Harold R. Behrman

Ovulation blockade by aspirin or indomethacin - in vivo evidence for a role of prostaglandin in gonadotrophin secretion

Abstract Inhibitors of prostaglandin (PG) synthesis, aspirin and indomethacin, were used to assess the role of PG in gonadotrophin secretion. Both drugs reduced plasma PGF content and indomethacin reduced pituitary and hypothalamic concentration of PGF measured by a radioimmunoassay procedure reported herein. Chronic and acute administration of either indomethacin or aspirin blocked ovulation but indomethacin was effective at 1/30 the dose of aspirin. Injection of either LH or a mixture of PGE 2 and PGF 2α at the time of the expected ovulatory surge of LH was effective in reversing the blockade of ovulation that occurred after a single injection of indomethacin administered about 3 hours before the expected ovulatory surge of LH. However, LH did not reverse the blockade of ovulation produced when indomethacin was administered chronically beginning 30 hours before the expected ovulatory LH-surge. These data support the hypothesis that prostaglandins play a functional role in regulating the release of LH necessary for ovulation in the rat.

Radioimmunoassay of the A prostaglandins.

Antibodies to the (PGA) prostaglandin A were produced in rabbits immunized with a conjugate of PGE2 covalently linked to (BSA) bovine serum albumin by reaction with carbodiimide reagent. A radioimmunoassay was developed using dextran-coated charcoal to separate the free from antibody bound PGA1-3H. The sensitivity of the method was found to be 100 picograms/ml of plasma. Ethyl acetate was used for extraction of plasma and the various classes of PGs were separated by silicic acid column chromatography. Recovery of PGA1-3H throughout the entire procedure was 65-75%. The antibody showed progressively decreasing affinity to PGA2, PGA1, PGE2, PGE1, PGB2, and PGF2alpha, respectively. The mean plasma PGA level in adult males (N=13) was found to be 1.39 + or - 0.55 ng/ml, and 1.62 + or - 0.52 ng/ml in adult females (N=7). Corresponding plasma and serum samples were found to give essentially similar results. Plasma PGA levels in adult males treated with indomethacin for rheumatoid arthritis were 0.18 + or - 0.15 ng/ml (P 0.001 in comparison with the normal adult males). This method is sufficiently sensitive, precise, and rapid to allow the routine estimation of the PGAs in biological samples.

Regulation of ovarian cholesterol esters: Evidence for the enzymatic sites of prostaglandin-induced loss of corpus luteum function

The regulation of cholesterol ester synthetase and cholesterol esterase by prostaglandins and gonadotrophins in luteinized ovaries of the rat was studied. Prostaglandin F2α (PGF2α) depressed ovarian cholesterol esters by 75% (p<.025) within 48 hr. Hypophysectomy (APX) produced a similar effect; prolactin administration to this group maintained cholesterol esters at a higher level than in the APX group but the trophic effect of prolactin was abolished by simultaneous PGF2α treatment. Progesterone output in incubated ovarian slices was reduced 50% by PGF2α treatment in vivo (p<.005), an effect similar to that produced by APX. Prolactin administration in vivo maintained the ability of the incubated tissue to synthesize progesterone at an elevated rate in APX rats but simultaneous PGF2α treatment abolished this action of prolactin. Cholesterol ester synthetase activity was severely depressed (p<.005) by PGF2α treatment to animals with intact pituitaries, a decrease similar to that produced by APX alone. The effect of APX on synthetase activity was reversed by prolactin treatment but not when PGF2α was administered with prolactin. Esterase activity, also maintained by prolactin in APX animals (p<.005), was not affected to the same extent by PGF2α although a decrease in activity was produced in both the intact and the APX+ prolactin group by PGF2α (p<.10). However simultaneous administration of luteinizing hormone (LH) reversed the effect of PGF2α in the APX+ prolactin +PGF2α group on esterase activity. These data indicate that the luteolytic action of PGF2α is directly on the corpus luteum and this action appears to be mediated by a neutralization of prolactin activity. The loss in synthetase activity and to some extent in esterase activity, induced by PGF2α depressed ovarian cholesterol ester turnover and the availability of cholesterol for conversion to progesterone.

