Studies on reproduction in rats with meclofenamate sodium, a nonsteroidal anti-inflammatory agent.

Reproduction and teratology studies were performed in rats given meclofenamate sodium, a nonsteroidal anti-inflammatory agent. Dosages of 0, 3, 6, and 9 mg/kg were administered orally as dietary admixtures in the Fertility and Perinatal-Postnatal studies. In the Teratology study, dosages of 10, 12, 15, and 20 mg/kg were administered by intragastric intubation. In the Male-Fertility study no adverse effects on fertility or litter and offspring parameters were observed in two generations. In the Female-Fertility and Perinatal-Postnatal studies, maternal toxicity (death associated with intestinal ulceration and adhesions) was particularly evident during lactation. Prolonged gestation periods, decreased weanling weights, and increased weanling mortality were evident at dosages of 6 and 9 mg/kg. Increased postimplantation loss occurred at 6 and 9 mg/kg in the Term Sacrifice subgroup of the Female-Fertility study. Fertility rates were unaffected and all other litter and offspring parameters of the F1 and F2 generations appeared normal. In the Teratology study no adverse effects on embryonic or fetal development were evident at maternally toxic dosages up to 20 mg/kg.

Teratology study with the synthetic prostaglandin ONO-802 given intravaginally to rabbits.

ONO-802, a synthetic E1 prostaglandin, was administered intravaginally via pessaries to Dutch belted rabbits at doses of 250, 62.5, and 12.5 micrograms/kg on days 6 through 18 of gestation. Rabbits in a vehicle control group were treated with pessaries that did not contain ONO-802 during the same period. Another group of animals remained untreated throughout gestation. Necropsies were performed on rabbits found dead and on those killed on gestation day 30. Body weight, food and water consumption, and clinical signs were monitored during the experiment. Major organs were weighed when the dams were necropsied on gestation day 30, and litter and fetal data were collected. Abortion and maternal deaths occurred in drug-treated groups. Body weight gains and food and water consumption were adversely affected by treatment particularly at the 250 and 12.5 micrograms/kg dose levels. Wastage (postimplantation loss) was significantly increased among treated groups (all dose levels), while other litter and fetal parameters were unaffected. ONO-802 was not teratogenic at maternal and embryotoxic dose levels.

Two-phase teratology study with the synthetic prostaglandin ONO-802 given intravaginally to rats.

The synthetic prostaglandin ONO-802 was administered intravaginally to Sprague Dawley rats at doses of 1.0, 0.5, and 0.125 mg/kg on days 6 through 15 of gestation. A vehicle control group was treated with pessaries that did not contain the drug while another group remained untreated. Body weight, food, water consumption, and clinical signs were monitored during the experiment. In Phase One, 20 pregnant animals from each group were sacrificed at term, major organs were weighted, and litter and fetal data were collected. In Phase Two ten dams per group were allowed to deliver their litters, and the offspring were evaluated for survival, growth, developmental signs, and physiological function. Selected F1 offspring were retained to assess learning and emotional behavior or reproductive capacity. Administration of either 0.5 or 1.0 mg/kg of ONO-802 resulted in a slight reduction in food consumption and body weight gain. Water consumption was increased both during and after the dosing period for the mid and high dose dams. Significantly increased weights for the heart, lungs, liver, adrenals, and ovaries and decreased weights for the thymus gland were noted at term sacrifice of the 1.0 mg/kg dams, whereas the 0.5 mg/kg group had increased weights of the adrenals and ovaries only. Litter parameters were unaffected by treatment. Weights of the female fetuses of the 1.0 and 0.5 mg/kg groups were significantly reduced when compared to controls. There were no significant drug-related abnormalities among the F1 offspring and no evidence that treatment of the F0 dams affected the development, behavior, or reproductive performance of the F1 offspring. Thus, ONO-802 was not teratogenic when given to rats by the intravaginal route.

Teratogenesis of calcium valproate in rats.

Studies were conducted to determine the teratogenic potential of the calcium salt of valproic acid in rats when given orally at doses of 600, 150, and 50 mg/kg on days 6--15 of gestation. The sodium salt of valproic acid was used as a reference agent at a dose level of 600 mg/kg. The administration of 600 mg/kg/day of either calcium or sodium valproate resulted in transient, severe sedation in the dams. Four dams receiving 600 mg/kg of either salt died during the experiment, with deaths occurring between day 7 and 11 of gestation. Food consumption and body weight gain were significantly reduced during the dosing period with both salts at dose levels of 600 mg/kg. Embryotoxicity at the high doses (600 mg/kg) with either salt was manifested by increases in fetal resorption, reduced body weights, and significantly increased incidence of supernumerary ribs and bifid vertebral centra among the surviving fetuses. A teratogenic effect was evident at 600 mg/kg with either salt of valproic acid. Seven of 16 fetuses from dams given the calcium salt were abnormal. Findings included one with omphalocele and six others with skeletal malformations. Eleven of 24 fetuses from dams given the sodium salt were abnormal: three littermates had bilateral ectrodactyly of the rear feet and malformed vertebral centra and eight others had skeletal malformations. No teratogenic effect was evident among the fetuses from dams given 150 mg/kg calcium salt. Embryotoxicity was demonstrated by a significant increase in the incidence of supernumerary ribs. No adverse effect was observed among the fetuses from dams given 50 mg/kg of the calcium salt.

