Abstract
Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.
Highlights
Cyclic adenosine monophosphate is a ubiquitous secondary messenger that is synthesized in response to the stimulation of G-protein-coupled receptors that mediate a wide variety of important cellular functions
Reverse-transcription PCR was performed on RNA extracted from granulosa cells, cumulus-oocyte complexes (COCs), cumulus cells, and oocytes using PDE8A-F and PDE8A-R primers (Table 1)
The use of western blots revealed immuno-reactive bands in granulosa cells, COCs, cumulus cells and oocytes at the molecular weights of 92, 74, 70, 58, and 56 kDa corresponding to PDE8A15,26
Summary
Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that is synthesized in response to the stimulation of G-protein-coupled receptors that mediate a wide variety of important cellular functions. To maintain high levels of oocyte cAMP, PDE3A activity must remain low[6] These conditions are possible with cGMP synthesized by granulosa cells and cumulus cells[5], since PDE3A is a cGMP-inhibited PDE4. The PDE2A identified from the brain and liver mitochondria of rats appear to be involved in the regulation of respiratory chain activity[12] This is of particular interest since the mitochondrial PDE2A in cardiac myocytes has been known to regulate local cAMP levels, mitochondrial morphology and apoptosis[22]. The PDE8 family is one type of PDE that was recently discovered, known to be insensitive to the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX)[24], and, inhibited by PF-0495732523 It remains one of the least studied PDEs, we have claimed the presence of PDE8A in bovine follicular cells[25]. The aim of the present study was to assess the mitochondrial sub-cellular localization of PDE8A in ovarian follicular cells using swine as an animal model
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