Yanwen Jiang

MicroRNA-16 Modulates Melatonin-Induced Cell Growth in the Mouse-Derived Spermatogonia Cell Line GC-1 spg Cells by Targeting Ccnd1

ABSTRACT Melatonin exerts a range of physiological effects. However, the functional significance of melatonin in spermatogenesis and the underlying mechanisms remain unclear. MicroRNAs (miRNAs) are essential in the initiation and progression of testicular development, including spermatogenesis. Thus far, limited information is known about the role of miRNAs in melatonin-mediated spermatogenesis. In this study, the expression levels of testicular miRNA machinery genes, namely, Dgcr8 and Xpo5, were significantly increased by melatonin. The miRNA expression profile was identified in the testes of melatonin-treated mice. Ten miRNAs were significantly up-regulated, and 15 miRNAs were down-regulated. Melatonin (25 μM) enhanced cell growth and reduced apoptosis in GC-1 spg cells. Among the down-regulated miRNAs, miR-16 expression was influenced by melatonin in GC-1 spg cells. The miR-16 mimics in GC-1 spg cells significantly suppressed cell growth and promoted cell apoptosis. Conversely, transfection of the miR-16 inhibitor significantly increased cell growth and decreased cell apoptosis. The protein expression level of CCND1 (Cyclin D1) in GC-1 spg cells was decreased by the miR-16 mimics and increased by knockdown of miR-16. Moreover, bioinformatics and reporter activity analyses showed that Ccnd1 was a potential target of miR-16. These results suggested that miR-16 may function as a novel regulator of testicular functions during melatonin stimulation by targeting Ccnd1.

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS.

Potential role of retinoids in ovarian physiology and pathogenesis of polycystic ovary syndrome

Retinoids (retinol and its derivatives) are required for maintaining vision, immunity, barrier function, reproduction, embryogenesis, cell proliferation and differentiation. Furthermore, retinoid signaling plays a key role in initiating meiosis of germ cells of the mammalian fetal ovary. Recently, studies indicated that precise retinoid level regulation in the ovary provides a molecular control of ovarian development, steroidogenesis and oocyte maturation. Besides, abnormal retinoid signaling may be involved in the pathogenesis of polycystic ovary syndrome (PCOS), one of the most common ovarian endocrinopathies in reproductive-aged women worldwide. This review primarily summarizes recent advancements made in investigating the action of retinoid signaling in ovarian physiology as well as the abnormal retinoid signaling in PCOS.

Melatonin promotes the proliferation of GC-1 spg cells by inducing metallothionein-2 expression through ERK1/2 signaling pathway activation

Synthesized by the pineal gland, melatonin is a neurohormone implicated in diverse physiological functions via several mechanisms. However, the role of melatonin in spermatogenesis and its underlying mechanisms have yet to be completely understood. In the present study, transcriptome sequencing was performed to characterize the mechanism of melatonin-induced GC-1 spg proliferation. Gene ontology (GO) enrichment and pathway analyses were also conducted to identify the signaling pathways and biological processes involved in differential mRNA expression. Results revealed 28 differential genes. Of these genes, 11 were upregulated and 17 were downregulated. Melatonin increased the expression of metallothionein-2 (Mt2), a gene that acts as a protector to sequester nonessential toxic heavy metals. Functional investigations demonstrated that Mt2 overexpression promoted the proliferation of GC-1 spg cells, but Mt2 knockdown significantly suppressed their proliferation and increased their apoptosis. Mechanistic analysis indicated that the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway participated in melatonin-promoted proliferation of GC-1 spg cells. Therefore, melatonin induces the proliferation of GC-spg 1 cells by stimulating Mt2 expression, and this process is mediated by the ERK1/2 signaling pathway.

4-Methylcatechol inhibits cell growth and testosterone production in TM3 Leydig cells by reducing mitochondrial activity.

4‐Methylcatechol (4‐MC) is a potential neuroprotective drug because it stimulates the synthesis of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in neurons. The present study explored the effect of 4‐MC on cell growth and testosterone synthesis in the TM3 Leydig cells of mice. 4‐MC did not enhance expression of both BDNF and NGF in these cells. However, this compound significantly inhibited cell proliferation and increased the number of apoptotic cells in a dose‐dependent manner. The expression profile of Bax/Bcl‐2 gene was altered considerably, and mitochondrial activity was significantly decreased in cells. 4‐Methylcatechol also inhibited testosterone synthesis in TM3 Leydig cells. The inhibitory roles of this compound in relation to growth and testosterone synthesis in TM3 Leydig cells maybe associated with increased Bax gene expression and decreased mitochondrial activity. As a result, caspase cascade is activated.

Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain-derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.

