Rat Uterine Weight Research Articles

Mechanism of quercetin on the improvement of ovulation disorder and regulation of ovarian CNP/NPR2 in PCOS model rats

To investigate the effects of quercetin on ovulation disorder and expression of androgen receptor (AR) and C-type natriuretic peptide (CNP) / Natriuretic Peptide Receptor 2 (NPR2) in dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) rat model. DHEA was used to construct the PCOS rat model. After intervention with quercetin, metformin, and AR, the estrous cycle, ovarian and uterine weight of rats were measured. The morphological changes of ovarian and uterine were detected by hematoxylin-eosin staining (HE staining). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by Enzyme linked immunosorbent assay (ELISA). Immunohistochemical detection of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), B-cell lymphoma-2 (BCL-2), BCL2-Associated X (Bax) and AR expression in ovarian. Determination of the expression of CNP and NPR2 mRNA by qRT-PCR. Chromatin immunocoprecipitation (ChIP) was used to detect the ability of AR to bind to CNP or NPR2 promoter. The results showed that quercetin could significantly reduce the expression of Testosterone (T) , Estradiol (E2) , LH, Bax, IL-1β, IL-6 and TNF-α, increase the expression of FSH and Bcl-2, inhibit the expression of AR, regulate the expression of CNP / NPR2 gene and protein by affecting the combination of AR with the specific sequence of CNP and NPR2 gene promoters, restore the maturation of oocyte and ovulation. The results suggest that quercetin can alleviate the hormone, metabolic and ovulatory aberrations caused by PCOS, and provide experimental basis for the clinical application of quercetin in PCOS.

Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test

Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity. Moreover, ERβ knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERβ-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERβ in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken together these data suggest that ERβ may play an important role in modulating mood and the ERβ specific compounds described herein will be useful tools for probing the utility of an ERβ agonist for treating neuroendocrine-related mood disturbance and menopausal symptoms.

Endocrine-Mediated Effects of a Benzophenone Related Chemical, 2,3,4,4'-Tetrahydroxybenzophenone, Based on Uterotrophic Assay, Hershberger Assay, and Subacute Oral Toxicity Study

The purpose of this study was to investigate the endocrine-mediated effects of a benzophenone-relative compound, 2,3,4,4’-tetrahydroxybenzophenone, based on the OECD protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 300 and 1,000 mg/kg doses of the test chemical increased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 60, 200, and 600 mg/kg/day to castrated male SD rats for 10 consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 60, 200, and 600 mg/kg/day for at least 28 days, serum T4 values decreased in male rats in the 600 mg/kg group. This suggests a mechanism of action related to the inhibition of thyroid peroxidase. The uterotrophic assay used in this study showed that the chemical has estrogen-agonist properties, however, estrogenic effects were not observed in a 28-day repeated-dose toxicity study. On the other hand, endocrine-mediated effects such as thyroid hormone dysfunction were detected in growing rats based on the results of the OECD test guideline No. 407.

Effect of the herbicide pendimethalin on rat uterine weight and gene expression and in silico receptor binding analysis

The endocrine disrupting potential of the herbicide pendimethalin was investigated in vivo on the uterotrophic response and on the expression of estrogen-regulated genes examined by quantitative real-time RT PCR. Receptor binding characteristics of pendimethalin were analyzed by an in silico method. Pendimethalin (150, 225, 300 and 600 mg/kg/day) was administered by oral gavage to immature female rats for 3 days, with ethinylestradiol (0.001 mg/kg/day) as positive control. Pendimethalin caused a small but significant increase in absolute uterine weight at and above 300 mg/kg/day and in relative uterine weight at 600 mg/kg/day. Estrogen receptor (ER)-alpha mRNA levels were not affected, whereas ER-beta mRNA was up-regulated at the highest dose. Progesterone receptor mRNA level was not significantly changed, while insulin-like growth factor-I mRNA was reduced, significantly at 225 mg/kg/day to 65% of control. Androgen receptor (AR) mRNA showed a marked down-regulation at doses of 225 mg/kg/day and above. The expression pattern differed from that of ethinylestradiol. In silico analysis revealed potential binding of pendimethalin to ER-beta and AR, but virtually no binding to ER-alpha. These data demonstrate that pendimethalin exhibits estrogenic activity also in vivo. However, its uterotrophic effect, which is an ER-alpha-mediated response, is very small, and it appears that in vivo actions should rather be sought in ER-beta-regulated functions.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4´-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols

