Chemotaxonomy of the pantropical genus Merremia (Convolvulaceae) based on the distribution of tropane alkaloids.

The occurrence and distribution of tropane and biogenetically related pyrrolidine alkaloids in 18 Merremia species of paleo-, neo-, and pantropical occurrence have been studied. The extensive GC-MS study included members of almost all sections of the genus and has been carried out with epigeal vegetative parts as well as with roots. It comprises altogether 74 tropanes and 13 pyrrolidines including nicotine. Along with datumetine known already from a solanaceous species, the study led to the isolation (from M. dissecta and M. guerichii, respectively) and structure elucidation (spectral data) of four novel 3alpha-acyloxytropanes, merresectines A-D: 3alpha-(4-methoxybenzoyloxy)nortropane (A), 3alpha-kurameroyloxytropane (B), 3alpha-nervogenoyloxytropane (C), 3alpha-[4-(beta-D-glucopyranosyloxy)-3-methoxy-5-(3-methyl-2-butenyl)benzoyloxy]tropane (beta-d-glucoside of D). Moreover, the novel 3alpha,6beta-di-(4-methoxybenzoyloxy)tropane (merredissine) has been isolated from M. dissecta and structurally elucidated. In addition the structures of datumetine and merresectine A could be confirmed by synthesis. Spectral data for two known 3alpha-acyloxytropanes (merresectine E beta-D-glucoside, 4'-dihydroconsabatine) and one known 3beta-acyloxytropane (concneorine) are documented for the first time. The structures of three further merresectines (F-H) have been determined by mass spectrometry. Furthermore, the linkage (2',3- and 2',4-, respectively) of two position isomer N-methylpyrrolidinylhygrines was proven by synthesis. The results of the study contribute to the solution of infrageneric taxonomic problems. Whereas all species yield pyrrolidine alkaloids without suitably differentiating results the diverging occurrence of tropane alkaloids leads to three groups of sections: (1) taxa free of tropanes, (2) taxa with simple tropanes, and (3) taxa with merresectines in addition to simple tropanes.

Ovarian follicular development and oocyte quality in anestrous ewes treated with melatonin, a controlled internal drug release (CIDR) device and follicle stimulating hormone.

The objective of the current study was to determine the effects of hormonal treatments on ovarian follicular development and oocyte quality in anestrous ewes. Multiparous crossbred (RambouilletxTarghee) ewes were given melatonin implants (MEL) and/or controlled internal drug release (CIDR) devices in conjunction with follicle stimulating hormone (FSH) during anestrus (March-May). In Experiment 1, ewes (n=25) were assigned randomly to four groups (n=4-7/group) in a 2x2 factorial arrangement [+/-MEL and +/-CIDR], resulting in Control (no treatment), CIDR, MEL, and MEL/CIDR groups, respectively. Ewes received an implant containing 18 mg of melatonin (Melovine) on Day 42 and/or a CIDR from Days 7 to 2 (Day 0: oocyte collection). In Experiment 2, ewes (n=12) were assigned randomly to two groups (n=6/group; 1CIDR or 2CIDR) and received the same type of melatonin implant on Day 60. All ewes received a CIDR device from Days -22 to -17 and 2CIDR ewes received an additional CIDR device from Days -10 to -2. In both experiments, ewes were given FSH im twice daily (morning and evening) on Days -2 and -1 (Day -2: 5 units/injection; Day -1: 4 units/injection). On the morning of Day 0, ovaries were removed, follicles>or=1 mm were counted, and oocytes were collected. Thereafter oocytes were matured and fertilized in vitro. In Experiment 1, the number of visible follicles and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for Control, CIDR, MEL and MEL/CIDR (overall 29.7+/-2.9%, 89.9+/-7.1% and 95.0+/-2.0%, respectively). The rates of in vitro fertilization (IVF) were lower (P<0.01) for CIDR and MEL/CIDR than for Control and MEL groups (10.3% and 10.1% versus 20.0% and 18.5%, respectively). In Experiment 2, the number of visible follicles, and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for 1CIDR and 2CIDR groups (overall 27.3+/-3.2%, 92.1+/-2.7% and 90.2+/-1.9%, respectively). However, the rates of IVF were lower (P<0.01) for 2CIDR than 1CIDR group (30.2% versus 58.0%, respectively). In summary, when treatment with P4 commenced only 2 d before oocyte collection, rates of IVF were reduced in both experiments. Therefore, progestin treatment protocols used in ovine IVF programs should be carefully designed to minimize adverse effects on fertilization rates. In addition, melatonin treatment did not affect follicular development and oocyte quality for anestrous ewes.

Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep.

To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.

Ipobscurines C and D: macrolactam-type indole alkaloids from the seeds of Ipomoea obscura.

Separation of the methanolic seed extract of Ipomoea obscura afforded five indole alkaloids, three of them (ipobscurines B-D) being new natural products of a unique structural type characterized as serotonin hydroxycinnamic acid amide-type conjugates with a second phenylpropanoid moiety forming an ether with the 5-OH position of the indole nucleus. Due to an oxidative phenolic coupling between the two phenylpropanoid moieties of the supposed precursor ipobscurine B two 21-membered macrolactams with a phenol ether partial structure are formed: the trans-cis isomers ipobscurines C and D. Their structures were established on the basis of spectral data. Moreover, total synthesis of the racemic erythro- and threo-ipobscurine B 4',4"-dimethyl ethers and the comparison with the corresponding derivative of natural (-)-ipobscurine B proved an erythro configuration of the latter.