Objective

Barrett's esophagus (BE) is transition from squamous to columnar mucosa as a result of gastroesophageal reflux disease (GERD). The role of microRNA during this transition has not been systematically studied.

Design

For initial screening, total RNA from 5 GERD and 6 BE patients was size fractionated. RNA <70 nucleotides was subjected to SOLiD 3 library preparation and next generation sequencing (NGS). Bioinformatics analysis was performed using R package “DEseq”. A p value<0.05 adjusted for a false discovery rate of 5% was considered significant. NGS-identified miRNA were validated using qRT-PCR in an independent group of 40 GERD and 27 BE patients. MicroRNA expression of human BE tissues was also compared with three BE cell lines.

Results

NGS detected 19.6 million raw reads per sample. 53.1% of filtered reads mapped to miRBase version 18. NGS analysis followed by qRT-PCR validation found 10 differentially expressed miRNA; several are novel (-708-5p, -944, -224-5p and -3065-5p). Up- or down- regulation predicted by NGS was matched by qRT-PCR in every case. Human BE tissues and BE cell lines showed a high degree of concordance (70–80%) in miRNA expression. Prediction analysis identified targets that mapped to developmental signaling pathways such as TGFβ and Notch and inflammatory pathways such as toll-like receptor signaling and TGFβ. Cluster analysis found similarly regulated (up or down) miRNA to share common targets suggesting coordination between miRNA.

Conclusion

Using highly sensitive next-generation sequencing, we have performed a comprehensive genome wide analysis of microRNA in BE and GERD patients. Differentially expressed miRNA between BE and GERD have been further validated. Expression of miRNA between BE human tissues and BE cell lines are highly correlated. These miRNA should be studied in biological models to further understand BE development.

Objective

A synonymous variant within scavenger receptor class B type I gene (SCARB1), exon 8 rs5888, has been associated with altered lipid levels and cardiovascular risk in humans. The objective was to determine if rs5888 decreased SR-BI protein expression and function in vitro.

Methods

SR-BI RNA secondary structure, turnover, polysomal distribution and protein expression were examined in COS cells transfected with wild-type or rs5888-SR-BI plasmids by selective 2’-hydroxyl acylation and primer extension assays, actinomycin D inhibition, polysomal profiling, and western blotting. SR-BI function in murine macrophages stably expressing wild-type or rs5888-SR-BI was assessed by measuring the specific cell association of 125I,3H-cholesteryl ester (CE) radiolabeled HDL.

Results

Rs5888 changed RNA secondary structure and led to marked differences in the polysomal profiles compared with wild-type transcript (p<0.02). As compared to wild-type cells, COS cells expressing rs5888 had significantly lower SR-BI protein expression (p<0.04), but no difference in total RNA transcript levels. There were no differences in SR-BI RNA turnover in murine macrophages, whereas specific cell association of 125I (p<0.0001) or 3H-CE (p<0.00001) was significantly lower in rs5888 cells.

Conclusions

The rs5888 variant affected SR-BI RNA secondary structure, protein translation, and was significantly associated with reduced SR-BI protein expression and function in vitro.

MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNAs in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2′-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides, and mature Mir21 expression decreased to one ninth and one twenty-seventh of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/human chorionic gonadotropin surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phospho thioate-modified LNA-21 oligonucleotide, and granulosa cell apoptosis was examined. Apoptosis increased in LNA-21-treated ovaries, and ovulation rate decreased in LNA-21-treated ovaries, compared with their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently, none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the antiapoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function.

In vivo and in vitro loss of microRNA 21, an LH-induced microRNA, results in mouse granulosa cell apoptosis.

Dicer is an RNAse III endonuclease that is essential for the biogenesis of microRNAs and small interfering RNAs. These small RNAs post-transcriptionally regulate mRNA gene expression through several mechanisms to affect key cellular events including proliferation, differentiation, and apoptosis. Recently, the role of Dicer function in female reproductive tissues has begun to be elucidated through the use of knock-out mouse models. Loss of Dicer within ovarian granulosa cells, luteal tissue, oocyte, oviduct, and potentially the uterus render females infertile. This review discusses these early studies and other data describing the current understanding of microRNAs and small interfering RNAs in female reproduction.

