Article

Roles of gene transcription and PKA subtype activation in maturation of murine oocytes.

Department of Animal Science, North Carolina State University, 231B Polk Hall, Raleigh, NC 27695-7621, USA.
Reproduction (Cambridge, England) (Impact Factor: 3.56). 07/2002; 123(6):799-806. DOI: 10.1530/rep.0.1230799
Source: PubMed

ABSTRACT The aims of this study were to examine the role of transcription and the coincident involvement of type I and type II protein kinase A (PKA) in the resumption of meiosis in murine cumulus-oocyte complexes (COCs) using the transcriptional inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. The first series of experiments was designed to: (i) characterize the role of transcription in gonadotrophin-mediated and spontaneous maturation of murine oocytes; (ii) examine the roles of specific gonadotrophins (FSH versus hCG) and cumulus cells in transcriptionally mediated oocyte maturation; and (iii) determine the reversibility of the transcriptional arrest of meiosis. In the presence of FSH, transcriptional inhibitors arrested germinal vesicle breakdown (GVBD) (DRB: 2 +/- 2% and control: 76 +/- 2%; alpha-amanitin: 4 +/- 4% and control: 70 +/- 4%). Furthermore, cumulus cells were required for transcriptional inhibitors to arrest GVBD (DRB with cumulus cells: 0 +/- 15%; DRB without cumulus cells: 94 +/- 13%; alpha-amanitin with cumulus cells: 15 +/- 2%; alpha-amanitin without cumulus cells: 99 +/- 2%). Thus, in mice, FSH-mediated GVBD uses a transcriptional mechanism, which probably occurs within the cumulus cell compartment. In a second series of experiments, the role of transcription in mediating the resumption of meiosis after activation of either type I or type II PKA was examined. Activation of type I PKA in murine COCs resulted in an arrest of GVBD that was independent of a transcriptional event (with DRB: 7 +/- 9% GVBD; without DRB: 11 +/- 9% GVBD). In contrast, activation of type II PKA resulted in a resumption of meiosis, which required the occurrence of gene transcription (with DRB: 12 +/- 9% GVBD; without DRB: 80 +/- 9% GVBD). As FSH binding to cumulus cells activates the PKA second messenger system, our results indicate that, in cultured murine COCs, FSH binding to cumulus cells results in the activation of type II PKA, which, in turn, mediates a downstream transcriptional event required for the initiation of GVBD.

0 Bookmarks
 · 
76 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: There is a constant increase in demand for large numbers of high-quality oocytes of domestic mam- mals for various reproductive biotechnologies. Prolonged in vitro culture represents one of the many possible ways of meeting this demand. However, oocytes further cultured in vitro after reaching the stage of metaphase II undergo the complex process called "ageing", which decreases the quality of oocytes. The aim of the present study was to improve the culture conditions for in vitro matured porcine oocytes by supplement of the epidermal growth factor (EGF) or the insulin-like growth factor 1 (IGF-1) in order to reduce the adverse effects of ageing. Supple- ment of either EGF or IGF-1 had no significant effect on the ageing of porcine oocytes. Significant effects were demonstrated when the oocytes were cultured with both EGF and IGF-1. A combination of 10 µg EGF/ml with 25 ng IGF-1/ml was the most effective. Oocytes aged under these conditions retained very good developmental competence. We observed development to the morula (21%) or blastocyst (25%) stage in oocytes aged for 1 day with EGF and IGF-1. Porcine oocytes matured in vitro are more resistant to the ageing when cultured in the presence of both EGF and IGF-1 and these conditions retain an elevated developmental competence for a certain time.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3'UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.
    PLoS ONE 01/2014; 9(5):e97324. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The use of gonadotropins to trigger oocyte maturation both in vivo and in vitro has provided precious and powerful knowledge that has significantly increased our understanding of the ovarian function. Moreover, the efficacy of most assisted reproductive technologies (ART) used in both humans and livestock species relies on gonadotropin input, mainly FSH and LH. Despite the significant progress achieved and the huge impact of gonadotropins, the exact molecular pathways of the two pituitary hormones, FSH and LH, still remain poorly understood. Moreover, these pathways may not be the same when moving from the in vivo to the in vitro context. This misunderstanding of the intricate synergy between these two hormones leads to a lack of consensus about their use mainly in vitro or in ovulation induction schedules in vivo. In order to optimize their use, additional work is thus required with a special focus on comparing the in vitro versus the in vivo effects. In this context, this overview will briefly summarize the downstream gene expression pathways induced by both FSH in vitro and LH in vivo in the cumulus compartment. Based on micro array recent comparative analysis, we are reporting that in vitro FSH stimulation on cumulus cells appears to achieve at least part of the gene expression activity after in vivo LH stimulation. We are then proposing that the in vitro FSH-response of cumulus cells have similitudes with the in vivo LH-response.
    Journal of Ovarian Research 09/2013; 6(1):68. · 2.43 Impact Factor

Full-text (2 Sources)

Download
30 Downloads
Available from
May 21, 2014