Experimental induction of reduced ovarian reserve in a nonhuman primate model (Macaca fascicularis).

Chronic diseases including coronary heart disease and osteoporosis represent a substantial health burden to postmenopausal women, yet the initiation of these conditions and their relationships with reproductive aging remain poorly understood. This situation is due, in part, to the lack of animal models reflecting ovarian and hormonal characteristics of peri- and postmenopausal women. Ovaries of women approaching menopause are nearly depleted of primordial follicles but retain a pool of larger developing follicles and androgen-producing stroma, a condition known as reduced ovarian reserve (ROR). The long-term goal of the research presented here was to create a monkey model of reproductive aging, beginning with ROR and progressing to perimenopause and finally postmenopause. Here we sought to develop a method to reduce primordial follicles in cynomolgus monkeys (Macaca fascicularis) and document hormonal changes associated with follicle reduction or ROR. At 30 d after surgical placement of a biodegradable fiber containing approximately 200 mg of 4-vinlycyclohexene diepoxide (VCD) next to one ovary in each of 8 monkeys, primordial follicles were reduced by approximately 70%, with a corresponding decrease (83%) in antimüllerian hormone (AMH, a serum marker of ovarian follicle numbers). At 4 mo after VCD-treatment of both ovaries in 29 monkeys (approximately 200 mg VCD per ovary), AMH was reduced 56% from baseline, testosterone was unchanged, and follicular phase estradiol was slightly increased. These data indicate that VCD treatment markedly reduced primordial follicles while preserving larger estradiol- and testosterone-producing follicles and ovarian stroma, a condition that mimics ROR in women.

Competitive FRET-aptamer-based detection of methylphosphonic acid, a common nerve agent metabolite.

Competitive fluorescence resonance energy transfer (FRET)-aptamer-based assay formats are described for one-step detection of methylphosphonic acid (MPA; a metabolite of several organophosphorus (OP) nerve agents). AminoMPA was attached to tosyl-magnetic beads and used for DNA aptamer selection from which one dominant aptamer sequence emerged. Two different FRET approaches were attempted. In one approach, the complementary DNA sequence was used as a template for labeling the aptamer with Alexa Fluor 546 (AF 546)-14-dUTP by asymmetric PCR. Following 3-dimensional (3-D), molecular modeling of the aptamer-MPA complex, a series of three fluoresceinated aptamers labeled at positions 50, 51, and 52 in the putative optimal binding pocket were synthesized. In both FRET formats, aminoMPA was linked to Black Hole Quencher (BHQ-1 or BHQ-2)-succinimides and allowed to bind the fluorescein or AF 546-labeled MPA aptamer. Following gel filtration to purify the labeled MPA aptamer-BHQ-aminoMPA FRET complexes, the complexes were competed against various concentrations of unlabeled MPA, MPA derivatives, and unrelated compounds in titration and cross-reactivity studies. Both approaches yielded low microgram per milliliter detection limits for MPA with generally low levels of cross-reactivity for unrelated compounds. However, the data suggest a pattern of traits that may effect the direction (lights on or off) and intensity of the FRET.