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Comment |
Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells. Lounas A et al. (2019) 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.//////////////////
Multiple cAMP phosphodiesterases act together to prevent premature oocyte meiosis and ovulation. Vigone G et al. (2018) 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 cAMP in the granulosa cells, and activity of the cAMP phosphodiesterase PDE4 contributes to preventing premature responses. However, 2 other cAMP-specific phosphodiesterases, PDE7 and PDE8, are also expressed at high levels in the granulosa cells, raising the question of whether these phosphodiesterases also contribute to preventing uncontrolled activation of meiotic resumption and ovulation. Using 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 3 inhibitors elevates cAMP to a level comparable to 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 3 cAMP phosphodiesterases act in concert to suppress premature responses in preovulatory follicles.//////////////////
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Active 3'-5' cyclic nucleotide phosphodiesterases are present in detergent-resistant membranes of mural granulosa cells. Bergeron A et al. (2016) Lipids rafts are specialised membrane microdomains involved in cell signalling that can be isolated as detergent-resistant membranes (DRMs). The second messenger cyclic AMP (cAMP) has a central role in cell signalling in the ovary and its degradation is carried out by the phosphodiesterase (PDE) enzyme family. We hypothesised that PDEs could be functionally present in the lipid rafts of porcine mural granulosa cell membranes. PDE6C, PDE8A and PDE11A were detected by dot blot in the DRMs and the Triton-soluble fraction of the mural granulosa cells membrane and the cytosol. As shown by immunocytochemistry, PDEs showed clear immunostaining in mural granulosa cell membranes and the cytosol. Interestingly, cAMP-PDE activity was 18 times higher in the DRMs than in the Triton-soluble fraction of cell membranes and was 7.7 times higher in the cytosol than in the DRMs. cAMP-PDE activity in mural granulosa cells was mainly contributed by the PDE8 and PDE11 families. This study shows that PDEs from the PDE8 and PDE11 families are present in mural granulosa cells and that the cAMP-PDE activity is mainly contributed by the cytosol. In the cell membrane, the cAMP-PDE activity is mainly contributed by the DRMs. In addition, receptors for prostaglandin E2 and LH, two G-protein-coupled receptors, are present in lipid rafts and absent from the non-raft fraction of the granulosa cell membrane. These results suggest that in these cells, the lipid rafts exist as a cell-signalling platform and PDEs are one of the key enzyme families present in the raft.//////////////////
Phosphodiesterases in the rat ovary - effect of cyclic AMP in primordial follicles. Petersen TS et al. (2015) Phosphodiesterases (PDE) are important regulators of the intracellular cyclic adenosine monophosphate (cAMP) concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity, and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67, or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67, and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation.//////////////////
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Mutations |
4 mutations
Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: Gene dosage as a relevant mechanism contributing to the determination of ovarian function in Turner syndrome. Castronovo C 2013 et al.
STUDY QUESTION
What is the burden of X chromosome mosaicism in the occurrence of spontaneous menarche (SM) in Turner syndrome (TS)?
SUMMARY ANSWER
SM was significantly associated with X chromosome mosaicism in the TS patients; a mosaicism with around 10% euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques on uncultivated tissues.
WHAT IS KNOWN ALREADY
Spontaneous puberty can be observed in a minority of patients with TS, more frequently, but not exclusively, in those with a high level of 46,XX/45,X mosaicism at standard karyotype. The genetic mechanisms contributing to ovarian function in TS patients are still not determined. However, submicroscopic X-linked and autosomal copy number variations (CNVs) have recently emerged as an important genetic risk category for premature ovarian insufficiency and may be involved in modulating the TS ovarian phenotype.
STUDY DESIGN, SIZE, DURATION
A group of 40 patients with a diagnosis of TS at conventional karyotyping participated in the study; 6 patients had SM and 34 patients had primary amenorrhoea (PA). All clinical data and the patients' DNA samples were collected over the years at a single paediatric clinic.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The patients' samples were used to perform both genetic (Copy Number Assay) and molecular-cytogenetic (array-CGH and iFISH, interphase-FISH) analyses in order to evaluate the X chromosome mosaicism rate and to detect possible rare CNVs of genes with a known or predicted role in female fertility.
