This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents the precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C, Nppc, mRNA while cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC elevated cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.Meiosis is a germ cell-specific process that reduces the number of chromosomes from the diploid to the haploid number. It begins in human and mouse ovaries during fetal life but meiotic progression becomes arrested for prolonged periods at the diplotene stage of meiotic prophase. Fully-grown mammalian oocytes in Graafian follicles are maintained in meiotic prophase arrest until the preovulatory surge of luteinizing hormone (LH) triggers the resumption of meiosis and ovulation. The mature oocytes (eggs) are then available for fertilization within the oviduct. The somatic cell compartment of Graafian follicles, consisting of mural granulosa cells lining the inside of the follicle wall and cumulus cells surrounding the oocyte, plays a crucial role in maintaining oocyte meiotic arrest in mammals since removal of the oocyte-cumulus cell complex from these follicles results in gonadotropin-independent meiotic resumption in culture (1,2). Cyclic nucleotides cAMP and cGMP are crucial to the maintenance of meiotic arrest. Cyclic AMP is generated within oocytes downstream of GPR3 and GPR12, regulators of Gs proteins controlling adenylyl cyclase (3,4). Inability to sustain oocyte cAMP levels leads to precocious gonadotropinindependent resumption of meiosis, which interrupts the synchrony between oocyte maturation and ovulation and compromises female fertility (3-5). PDE3A, an oocytespecific phosphodiesterase, becomes activated after the LH-surge to decrease cAMP levels in oocytes and thereby initiates pathways governing meiotic resumption (6). Before the LHsurge, cGMP, originating in granulosa cells of the follicular somatic compartment and transferred to the oocyte via gap junctions, inhibits activity of PDE3A in the oocyte (7,8). Therefore, control of cGMP production by granulosa cells is crucial for maintaining meiotic arrest in fully-grown oocytes. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptExploration of the mouse cumulus cell transcriptome for mRNAs encoding guanylyl cyclases using microarray analysis (9) revealed abundant expression of natriuretic peptide receptor 2 (Npr2, also called GC-B) mRNA. The presence of this guanylyl cyc...

*Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15-/-and Bmp15 -/-Gdf9 +/-double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production.

There is massive destruction of transcripts during the maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using a microarray approach. A system for oocyte transcript amplification using both internal and 3'-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using the Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among the stable transcripts.

Bidirectional communication between oocytes and the companion granulosa cells is essential for the development and functions of both compartments. Oocytes are deficient in their ability to transport certain amino acids and in carrying out glycolysis and cholesterol biosynthesis. Cumulus cells must provide them with the specific amino acids and the products in these metabolic pathways. Oocytes control metabolic activities in cumulus cells by promoting the expression of genes in cumulus cells encoding specific amino acid transporters and enzymes essential for the oocyte-deficient metabolic processes. Hence oocytes outsource metabolic functions to cumulus cells to compensate for oocyte metabolic deficiencies. Oocyte control of granulosa cell metabolism may also participate in regulating the rate of follicular development in coordination with endocrine, paracrine, and autocrine signals. Oocytes influence granulosa cell development mainly by secretion of paracrine factors, although juxtacrine signals probably also participate. Key oocyte-derived paracrine factors include growth differentiation factor 9, bone morphogenetic protein 15, and fibroblast growth factor 8B.