Dataset Information

Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

ABSTRACT: Cytochrome P450 aromatase 19A1 (CYP19A1) is a critical enzyme in estrogen synthesis. However, the effect of CYP19A1 on cell growth and hormone secretion of buffalo follicular granulosa cells (BFGCs) is poorly understood. The objective of this study was to assess the role of CYP19A1 in cell proliferation and hormone secretion of BFGCs by knocking down CYP19A1 mRNA expression. The mRNA expression level of CYP19A1 gene was knocked down in BFGCs using the siCYP19A1-296 fragment with the best interference efficiency of 72.63%, as affirmed by real-time quantitative PCR (qPCR) and cell morphology analysis. The CYP19A1 knockdown promoted the proliferation of BFGCs through upregulating the mRNA expression levels of six proliferation-related genes (CCND1, CCNE1, CCNB1, CDK2, CDKN1A, and CDKN1B). Moreover, CYP19A1 knockdown increased (P < 0.05) the concentrations of progesterone secretion (P4) in BFGCs through increasing the mRNA expression levels of three steroidogenic genes (HSD17B1, HSD17B7, and CYP17A1). Our data further found that the FSH could inhibit the mRNA expression level of CYP19A1 in BFGCs, while LH obtains the opposite effect. These findings showed that the CYP19A1 knockdown had a regulatory role in the hormone secretion and cell proliferation in BFGCs.

SUBMITTER: Lu X

PROVIDER: S-EPMC7545956 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

Lu Xingrong X Duan Anqin A Ma Xiaoya X Liang Shasha S Deng Tingxian T

Frontiers in veterinary science 20200925

Cytochrome P450 aromatase 19A1 (CYP19A1) is a critical enzyme in estrogen synthesis. However, the effect of CYP19A1 on cell growth and hormone secretion of buffalo follicular granulosa cells (BFGCs) is poorly understood. The objective of this study was to assess the role of CYP19A1 in cell proliferation and hormone secretion of BFGCs by knocking down CYP19A1 mRNA expression. The mRNA expression level of CYP19A1 gene was knocked down in BFGCs using the siCYP19A1-296 fragment with the best interfe ...[more]

PMID: 33102564

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Project description:Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.

Project description:BACKGROUND:The complex regulatory mechanism involved in ovarian follicular development is not completely understood. Neuronal neuropeptide Y (NPY) is involved in the regulation of feeding behavior, energy homeostasis, and reproduction behavior, while its function in ovarian follicular development is not clear. The objective of this study was to investigate if and how NPY regulates follicle development in the ovary. METHODS:All experiments were performed using Sprague Dawley rats. To understand NPY expression pattern at different stages of follicular development, NPY content was assessed using immunohistochemistry in individual follicles. NPY and its receptors expression pattern were evaluated in granulosa cells isolated from preantral (PA), early antral (EA) and late antral follicles (LAF). The influence of NPY on granulosa cell proliferation and apoptosis were further assessed in vitro, using Ki67- and TUNEL-positivity assays. To investigate whether NPY induced-proliferation in EA granulosa cells is mediated through the activation of NPY receptor Y5 (NPY5R) and Mitogen-activated protein kinase (MEK) signal pathway, EA granulosa cells were treated with NPY5R antagonist (CGP71683) and MEK inhibitors (PD98059 and U0126), and Ki67-positive cells were assessed. RESULTS:NPY protein expression was follicular stage-dependent and cell type-specific. NPY signal intensity in EA was higher than those in PA and LAF. Antral granulosa cells showed the highest signal intensity compared to mural granulosa cells, cumulus cells and theca cells. Granulosa cells NPY protein content and mRNA abundance were higher in EA than in LAF. NPY receptor contents in granulosa cells were follicular stage-dependent. While NPY reduced apoptosis of EA granulosa cells, it increased the proliferation through NPY5R and MEK pathway. In contrast, in LAF granulosa cells, NPY reduced proliferation and increased the number of apoptotic cells, with no significant effects on PA granulosa cells. CONCLUSION:This study is the first to evaluate the intraovarian role of NPY in granulosa cells at various stage of follicular development. These results indicate that NPY regulates granulosa cells proliferation and apoptosis in a follicular stage-dependent and autocrine manner. NPY may play a role in pathogenesis of ovarian follicular disorders.

Dataset Information

Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

Publications

Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

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