No abstract

The effect of insulin-like growth factor-I (IGF-I) on the concentrations of follicular fluid factors during follicle deviation and the development of dominance was studied in mares in two experiments. Transvaginal ultrasound guidance was used for intrafollicular injection and subsequent sequential sampling of follicular fluid. Treatment involved a single injection of IGF-I into the second-largest follicle (F2) at the expected beginning of deviation (Hour 0) based on diameter (> or =20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 microg (experiment 1) or 250, 25, or 2.5 microg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-microg doses stimulated growth, leading to ovulation of F2 in 10 of 10 and 4 of 5 mares in the two experiments, respectively, compared to 4 of 12 and 0 of 5 in saline-injected controls. These doses prevented (P < 0.05) the increase in IGF binding protein-2 and androstenedione that occurred in F2 of controls and increased (P < 0.05) the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF). The 500-microg dose stimulated higher (P < 0.05) concentrations of estradiol, but not until Hour 48, whereas the lower doses were ineffective. In experiment 2, free IGF-I concentrations in F2 at Hour 24 decreased progressively as the dose decreased so that concentrations for the 2.5-microg dose were higher (P < 0.05) than in F2 of controls and similar (not significantly different) to endogenous concentrations in F1. Correspondingly, concentrations of androstenedione in F2 at Hour 24 were lower (P < 0.05) and concentrations of activin-A, inhibin-A, and VEGF were higher (P < 0.05) after treatment of F2 with the 2.5-microg dose than in F2 of controls and were similar to concentrations in F1. Hence, a physiologic intrafollicular dose of IGF-I did not stimulate estradiol production but reduced the production of androstenedione and stimulated the production of activin-A, inhibin-A, and VEGF during follicle selection in mares.

No abstract

Prostanoid synthesis via cyclooxygenase (COX)-2 induction during urothelial stretch is central to nociception, inflammation, contractility, and proliferation caused by urinary tract obstruction. We used our primary human urothelial cell stretch model published previously to evaluate the signaling mechanisms responsible for stretch-induced COX-2 expression in urothelial cells. To determine intracytosolic calcium concentrations ([Ca(2+)](i)), primary human urothelial cells were grown on flexible membranes and loaded with Fura-2 acetoxymethyl ester (AM). We determined [Ca(2+)](i) using a fluorescent scope during stretch. Additional cells were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, stretched, and COX-2 mRNA and protein were evaluated by real-time polymerase chain reaction and immunoblotting. To evaluate protein kinase C (PKC) in this system, cells were stretched and fractionated into membrane, cytosol, and nucleus. Fractions were immunoblotted for PKCalpha, beta1, and zeta, the predominant isoforms in urothelial cells. We treated additional cells with increasing concentrations of either bisindolylmaleimide-I or a peptide PKC pseudosubstrate inhibitor, and COX-2 mRNA and protein were evaluated after stretching. Furthermore, we transfected urothelial cells with siRNA against each of the inducible PKC isoforms in these cells and evaluated the stretch-induced COX-2 response. Stretch of urothelial cells activated calcium flux and PKC translocation to membrane and nucleus. Pharmacological inhibition indicated that stretch-induced COX-2 expression is dependent on calcium and PKC, and biochemical knockdown experiments indicated that PKCzeta is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.

ContentsA GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles ‡ 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11-13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.