (9), the function of this enzyme in other organ systems, including the embryonic testes, has not been studied extensively. Cyp26b1 is expressed in somatic cells of the embryonic testes (10, 11), whereas Aldh1a2 and Aldh1a3 are detected in the mesonephros (12, 13). Organ culture experiments have shown that embryonic testes grown in the presence of RA express Stra8, which normally is expressed in embryonic germ cells of ovaries (14). Furthermore, expression of Stra8 and Scp3 is up-regulated in embryonic d (E) 13.5 testes in Cyp26b1 null embryos, raising the possibility that Cyp26b1 expression prevents entry of male germ cells into meiosis (11). We have generated a transgenic mouse line lacking Cyp26b1 and observed that in addition to preventing meiotic entry of male germ cells, CYP26B1 is a critical survival factor for germ cells in the embryonic testis. Analysis of Cyp26b1–/– mice revealed increased levels of RA in the embryonic testes, resulting in initiation of meiosis and increased apoptosis of male germ cells as early as E13.5. Testes of neonatal Cyp26b1–/– animals are largely devoid of germ cells, whereas somatic (Sertoli and Leydig) cell differentiation appears unaffected.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ES cells with one targeted Cyp26b1 allele (L3/+) were injected into blastocysts as described elsewhere (7). Agouti mice with germ-line transmission of the L3 allele were bred with CMV-Cre mice, which act as a deletor strain for floxed alleles (16). Resultant offspring were genotyped by PCR for excision of exons 3–6 corresponding to the Cyp26b1 allele. Cyp26b1+/– mice were then crossed to generate Cyp26b1–/– animals.
PCR was used to genotype Cyp26b1 mice using DNA isolated from either tail clips or yolk sacs. A standard PCR was performed using Taq polymerase (Sigma-Aldrich, Oakville, Ontario, Canada) and 3 primers, P1: 5'-CAGTAGATGT TTGAGTGACACAGCC, P2:5'-GAGGAAGT GTCAGGAGAAGTGG, and P3:5'-GGGCCAC CAAGGAAGATGCTGAGG. P1 and P2 amplify a product of 223 bp from the wild-type allele or a product of 284 bp from a targeted (L3) allele but will not amplify a null allele. P1 and P3 will amplify a 364-bp product from only the excised allele. All reactions included 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 mM P1, and 0.2 mM P2 and P3. Each PCR consisted of 30 cycles of 94 C for 20 sec, 58 C for 45 sec, and 72 C for 50 sec. Resultant products were visualized by ethidium bromide staining after electrophoresis in a 2% agarose gel.
Embryo collection and histology
Timed matings were established by housing female mice with males overnight. Females were checked for vaginal plugs, and noon of the day that a plug appeared was denoted as E0.5. Freshly dissected gonads from wild-type and mutant mice were fixed for 1 h in Bouin’s solution and paraffin embedded. Sections (5 µm) were cut, dewaxed, and stained with hematoxylin and eosin as previously described (17). At least four gonads from embryos of each genotype were examined at every developmental stage.
All animal experimentation was reviewed and approved by the University’s Animal Care and Use Committee.
Detection of RA activity
For RA detection, a previously described RA reporter cell line was used (18). The reporter cell line was derived from stable transfection of mouse P19 teratocarcinoma cells with a transgene consisting of the murine Cyp26a1 promoter fused to a firefly luciferase gene. Eight XY gonads from E12.5 Cyp26b1+/+, Cyp26b1+/–, or Cyp26b1–/– embryos were minced finely in MEM (Life Technologies, Inc., Invitrogen Corp., Burlington, Ontario, Canada), subjected to freeze/thaw, and centrifuged at 12,000 g for 10 min at 4 C. The supernatant was added to confluent P19 reporter cells growing in a 24-well plate wrapped in aluminum foil and incubated for 24 h at 37 C, 5% CO2. The medium was then aspirated, and cells were washed twice with PBS. Cells were lysed by adding 50 µl Passive Lysis Buffer (Promega, San Luis Obispo, CA), and cell lysates were transferred to a 96-well assay plate. Readings were taken using a Berthold MicroLumat Plus LB 96V luminometer in the presence of Firefly Luciferase Assay Substrate (Promega). Each sample pool was assayed in triplicate.
Immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis
For immunohistochemistry, sections were blocked in PBS with 10% goat or rabbit serum and 0.1% Triton X-100 for 30 min at room temperature before staining with primary antibodies. Sections were incubated overnight at 4 C with antibodies directed against Müllerian-inhibiting substance (MIS) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse vasa homolog protein (MVH) (1:5000; kindly provided by Dr. Toshiaki Noce, Mitsubishi Kagaku of Life Sciences, Tokyo, Japan), 3ß-hydroxysteroid dehydrogenase (3ßHSD) (1:5000; a kind gift from Dr. Anita Payne, Stanford University, Stanford, CA), or activated caspase-3 (1:200; Cell Signaling Technology, Inc., Danvers, MA). After three washes in PBS, the sections were incubated for 30 min at room temperature with biotinylated antirabbit or antigoat secondary antibody at a dilution of 1:500 (Vector Laboratories, Burlingame, CA). Sections were washed three times in PBS for 5 min each and then incubated with peroxidase-conjugated streptavidin (Zymed Laboratories, South San Francisco, CA) for 10 min at room temperature. Antibody localization was determined by application of diaminobenzidine (Zymed Laboratories). Negative controls, omitting the primary antisera, were included in each experiment.
For the TUNEL assay, sections were dewaxed and boiled for 10 min in 10 mM citrate buffer. TUNEL was performed using the DeadEnd Colorimetric TUNEL System (Promega), and sections were counterstained with hematoxylin. The TUNEL-positive cells in the entire cross section were counted using a light microscope. At least six cross sections were analyzed for each developmental stage.
Chromosome spreading, preparation, and immunofluorescence
For analysis of male meiotic chromosomes, embryonic testes were removed between E12.5 and E16.5, and chromosome spreads were prepared as described previously (19). Slides containing chromosome spreads were subjected to immunofluorescent staining as described (20). Polyclonal rabbit anti-SCP3 antiserum (1:500; a kind gift from Dr. Christa Heyting, Wegeningen University, Wegeningen, The Netherlands) was applied overnight at room temperature. Slides were then washed three times in PBS for 5 min each and incubated with goat antirabbit Alexa 488 (Molecular Probes, Eugene, OR) diluted 1:500 for 60 min at 37 C. After a further three washes in PBS, slides were mounted with Fluorescence Mounting Medium (Dako, Carpinteria, CA) and analyzed by confocal microscopy with a Leica TCS MP2 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Organ culture of embryonic gonads
E12.5 genital ridges were collected from wild-type embryos and cultured as described previously (21). Briefly, paired genital ridges were dissected and cultured on Millicell CM filters (Millipore, Bedford, MA) floating on the surface of DMEM (Invitrogen Corp.) supplemented with penicillin/streptomycin (25 U/ml) and incubated at 37 C with 5% CO2. One genital ridge was cultured for 48 h in medium containing 1 nM of the synthetic retinoid Am580 (Biomol Research Lab, Plymouth Meeting, PA). The other genital ridge from the same embryo was cultured in medium containing an equivalent volume of dimethyl sulfoxide (solvent for Am580) as control. Two independent organ culture experiments were conducted, with each experiment consisting of two pairs of genital ridges.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Cyp26b1+/– mice were fertile and phenotypically indistinguishable from wild-type littermates. When Cyp26b1+/– mice were intercrossed, Cyp26b1–/– offspring were born at the expected mendelian ratio but died within hours of birth due most likely to respiratory distress. Cyp26b1–/– embryos exhibit truncated limbs and severe craniofacial abnormalities, as have been previously reported (9).
|
).
|
In contrast to Cyp26b1+/+ testes, in which many germ cells were observed (Fig. 3, A and C), virtually no germ cells were found in Cyp26b1–/– testes (Fig. 3, B and D). Neonatal ovaries were also examined, and there were comparable numbers of germ cells in Cyp26b1–/– and wild-type littermates (results not shown).
