4-4-dimethylcholesta-8-14-24-trienol and 4-4-dimethylcholesta-8-14-dien-3-ol

Meiosis activating sterol (MAS) have been found to be able to promote oocytes meiotic maturation of small animals in vitro, such as mouse, rat and rabbit. But in large animals, whether MAS play the same function, especially the physiological mechanisms of MAS on oocytes maturation are not clear. To our knowledge, this is the first time to investigate the role and signal pathway of MAS on FSH-induced porcine oocytes meiotic resumption. Porcine cumulus-enclosed oocytes (CEOs) isolated from 3 to 5mm follicles were cultured in the FSH-medium for 24h supplemented with 0-50 microM RS21745 or 0-100 microM RS21607 (two specific inhibitors of lanosterol 14alpha-demethylase that converts lanosterol to FF-MAS), or cultured in FSH-medium with 25 microM RS21745 for 0-24h firstly, then transferred into a new FSH-medium (the total culture time is 24h). The results revealed that RS21745 or RS21607 could inhibit FSH-induced porcine CEOs meiotic resumption in a dose and time-dependent manner. Meanwhile, FSH-induced cumulus expansion could also be inhibited dose-dependently by RS21745 or RS21607. Otherwise, AY9944-A-7, an inhibitor of Delta14-reductase which promotes cholesterol accumulation from FF-MAS, had no effect on both denuded oocytes (DOs) cultured for 24 or 44 h and CEOs cultured for 24h meiotic resumption, but it could promote CEOs meiotic resumption after 44 h culture. In addition, we got that 10(-8) to 10(-6)M PMA, an activator of PKC pathway, could reverse the inhibiting effect of RS21745 on FSH-induced CEOs meiotic resumption and enhance the rate of germinal vesicle breakdown (GVBD) of CEOs cultured in medium with hypoxanthine (HX). Moreover, 5-10 microM chelerythrine chloride, an inhibitor of PKC, could enhance the inhibitory effect of RS21745 on FSH-induced porcine oocytes resumption of meiosis. All the data of this study support that endogenous FF-MAS takes part in the FSH-induced porcine oocytes meiotic resumption and might play an active role via PKC signal pathway.

Topics: Aniline Compounds; Animals; Cholestadienols; Cholestenes; Cytochrome P-450 Enzyme Inhibitors; Female; Follicle Stimulating Hormone; Meiosis; Oocytes; Ovarian Follicle; Oxidoreductases; Protein Kinase C; Sterol 14-Demethylase; Sterols; Sulfides; Swine; trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride

This is the first study evaluating whether oocyte development and fertilization competence are related to intrafollicular concentration of cholesterol, meiosis-activating sterols and progesterone, after human chorionic gonadotrophin (HCG) administration of women with polycystic ovarian syndrome (PCOS). The concentration of follicular fluid meiosis-activating sterol (FF-MAS) significantly increased in the periovulatory period from 10-14 to 34-38 h after HCG administration, while the concentration of testis meiosis-activating sterol (T-MAS) decreased, suggesting a HCG-dependent inhibition of sterol Delta14-reductase. There was no correlation between follicular lanosterol, FF-MAS, T-MAS, and progesterone concentrations and the presence or absence of MII oocytes. Interestingly, free cholesterol level was significantly lower and FF-MAS/cholesterol and progesterone/cholesterol ratios significantly higher in follicles containing MII oocytes compared to follicles from which oocytes were not retrieved. Yet, fertilization and embryo quality did not correlate with follicular sterols. This knowledge should be beneficial for the implementation of protocols for in vitro maturation process, usually used in PCOS patients.

Topics: Cholestadienols; Cholestenes; Cholesterol; Chorionic Gonadotropin; Embryonic Development; Female; Fertilization in Vitro; Humans; Lanosterol; Metaphase; Oocytes; Ovarian Follicle; Ovulation Induction; Polycystic Ovary Syndrome; Progesterone

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Topics: Cholestadienols; Cholestenes; Genetic Engineering; Saccharomyces cerevisiae; Sterols