MAIN RESULTS AND THE ROLE OF CHANCE
All TS patients showed variable percentages of the 46,XX lineage, but these percentages were higher in the SM group (P < 0.01). A mosaicism around 10% for the euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques performed in uncultivated tissues. A few CNVs involving autosomal and X-linked ovary-related loci were identified by array-CGH analysis and confirmed by real-time quantitative PCR, including a BMP15 gene duplication at Xp11.22, a deletion interrupting the PAPPA gene at 9q33.1, and an intragenic duplication involving the PDE8A gene at 15q25.3.
LIMITATIONS, REASONS FOR CAUTION
This is a pilot study on a relatively small sample size and confirmation in larger TS cohorts may be required. The ovarian tissue could not be studied in any patients and in a subgroup of patients, the mosaicism was estimated in tissues of different embryonic origin.
WIDER IMPLICATIONS OF THE FINDINGS
The combined determination of X chromosome mosaicism by molecular and molecular-cytogenetic techniques may become useful for the prediction of SM in TS. The detection of CNVs in both X-linked and autosomal ovary-related genes further suggests gene dosage as a relevant mechanism contributing to the ovarian phenotype of TS patients. These CNVs may pinpoint novel candidates relevant to female fertility and generate further insights into the mechanisms contributing to ovarian function.
STUDY FUNDING/COMPETING INTEREST(S)
This study was funded by Telethon Foundation (grant no: GGP09126 to L.P.), the Italian Ministry of the University and Research (grant number: 2006065999 to P.F.) and a Ministry of Health grant 'Ricerca Corrente' to IRCCS Istituto Auxologico Italiano (grant number: 08C704-2006). The authors have no conflict of interest to declare.
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Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: PDE8A Genetic Variation, Polycystic Ovary Syndrome and Androgen Levels in Women. Chen C et al. Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of adenosine 3', 5'-cyclic monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and cAMP degradation by phosphodiesterases (PDEs). PDE8A, a high-affinity cAMP-specific PDE is expressed in ovary and testis cells. Leydig cells from mice with a targeted mutation in the PDE8A gene are sensitized to the action of LH in terms of testosterone production. These observations led us to evaluate the human PDE8A gene as a PCOS candidate gene and the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. We identified a rare variant (R136Q; NM_002605.2 c.407 G>A) and studied another known single nucleotide polymorphism (SNP) (rs62019510, N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new SNP in the promoter region (NT_010274.16:g.490155G>A). Although PDE8A kinetics were consistent with reduced activity in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. Cellular localization of the enzyme was also not different among the coding sequence variants. The PDE8A promoter SNP and a previously described promoter SNP did not affect promoter activity in vitro assays. The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our transmission/disequilibrium test (TDT)-based analysis, nor where they associated with total testosterone or hydroepiandrosterone sulphate (DHEAS) levels. These findings exclude a significant role for PDE8A as a PCOS candidate gene and a major determinant of androgen levels in women.
Species: mouse
Mutation name:
type: null mutation
fertility: None
Comment: Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A. Vasta V et al. (2007) Leydig cells produce testosterone in the testes under the pulsatile control of pituitary luteinizing hormone (LH). cAMP is the intracellular messenger for LH action on steroidogenesis, and pharmacological evidence indicates that the response to LH can be modulated by cyclic nucleotide phosphodiesterases (PDEs). However the types and roles of the PDEs present in Leydig cells have not been fully defined. We report here that PDE8A is expressed in Leydig cells, and using PDE8A knockout mice we provide evidence that PDE8A is a key regulator of LH signaling and steroidogenesis. A 4-fold increase in the sensitivity to LH for testosterone production was detected in Leydig cells isolated from PDE8A knockout mice. In Leydig cells from wild-type mice, 3-isobutyl-1-methylxanthine, a compound that inhibits all cAMP PDEs except PDE8A, elicited only a small increase in the sensitivity of testosterone production to LH. However, in the PDE8-null mice, the effect of this inhibitor is much more pronounced. These observations indicate that PDE8A and at least one other PDE control the same or a complementary pool of cAMP that mediates LH-regulated steroidogenesis. Overall, these results suggest that pharmacological manipulation of PDE8A, alone or in combination with other PDEs present in Leydig cells, may be exploited to modulate testosterone synthesis and possibly to treat various conditions where the local levels of this androgen need to be altered.//////////////////
Species: mouse
Mutation name:
type: null mutation
fertility: None
Comment:
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