Catherine J. Norris

Effect of indomethacin, cycloheximide, aminoglutethimide on ovarian steroid and prostanoid levels during ovulation in the gonadotropin-primed immature rat

It has become popular to use the gonadotropin-primed immature rat to study ovulation. The ovarian content of progesterone, estradiol, PGE2, PGF2 alpha, and 6-keto-PGF1 alpha during the ovulatory process was determined in this model. Also, the effect of three anti-ovulatory agents on the ovarian levels of the above substances was determined. At 23 days of age, Wistar rats were primed with pregnant mares serum gonadotropin (PMSG) sc, and two days later the ovulatory process was initiated with human chorionic gonadotropin (hCG) sc. The ovarian follicles began rupturing 12 h later. Ovaries were assayed for the two steroids and prostanoids at 2-h intervals before and several 4-h intervals after ovulation. The ovarian estradiol level increased slightly between 0 and 2 h after hCG, while the progesterone level increased sharply between 2 and 4 h after hCG--at a time when the estradiol declined markedly. All three prostanoids increased concomitantly with progesterone. When the PG synthesis was blocked by indomethacin treatment at 1 h before hCG, ovarian progesterone levels still increased. In contrast, when steroidogenic activity was inhibited by aminoglutethimide, the ovarian prostanoid levels also decreased. Cycloheximide had little effect on the steroids and prostanoids. It is concluded that ovarian prostanoid synthesis might be influenced by ovarian steroid output.

Effect of indomethacin in vivo on prostaglandin content of several rabbit tissues.

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle in vivo. PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.

Dependence of 3',5'-cyclic adenosine monophosphate--stimulated gonadotropin-releasing hormone release on intracellular calcium levels and L-type calcium channels in superfused GT1-7 neurons.

Objective: Immortalized GT1-7 neurons were used to characterize the interactive roles of adenylate cyclase-3′,5′-cyclic adenosine monophosphate (cAMP) and L-type calcium channels on gonadotropin-releasing hormone (GnRH) release. Methods: Dibutyryl (db)-cAMP was used as an active analog of endogenous cAMP, and forskolin was used to activate adenylate cyclase. Extracellular calcium was chelated using EGTA and L-type calcium channels were blocked using nimodipine. The selective Ca2+ ionophore A23187 was employed to increase intracellular calcium levels. GT1-7 neurons were grown on Cytodex-3 beads (Pharmacia Biotech, Uppsala, Sweden) and placed in special superfusion microhambers. The cells were superfused at a rate of 6.2 mL/h with media 199 (M-199; Gibco, Grand Island, NY; pH 7.35, 37C); effluent fractions were collected at 5-minute intervals for analysis of GnRH concentrations by radioimmunoassay. Results: Basal GnRH release from superfused GT1-7 neurons ranged from 10 to 62 pg · min-1. mL-1. Coexposure of the cells to forskolin and A23187 produced an additive effect on stimulated release of GnRH. Cells exposed to 1 μM of forskolin (an activator of adenylate cyclase) for 5 minutes showed a 2.6-fold increase in GnRH release. Likewise, the addition of 100 μM of db-cAMP to the superfusion for 5 minutes demonstrated a 2.3-fold increase in the amplitude of GnRH secretion. Maintaining the superfused cells in medium containing 5 mM EGTA had no obvious effect on basal GnRH release but blocked the effect of db-cAMP to increase GnRH release. Similarly, the addition of 10 μM nimodipine to the superfusion medium blocked db-cAMP-stimulated GnRH release. Conclusions: These findings provide additional evidence that cAMP-mediated GnRH release from GT1-7 neurons is dependent on influx of extracellular calcium via L-type Ca2+ channels.

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1–7 neurons in vitro

Immortalized GT1-7 neurons were used to characterize the effect of muscimol, a GABAA receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1-7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml x h-1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 microM muscimol or 5 microM muscimol+20 microM of the GABAA receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1-7 neurons was pulsatile with an average interpulse interval of 45.4+/-0.5 min and an average pulse amplitude of 191.5+/-22.6 pg x min x ml-1. Our results also demonstrated that the GABAA receptor agonist, muscimol, enhances pulsatile GnRH release from GT1-7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC50 of 0.47 microM. The effects of 5 microM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABAA receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7+/-1.8 min, 32.5+/-2.9 min, 30.6+/-0.7 min and 25.5+/-0.4 min in the period following exposure to 0.35, 1, 5 and 10 microM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABAA receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABAA receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.

[3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts.

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)

Spermicidal effect of antagonists of platelet-activating factor**Supported in part through a subproject agreement No. 012 with CONRAD and by National Institutes of Health grant HD14048.

A variety of antagonists of platelet-activating factor (PAF) have been examined for their ability to prevent pregnancy, by admixture with spermatozoa in vitro followed by insemination, or in vivo by administration to female rabbits. When the antagonists were added to the ejaculate at a concentration of 10(-4) M 30 minutes before insemination of the females, a significant failure of fertilization was seen only with CV-3988, U66985, and SRI 63-441--all structural analogs of PAF. Antagonists that were not structural analogs were not effective. All compounds to a lesser or greater extent caused some qualitative agglutination and loss of sperm motility, but these effects were not correlated with inhibition of fertilization. SRI 63-441 was the most effective compound and was subjected to further study. When given intravenously prior to ovulation (5 mg/kg at 1, 5, and 9 hours after the ovulating injection), no effect on fertilization was seen. If SRI 63-441 (40 mg in 0.5 ml aqueous solution) was instilled into the vagina 2 minutes before insemination, a highly significant reduction in the fertilization rate was achieved. It is concluded that these compounds act by an action on the sperm membrane rather than by direct PAF antagonism on spermatozoa.