P.K. Mehrotra

Expression of αVβ3 integrin in rat endometrial epithelial cells and its functional role during implantation

The alpha(V)beta(3) integrin as a marker of endometrial receptivity has been well established in human and other mammalian species; however, its expression is still not known in rats. Our objective was to establish the expression of alpha(V)beta(3) integrin as a marker of endometrial receptivity in rat and to further prove its role in implantation by function-blocking studies in this species. Immunocytochemical, immunohistochemical and flow-cytometric studies were performed in rat endometrial epithelial cells (EEC) to demonstrate the expression of alpha(V)beta(3) integrin during non-receptive, pre-receptive and receptive phases of the uterus. Results revealed positive immunocytochemical staining for alpha(v) and beta(3) subunits on the surface of EEC of days 4 and 5p.c. (post-coitum), but the intensity was higher in cells of day 5p.c. Flow-cytometric study revealed higher level of alpha(V)beta(3) on day 5p.c. as compared to day 4p.c. and non-pregnant animals. Immunohistochemical analysis of uterine tissue also revealed that the alpha(V)beta(3) expression in LE was higher on day 5p.c. morning as compared to that observed on day 4p.c. In addition, the expression of beta(3) subunit was not evident in rats receiving ormeloxifene, an agent known to inhibit the uterine receptivity. Immunoblotting experiments also revealed higher expression of uterine beta(3) on day 5p.c. On day 6, expression of beta(3) was high in implantation sites than on inter-implantation sites. In immature ovariectomized rats, alpha(V)beta(3) was up-regulated by progesterone and by a combination of estrogen and progesterone. The expression of alpha(V)beta(3) was also up-regulated in EEC co-cultured with blastocysts. All the agents used for function-blocking studies showed significant reduction in the number of implantation sites in treated horn as compared to sham control horn. The present study has successfully demonstrated the expression of alpha(V)beta(3) in rat EEC as a marker of endometrial receptivity and showed that this molecule is indispensable for the process of implantation in this species.

Search for new chemical entities as menses inducing agents

In continuation of an ongoing program on developing nonsteroidal pregnancy interceptives to be used as a menses regulating agent, a new class of compounds belonging to 3-substituted amino-1-aryl-6-hydroxy-hex-2-ene-1-ones series has been investigated for pregnancy interceptive activity in the hamster and rat. The compounds were administered (subcutaneous) on days 4-8 (hamster) and 5-9 (rat) post coitum (PC). The animals were laparotomized on days 12 (hamster) and 16 (rat) PC. To derive percent efficacy, the total number of implantation was divided by the number of normal and resorbed implantations. Among the 14 compounds evaluated, three were found to intercept pregnancy by 100%. Another compound was active by 75%, whereas the rest were inactive. None of the active compounds were, however, active in rat with this schedule. Results indicate that the observed species- and schedule-specific activity owes its origins to differences in the implantation physiology and early post-implantation development between the two species. The study, nevertheless, offers an insight to the new class of compounds for this activity.

Pregnancy interceptive efficacy and biological profile of 3-amino-6,7-dimethoxy-1H-pyrazolo [3,4-b] quinoline (compound 85/83) in rodents

Administration of compound 85/83 during the peri- and post-implantation period intercepted pregnancy in hamster and guinea pig by parenteral route and in hamster by oral route also. The m.e.d. for hamster and guinea pig was 10 and 20 mg/kg, respectively; lower doses were less effective. Restricting the administration to early post-implantation schedule interrupted pregnancy partially in both species. The compound was, however, ineffective in rat and in the pre-implantation schedule (days 1-4 post-coitum) in hamster. When tested in vitro on growing trophoblasts at 13.8 x 10(-5) M concentration, it prevented growth and caused degeneration of the cells within 24 h; lower concentration (9.2 x 10(-5) M) was less effective. The compound was found to be devoid of estrogenic, antiestrogenic, progestational and antiprogestational properties in conventional bioassays. In hormone competition assays, its relative binding affinity (RBA) to estrogen receptor was negligible (0.002% of estradiol-17 beta), while for uterine cytosol progesterone receptors in rabbit and hamster was 0.06 and 0.08% of progesterone, respectively. The compound 85/83 appears to intercept pregnancy by interfering with development of trophoblast cells.

Effect of inhibitors of enzymes involved in polyamine biosynthesis pathway on pregnancy in mouse and hamster

The syntheses of polyamines, which play a definitive role in cell proliferation and tissue growth during early embryogenesis, are believed to be principally regulated by two enzymes: ornithine decarboxylase (ODC) and polyamine oxidase (PAO). However, when the level of enzyme ODC goes down, another enzyme known as S-adenosyl methyl decarboxylase (SAMDC) is believed to up-regulate the biosynthetic pathway. In the present study, the effects of inhibitors of enzymes SAMDC and PAO were determined in mated hamster and mouse in peri-and immediate postimplantation stages of pregnancy. It was noticed that, whereas the SAMDC inhibitor (MDL-73815) failed to arrest the pregnancy in the hamster in spite of raising the dose to 100 mg/kg, the PAO inhibitor (MDL-72527DA) was found to be 100% effective at 75 mg/kg in both species. The results confirmed the role of enzyme PAO in promoting early development of the embryo.

