Sex-specific expression of CTNNB1 in the gonadal morphogenesis of the chicken
- Seung-Min Bae†1,
- Whasun Lim†1,
- Wooyoung Jeong1,
- Jin-Young Lee1,
- Jinyoung Kim2,
- Fuller W Bazer3 and
- Gwonhwa Song4Email author
© Bae et al.; licensee BioMed Central Ltd. 2013
Received: 31 May 2013
Accepted: 5 September 2013
Published: 11 September 2013
Beta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system.
To examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated.
Results of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes.
Collectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.
Beta-catenin (CTNNB1), the vertebrate homolog of Armadillo in Drosophila melanogaster, plays important roles as a transcriptional co-activator in the WNT signal transduction cascade as well as linkage of the cadherin complex to the actin filament network during early embryogenesis and adult homeostasis . In general, in the canonical WNT signaling pathway, cytoplasmic CTNNB1 is activated after binding of the WNT ligand to its transmembrane receptor Frizzled and its co-receptor, low-density lipoprotein receptor related protein 6. The activated CTNNB1 is shuttled into the nucleus to act as a transcriptional stimulator of the TCF/LEF family of DNA-bound transcription factors of the promoter regions of WNT target genes. Without WNT ligand, CTNNB1 is phosphorylated at serine residue 33 and 37 and provides a binding site for the E3 ubiquitin ligase, resulting in CTNNB1 ubiquitination and degradation by the destruction complex composed of Axin, adenomatous polyposis coli gene product (APC), glycogen synthase kinase 3 (GSK3), protein phosphatase 2A (PP2A), and casein kinase 1 (CK1) . Thus, mutant CTNNB1 via mutation at the phosphorylation sites continuously activates WNT signaling cascades, leading to various diseases such as cancer . For instance, it is well known that deregulation of the WNT/CTNNB1 signal transduction cascade results in colorectal carcinogenesis through excessive proliferation or renewal of stem cells resulting from a CTNNB1 mutation .
In the WNT signaling pathway, CTNNB1 also acts as a key player in a number of biological processes including development and differentiation of embryonic organs. For instance, during mouse embryonic development, Ctnnb1−/− embryos fail to develop mesoderm and initiate gastrulation  and they have defects in anterior-posterior axis formation and fail to generate head structures . On the other hand, CTNNB1 is involved in sex determination and differentiation in mice. Recently, Liu and colleagues reported that Ctnnb1 is essential only for ovarian differentiation, but not necessary for testicular development in a conditional knockout mouse model (Ctnnb1+/−) . However, little is known about the expression and function CTNNB1 in development of the chicken embryo, especially gonadal development, sex determination and differentiation. Indeed, in chickens, sex determination and differentiation is decided by a genetic ZZ/ZW system in which the male is homogametic (ZZ) and the female is heterogametic (ZW). In the chicken embryo (21-day embryonic period), the gonads on embryonic day (E) 3.5 exist in a bipotential undifferentiated state and are initially visible on the ventral surface of the embryonic kidneys . The first signs of sex determination and differentiation occur at E6.5 when morphological changes include proliferation and thickening of the cortex of the left gonad as well as regression of the right gonad in the female. In contrast, the male embryo undergoes histological changes associated with formation of seminiferous cords in the medullary region and a reduction in size of the cortex without any morphological differences between the two gonads . In mammals, the sex determining region Y (SRY), a well-known sex determining gene, Sry-associated HMG-box gene 9 (SOX9) and anti-Müllerian hormones (AMH) are known molecular markers for fetal Sertoli cells of the differentiating embryonic testes . Likewise, double-sex and mab-3 related transcription factor 1 (DMRT1) is the avian Z-linked gene that acts as a vital factor for testis determination in chickens . However, the sex-specific role of CTNNB1 in chicken gonadal development is unknown; therefore, we focused our investigation on that gene. Results of the present study indicate that CTNNB1 plays essential roles in development of the testes and, specifically, differentiation of Sertoli cells during formation of the seminiferous tubules.