Synthesis and biological activity of peptide antagonists of luliberin (luteinizing hormone-releasing hormone).

A series of des-His2 octa- and nonapeptide analogues of luliberin (luteinizing hormone-releasing hormone) with modifications in the 1 and 6 positions, and in some instances the 10 position, has been prepared. Some of these analogues are potent inhibitors of luliberin in vitro and in vivo. The use of ultraviolet absorption measurements for evaluating peptides containing tyrosine and tryptophan is described. An efficient synthesis of O-methyl-d-tyrosine is reported.

Competitive progesterone antagonists: receptor binding and biologic activity of testosterone and 19-nortestosterone derivatives.

Testosterone and 19-nortestosterone derivatives were evaluated in a developmental scheme designed to identify competitive progesterone antagonists having abortifacient activity. Compounds that displayed significant binding to the rabbit uterine progesterone receptor were followed in biologic tests for progestational, antiprogestational, and abortifacient activities. Of the seven compounds tested for both progestational and antiprogestational activity, only one, 5 alpha-dihydronorethindrone, behaved exclusively as an antagonist. Five other 19-nortestosterones (19-nortestosterone, 17 beta-hydroxyestra-4, 9(10)-dien-3-one, norethindrone, norethindrone acetate, and R 2323) proved to be mixed agonists/antagonists. 5 alpha-Dihydronorethindrone, norethindrone, and 19-nortestosterone terminated pregnancy during the postnidatory period in rats; in addition, the latter two compounds inhibited progesterone-supported pregnancy in spayed rats and curtailed pregnancy during the postnidatory period in hamsters. These results demonstrate that several 19-nortestosterone derivatives bind to the uterine progesterone receptor and behave either as antagonists or mixed agonists/antagonists.

Developmental studies on an apparent cell-lethal mutant gene-ut-in the Mexican axolotl, Ambystoma mexicanum.

The discovery of a new mutant gene in stocks of the Mexican axolotl derived from breeding stock of the Hubrecht Laboratory, the Netherlands, is described. The gene appears to be a simple recessive and displays complete penetrance. ut/ut larvae develop normally through hatching, but begin to lag in growth and display characteristics defects in gill and limb formation shortly thereafter. The results of parabiosis of normal and mutant embryos, as well as embryological transplants of mutant limb and branchial rudiments, support the conclusion that the gene ut is expressed as an 'autonomous-cell lethal'. Despite gross morphological defects in ut/ut larvae, comparisons between normal and mutant animals of the protein spectra of various tissues and organs revealed no substantial differences. A subtle change in the metabolism of ut/ut larvae apparently, therefore, leads to developmental arrest.

A lethal mutant gene in the Mexican axolotl.

Gene ph was discovered in a wild-type axolotl male received from Mexico City. Larvae homozygous for this gene become recognizable by their lighter color at hatching or shortly after. The development of their forelimbs is retarded, and all limbs are of subnormal length because of the reduction in length of their long bones. Many affected larvae die without feeding, and very few survive beyond their third month. At death, older larvae usually show abnormalities of the renal system, edema, ascites, or adhesions of the viscera. The gene is apparently a simple recessive with full penetrance.

Genetic and experimental studies on three associated mutant genes in the Mexican axolotl: st (for stasis), mi (for microphthalmic) and h (for hand lethal).

Three mutant genes, st, mi, and h, were discovered in an axolotl male received from Mexico City. All three are recessive to their normal alleles, and appear to segregate independently. Larvae homozygous for st (for stasis) suffer blockage of the circulation at hatching or shortly after, and the majority soon die; any surviving live only a few weeks at most. The mi/mi (microphthalmic) can be identified at the feeding stage. None survives more than a few days. The h/h (hand lethals) live until the digits have appeared on the forelimb. They may then be recognized by the thumb-like orientation of digit 1. Transplants from st/st embryos into normal produce normal structures which persist indefinitely. Those from mi/mi and h/h donors do not survive. The structures (forelimb, gills) derived from h/h donors grow for a relatively long time, and their final death and degeneration result in defects leading to death of most of the recipients. Parabiosis is of no benefit to st/st or h/h mutants and leads to the death of the normal twin; mi/mi mutants undergo a gradual absorption by the normal twin.

The effects of luteinizing hormone releasing hormone (LH-RH) in pregnant rats. I. Postnidatory effects.

PIP: The effects of LH-RH on pregnancy in rats were investigated. A single 500 mcg injection of LH-RH on Days 9, 10, or 11 of pregnancy terminated pregnancy, whereas injection on Days 6-8 or 13-16 had little or no effect. The ED 50 on Day 10 for b.i.d. administration was 150 mcg and 550 mcg for a single injection. Administration on Day 9 was followed by a decrease in circulating progesterone levels on Days 10 and 11. The administration of large doses of progesterone reversed the effects of LH-RH administration on Days 7-12. Treatment with estradiol-17beta did not potentiate the effect of progesterone, but appeared to slightly retard fetal resorption when administered alone. The results suggest that the antifertility effect of LH-RH is mediated via functional luteolysis.^ieng

Factors influencing ovulation in the Mexican axolotl as revealed by induced spawnings.

