miR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells.

(1) Background: DNA damage in cumulus cells hinders oocyte maturation and affects steroid hormone secretion. It is crucial to identify the key factors that regulate cellular DNA damage and steroid hormone secretion. (2) Methods: Treatment of bovine cumulus cells with bleomycin to induce DNA damage. The effects of DNA damage on cell biology were determined by detecting changes in DNA damage degree, cell cycle, viability, apoptosis, and steroid hormones. It was verified that mir-302d targeted regulation of CDKN1A expression, and then affected DNA damage and steroid hormone secretion in cumulus cells. (3) Results: Bleomycin induced increased DNA damage, decreased G1-phase cells, increased S-phase cells, inhibited proliferation, promoted apoptosis, affected E2 and P4 secretion, increased CDKN1A expression, and decreased miR-302d expression. Knockdown of CDKN1A reduced DNA damage, increased G1-phase cells, decreased G2-phase cells, promoted proliferation, inhibited apoptosis, increased E2 and P4 secretion, and increased the expression of BRCA1, MRE11, ATM, CDK1, CDK2, CCNE2, STAR, CYP11A1, and HSD3B1. The expression of RAD51, CCND1, p53, and FAS was decreased. Overexpression of CDKN1A resulted in the opposite results. miR-302d targets CDKN1A expression to regulate DNA damage and then affects the cell cycle, proliferation, apoptosis, steroid hormone secretion, and the expression of related genes. (4) Conclusions: miR-302d and CDKN1A were candidate molecular markers for the diagnosis of DNA damage in bovine cumulus cells.

Sequencing of the Pituitary Transcriptome after GnRH Treatment Uncovers the Involvement of lncRNA-m23b/miR-23b-3p/CAMK2D in FSH Synthesis and Secretion.

The pituitary gland is a key participant in the hypothalamic-pituitary-gonadal axis, as it secretes a variety of hormones and plays an important role in mammalian reproduction. Gonadotrophin-releasing hormone(GnRH) signaling molecules can bind to GnRH receptors on the surfaces of adenohypophysis gonadotropin cells and regulate the expression of follicle-stimulating hormone(FSH) and luteinizing hormone(LH) through various pathways. An increasing number of studies have shown that noncoding RNAs mediate the regulation of GnRH signaling molecules in the adenohypophysis. However, the expression changes and underlying mechanisms of genes and noncoding RNAs in the adenohypophysis under the action of GnRH remain unclear. In the present study, we performed RNA sequencing (RNA-seq) of the rat adenohypophysis before and after GnRH treatment to identify differentially expressed mRNAs, lncRNAs, and miRNAs. We found 385 mRNAs, 704 lncRNAs, and 20 miRNAs that were significantly differentially expressed in the rat adenohypophysis. Then, we used a software to predict the regulatory roles of lncRNAs as molecular sponges that compete with mRNAs to bind miRNAs, and construct a GnRH-mediated ceRNA regulatory network. Finally, we enriched the differentially expressed mRNAs, lncRNA target genes, and ceRNA regulatory networks to analyze their potential roles. Based on the sequencing results, we verified that GnRH could affect FSH synthesis and secretion by promoting the competitive binding of lncRNA-m23b to miR-23b-3p to regulate the expression of Calcium/Calmodulin Dependent Protein Kinase II Delta(CAMK2D). Our findings provide strong data to support exploration of the physiological processes in the rat adenohypophysis under the action of GnRH. Furthermore, our profile of lncRNA expression in the rat adenohypophysis provides a theoretical basis for research on the roles of lncRNAs in the adenohypophysis.

Integrative Proteomics and Phosphoproteomics Analysis of the Rat Adenohypophysis after GnRH Treatment.

The regulation of mammalian reproductive activity is tightly dependent on the HPG axis crosstalk, in which several reproductive hormones play important roles. Among them, the physiological functions of gonadotropins are gradually being uncovered. However, the mechanisms by which GnRH regulates FSH synthesis and secretion still need to be more extensively and deeply explored. With the gradual completion of the human genome project, proteomes have become extremely important in the fields of human disease and biological process research. To explore the changes of protein and protein phosphorylation modifications in the adenohypophysis after GnRH stimulation, proteomics and phosphoproteomics analyses of rat adenohypophysis after GnRH treatment were performed by using TMT markers, HPLC classification, LC/MS, and bioinformatics analysis in this study. A total of 6762 proteins and 15,379 phosphorylation sites contained quantitative information. Twenty-eight upregulated proteins and fifty-three downregulated proteins were obtained in the rat adenohypophysis after GnRH treatment. The 323 upregulated phosphorylation sites and 677 downregulated phosphorylation sites found in the phosphoproteomics implied that a large number of phosphorylation modifications were regulated by GnRH and were involved in FSH synthesis and secretion. These data constitute a protein-protein phosphorylation map in the regulatory mechanism of "GnRH-FSH," which provides a basis for future studies on the complex molecular mechanisms of FSH synthesis and secretion. The results will be helpful for understanding the role of GnRH in the development and reproduction regulated by the pituitary proteome in mammals.

TRH Regulates the Synthesis and Secretion of Prolactin in Rats with Adenohypophysis through the Differential Expression of miR-126a-5p.

Prolactin (PRL) is an important hormone that is secreted by the pituitary gland and plays an important role in the growth, development and reproduction of organisms. Thyrotropin-releasing hormone (TRH) is a common prolactin-releasing factor that regulates the synthesis and secretion of prolactin. In recent studies, microRNAs (miRNAs) have been found to play a key role in the regulation of pituitary hormones. However, there is a lack of systematic studies on the regulatory role that TRH plays on the pituitary transcriptome, and the role of miRNAs in the regulation of PRL synthesis and secretion by TRH lacks experimental evidence. In this study, we first investigated the changes in PRL synthesis and secretion in the rat pituitary gland after TRH administration. The results of transcriptomic analysis after TRH treatment showed that 102 genes, including those that encode Nppc, Fgf1, PRL, Cd63, Npw, and Il23a, were upregulated, and 488 genes, including those that encode Lats1, Cacna2d1, Top2a, and Tfap2a, were downregulated. These genes are all involved in the regulation of prolactin expression. The gene expression of miR-126a-5p, which regulates the level of PRL in the pituitary gland, was screened by analysis prediction software and by a dual luciferase reporter system. The data presented in this study demonstrate that TRH can regulate prolactin synthesis and secretion through miR-126a-5p, thereby improving our understanding of the molecular mechanism of TRH-mediated PRL secretion and providing a theoretical basis for the role of miRNAs in regulating the secretion of pituitary hormones.

Circ-ERC2 Is Involved in Melatonin Synthesis by Regulating the miR-125a-5p/MAT2A Axis.

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.

LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells.

Long noncoding RNAs (lncRNAs) are expressed in different species and different tissues, and perform different functions, but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone (FSH). In general, we have revealed lncRNA|‒|microRNA (miRNA)|‒||messenger RNA (mRNA) interactions that may play important roles in rat primary pituitary cells. In this study, a new lncRNA was identified for the first time. First, we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization, which indicated that this lncRNA was distributed mainly in the cytoplasm. Next, we used RT-qPCR and enzyme-linked immunosorbent assay (ELISA) to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion. In addition, mothers against decapentaplegic homolog 2 (Smad2) was highly expressed in our sequencing results. We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2. We used RNA immunoprecipitation-qPCR (RIP-qPCR) and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2. Fluorescence in situ hybridization (FISH) showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm. Finally, we determined the relationship among lncRNA-m18as1, miR-18a-5p, and the Smad2/3 pathway. Overall, we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.