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Dynamic Changes in the Follicular Transcriptome and Promoter DNA Methylation Pattern of Steroidogenic Genes in Chicken Follicles throughout the Ovulation Cycle.

Zhu G, Mao Y, Zhou W, Jiang Y - PLoS ONE (2015)

Bottom Line: The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species.Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation.Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology Science and Technology, Taishan University, Taian 271021, China.

ABSTRACT
The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species. In this study, we used RNA-seq to study the gene expression profiles of the chicken follicles from different developmental stages (pre-hierarchical, pre-ovulatory and post-ovulatory). Transcriptomic analysis revealed a total of 1,277 and 2,310 genes were differentially expressed when follicles progressed through the pre-hierarchical to hierarchical and pre-ovulatory to post-ovulatory transitions, respectively. The differentially expressed genes (DEG) were involved in signaling pathways such as adherens junction, apoptosis and steroid biosynthesis. We further investigated the transcriptional regulation of follicular steroidogenesis by examining the follicle-specific methylation profiles of Star (steroidogenic acute regulatory protein), Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1) and Hsd3b (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1), genes encoding the key enzymes for progesterone synthesis. The varied patterns of DNA methylation in proximal promoters of Star and Cyp11a1but not Hsd3b in different follicles could play a major role in controlling gene expression as well as follicular steroidogenic activity. Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation. Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species.

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Methylation patterns of the Star, Cyp11a1 and Hsd3b promoters in chicken follicles.Site-specific methylation levels of the proximal promoters of Star, Cyp11a1 and Hsd3b from SWF, F1 and POF1 follicles were compared. The Sequenom MassARRAY platform was used for the quantitative methylation analysis. The CpG units locations are as defined in S1 Fig. Data are expressed as mean ± SEM. n = 3 animals.
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pone.0146028.g001: Methylation patterns of the Star, Cyp11a1 and Hsd3b promoters in chicken follicles.Site-specific methylation levels of the proximal promoters of Star, Cyp11a1 and Hsd3b from SWF, F1 and POF1 follicles were compared. The Sequenom MassARRAY platform was used for the quantitative methylation analysis. The CpG units locations are as defined in S1 Fig. Data are expressed as mean ± SEM. n = 3 animals.

Mentions: Ovarian follicles are able of producing steroid hormones, such as estrogen and progesterone, and this essential ability changes according to their developmental status[14]. The progesterone synthesis is initiated with the transportation of cholesterol into the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR), then cholesterol is converted to pregnenolone by cytochrome P450 side-chain cleavage enzyme (Cyp11a1), transported out of the mitochondria and catalyzed to progesterone by 3β-hydroxysteroid dehydrogenase (Hsd3b)[15]. The lipid-soluble steroids easily diffuse through cells and the steroids synthesis directly reflects the levels of steroidogenic enzymes, which in turn were regulated primarily through gene transcription[16,17].Our RNA-seq and qPCR results show that Star, Cyp11a1 and Hsd3b were differentially expressed in different chicken follicles and could have undergone an increase or decrease in DNA methylation. For the analysis of methylation status of the Star, Cyp11a1 and Hsd3b genes, several amplicons spanning the corresponding proximal promoter regions were designed. A total of 89 CpG sites were covered and genotyped by using Sequenom Technology (S1 Fig). The methylation status of individual CpGs in proximal promoters of Star, Cyp11a1 and Hsd3b were shown in Fig 1. We found no significant changes in the average methylation frequency of Star promoter between SWF and F1 follicles (p = 0.1878, Wilcoxon matched pairs test), whereas the methylation level of Cyp11a1 promoter was much lower in F1 follicles than in SWF follicles (p<0.0033). Both Star (p = 0.0061) and Cyp11a1 (p<0.0093) gene promoters exhibited significant higher methylation levels in post-ovulatory than pre-ovulatory follicles. However, methylation analysis of Hsd3b revealed that most of the proximal promoter region was hypomethylated in all ovarian follicular samples investigated (Fig 1).The overall DNA methylation patterns of proximal promoters of Cyp11a1 and Star but not Hsd3b were in negative correlation with the respective transcript abundance levels in different follicles (Table 3).


