Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells

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.

The follicle-stimulating hormone triggers rapid changes in mitochondrial structure and function in porcine cumulus cells

“…The supplementation of FSH in the IVM medium induces important metabolic changes in cumulus cells, including steroid synthesis, glucose uptake, and glycogen consumption 29 , 30 . To determine how FSH affects mitochondrial response, we first determined mitochondrial structure in COCs (10 COCs by replicate for each culture condition, n = 3) incubated for different times in the absence or presence of FSH (at 0.01 µg/ml which has been optimized for cumulus cells expansion and the percentage of meiotic resumption measured as germinal vesicle breakdown (GVBD)) 31 . Mitochondria were longer in cumulus cells (120 cumulus cells by 10 COCs per replicate for each culture condition, n = 3) after 2 h of culture with FSH compared to incubation in the absence of the hormone, as mitochondria longer than 2 µm increased from 54% ± 0.54 to 65% ± 0.71 in the presence of FSH ( p = 0.0001, t-test) (Fig.…”

“…It is also known that high cAMP concentrations within the COC maintain gap junction communication and nutrient transfer to the oocyte, resulting in oocyte quality improvement 2 . Here, we investigated the effects of a pre-IVM culture step using high cAMP levels, raised by specific phosphodiesterase (PDE) inhibitors (PF-04957325 (300 nM) 31 + 3-Isobutyl-1-methylxanthine (IBMX, 500 µM) 39 ), on FSH-dependent mitochondrial responses 40 . The morphology of active mitochondria was studied in cumulus cells (120 cumulus cells by 10 COCs per replicate for each culture condition, n = 3) using confocal images of TMRM-stained mitochondria after 2 and 24 h of culture with FSH, combined or not with PDE inhibitors.…”

Expression of sirtuins in ovarian follicles of postnatal mice

“…Mitochondrial function in granulosa cells is essential for normal follicular development and for the generation of high‐quality oocytes (Lounas, Vernoux, Germain, Tremblay, & Richard, 2019; W. P. Zhao et al, 2019). Mitofusins (Mfns) are GTPases located on the outer mitochondrial membrane, and their primary function is to promote the fusion of the outer mitochondrial membrane, thus directly affecting mitochondrial function and regulating cellular energetics, mitophagy, and apoptosis (M. Zhang et al, 2019a, 2019b).…”

A 3D analysis revealed complexe mitochondria morphologies in porcine cumulus cells

“…The COC were prepared according to an adapted protocol as described previously with some modifications 54 . Immediately after recovery, two hundred fresh COC were fixed in 3.5% acrolein solution for 30 min at room temperature and washed twice with PBS.…”