Experimental animals and animal care
The experimental use of chickens for this study was approved by the Animal Care and Use Committee of Dankook University. White Leghorn (WL) chickens were exposed to a light regimen of 15 h light and 9 h dark with ad libitum access to feed and water, and subjected to standard poultry husbandry guidelines.
Freshly laid eggs were incubated with intermittent rocking at 37°C under 60-70% relative humidity. Sex was determined on embryonic day E6.5. Approximately 0.2 ul of embryonic blood was collected from the dorsal aorta, diluted in 15 ul of 1× phosphate buffered saline (PBS, pH 7.4), and boiled at 95°C for 10 min to prepare the DNA template for PCR. Each 20 ul PCR reaction contained 2 ul of DNA template, 2 ul of PCR buffer, 1.6 ul of 2.5 mM dNTP mixture, 10 pmol of each forward and reverse primer of chicken W chromosome (F: 5’-CTA TGC CTA CCA CAT TCC TAT TTG C-3’ and R: 5’-AGC TGG ACT TCA GAC CAT CTT CT-3’), and 1 unit of Taq DNA polymerase. The thermal conditions for 35 cycles were 95°C for 30 sec, 66°C for 30 sec, and 72°C for 30 sec. Male or female sex was determined based on the strong bands detected for the W chromosome in the agarose gel after separation of PCR products by gel electrophoresis.
We collected left and right gonads apart from mesonephric kidney of chicken embryos at E6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens. After collecting gonads, tissue samples were stored at −80°C for extracting RNA or fixed in freshly prepared 4% paraformaldehyde in PBS (pH 7.4). After 24 h, embryos fixed in 4% paraformaldehyde were changed to 70% ethanol for 24 h and then dehydrated in a graded series of increasing concentrations of ethanol. Embryos were incubated in xylene for 3 h and embedded in Paraplast-Plus (Leica Microsystems, Wetzlar, Germany). Paraffin-embedded tissues were sectioned at 5 μm for further analyses.
Total cellular RNA was isolated from frozen tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.
Quantitative RT-PCR analysis
Total RNA was extracted from each segment of oviduct at each time point using TRIzol (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized using a Superscript® III First-Strand Synthesis System (Invitrogen). Gene expression levels were measured using SYBR® Green (Biotium, Hayward, CA, USA) and a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene was analyzed simultaneously as a control and used for normalization of data. GAPDH expression is assumed to be most stable among other house-keeping genes, so it is commonly used for normalizing for variations. Each target gene and GAPDH was analyzed in triplicate. Using the standard curve method, we determined expression of the examined genes using the standard curves and Ct values, and normalized them using GAPDH expression. The PCR conditions were 95°C for 3 min, followed by 40 cycles at 95°C for 20 sec, 60°C for 40 sec, and 72°C for 1 min using a melting curve program (increasing the temperature from 55°C to 95°C at 0.5°C per 10 sec) and continuous fluorescence measurement. ROX dye (Invitrogen) was used as a negative control for the fluorescence measurements. Sequence-specific products were identified by generating a melting curve in which the Ct value represented the cycle number at which a fluorescent signal was statistically greater than background, and relative gene expression was quantified using the 2–ΔΔCt method . For the control, the relative quantification of gene expression was normalized to the Ct value for the control oviduct.