Axolotl females may be induced to spawn by injecting them with small doses of FSH prior to mating. Data on the different categories of spawnings obtained by this procedure have established the following points. (1) Spawnings of injected females completely without fertile eggs are small (173 eggs average). Courtship occurred in some of the matings (spermatophores found) but not in all. Spawnings of injected but uninseminated females re-mated with a second (or third) male, not becoming inseminated, but all having some eggs fertilized by contact with spermatophores (showing courtship) are likewise small (219). It can be concluded that courtship of the injected female, even by two successive males, does not stimulate ovulation of additional eggs. (2) Insemination of injected females induced additional ovulation more or less frequently, depending upon whether the female was inseminated by the first, or a subsequent male with whom she was mated. Insemination by the first male, within 15 hours after the female had been injected with FSH, resulted in stimulation of ovulation in 36 of 54 spawnings (67%). The 36 spawnings averaged 593 eggs per spawning. In contrast, insemination by a second or third male, more than 15 hours after the female was injected with FSH, resulted in stimulation of additional ovulation in only 4 of 21 spawnings (19%). (3) Survival time of spermatozoa in inseminated females has been established. Considerable numbers of spermatozoa capable of fertilizing eggs survive for about one week. A few survive for 11 to 12 days, but none was found after 14 days.

Postnidatory effects of luteinizing hormone releasing hormone (LHRH) in hamsters.

Whereas the administration of LHRH to pregnant hamsters has no effect during the prenidatory period, the hormone is effective in terminating pregnancy when given after implantation (days 6-10). The ED50 for pregnancy termination over this period approximates a dose of 0.35-0.4 mg b.i.d. When given to pregnant females in a second study, the effects of LHRH at this dose were completely reverse by minute doses of progesterone (30 microgram and above). Finally, administration of LHRH at 1.5 mg b.i.d., from days 6-10 was followed by daily sacrifice through day 12; bloods were sampled at autopsy for progesterone evaluation. Autopsies on days 7 and 8 showed few differences between controls and LHRH-treated hamsters, although decreased weights of the uterine/conceptus units signaled the initation of resorption. Significant LHRH-induced decreases in circulating progesterone were seen by day 9. Fetal resorption continued and was essentially complete by day 11, while progesterone levels continued depressed through the end of the study.

PIP: Postnidatory effects of LH-RH were studied in hamsters. The administration of LH-RH to pregnant hamsters had no effect during the prenidatory period, however, the hormone was effective in terminating pregnancy when given after implantation (Days 6-10). The effective-dose-50 for pregnancy termination over this period approximated a dose of .35-.4 mg twice/day. When given to pregnant females in a 2nd study, the effects of LH-RH at this dose were completely reversed by minute doses of progesterone (30 mcg and above). Finally, administration of LH-RH at 1.5 mg twice/day from Days 6 to 10 was followed by daily sacrifice through Day 12. Blood was sampled at autopsy for progesterone evaluation. Autopsies on Days 7 and 8 showed few differences between controls and LH-RH-treated hamsters, although decreased weights of the uterine conceptus units signaled the initiation of resorption. Significant (p .05) LH-RH-induced decreases in circulating progesterone were seen by Day 9. Fetal resorption continued and was essentially complete by Day 11, while progesterone levels continued depressed throughout the study.

Absence of a prenidatory effect of luteinizing hormone releasing hormone (LHRH) in hamsters.

Luteinizing hormone releasing hormone at high doses will terminate gestation in rats during early and midpregancy (ED50 approximately equal to 100 microgram/day) and rabbits during early pregnancy. Early pregnancy in hamsters, in contradistinction, seems refractory to this effect. Administration of LHRH up to massive doses (10 mg/day) over the first 3 or 7 days of pregnancy failed to affect the pregnancies in meaningful fashion. Further, a single injection (100 mg) on day 5 had no effect on pregnancy; this system has been employed for the assay of prostaglandins because hamsters are remarkably sensitive to PG's (PGF2alpha, ED50 approximately equal to 17 microgram, PGE2, ED50 approximately equal to 210 microgram). The absence of response of hamsters to LHRH cannot be interpreted at present.

Luteinizing hormone-releasing hormone versus human chorionic gonadotropin: differential effect on the development of ovulatory refractoriness and antibodies.

Cyclic intramuscular injections of 25 IU of human chorionic gonadotropin (HCG) at 3-week intervals induced ovulatory refractoriness and HCG antibodies after five to eight treatment cycles. Two of fifty rabbits failed to ovulate following two successive injections of luteinizing hormone-releasing hormone (LH-RH); however, no LH-RH antibodies were detected in the sera of these two animals, suggesting that these observations were due to chance alone. Thus, 0.5 mug of LH-RH injected intramuscularly at 3-week intervals did not induce ovulatory refractoriness or antibody formation after as many as 18 successive treatment cycles.