Follicle-stimulating hormone (FSH) promotes retinol uptake and metabolism in the mouse ovary

BackgroundRetinoids (retinol and its derivatives) are required for the development and maintenance of normal physiological functions of the ovary. However, the mechanisms underlying the regulation of ovarian retinoid homeostasis during follicular development remain unclear.MethodsThe present study determined retinoid levels and the expression levels of genes involved in the retinol uptake and its metabolic pathway in the ovaries of follicle-stimulating hormone (FSH)-treated mice and in granulosa cells treated with FSH using ultra performance liquid chromatography (UPLC) combined with quadrupole time-of-flight high-sensitivity mass spectrometry (Q-TOF/HSMS) and real-time PCR analysis.ResultsThe levels of total retinoids and retinoic acid (RA) and expressions of retinol-oxidizing enzyme genes alcohol dehydrogenase 1 (Adh1) and aldehyde dehydrogenase (Aldh1a1) are increased in the ovaries of mice treated with FSH; in contrast, the retinyl ester levels and retinol-esterifying enzyme gene lecithin: retinol acyltransferase (Lrat) expression are diminished. In FSH-treated granulosa cells, the levels of retinyl esters, retinaldehyde, and total retinoids are augmented; and this is coupled with an increase in the expressions of stimulated by retinoic acid 6 (Stra6) and cellular retinol-binding protein 1 (Crbp1), genes in the retinol uptake pathway, and Adh1, Adh7, and Aldh1a1 as well as a diminution in Lrat expression.ConclusionsThese data suggest that FSH promotes retinol uptake and its conversion to RA through modulating the pathways of retinol uptake and metabolism in the mouse ovary. The present study provides a possible mechanism for the regulation of endogenous RA signaling in the developing follicles.

Effects of ammonia on apoptosis and oxidative stress in bovine mammary epithelial cells

Ammonia, produced mainly from the deamination of amino acids and glutamine, is one of the major toxic components in blood and tissues that may affect bovine health. However, the physiological and pathological roles of ammonia in the mammary glands are not understood clearly. In the present study, the bovine mammary epithelial cell line (MAC-T) was utilised as an in vitro model to determine the effects of ammonia on bovine mammary gland. We demonstrated that ammonia stimulated the production of intracellular reactive oxygen species, decreased mitochondrial membrane potential, interrupted intracellular calcium ion (Ca2+) homeostasis and induced cell apoptosis. Ammonia also significantly reduced cell viability and increased the proportion of apoptotic cells through enhancing the level of p53 phosphorylation and increasing the expressions of BAX, caspase 8, caspase 9, caspase 3. Interestingly, bumetanide, a specific Na+ K+ 2Cl--cotransporter inhibitor, dramatically abolished the damaging effects of ammonia on the cells. These data suggest that ammonia exposure induces apoptosis in bovine mammary epithelial cells via activation of the p53 pathway and the mitochondrial apoptotic pathway, and that these effects involved the Na+ K+ 2Cl--cotransporter.

Analysis of miRNA expression profiles in melatonin-exposed GC-1 spg cell line

Melatonin is an endocrine neurohormone secreted by pinealocytes in the pineal gland. It exerts diverse physiological effects, such as circadian rhythm regulator and antioxidant. However, the functional importance of melatonin in spermatogenesis regulation remains unclear. The objectives of this study are to: (1) detect melatonin affection on miRNA expression profiles in GC-1 spg cells by miRNA deep sequencing (DeepSeq) and (2) define melatonin affected miRNA-mRNA interactions and associated biological processes using bioinformatics analysis. GC-1 spg cells were cultured with melatonin (10-7M) for 24h. DeepSeq data were validated using quantitative real-time reverse transcription polymerase chain reaction analysis (qRT-PCR). A total of 176 miRNA expressions were found to be significantly different between two groups (fold change of >2 or <0.5 and FDR<0.05). Among these expressions, 171 were up-regulated, and 5 were down-regulated. Ontology analysis of biological processes of these targets indicated a variety of biological functions. Pathway analysis indicated that the predicted targets were involved in cancers, apoptosis and signaling pathways, such as VEGF, TNF, Ras and Notch. Results implicated that melatonin could regulate the expression of miRNA to perform its physiological effects in GC-1 spg cells. These results should be useful to investigate the biological function of miRNAs regulated by melatonin in spermatogenesis and testicular germ cell tumor.

Differential expression of microRNAs in luteinising hormone‐treated mouse TM3 Leydig cells

Testosterone is primarily produced by Leydig cells of the mammalian male gonads. The cellular functions of Leydig cells are regulated by the hypothalamus–pituitary–gonad axis, whereas the microRNA (miRNA) changes of LH‐treated Leydig cells are unknown. Mouse TM3 Leydig cells were treated with LH, and deep sequencing showed that 29 miRNAs were significantly different between two groups (fold change of >1.5 or <0.5, p < .05), of which 27 were upregulated and two were downregulated. The differential expression of miR‐29b‐3p, miR‐378b, miR‐193b and miR‐3695 was confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs were involved in the Wingless and INT‐1, adenosine 5′‐monophosphate‐activated protein kinase, NF‐kappa B and Toll‐like receptor signalling pathways. Results showed that miRNAs might be involved in the regulation of LH to Leydig cells.