In situhybridization analysis
For hybridization probes, PCR products were generated from cDNA with the primers used for RT-PCR analysis. The products were extracted from the gel and cloned into pGEM-T vector (Promega, Madison, WI). After verification of the sequences, plasmids containing gene sequences were amplified with T7- and SP6-specific primers (T7:5’-TGT AAT ACG ACT CAC TAT AGG G-3’; SP6:5’-CTA TTT AGG TGA CAC TAT AGA AT-3’) then digoxigenin (DIG)-labeled RNA probes were transcribed using a DIG RNA labeling kit (Roche Applied Science, Indianapolis, IN). Tissues were collected and fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5 μm on APES-treated (silanized) slides. The sections were then deparaffinized in xylene and rehydrated to diethylpyrocarbonate (DEPC)-treated water through a graded series of alcohol. The sections were treated with 1% Triton X-100 in PBS for 20 min and washed two times in DEPC-treated PBS. After washing in DEPC-treated PBS, the sections were digested with 5 μg/ml Proteinase K (Sigma-Aldrich, St. Louis, MO) in TE buffer (100 mM Tris–HCl, 50 mM EDTA, pH 8.0) at 37°C. After post-fixation in 4% paraformaldehyde, sections were incubated twice for 5 min each in DEPC-treated PBS and incubated in TEA buffer (0.1 M triethanolamine) containing 0.25% (v/v) acetic anhydride. The sections were incubated in a prehybridization mixture containing 50% formamide and 4× standard saline citrate (SSC) for at least 10 min at room temperature. After prehybridization, the sections were incubated overnight at 42°C in a humidified chamber in a hybridization mixture containing 40% formamide, 4× SSC, 10% dextran sulfate sodium salt, 10 mM DTT, 1 mg/ml yeast tRNA, 1 mg/ml salmon sperm DNA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.2 mg/ml RNase-free bovine serum albumin and denatured DIG-labeled cRNA probe After hybridization, sections were washed for 15 min in 2× SSC at 37°C, 15 min in 1× SSC at 37°C, 30 min in NTE buffer (10 mM Tris, 500 mM NaCl and 1 mM EDTA) at 37°C and 30 min in 0.1× SSC at 37°C. After blocking with a 2% normal sheep serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), the sections were incubated overnight with sheep anti-DIG antibody conjugated to alkaline phosphatase (Roche, Indianapolis, IN). The signal was visualized following exposure to a solution containing 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate, 0.4 mM nitroblue tetrazolium, and 2 mM levamisole (Sigma Chemical Co., St. Louis, MO).
Immunocytochemical localization of CTNNB1 protein was performed as described previously using a rabbit polyclonal antibody to CTNNB1 (catalog number #9582; Cell Signaling Technology) at a final dilution of 1:100. Negative controls included substitution of the primary antibody with purified non-immune rabbit IgG at the same final concentration.
All quantitative data were subjected to analysis of variance (ANOVA) according to the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC). All tests of significance were performed using the appropriate error terms according to the expectation of the mean square for error. Data are presented as mean ± SEM unless otherwise stated. Differences in the variances between E6 and E9 for each gonad were analyzed using the F test, and differences in the means were subjected to Student’s t test. Differences were considered significant at P < 0.05.
Differential expression of CTNNB1 mRNA and protein during the male gonadal development and differentiation
Differential expression of CTNNB1 mRNA and protein during the female gonadal development and differentiation
Expression of sertoli cell development-related genes, AMH, ESR1, CCND1 and CDH2 during testicular development
Localization of sertoli cell development-related genes, AMH, ESR1, CCND1 and CDH2 during testis development
Results of the present study provide comprehensive information on expression of CTNNB1 during embryogenesis and in the maturation of male and female gonads of chickens. Our results also indicate stage of development and cell-specific patterns of expression of AMH, ESR1 and CCND1 during embryonic development, as well as during post-hatching sexual maturation of roosters that are very similar to that for CTNNB1 in testes of the rooster. These results support our hypothesis that CTNNB1 plays important roles in differentiation of Sertoli cells and in the formation of seminiferous tubules during testes development.