Dynamic Changes in the Follicular Transcriptome and Promoter DNA Methylation Pattern of Steroidogenic Genes in Chicken Follicles throughout the Ovulation Cycle.

Zhu G, Mao Y, Zhou W, Jiang Y - PLoS ONE (2015)

Methylation patterns of the Star, Cyp11a1 and Hsd3b promoters in chicken follicles.Site-specific methylation levels of the proximal promoters of Star, Cyp11a1 and Hsd3b from SWF, F1 and POF1 follicles were compared. The Sequenom MassARRAY platform was used for the quantitative methylation analysis. The CpG units locations are as defined in S1 Fig. Data are expressed as mean ± SEM. n = 3 animals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696729&req=5

pone.0146028.g001: Methylation patterns of the Star, Cyp11a1 and Hsd3b promoters in chicken follicles.Site-specific methylation levels of the proximal promoters of Star, Cyp11a1 and Hsd3b from SWF, F1 and POF1 follicles were compared. The Sequenom MassARRAY platform was used for the quantitative methylation analysis. The CpG units locations are as defined in S1 Fig. Data are expressed as mean ± SEM. n = 3 animals.
Mentions: Ovarian follicles are able of producing steroid hormones, such as estrogen and progesterone, and this essential ability changes according to their developmental status[14]. The progesterone synthesis is initiated with the transportation of cholesterol into the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR), then cholesterol is converted to pregnenolone by cytochrome P450 side-chain cleavage enzyme (Cyp11a1), transported out of the mitochondria and catalyzed to progesterone by 3β-hydroxysteroid dehydrogenase (Hsd3b)[15]. The lipid-soluble steroids easily diffuse through cells and the steroids synthesis directly reflects the levels of steroidogenic enzymes, which in turn were regulated primarily through gene transcription[16,17].Our RNA-seq and qPCR results show that Star, Cyp11a1 and Hsd3b were differentially expressed in different chicken follicles and could have undergone an increase or decrease in DNA methylation. For the analysis of methylation status of the Star, Cyp11a1 and Hsd3b genes, several amplicons spanning the corresponding proximal promoter regions were designed. A total of 89 CpG sites were covered and genotyped by using Sequenom Technology (S1 Fig). The methylation status of individual CpGs in proximal promoters of Star, Cyp11a1 and Hsd3b were shown in Fig 1. We found no significant changes in the average methylation frequency of Star promoter between SWF and F1 follicles (p = 0.1878, Wilcoxon matched pairs test), whereas the methylation level of Cyp11a1 promoter was much lower in F1 follicles than in SWF follicles (p<0.0033). Both Star (p = 0.0061) and Cyp11a1 (p<0.0093) gene promoters exhibited significant higher methylation levels in post-ovulatory than pre-ovulatory follicles. However, methylation analysis of Hsd3b revealed that most of the proximal promoter region was hypomethylated in all ovarian follicular samples investigated (Fig 1).The overall DNA methylation patterns of proximal promoters of Cyp11a1 and Star but not Hsd3b were in negative correlation with the respective transcript abundance levels in different follicles (Table 3).

Bottom Line: The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species.Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation.Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology Science and Technology, Taishan University, Taian 271021, China.

ABSTRACT
The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species. In this study, we used RNA-seq to study the gene expression profiles of the chicken follicles from different developmental stages (pre-hierarchical, pre-ovulatory and post-ovulatory). Transcriptomic analysis revealed a total of 1,277 and 2,310 genes were differentially expressed when follicles progressed through the pre-hierarchical to hierarchical and pre-ovulatory to post-ovulatory transitions, respectively. The differentially expressed genes (DEG) were involved in signaling pathways such as adherens junction, apoptosis and steroid biosynthesis. We further investigated the transcriptional regulation of follicular steroidogenesis by examining the follicle-specific methylation profiles of Star (steroidogenic acute regulatory protein), Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1) and Hsd3b (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1), genes encoding the key enzymes for progesterone synthesis. The varied patterns of DNA methylation in proximal promoters of Star and Cyp11a1but not Hsd3b in different follicles could play a major role in controlling gene expression as well as follicular steroidogenic activity. Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation. Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species.

Show MeSH
Related in: MedlinePlus