We performed an uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 1,000 mg/kg dose of the test chemical plus ethinyl estradiol decreased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 100, 300, and 1,000 mg/kg day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 100, 300, and 1,000 mg/kg day for at least 28 days, a decrease in LH values in rats of both sexes and a decrease in FSH and estradiol values in female rats were detected in the 1,000 mg/kg group, and abnormal estrous cycles, uterine glandular atrophy, persistence of ovarian corpora lutea, vaginal epithelial mucification, and mammary glandular hyperplasia were also observed in one female rat in the 1,000 mg/kg group. Therefore, the uterotrophic assay used in this study showed that the chemical has the estrogen-antagonist properties, and some potentially endocrine-mediated effects were detected in growing rats based on the results of the enhanced OECD test guideline No. 407. However, the changes were observed in rats given a high dose of the chemical, 1,000 mg/kg day.

Estrogen receptor ligands. Part 10: Chromanes: old scaffolds for new SERAMs

The discovery, synthesis, and SAR of chromanes as ERα subtype selective ligands are described. X-ray studies revealed that the origin of the ERα-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx’d adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).

Antiestrogenic effects of motorcycle exhaust particulate in MCF-7 human breast cancer cells and immature female rats.

The emissions from 2- and 4-stroke motorcycles pollute the air of urban areas where motorcycle is a popular means of transportation. This study aimed to determine the endocrine-disrupting activity of motorcycle exhaust particulate (MEP) using MCF-7 human breast cancer cells and immature female Wistar rats treated with organic extracts of MEP. Treatments with 1, 10, and 50 microg/ml MEP extract for 2 and 4 days produced dose-dependent inhibition of thymidine incorporation and cell growth, respectively, in untreated and 1 nM 17beta-estradiol (E2)-treated cells. Treatments of MCF-7 cells with MEP extract replaced [3H]E2 from the estrogen receptor in a time- and concentration-dependent manner. These antiestrogenic and receptor binding properties of MEP extract were blocked by cotreatment of the cells with 2 microM alpha-naphthoflavone, a cytochrome P450 inhibitor and aryl hydrocarbon receptor antagonist. E2 metabolism and HPLC analysis showed that treatment of MCF-7 cells with 50 microg/ml MEP extract for 24 h increased E2 2- and 4-hydroxylation in microsomes. The MEP-mediated increase in E2 2-hydroxylation was inhibited by the addition of 1 microM alpha-naphthoflavone to MCF-7 microsomes. Cotreatment of immature female rats with 10 microg/kg E2 and 10 mg/kg MEP extract intraperitoneally for 3 days decreased the E2-induced uterine weights. MEP extract alone showed no effect on rat uterine weight. The endocrine-disrupting activity of MEP extract was further confirmed in parallel experiments using MCF-7 cells and immature female rats treated with benzo(a)pyrene, an MEP constituent compound. The present findings demonstrate that MEP extract is antiestrogenic in vitro and in vivo and cytochrome P450 induction is an underlying mechanism.

Estrogen receptor ligands. Part 4: The SAR of the syn-dihydrobenzoxathiin SERAMs

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/ anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERα selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.

Estradiol-16alpha-carboxylic acid esters as locally active estrogens.

We attempted to design analogues of estradiol to act as locally active estrogens without significant systemic action. We synthesized a series of 16alpha-carboxylic acid substituted steroids and their esters and tested their action in several assays of estrogenic action, including estrogen receptor (ER) binding, estrogenic potency in Ishikawa cells (human endometrial carcinoma), rat uterine weight (systemic action), and mouse vaginal reductases (local action). All of the estradiol substituted carboxylic acids (formic, acetic and propionic acids) were devoid of estrogenic action. To the contrary, many of the esters had marked estrogenic potency in the receptor and the Ishikawa assays. The esters of the 16alpha-formic acid series had the highest ER affinity with little difference between the straight-chain alcohol esters (from methyl to n-butyl). However, estrogenic action in the Ishikawa assay decreased precipitously with esters longer than the ethyl ester. This decrease correlated well with the increased rate of esterase hydrolysis of longer esters as determined in incubations with rat hepatic microsomes. The most promising candidates, the methyl, ethyl, and fluoroethyl esters of the formate series, were tested for systemic and local action in the in vivo models. All three, especially the fluoroethyl ester, showed divergence between systemic and local estrogenic action. These metabolically labile estrogens will be extremely useful for the therapeutic treatment of the vaginal dyspareunia of menopause in women for whom systemic estrogens are contraindicated.