CTNNB1, a member of the armadillo family of proteins, has the classical armadillo repeat domain and the C-terminal domain. Interestingly, the N-terminal region (known as a Helix C) of the C-terminal domain connects with the end of the armadillo repeat domain resulting in formation of a CTNNB1 superhelical core that is necessary to recruit and bind various CTNNB1 co-activators for increasing levels of transactivation in the WNT signal transduction cascades . In addition, the sequence of CTNNB1 is highly conserved among various species. For instance, chicken CTNNB1 amino acid sequence shares 99-, 99- and 85% identity with mouse, human and Xenopus, respectively . In the present study, we assessed sex-specific, cell-specific and temporal expression of CTNNB1 in gonads on selected embryonic days from female and male embryos. In male gonads, CTNNB1 mRNA and protein were expressed predominantly in the seminiferous cords of developing testes between E6 and E14 (Figure 1). But, in female gonads, expression of CTNNB1 mRNA and protein were mainly localized to the medullary region of embryonic gonads from E6 to E9 and weakly detected in the cortex region of gonads from E14 to E18 (Figure 2). In chickens, undifferentiated gonads consist of an outer cortex and underlying medulla, and most mesenchymal cells are localized in the medullary region and most primordial germ cells are in the cortical region . However, during morphological differentiation after onset of sex determination, medullary cords transform into seminiferous cords in male embryonic gonads and the cortex is diminished in developing testes . Interestingly, CTNNB1 mRNA and protein were abundant in Sertoli cells of testes from immature and mature male chickens (Figure 1). In the chicken oviduct, CTNNB1 mRNA and protein were predominantly detected in luminal epithelial cells, but not in any other cell-type (Figure 2). However, little or no expression of CTNNB1 mRNA was detected in ovaries from either immature or mature female chickens. Therefore, CTNNB1 likely has an essential role in testis development and Sertoli cell differentiation, but it is not required for development and differentiation of the ovary in female chickens.
Morphological and functional aspects of the reproductive organs (i.e. two gonads, and Müllerian and Wolffian ducts) of mammalian male and female embryos are initially indistinguishable. But, with initiation of sexual differentiation occurs in response to testicular hormones such as testosterone and anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance . AMH, a glycoprotein in the transforming growth factor-beta family, is secreted by Sertoli cells and transduces its signal via transmembrane receptors such as AMH type II receptor, after the onset of testis determination in the male fetus . As a result, there is regression of the Müllerian duct and testosterone produced by Leydig cells, promotes Wolffian duct development . AMH induces accumulation of CTNNB1 in the cytoplasmic and nuclear compartments of peri-Müllerian mesenchymal cells which likely alters target gene expression patterns in the cells leading to regression of the Müllerian ductal epithelial cells . This result indicates that CTNNB1 might be a critical regulator in the AMH signaling pathway . Thus, we examined the expression AMH in gonads of male chicken embryos in this study and demonstrated that it was expressed predominantly in seminiferous cords from E9 to hatching (Figures 3 and 4). In addition, AMH mRNA was expressed in seminiferous tubules in testes from 12-weeks-old chickens during development and differentiation of Sertoli cells in seminiferous tubules, but not in Sertoli cells in testes of sexually mature male chickens. These results support the idea that AMH initially produced by the Sertoli cells of the embryonic testes in the fetal male remain elevated levels during childhood, but decreases to low levels from puberty to mature adult male stage in life . Therefore, results of our current study suggest that AMH-activated CTNNB1 plays an important role in sex determination, differentiation and testes development in male chickens.
