The ovarian follicle has a three-dimensional (3D) structure in which the oocyte is surrounded by tightly connected follicle cells that mediate the action of external signals and nourish the gamete during its maturation. Thus, the maintenance of follicle organization during the whole growth process is crucial for the correct acquisition of developmental competence. In recent years, much attention has been given to in vitro culture systems capable of maintaining follicle architecture. With the aim of providing a quick reference guide, in this review we will summarize the techniques developed for the 3D culture of mouse follicles.

Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.

BackgroundThe cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts.ResultsGlobal gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR.ConclusionsThe developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte.

Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species.

Luteinizing hormone (LH) acts on the granulosa cells that surround the oocyte in mammalian preovulatory follicles to cause meiotic resumption and ovulation. Both of these responses are mediated primarily by an increase in cyclic adenosine monophosphate (cAMP) in the granulosa cells, and the activity of cAMP phosphodiesterases (PDEs), including PDE4, contributes to preventing premature responses. However, two other cAMP-specific PDEs, PDE7 and PDE8, are also expressed at high levels in the granulosa cells, raising the question of whether these PDEs also contribute to preventing uncontrolled activation of meiotic resumption and ovulation. With the use of selective inhibitors, we show that inhibition of PDE7 or PDE8 alone has no effect on the cAMP content of follicles, and inhibition of PDE4 alone has only a small and variable effect. In contrast, a mixture of the three inhibitors elevates cAMP to a level comparable with that seen with LH. Correspondingly, inhibition of PDE7 or PDE8 alone has no effect on meiotic resumption or ovulation, and inhibition of PDE4 alone has only a partial and slow effect. However, the fraction of oocytes resuming meiosis and undergoing ovulation is increased when PDE4, PDE7, and PDE8 are simultaneously inhibited. PDE4, PDE7, and PDE8 also function together to suppress the premature synthesis of progesterone and progesterone receptors, which are required for ovulation. Our results indicate that three cAMP PDEs act in concert to suppress premature responses in preovulatory follicles.