|
Histological abnormalities of germ cells in embryonic Cyp26b1–/– testes
Because virtually no germ cells were found in Cyp26b1–/– neonatal testes, embryonic testes were examined at various stages. The morphology of the testis cords in Cyp26b1–/– mice was normal at all stages examined, whereas there were significant germ cell abnormalities. At E12.5, Cyp26b1–/– testes were indistinguishable from wild-type testes (data not shown). In mutants at E13.5, some germ cells exhibited dark, condensed nuclei (Fig. 4B), typical of apoptotic cells, whereas such cells were rarely observed in the testes of wild-type littermates (Fig. 4A). By E14.5, some germ cells in Cyp26b1–/– testes appeared to be apoptotic cells, whereas others appeared to be in the zygotene stage of meiosis (Fig. 4D). At E16.5, there were fewer germ cells in Cyp26b1–/– mice (Fig. 4F) compared with control mice (Fig. 4E). Cells that were present appeared to be either apoptotic or in the zygotene/pachytene stage of meiosis.
|
, A, C, and E), whereas Cyp26b1–/– testes had significantly more TUNEL-positive cells at all stages examined (Fig. 5, B, D, F, and G). Most TUNEL-positive cells were found within the testis cords, indicating that apoptotic cells were germ cells. Similar results were obtained using a marker of apoptotic death, the active form of caspase-3 (Fig. 5, insets).
|
), whereas at E13.5, there was diffuse SCP3 labeling indicative of leptotene stage (Fig. 6B). At E14.5, in addition to cells in leptotene, samples with thickened threads characteristic of zygotene stage were detected (Fig. 6C). Finally, by E16.5, some germ cells were in zygotene, whereas others had progressed to the pachytene stage with distinct threads representative of paired chromosomes (Fig. 6D).
|
), suggesting that Sertoli and Leydig cells develop normally in Cyp26b1–/– testes.
|
, B, D, and F), whereas such changes were rarely seen in control testes cultured with vehicle (dimethyl sulfoxide) only (Fig. 8, A, C, and E). This suggests that ectopic RA, rather than the lack of specific CYP26B1-generated metabolites, is responsible for phenotypes observed in Cyp26b1–/– testes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In Cyp26b1–/– testes at E13.5, meiotic germ cells were observed, which is the same time that germ cells in ovaries enter meiosis (26). It has been proposed that embryonic germ cells are intrinsically programmed to enter meiosis (26). Germ cells enter meiosis around E13.5 not only in ovaries, but also in regions outside the gonad such as the intervening mesonephric region and adrenal primordia of male embryos (26, 27). Recently, it was speculated that Cyp26B1 prevents premature meiosis in embryonic male germ cells, based on the observation that expression of Stra8 and Scp3 were up-regulated in Cyp26b1–/– testes (11). In the present study, we have categorically demonstrated that male germ cells progressed through meiotic prophase in embryonic Cyp26b1–/– testes. These results provide additional evidence that CYP26B1 acts as a meiotic inhibitor in embryonic testes.
Furthermore, we have observed the striking loss of testicular germ cells due to apoptosis in the absence of Cyp26b1. Increased apoptosis is observed at the same time (E13.5), when Cyp26b1–/– male germ cells enter meiosis. As a result, male germ cells were essentially absent in newborn Cyp26b1–/– testes. This finding is significant because it shows that in addition to preventing meiosis in germ cells, CYP26B1 activity is required for germ cell survival in the embryonic testes. We also show that RA is present in Cyp26b1–/– testes at E12.5, and yet no morphological abnormalities are detected in germ cells until E13.5. This may reflect an indirect effect of RA or a delay between RA exposure and triggering of meiosis and apoptosis.
CYP26B1 metabolizes RA into polar derivatives, which appear to be inactive as shown by genetic and biochemical analysis (28). However, it has been suggested that 4-oxo-RA may have some specific biological role, particularly in lower vertebrates (29, 30). Thus, one possible explanation for our observations is that metabolites generated by CYP26B1 may prevent the apoptosis and entry of male germ cells into meiosis. However, this is unlikely because the synthetic retinoid Am580 is not metabolized by CYP26B1, and yet can still induce meiosis and apoptosis in cultured embryonic testes. This suggests that RA is responsible for the abnormal testicular phenotypes in Cyp26b1–/– embryos.
Finally, we observed that testis cord formation and somatic cell differentiation appear essentially normal in Cyp26b1–/– embryonic testes. The male pathway of gonad development, which is determined by the expression of Sry in somatic cells, occurs within a narrow window (between E11.5 and E12.5). Although Sry was discovered a decade ago, no direct target genes of SRY have been identified (31). Cyp26b1 is first detected in embryonic gonads of both sexes by E11.5 and becomes male-specific by E12.5, shortly after the onset of Sry transcription, indicating that Cyp26b1 could act downstream of Sry. Although Sry is essential for testis cord formation, and Sertoli and Leydig cell differentiation, these male-specific events occur normally in Cyp26b1–/– mice. These observations suggest that Sry and Cyp26b1 act along different pathways.