In the male reproductive system, androgens from Leydig cells are metabolized to estrogens by the P450 aromatase enzyme localized mainly to Sertolic cells and germ cells and estrogen is involved in various biological and physiological functions of testes . In general, estrogen mediates various physiological effects via specific estrogen receptors, particularly estrogen receptor alpha (ESR1) and –beta (ESR2) that accompany activation of SRC and several signaling cascades, such as the MAPK and EGFR cell signaling pathways. Hence, this activation of estrogen-dependent signal transduction likely contributes to the proliferation of Sertoli cells and interactions between Sertoli cells and germ cells in mice. For instance, Esr1 knockout mice have impaired spermatogenesis with reduced sperm numbers and abnormal sperm function leading to infertility [27, 28]. In addition, Esr1−/− and Esr1−/−/Esr2−/− mice exhibit depletion of germ cells in the seminiferous tubules and defective structural changes such as a marked dilation of straight tubules and rete testis . These results indicate that estrogen and its receptor are essential for normal spermatogenesis, development of seminiferous tubules and fertility in males. In the present study, ESR1 was expressed predominantly in seminiferous cords at E9 and then its expression decreased until after hatching. ESR1 was abundant in seminiferous tubules of testes of male chickens at 12 weeks of age and expressed specifically in Sertoli cells of adult testes; changes are coordinate with abundant expression of CTNNB1. Indeed, there are many reports that estrogen induces WNT/CTNNB1 signaling pathways in various organs and disease states. For example, when estrogen levels increase or decrease, there is coordinate accumulation or reductions in amounts of activated CTNNB1 proteins in the nucleus during the proliferative and secretory phases of the menstrual cycle in women . Moreover, administration of exogenous estrogen to mice increased nuclear localization of Ctnnb1 in epithelial cells of the mouse uterus . Therefore, our current results suggest that estrogen-ESR1 signaling via CTNNB1 is likely to be involved in development and differentiation of seminiferous tubules and Sertoli cells in male chickens.
In the Sertoli cells, estrogen also regulates expression of cyclin D1 (CCND1), as a well-known D-type cell cycle regulator, through SRC/MAPK pathways , that plays a pivotal roles in the G1/S phase during spermatogonial cells proliferation [33, 34]. Indeed, CCND1 is one of the essential factors in cell adhesion. In the testis, integrins mediate formation of Sertoli cell tight junction complexes and several studies suggested that CCND1 plays a key role in integrin-mediated cell adhesion . In addition, expression of CCND1 is stimulated by focal adhesion kinase in response to cell adhesion signals  and expression is directly increased by integrin-liked kinase which is a required for cell adhesion and interactions with integrin beta subunits . Interestingly, over-expression of CTNNB1 increased CCND1 protein levels and consequently induced rapid progression of the cell cycle . On the other hand, in the non-canonical WNT pathway, CTNNB1 and N-cadherin participate in cell-cell adhesion . Of particular note, N-cadherin can mediate adhesion between Sertoli cells and spermatogenic cells and also serve as a marker of Sertoli cells. Moreover, CTNNB1 has an important function to regulate adhesion and signaling pathways at Sertoli cell-germ cell junctions [39–41]. In the present study, CCND1 was specifically and abundantly expressed in the seminiferous cords of male gonads between E6 and E9 and in seminiferous tubules of testes at 12 weeks of age. Interestingly, CCND1 was predominantly expressed in Sertoli cells within the seminiferous tubules of adult testes. Therefore, based on previous reports and our present results, we suggest that CCND1 expression increases coordinately with increases in expression of CTNNB1 to play important roles in increasing proliferation of seminiferous epithelial cells and Sertoli cells within the seminiferous tubules.
Collectively, results of the present study provide the first molecular and cellular evidence for distinct sex-specific, cell-specific and temporal expression of CTNNB1 during gonadal development from embryonic stages to maturation of the reproductive organs, specifically the gonads, in chickens. Further, our results revealed coordinate changes in expression of CTNNB1, along with AMH, ESR1 and CCND1 during development of the seminiferous tubules and differentiation of Sertoli cells in testes of male chickens. Therefore, results of the present study provide new insights into sex-specific expression of CTNNB1 during sex determination and differentiation as well as during testicular development during post-hatching stages of development in male and female chickens.
This research was funded by the Ministry of Education, Science, and Technology, and also by a grant from the Next-Generation BioGreen 21 Program (No. PJ008142), Rural Development Administration, Republic of Korea.
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