Modulation by Estrogen-Receptor Directed Drugs of 5-Hydroxytryptamine-2A Receptors in Rat Brain

Hormonal specificity of modulation of brain 5-HT2A receptors was investigated by comparing activity of compounds with varying effects on estrogen response in breast, bone, and uterus. A two-week estradiol treatment stimulated the decreased uterine weight of ovariectomized rats to intact rat values whereas an increase of 29% with tamoxifen and 16% with raloxifene was observed compared to vehicle-treated ovariectomized rats. In 18 assayed brain regions, ovariectomy decreased 5-HT2A receptor binding and mRNA levels in anterior cingulate and frontal cortices, striatum, and nucleus accumbens; estradiol restored this decrease to intact rat values. Dehydroepiandrosterone (DHEA) increased ovariectomized rats 5-HT2A receptor expression only in striatum and cortical amygdala. Tamoxifen increased 5-HT2A receptor density only in striatum. Raloxifene, an uterine estrogen receptor (ER) antagonist, increased, like estradiol, 5-HT2A receptor density and expression in cingulate and frontal cortices, striatum, and nucleus accumbens. Brain regional specificity of estradiol, DHEA, tamoxifen, and raloxifene on 5-HT2A receptors was observed which can be dissociated from peripheral activity.

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3- O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3- O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3- O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.

Screening for antiproliferative actions of mifepristone: Differential endometrial responses of primates versus rats

This laboratory has previously shown the capability of the antiprogestin, mifepristone, to noncompetitively inhibit estrogen-induced endometrial proliferation in nonhuman primates. In the following study, use of the rat uterine weight bioassay was compared against a primate ( Macaca fascicularis) uterine bioassay to identify the noncompetitive/antiproliferative effects of mifepristone. These uterine bioassays were contrasted for reasons of identifying a comparative laboratory rodent model that could substitute for the need to use primate models in the screening of potential antiprogestins, thereby saving time, cost, and primate resources. Results of the primate experiment showed that mifepristone decreased endometrial proliferation in a dose-dependent manner; importantly, this decrease occurred in the presence of sustained physiologic serum 17β-estradiol (E 2) levels. However, in the rat model, results showed that mifepristone altered uterine wet weight and blotted weight values only in those animals receiving pharmacological doses of E 2 (p <0.05). Based on the results summarized herein, use of this rat uterine weight bioassay as a substitute for primate models is not recommended for screening and identification of “interesting” antiprogestins. Apparently, the endometrial noncompetitive antiestrogenic/antiproliferative effects of mifepristone, observed repeatedly in these laboratory primates, do not operate in the rat uterine tissue.

The potent and selective inhibition of estrogen production by non-steroidal aromatase inhibitor, YM511.

YM511 inhibited aromatase activities in microsomes from rat ovary and human placenta competitively (IC50s: 0.4 and 0.12 nM, respectively). YM511 was about 3 times more potent than other aromatase inhibitors, such as CGS 16949A, CGS 20267 and R 76713. YM511 decreased the contents of estradiol stimulated by pregnant mare's serum gonadotropin in rat ovary with an ED50 of 0.002 mg/kg, indicating that YM511 was equipotent to CGS 20267 and 3 times more potent than the other two inhibitors. Serum estradiol levels in female rats were reduced by YM511 at 0.01 mg/kg into the ovariectomized range. YM511 at 1 mg/kg for 2 weeks decreased rat uterine weight to levels comparable to ovariectomy, showing it was 10 times more potent than other inhibitors. But the maximal inhibitory effect of tamoxifen failed to reach ovariectomized level. YM511 slightly inhibited production of other steroid hormones in vitro and in vivo. The IC50s of YM511 for aldosterone and cortisol production from adrenal cells were from 5500 to 9800 times higher than that for rat ovarian aromatase and 130,000 times higher for testosterone production, indicating that YM511 is a highly specific aromatase inhibitor. The data suggest that YM511 may be a potent and selective agent for suppressing estrogen-dependent action without affecting serum levels of other steroid hormones.