RA is synthesized in the mesonephros by ALDH1A2 and ALDH1A3, and degraded by CYP26B1, which is expressed in somatic cells of the male gonad. Under these conditions, germ cells develop normally and are maintained in a mitotic state (Fig. 9) until E13.5, when they arrest in quiescence. However, in the absence of Cyp26b1, RA diffuses from the mesonephros, across the basal lamina, where it either directly or indirectly induces meiosis and apoptosis in male germ cells. Although it appears that meiosis precedes apoptosis in germ cells, we cannot exclude the possibility that some germ cells undergo apoptosis without entering meiosis. Two potential mechanisms for these observations are presented in Fig. 9; RA can either act on germ cells directly (Fig. 9B), or induce or repress expression of a secreted factor(s) from Sertoli cells that in turn modulates germ cell meiosis and apoptosis (Fig. 9C). We presently favor the former explanation because Cyp26b1 expression in Sertoli cells enclosing germ cells could provide a barrier to prevent RA from reaching the interior of the seminiferous cord. Furthermore, RARs have been detected in male germ cells (11), and female germ cells appear to enter meiosis in response to RA in vivo. However, it is also possible that meiotic germ cells undergo apoptosis in response to a factor secreted by male somatic cells. Future research will focus on discriminating between these two models, and identifying genes that are responsible for initiating germ cell entry into meiosis and subsequent extinction in the presence of RA.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online June 21, 2007
1 G.M. and H.L. contributed equally to this work. 
Abbreviations: CYP, Cytochrome P450; E, embryonic d; ES, embryonic stem; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; MIS, Müllerian-inhibiting substance; MVH, mouse vasa homologue protein; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Received April 17, 2007.
Accepted for publication June 8, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylase/C17–20 lyase, and P450 aromatase in fetal mouse gonads. Endocrinology 135:362–368This article has been cited by other articles:
 |
|
 |
|
  H. Li and M. Clagett-Dame Vitamin A Deficiency Blocks the Initiation of Meiosis of Germ Cells in the Developing Rat Ovary In Vivo Biol Reprod, November 1, 2009; 81(5): 996 - 1001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Edson, A. K. Nagaraja, and M. M. Matzuk The Mammalian Ovary from Genesis to Revelation Endocr. Rev., October 1, 2009; 30(6): 624 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kocer, J. Reichmann, D. Best, and I. R. Adams Germ cell sex determination in mammals Mol. Hum. Reprod., April 1, 2009; 15(4): 205 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Park, E.-J. Lee, D. Emge, C. L. Jahn, and J. L. Jameson A Phenotypic Spectrum of Sexual Development in Dax1 (Nr0b1)-Deficient Mice: Consequence of the C57BL/6J Strain on Sex Determination Biol Reprod, December 1, 2008; 79(6): 1038 - 1045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Laue, M. Janicke, N. Plaster, C. Sonntag, and M. Hammerschmidt Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development Development, November 15, 2008; 135(22): 3775 - 3787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Nowickyj, J. V. Chithalen, D. Cameron, M. G. Tyshenko, M. Petkovich, G. R. Wyatt, G. Jones, and V. K. Walker Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect PNAS, July 15, 2008; 105(28): 9540 - 9545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Otsuka, A. Konno, Y. Hashimoto, N. Sasaki, D. Endoh, and Y. Kon Oocytes in Newborn MRL Mouse Testes Biol Reprod, July 1, 2008; 79(1): 9 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Suzuki and Y. Saga Nanos2 suppresses meiosis and promotes male germ cell differentiation Genes & Dev., February 15, 2008; 22(4): 430 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. S. Blaner and C. L. Mendelsohn Retinoid Inactivation: Survival Factor for Male Germ Cells Endocrinology, October 1, 2007; 148(10): 4557 - 4559. [Full Text] [PDF]
|
|
|
|
|
|
J. Bowles and P. Koopman Retinoic acid, meiosis and germ cell fate in mammals Development, October 1, 2007; 134(19): 3401 - 3411. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
