The second messenger cyclic adenosine 5'monophosphate (cAMP) has been implicated in controlling meiotic maturation. To date, there have been no direct measurements of cAMP in living mammalian oocytes. Here, we have used the fluorescently labelled cAMP-dependent protein kinase A (PKA), FlCRhR, to monitor cAMP in mouse oocytes. In cumulus-enclosed oocytes, follicle-stimulating hormone (FSH) stimulated an increase in the oocyte [cAMP] that was prevented by using the gap junction inhibitor, carbenoxolone. The FSH-induced increase in oocyte [cAMP] was suppressed in a time-dependent manner by prior exposure to ATP, while epidermal growth factor had no effect on basal or stimulated levels of cAMP. Finally, using confocal microscopy, we show that the regulatory and catalytic subunits of the microinjected PKA are distributed in a punctate manner with a stronger accumulation in the perinuclear region. On an increase in [cAMP], in response to phosphodiesterase inhibition or FSH, the catalytic subunit diffused throughout the cytoplasm and germinal vesicle, while the regulatory subunit remained anchored. These experiments show that increases in cAMP in ovarian somatic cells are communicated via gap junctions to the oocyte, where it can lead to a redistribution of the catalytic subunit of PKA.

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development. Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11b-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111G6 vs 2041G115 fmol cortisone/oocyte.24 h; P!0.001). Likewise, HSD11B activities were 17G1 fold higher in DOs from expanded COCs than in those from compact COCs (P!0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146G18 to 1857G276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P!0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation. Reproduction (2008) 136 725-732

Context: Altered hepatic cortisol-cortisone metabolism by type 1 11␤-hydroxysteroid dehydrogenase (11␤HSD1) has previously been linked with polycystic ovary (PCO) syndrome (PCOS).Objectives: Our objectives were to establish whether ovarian 11␤HSD activities are also altered in PCOS and to determine whether any changes in ovarian cortisol metabolism might reflect exposure to elevated concentrations of insulin or androgens.Design: Cortisol and cortisone concentrations were measured in follicular fluid aspirated from size-matched follicles dissected from normal, ovulatory, and anovulatory PCOs. Human granulosalutein cells, recovered during oocyte retrieval for assisted conception, were maintained in primary culture for 4 days, after which 11␤HSD1 activities were measured as the net oxidation of(100 nmol/L) in the absence and presence of insulin (100 nmol/L) with or without metformin (1 mol/L) or a range of androgens/oxy-androgen metabolites (0.01-10 mol/L).Results: Intrafollicular cortisol to cortisone ratios were elevated in anovulatory PCOs (2.1 Ϯ 0.4, P Ͻ .05, n ϭ 13) but did not differ between follicles from ovulatory PCOs (1.6 Ϯ 0.1, n ϭ 24) and normal ovaries (1.2 Ϯ 0.1, n ϭ 14). 11␤HSD1 activities were lower in granulosa-lutein cells recovered from patients with PCOS compared with all other causes of infertility (median ϭ 5.8 vs 14.9 pmol cortisone/4 h, respectively; P Ͻ .05). Cortisol oxidation was unaffected by insulin with or without metformin, dehydroepiandrosterone, and androstenedione, but was inhibited in a concentrationdependent manner by testosterone, 11␤-hydroxyandrostenedione, and 7␣-and 7␤-hydroxy-dehydroepiandrosterone (P Ͻ .01). Conclusions:There is decreased inactivation of cortisol in follicles from anovulatory PCOS. This may reflect inhibition of 11␤HSD1 by androgens and their 7/11-oxy-metabolites, local concentrations of which are increased in PCOS, and may contribute to the block to folliculogenesis seen in PCOS.(J Clin Endocrinol Metab 98: 3375-3383, 2013) P olycystic ovary (PCO) syndrome (PCOS) is the predominant cause of anovulatory infertility (1). This endocrine disorder, which affects up to 10% of premenopausal women (2), is characterized by PCOs accompanied by elevated plasma LH, hyperandrogenism, and peripheral insulin resistance/hyperinsulinemia (3). Recent studies have explored the role of glucocorticoids in the pathophysiology of PCOS. Within potential target cells, the

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process.