Contraceptive and hormonal properties of a new 1,4-dihydro-2-oxoquinoline derivative (compound 84–182) in rodents and rhesus monkeys

Compound 84-182 prevented pregnancy when administered subcutaneously at 10 mg/kg dose on days 3-8 post-coitum in hamsters and on days 6-10 post-coitum in guinea pigs. At lower doses, while in hamsters there was a marked reduction in implantation number, majority of implantations in guinea pigs showed signs of resorption. The compound was ineffective when administered at 10 mg/kg dose on days 1-3 or 6-7 post-coitum in hamsters and on days 1-5 or 4-8 post-coitum in rats. In rhesus monkeys, treatment with the compound at 5 and 10 mg/kg doses on days 16-21 of the menstrual cycle induced frank vaginal bleeding between days 21 and 24. Treatment on days 21-30 or after confirmation of pregnancy on days 32-36 was ineffective. In conventional bioassays, the compound was devoid of any estrogenic, antiestrogenic, progestational, antiprogestational, androgenic or antiandrogenic properties at the contraceptive dose. In competitive protein binding assay, the compound showed relative binding affinity (RBA) of less than 0.1% and 0.28% of progesterone, respectively, for rabbit and hamster uterine cytosol progesterone receptors. Its RBA for rat uterine cytosol estrogen receptors was less than 0.1% of estradiol-17 beta.

Estrogenicity of some nonsteroidal compounds

Abstract The estrogenicity of four nonsteroidal compounds found active as post-coital contraceptives in laboratory animals has been assessed on the basis of ponderal, histologic and biochemical changes in the uterus, cervix and the vagina of ovariectomized rats. The compounds are a benzofuran (2-phenyl-3-p-(β-pyrrolidinoethoxy) phenyl-6-methoxy benzofuran hydrochloride), a naphthofuran (2-phenyl-3-p-(β-pyrrolidinoethoxy) phenyl-naphtho (2,1-b) furan), a chroman (3,4-trans-2,2-dimethyl-3-phenyl-4-(p-(β-pyrrolidinoethoxy)-phenyl) 7-methoxy chroman) and a propiophenone (3-chloro-2, 3-diphenyl-propiophenone) derivative. It was observed that the estrogenicity of a compound varies depending upon the target organ and the type of parameter employed for assessment. It is pointed out that such difficulty might also arise as a result of different thresholds of the compound required for eliciting an estrogenic response, and also of its dissimilar absorption and retention by a particular target organ. Further, a compound might provoke an “estrogen-like” reaction which cannot be explained solely on the basis of its imputed estrogenicity.

Modified iridoid glycosides as anti-implantation agents ☆: Inhibition of cell adhesion as an approach for developing pregnancy interceptive agents

Structural modifications in iridoid glycosides and evaluation of their efficacy on adhering capability (in vitro) of immature hamster uterine epithelial cells to the substratum have been studied. Out of 31, eight compounds in vitro, five compounds in utero and two in vivo showed adhesion/implantation preventing activity, respectively. The results provide an indication for further exploration in the line of development of anti-adhesive agents.

Pregnane derivatives as pregnancy interceptive agents: Efficacy determination on growing trophoblasts (in vitro) and in pregnant hamsters (in vivo)

An in vitro test system was standardized to study potentiality of five hormonally inert pregnane derivatives on growing trophoblasts isolated from ectoplacental cone (EPC) of day 8 hamster embryo. Cells were incubated with different concentrations of respective compounds in surface droplets. The response was determined by analyzing the sequence of changes in cell morphology like attachment, growth, proliferation, differentiation and/or degeneration within 24 or 48 h following seeding. The in vivo efficacy of these compounds was determined in hamster during peri- and immediate post-implantation periods (days 3-8 post coitum). Two compounds 88/583 and 88/585 were found to inhibit not only growth and proliferation of the cells but caused total degeneration within 24 h. The same compounds induced partial to complete resorption of the foetuses in treated animals. Whereas, the other three compounds 88/506, 88/594 and 89/43 that showed lack of comparable potentiality in vitro were found to be equally ineffective in vivo. The results indicate a positive correlationship between in vitro and in vivo activity.

Phenotypical response in different cell types of rat uterus to centchroman: A qualitative analysis

To assess the alleged estrogenic and antiestrogenic profile of centchroman, a phenotypical response was studied in native uterine cells or migrant blood cells in adult ovariectomized rats under the influence of estradiol-dipropionate (EDP) and centchroman (CC) given per se and together. Histologic observations were divided arbitrarily into the following regions: luminal epithelium, superficial and deep stroma, and the two layers of muscularis. Glandular cell proliferation, mitoses, and vascularity were examined additionally. It was found that CC per se stimulated luminal epithelial cell height and glandular cell proliferation better than EDP. Leukocytic infiltration and vascularity were also more pronounced in the uteri of CC-treated rats. Furthermore, except for a slight reduction in glandular cell size and edematous condition of circular muscle layer, the changes remained unaffected after conjoint treatment of CC and EDP. The observations revealed that some of the nongenomic responses, elicited by the rat uterus after CC treatment, seem to be unrelated to the hormonal attributes of the molecule.

Isolation and culture of hamster ectoplacental cone trophoblasts: an in vitro study on the cell types and their growth pattern.

The method of isolation of trophoblast cell types from ectoplacental cone of hamster embryo of day 8 post coitum (plug day as day 1) and examination of their growth pattern in vitro is presented in this study. Based on their size, three types of trophoblast cells were identified: (1) the smaller cells having clear cytoplasm formed the major portion (70% to 80%) of the total cell population (2) moderately bigger cells having mono or binucleated appearance and containing minute granules in the nuclear region formed 5% to 10% and (3) extra large and multinucleated cells shared 10% to 20% of the total cell number. While the smaller and slightly bigger cells showed moderate growth the larger ones had extensive growth and were seen to acquire different shapes on extending the duration of culture, such as fusiform, dumb‐bell, polygonal, rectangular or flowery. The extremities of the cytoplasmic growth of these cells were seen to be thickened at one end giving the impression of a pad‐like structure. The significance of these adaptations are not known at present.