Meiosis activating sterols (MAS) are pre-cholesterol sterols that can be isolated from follicular fluid (FF-MAS) or testes (T-MAS). Meiosis activating sterols trigger the resumption of meiosis in cultured meiotically competent oocytes. In the present work MAS, cholesterol and progesterone were assayed by HPLC in follicular fluids collected from pony mares at fixed days after the last ovulation. Follicles were divided into two groups according to whether they were aspirated before or after Day 17 after the last ovulation. The latter group was further divided according to whether the follicle diameter was < or = 22 mm or > 27 mm. Both FF-MAS and T-MAS were detected in almost all samples. Overall, the total amount of MAS in the follicular fluids increased with the size of the follicles but was accompanied by a decrease in the amount of free cholesterol. The amounts of MAS and progesterone in > 27 mm follicles aspirated after Day 17 were significantly higher as compared to the other groups. A transversal cohort analysis showed that the largest follicle at the time of aspiration had the highest level of MAS after day 17 of the cycle, which was not always true for follicle samples aspirated before Day 17 of the cycle. The study demonstrates that the content of MAS in equine follicular fluids increased during follicular maturation concomitant with a decrease in the concentration of free cholesterol. Moreover, MAS concentration is higher in dominant follicles than in subordinate follicles. The MAS may therefore play an as yet unknown physiological role during pre-ovulatory maturation.

Topics: Animals; Cholestadienols; Cholestenes; Cholesterol; Estrus; Female; Follicular Fluid; Horses; Lanosterol; Ovarian Follicle; Progesterone; Statistics, Nonparametric

Follicular Fluid-Meiosis Activating Sterol (FF-MAS) is a compound important for maturation of gametes in mammals. Therefore, it may serve as a lead compound for a novel method of contraception. We studied the Molecular Electrostatic Potential of a series of active and inactive analogues of FF-MAS. We find that double bond configurations required for activity result in a local negative electrostatic potential which is larger as well as more dense compared to those of inactive molecules. We therefore hypothesize that the interaction energy of the double bond system of the MAS compounds with its receptor substantially contributes to the overall interaction energy. This notion is supported by interaction studies of the electrostatic potential originating from the double bonds in crystal structures of cholesterol and four MAS-derived Delta(8,14) structures synthesized and crystallized by us. In addition, we were able to derive a pharmacophore model that relates the local average ESP and its distance to the 3beta-OH oxygen atom to the activity of the molecules.

Topics: Animals; Cells, Cultured; Cholestadienols; Cholestenes; Cholesterol; Crystallography, X-Ray; Female; Hydroxylation; Menotropins; Mice; Models, Chemical; Molecular Conformation; Molecular Structure; Oocytes; Ovary; Sterols; Structure-Activity Relationship

The resumption of oocyte meiosis is triggered by a number of 4,4-dimethylsterols termed meiosis-activating sterols (MAS). The levels of meiosis active (follicular fluid [FF]-MAS and bull testes [T]-MAS) and inactive (lanosterol) 4,4-dimethylsterols, free cholesterol, and progesterone were determined in gonadotropin-primed prepubertal mouse ovaries in vivo by high-performance liquid chromatography. Ovaries responded to an ovulatory stimulation by increasing their content of 4,4-dimethylsterols but not of free cholesterol. The ovarian 4,4-dimethylsterol response was followed with regard to time and dose-response to the gonadotropins and the resumption of meiosis was evaluated using histologic sections. All 4,4-dimethylsterols accumulated in a time-dependent manner in gonadotropin-primed mice after a subsequent stimulation with hCG. The peak of 4,4-dimethylsterol accumulation appeared postmeiotically but coincided roughly with ovulation, and the resumption of meiosis was triggered when the intraovarian level of MAS was <20% of its maximum. The ovarian accumulation of progesterone preceded the 4,4-dimethylsterol accumulation. The FF-MAS accumulation displayed a dose-response maximum with respect to hCG, and a variation of the follicular priming regime revealed that, in contrast to progesterone production, 4,4-dimethylsterol accumulation is dependent on previous follicular growth beyond the gonadotropin-dependent stage. The FF-MAS was not liberated from esterified stores during the accumulatory response and appeared to be synthesized de novo from a precursor (or precursors) metabolically upstream to lanosterol. The data remain inconsistent with a model in which MAS is regarded as the physiological trigger of meiosis. The 4,4-dimethylsterol accumulation is suggested to influence maturation processes by affecting membrane sterol composition.

Topics: Animals; Cholestadienols; Cholestenes; Cholesterol; Chorionic Gonadotropin; Chromatography, High Pressure Liquid; Esterification; Female; Follicular Fluid; Gonadotropins; Kinetics; Lanosterol; Male; Meiosis; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Ovary; Ovulation; Progesterone