The cAMP Inducers Modify N-Acetylaspartate Metabolism in Wistar Rat Brain

Neuronal N-acetylaspartate production appears in the presence of aspartate N-acetyltransferase (NAT8L) and binds acetyl groups from acetyl-CoA with aspartic acid. Further N-acetylaspartate pathways are still being elucidated, although they seem to involve neuron-glia crosstalk. Together with N-acetylaspartate, NAT8L takes part in oligoglia and astroglia cell maturation, myelin production, and dopamine-dependent brain signaling. Therefore, understanding N-acetylaspartate metabolism is an emergent task in neurobiology. This project used in in vitro and in vivo approaches in order to establish the impact of maturation factors and glial cells on N-acetylaspartate metabolism. Embryonic rat neural stem cells and primary neurons were maturated with either nerve growth factor, trans-retinoic acid or activators of cAMP-dependent protein kinase A (dibutyryl-cAMP, forskolin, theophylline). For in vivo, adult male Wistar rats were injected with theophylline (20 mg/kg b.w.) daily for two or eight weeks. Our studies showed that the N-acetylaspartate metabolism differs between primary neurons and neural stem cell cultures. The presence of glia cells protected N-acetylaspartate metabolism from dramatic changes within the maturation processes, which was impossible in the case of pure primary neuron cultures. In the case of differentiation processes, our data points to dibutyryl-cAMP as the most prominent regulator of N-acetylaspartate metabolism.

Bisphenol A disturbs metabolism of primary rat adipocytes without affecting adipokine secretion

Bisphenol A (BPA) is an ubiquitous synthetic chemical exerting numerous adverse effects. Results of rodent studies show that BPA negatively affects adipose tissue. However, the short-term influence of this compound addressing adipocyte metabolism and adipokine secretion is unknown. In the present study, isolated rat adipocytes were exposed for 2 h to 1 and 10 nM BPA. Insulin-induced glucose conversion to lipids along with glucose transport was significantly increased in the presence of BPA. However, basal glucose conversion to lipids, glucose oxidation, and formation of lipids from acetate were unchanged in adipocytes incubated with BPA. It was also shown that BPA significantly increases lipolytic response of adipocytes to epinephrine. However, lipolysis stimulated by dibutyryl-cAMP (a direct activator of protein kinase A) and the antilipolytic action of insulin were not affected by BPA. Moreover, BPA did not influence leptin and adiponectin secretion from adipocytes. Our new results show that BPA is capable of disturbing processes related to lipid accumulation in isolated rat adipocytes. This is associated with the potentiation of insulin and epinephrine action. The effects of BPA appear already after short-term exposure to low doses of this compound. However, BPA fails to change adipokine secretion.

30 The importance of cumulus cells for the survival and timing of meiotic resumption of porcine oocytes vitrified at the immature stage

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4M sucrose. The oocytes were then cultured for 22h in a chemically defined porcine oocyte medium (POM) supplemented with 10ngmL−1 epidermal growth factor, 10IUmL−1 equine chorionic gonadotrophin, 10IUmL−1 human chorionic gonadotrophin, and 1mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P<0.05) than that of the DO group (160/191=84.7±3.4% vs. 153/237=65.0±6.2%, respectively) but reduced (P<0.05) compared with those in the control COC and DO groups (138/143=96.6±1.0% and 152/153=99.3±0.6%, respectively). The majority of the control COCs and DOs were at the GV stage with similar percentages (95.6±2.2% and 94.0±2.2%, respectively). In contrast, the percentages of oocytes at the GV stage in the vitrified COC and DO groups were reduced (71.6±9.4% and 45.7±10.5%, respectively; P<0.05) compared with the control groups, which were associated with increased frequencies of diakinesis and MI stages. Percentages of oocytes at the GV stage in the vitrified COC and DO groups were not significantly different (P=0.23). In conclusion, cumulus cells can prevent vitrification-related membrane damage of oocytes. Furthermore, vitrification induced premature meiosis both in the cumulus-enclosed and denuded oocytes even in the presence of the meiotic inhibitor, dibutyryl cAMP. Nevertheless, cumulus removal without vitrification did not induce premature meiosis in the oocytes. Therefore, disruption in communication with cumulus cells might not be the primary reason for premature meiosis in vitrified oocytes.

Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20–23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.