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Dysregulation of methylation and expression of imprinted genes in oocytes and reproductive tissues in mice of advanced maternal age. Paczkowski M et al. (2015) To evaluate reproductive outcomes in aged compared to young female mice, and determine associated methylation and expression of imprinted genes in reproductive tissues. Fetal, placental, and ovarian tissue were collected on d16.5 of pregnancy from young (4-5 weeks) and aged (15 months) mice. Uterine tissue and in vivo matured oocytes were collected from non-pregnant females. Methylation of imprinted genes was determined by restriction enzyme based assays, and transcript abundance of imprinted and nutrient supply genes were analyzed by quantitative PCR (qPCR). Maternal age was associated with fetal growth restriction and placental overgrowth. In maternally aged mice, methylation was minimally dysregulated in fetal tissue, while placental tissue showed aberrant methylation and transcript abundance of imprinted genes. Ovarian methylation and gene expression was severely dysregulated, although oocyte gene expression was only minimally altered. Abundance of Kcnq1 transcripts was significantly (P < 0.05) increased in oocytes obtained from aged females compared to young females. Gene expression was also severely dysregulated in the uterus, including nutrient transport genes. Fetal and placental growth abnormalities correspond to aberrant methylation and gene expression in reproductive tissues from maternally aged mice. Significant alterations in gene expression and methylation in the aged ovary suggests that the follicular environment may be compromised. Aberrant methylation and expression of imprinted genes in the aged uterus may contribute to reduced implantation. Maternal age negatively affects imprinted gene methylation and expression in both germ cells and somatic cells of the reproductive tract, contributing to the reduced fertility observed with advanced maternal age.//////////////////
Mason DE, et al 2002 reported the molecular basis of voltage-dependent potassium currents in
porcine granulosa cells.
The major objective of this study was to elucidate the molecular bases for K+
current diversity in porcine granulosa cells (GC). Two delayed rectifier K+
currents with distinct electrophysiological and pharmacological properties
were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly
activating, noninactivating current (I-Ks) antagonized by clofilium, 293B,
L-735,821, and L-768,673; and 2) an ultrarapidly activating, slowly
inactivating current (I-Kur) antagonized completely by clofilium and
4-aminopyridine and partially by tetraethylammonium, charybdotoxin,
dendrotoxin, and kaliotoxin. The molecular identity of the K+ channel genes
underlying I-Ks and I-Kur was examined using reverse transcription-polymerase
chain reaction and immunoblotting to detect K+ channel transcripts and
proteins. It was found that GC could express multiple voltage-dependent K+ (Kv)
channel subunits, including KCNQ1, KCNE1, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5,
Kv1.6, Kvbeta1.3, and Kvbeta2. Coimmunoprecipitation was used to establish the
hetero-oligomeric nature of granulosa cell Kv channels. KCNE1 and KCNQ1 were
coassociated in GC, and their expression coincided with the expression of
I-Ks. Extensive coassociation of the various Kv alpha- and beta-subunits was
also documented, suggesting that the diverse electrophysiological and
pharmacological properties of I-Kur currents may reflect variation in the
composition and stoichiometry of the channel assemblies, as well as
differences in post-translational modification of contributing Kv channel
subunits. These findings provide an essential background for experimental
definition of granulosa K+ channel function(s). It will be critical to define
the functional roles of specific GC K+ channels, because these proteins may
represent either novel targets for assisted reproduction or potential sites of
drug toxicity.
Ovarian acetylcholine and ovarian KCNQ channels: Insights into cellular regulatory systems of steroidogenic granulosa cells. Kunz L et al. Acetylcholine (ACh) may be an ovarian signaling molecule, since ACh is produced by non-neuronal granulosa cells (GCs) derived from the antral follicle, and likely also by their in vivo counterparts in the growing follicle. Furthermore, muscarinic ACh receptors (MR) are present in GC membranes and in cultured human GCs a number of MR-mediated actions have been described, including regulation of proliferation and gap junctional communication. Importantly, muscarinic stimulation elevates intracellular calcium levels, thereby opening a calcium-activated potassium channel (BK(Ca)) and causing membrane hyperpolarization. In the course of electrophysiological experiments with human GCs we also observed a reversible inhibitory action of an ACh analogue (carbachol) on an outward potassium current. This current is reminiscent of a so-called M-current described in neuronal systems, of which muscarinic regulation is well-known. Indeed, the current is sensitive to the specific KCNQ blocker XE991 and a possible underlying channel, KCNQ1 (K(v)7.1/K(v)LQT1) was detected by RT-PCR in GCs and by immunohistochemistry in large ovarian follicles. Pharmacological inhibition of the channel by XE991 blocked gonadotropin-stimulated steroid production and increased cell proliferation, i.e. fundamental processes of GCs in the ovary. Assuming a similar effect of ACh in vivo, this channel may be a pivotal regulator of physiological GC function linked to actions of the novel intraovarian signaling molecule ACh.
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