Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.

BackgroundGPRC6A 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 FindingsIn 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.

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.

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.