Luteinizing hormone (LH) acts on periovulatory granulosa cells by activating the PKA pathway as well as other cell signaling cascades to increase the transcription of specific genes necessary for ovulation and luteinization. Collectively, these cell signaling responses occur rapidly (within minutes), however, presently no high throughput studies have reported changes before 4 h after the LH surge. To identify early response genes that are likely critical for initiation of ovulation and luteinization, mouse granulosa cells were collected before and 1 h after hCG. Fifty-seven gene transcripts were significantly (p<0.05) upregulated and 3 downregulated following hCG. Twenty-four of these transcripts were known to be expressed after the LH/hCG surge at later time points, while 36 were unknown to be expressed by periovulatory granulosa cells. Temporal expression of several transcripts, including the transcription factors Nr4a1, Nr4a2, Egr1, Egr2, Btg1, and Btg2, and the EGF-like ligands Areg and Ereg, were analyzed by quantitative RT-PCR, and their putative roles in granulosa cell function are discussed. Epigen (Epgn), another member of the family of EGF-like ligands, was identified for the first time in granulosa cells as rapidly induced by LH/hCG. We demonstrate that Epgn initiates cumulus expansion, similar to the other EGF-receptor ligands Areg and Ereg. These studies illustrate that a number of changes in gene expression occur in vivo in response to LH, and that many of the differentially expressed genes are transcription factors that we would predict in turn modulate granulosa cell gene expression to ultimately impact the processes of ovulation and luteinization.

Background

The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.

Methods

Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization.

Results

Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as Gαs/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment.

Conclusions

Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3′ untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.

The ovulatory surge of luteinizing hormone induces the expression of microRNAs, which posttranscriptionally regulate the expression of a regulatory protein within mural granulosa cells of the periovulatory follicle.

Background

GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.

Methods/Principal Findings

In this study, we created and characterized the phenotype of GPRC6A−/− mice. We observed complex metabolic abnormalities in GPRC6A−/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A−/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A−/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.

Conclusions/Significance

GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

The CCAAT/enhancer binding protein (CEBP) family of transcription factors includes five genes. In the ovary, both Cebpa and Cebpb are essential for granulosa cell function. In this study we have explored the role of the Cebpd gene in ovarian physiology by expression and functional studies. Here we report that Cebpd (C/EBPδ) is expressed in the mouse ovary in a highly restricted temporal and spatial pattern. In response to luteinizing hormone (LH/hCG), CEBPD expression is transiently induced in interstitial cells and in theca cells of follicles from the primary to pre-ovulatory stage, and overlaps in part with expression of the alpha-smooth muscle actin protein. Efficient down-regulation of CEBPD was dependent on a functional Cebpb gene. Proliferating human theca cells in culture also express Cebpd. Cells from patients with polycystic ovarian syndrome (PCOS) exhibited higher Cebpd expression levels. However, deletion of Cebpd in mice had no overt effect on ovarian physiology and reproductive function. Very little is known at present about the molecular mechanisms underlying theca/interstitial cell functions. The expression pattern of CEBPD reported here identifies a novel functional unit of mouse theca cells of primary through tertiary follicles responding to LH/hCG together with a subset of interstitial cells. This acute stimulation of CEBPD expression may be exploited to further characterize the hormonal regulation and function of theca and interstitial cells.

The synthesis of progesterone by the corpus luteum is essential for the establishment and maintenance of early pregnancy. Regulation of luteal steroidogenesis can be broken down into three major events; luteinization (i.e., conversion of an ovulatory follicle), luteal regression, and pregnancy induced luteal maintenance/rescue. While the factors that control these events and dictate the final steroid end products are widely varied among different species, the composition of the corpus luteum (luteinized thecal and granulosa cells) and the enzymes and proteins involved in the steroidogenic pathway are relatively similar among all species. The key factors involved in luteal steroidogenesis and several new exciting observations regarding regulation of luteal steroidogenic function are discussed in this review.