AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro.

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 µM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 µM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 µM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 µM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.

Prevention of prion propagation by dehydrocholesterol reductase inhibitors in cultured cells and a therapeutic trial in mice.

In prion diseases, the normal cellular form of prion protein (PrP(C)) is converted into the disease-associated isoforms (PrP(Sc)) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrP(Sc) in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrP(Sc) from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 microM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.

Gonadotropin-induced delta14-reductase and delta7-reductase gene expression in cumulus cells during meiotic resumption of porcine oocytes.

Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of Delta14-reductase and Delta7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both Delta14-reductase mRNA and Delta7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either Delta14-reductase inhibitor, AY9944-A-7, or Delta7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 microM AY9944-A-7 or 20 microM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of Delta14-reductase and Delta7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.

Formation of 7-dehydrocholesterol-containing membrane rafts in vitro and in vivo, with relevance to the Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease typified by 7-dehydrocholesterol (7DHC) accumulation and depletion of cholesterol. Because cholesterol is a primary component of detergent-resistant membrane domains ("rafts"), we examined the compatibility of 7DHC with raft formation. Liposomes containing bovine brain phosphatidylcholine, sphingomyelin, cerebrosides, and either cholesterol, 7DHC, or coprostanol (the latter being incompatible with raft formation) were prepared. 7DHC was indistinguishable from cholesterol in its ability to become incorporated into membrane rafts, as judged by physical and chemical criteria, whereas coprostanol did not form rafts. The in vivo compatibility of 7DHC with raft formation was evaluated in brains of rats treated with trans-1,4-bis(2-dichlorobenzylamino-ethyl)cyclohexane dihydrochloride (AY9944), which mimics the SLOS biochemical defect. 7DHC/cholesterol ratios in rafts and whole brains from AY9944-treated rats were similar, indicating comparable efficiency of 7DHC and cholesterol incorporation into brain rafts. In contrast, dolichol (a nonsterol isoprenoid incompatible with raft formation) was greatly depleted in brain rafts relative to whole brain. Although brain raft fractions prepared from AY9944-treated and control rats yielded similar sterol-protein ratios, their gel electrophoresis profiles exhibited multiple differences, suggesting that altered raft sterol composition perturbs raft protein content. These results are discussed in the context of the SLOS phenotype, particularly with regard to the associated central nervous system defects.

Evolución de las inclusiones citoplasmáticas inducidas por Y-9944 en nervio periférico: análisis cuantitativo / Evolution of the cytoplasmic inclusions induced for AY-9944 in peripheral nerve: quantitative analysis

En el presente trabajo se estudia la evolución morfológica de inclusiones intracitoplasmáticas en ratas lactantes tratadas con AY-9944 (trans-1, 4-bis (2-clorobencilaminometil ciclohexano dihidroclórido), un inhibidor de la biosíntesis de colesterol. Para ello, ratas lactantes fueron inyectadas intraperitonealmente a los tres dias de edad con una dosis única de AY-9944, y fueron sacrificadas juntas con sus correspondientes controles a los 5,10 minutos, 1 y 8 dias de inyectadas. El análisis se basó en la cuantificación, a nivel de microscopía electrónica, del número de inclusiones totales, y del número de inclusiones de cada tipo morfológico que aparecían en cada uno de los tiempos estudiados. Los resultados han indicado: primero, un aumento progresivo del número total de inclusiones desde los 5 minutos hasta los 8 dias de inyectado el inhibidor; y segundo, una variación en los tipos morfológicos de inclusiones predominantes en los tiempos estudiados, en los animales inyectados; en comparación con la ausencia de inclusiones en los respectivos controles

The effect of AY-9944 on yeast sterol and sterol ester metabolism.

The effects of the hypocholesterolemic drug AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride) at two concentrations (10(-4) M and 5 X 10(-4) M) on the synthesis of sterols and sterol esters by Saccharomyces cerevisiae were investigated. Although growth was not markedly affected by the drug, there was a decrease in the free sterol to sterol ester ratio with increased drug concentration. A concomitant increase in the saturated fatty acids esterified to sterol relative to the unsaturated fatty acids was also noted in response to increased drug concentration. Ergosterol accounted for 94.7% of the free sterol in the control culture and for 87.8% of the 5 X 10(-4) M drug-treated culture, respectively. However, in the sterol ester fraction, the ergosterol content decreased from a value of 45.1% in the control culture to 2.4% in the 5 X 10(-4) M AY-9944 treated culture. The sterol ester fraction simultaneously showed increased levels of the delta 8 sterol, fecosterol, in response to increased drug concentration from a 7.4% control value to 57.4% in the 5 X 10(-4) M drug-treated culture. The accumulation of the delta 8 sterol suggests that the site of action of the drug is probably at the delta 8 to delta 7 isomerase step in the biosynthesis of ergosterol. The fact that ergosterol is retained as the major free sterol suggests a biological advantage to the retention of this particular sterol. In addition, the near normal growth in the presence of the drug, in spite of the occurrence of an altered sterol ester profile, indicates that the composition of the sterol ester fraction is not as critical as the free sterol fraction.

Changes in brain hydrolytic enzyme activities in rats treated with cholesterol biosynthesis inhibitor, AY9944.

Intraperitoneal administration of AY9944 causes accumulation of 7-dehydrocholesterol, appearance of abnormal neuronal cytoplasmic lamellar inclusions containing acid phosphatase activities, and degeneration of oligodendroglial cells. In the present study, we attempted to correlate appearance and disappearance of abnormal inclusions, oligodendroglial degeneration, activities of lysosomal enzymes, and accumulation of 7-dehydrocholesterol. One group of 5-day-old rats received daily injection of AY9944, 30 mg/kg, for 30 days. Other groups received the same daily dosage but only in 10 consecutive days, starting from 5, 15, 25, and 35 days, respectively. Activities of 13 brain hydrolytic enzymes were determined. In the first group, activities of most enzymes increased over the controls, reaching the peak between the 15th and 25th day, corresponding to the maximum appearance of the neuronal inclusions and degenerating oligodendroglia. Despite the continued administration of AY9944 and the high tissue concentration of 7-dehydrocholesterol, activities of most enzymes declined after 25 days to relatively steady levels somewhat higher than controls. In the group which received 10-day injections, enzyme activities reached the peak at the end of the injections and then rapidly returned to normal within 10 days thereafter, corresponding to the appearance and disappearance of the abnormal inclusions. However, 7-dehydrocholesterol continued to increase for 10 days after the drug administration was discontinued. AY9944 had no direct effect on any of the enzymes in vitro. There appears to be generalized lysosomal activation concomitant with formation of abnormal neuronal inclusions in the experimental conditions.