Effects of a cholesterol esterase inhibitor and of prostaglandin F2α on testis cholesterol and on plasma testosterone in mice

Abstract Administration of Phenylmethylsulfonylfluoride, an inhibitor of cholesterol esterase, to male mice caused an increase in the concentration of esterified cholesterol in the testis, a decrease in the weight of seminal vesicles and a decrease in the concentration of testosterone in peripheral plasma. It is suggested that hydrolysis of cholesterol esters present in the testis is required for normal production of androgenic steroids. Administration of prostaglandin F 2α to male mice lowered plasma testosterone levels and raised the concentration of esterified cholesterol in the testes. Apparently testicular steroidogenesis was inhibited.

Partial purification of human placental 15-hydroxyprostaglandin dehydrogenase: Kinetic properties

Abstract The enzyme system, 15-hydroxyprostaglandin dehydrogenase, which catalyzes the first inactivation step in the catabolism of the prostaglandins has been isolated and purified 107-fold from human placenta. Kinetic studies reveal different Michaelis-Menten constants for most of the naturally occurring prostaglandins. The Km for PGE2 was found to be 10 μM, for PGE1, 27 μM; for PGA2, 32 μM; for PGA1, 33 μM; and for PGF2α 59 μM. The enzyme has a sharp pH-optimum between 7.5 and 8.8. Prostaglandin dehydrogenase appears to be isoenzymic as judged by separation on polyacrylamide disc gel electrophoresis. Inhibition studies with the partially purified enzyme indicate that progesterone and estrogen may influence the conversion of biologically active prostaglandins into the biologically inactive 15-ketoprostaglandins. These findings offer evidence for the control of prostaglandin metabolism in the human placenta.

Effect of synthetic gonadotrophin-releasing hormone (Gn-Rh) on ovulation blockade by aspirin and indomethacin

Abstract To determine the site of the ovulation blocking action of indomethacin and aspirin, reversal of each drugs inhibitory effect by synthetic gonadotrophin-releasing hormone (Gn-RH) and luteinizing hormone (LH) was tested. Aspirin blockade of ovulation was consistently reversed by LH administration (10 μg;iv) 2 or 3 hours after drug injection and by Gn-RH (approximately 0.6 and 0.3 nanomoles, sc) given twice either 3 and 4 or 2 and 3 hours after drug injection. In contrast, ovulation blockade by indomethacin was not reversed by Gn-RH, nor was it reversed by exogenous LH given at the time of the expected LH surge if the indomethacin had been administered 2 hours earlier. Treatment with either drug resulted in a reduction in pituitary and hypothalamic PGF levels. The dosage of and injection schedule for Gn-RH was also effective in reversing phenobarbital-blocked ovulation. All animals, including those that did not ovulate after indomethacin and Gn-RH or LH treatment, exhibited loss of uterine fluid on the day after proestrus. Aspirin blocked ovulation at the hypothalamic level, whereas indomethacin may act at the ovarian level by interfering with the rupture of the follicle and release of the ovum.

Aminoglutethimide potentiation of estrogen-induced abortion

Abstract Treatment of day 8 pregnant rats for 3 days with 30 mg aminoglutethimide, an inhibitor of steroid biosynthesis, did not affect gestation but when administered with as little as 0.1 μg of estradiol-17β, abortion occurred in 50% of the animals. At 0.5 μg or higher doses of estradiol-17β and the same dose of aminoglutethimide, abortion occurred in all animals. This effect was reversed by simultaneous treatment with progesterone. AGP (30 mg) reduced progesterone, 20α-dihydroprogesterone and estrogen concentration in peripheral plasma by 30, 50 and 50%, 6 hours after a single injection.

Influence of prostaglandin F autoantibodies on the estrous cycle of the guinea pig

Adult female guinea pigs were actively immunized with prostaglandin F-2alpha conjugated to bovine serum albumin (BSA). Control animals, immunized against BSA continued to cycle normally, while the animals immunized against prostaglandin F-2alpha stopped cycling after one to three normal cycles. Laparotomy at 30 days after the last estrus revealed no recently formed corpora lutea. During the remaining 70 days of observation the antibody titer increased to 1:700, accompanied by increasing total serum estrogens (136 pg/ml at day 100) and a slow decline in circulating progesterone levels (0.6 ng/ml at day 100). The ovaries at day 100 contained degenerated corpora lutea and luteinized follicles. The suppression of the estrous cycle in the present experiments was interpreted as resulting from prolongation of luteal function as well as from inhibition of ovulation.