Estrogenicity, antiestrogenicity and estrone sulfatase inhibition of estrone-3-amine and estrone-3-thiol

Estrogen levels in breast tumors of post-menopausal women are at least 10 times higher than estrogen levels in plasma. The high level of estrogen in these tumors is postulated to be due to in situ formation of estrogen, possibly through conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for the treatment of hormone-dependent breast cancers. We designed and synthesized a series of estra-1,3,5(10)triene-17-one, 3-amino and estra-1,3,5(10)triene-17-one, 3-thio derivatives. We have shown previously that several of these compounds substantially inhibit estrone sulfatase, exceeding Danazol in their inhibitory activity. However, little is known about the metabolism of these compounds and the possible effects of their metabolites in vivo. Two probable metabolites of the synthetic estrone analogs are estra-1,3,5(10)triene-17-one, 3-amine (E1-NH2), and estra-1,3,5(10)triene-17-one, 3-thiol (E1-SH). We tested these two compounds for estrogenicity, antiestrogenicity and inhibition of estrone sulfatase activity using a combination of in vivo and in vitro assays. The ovariectomized rat uterine weight gain assay was used to test for estrogenicity. Neither E1-NH2 nor E1-SH were estrogenic, as indicated by a lack of uterine weight gain when given at 25 micrograms/day for 7 days. The test compounds also were not antiestrogenic, in that they did not block estrone-induced uterine weight gain when given (100 micrograms/day) simultaneously with estrone (2 micrograms/day). Both compounds showed low affinity for the estrogen receptor. Using rat uterine cytosol as a source of estrogen receptor, the compounds displaced only a small percentage of [3H]estradiol binding, even when present at 1000-fold excess. Inhibition of estrone sulfatase activity was tested using human placental microsomes as a source of estrone sulfatase. E1-NH2 and E1SH showed very low levels of estrone sulfatase inhibition (15.1 and 9.8%, respectively) under conditions where Danazol showed more than 60% inhibition. Our results indicate that neither of these two compounds would present significant problems if they were the primary metabolite in a treatment involving estrone sulfatase inhibition of estrogen-dependent breast cancer.

Progesterone and Estradiol Interaction in the Regulation of Rat Uterine Weight and Estrogen Receptor Concentration

Autologous down-regulation of hormone receptors has been shown for several steroid hormones. We have previously shown estradiol (E2) regulates estrogen receptor (ER) in ovariectomized (OVX) rats. These studies have been extended to investigate the interaction between progesterone (P) and E2 in the regulation of ER and uterine weight. We implanted Silastic capsules containing varying concentrations of E2 (0.0005 mg E2/ml to 5.0 mg E2/ml of sesame oil) into adult female Sprague-Dawley rats one week after OVX. Simultaneously with implantation, P injections were started (sc in sesame oil) at doses of 1-40 mg/day for three days. In the absence of P, the 0.05 mg E2/ml implant significantly increased total ER levels (measured by cytosol and nuclear exchange assays) by 25%, while the two highest concentrations of E2 (0.5 and 5.0 mg E2/ml) significantly decreased cytosol and total ER levels by at least 40%. No P dose altered ER levels in OVX rats or in rats given E2 implants of 0.01 mg E2/ml or lower. At E2 implant concentrations of 0.05 mg E2/ml and higher, P decreased total ER levels 30%-50% compared to the appropriate E2-only controls. P increased uterine weight by 25% in OVX controls and in rats treated with E2 implants of 0.01 mg E2/ml and below. In contrast, P inhibited uterine weight gain induced by 0.05-5.0 mg/E2 ml implants by 20%-30%; maximal inhibition occurred at 5 mg/day of P and above. These data demonstrate that P increases uterine weight but does not alter ER concentration in rats with low E2 levels (OVX or low E2 concentration implants) but decreases uterine weight and down-regulates ER at higher E2 levels, regardless of whether ER is up-regulated or down-regulated by E2.

Synthesis and biological activity of 17β -substituted estradiol

The dialkylaminoethoxy side chain in triphenylethylene antiestrogens is required for their antiestrogenic activity. Without this side chain the compounds lose their antiestrogenic activity and become essentially estrogenic. Estradiol substituted at the 17β -position with dialkylaminoethoxy, dialkylaminoethylamino, or dialkylaminoethylthiol were synthesized and tested for their ability to displace estradiol for its receptor. All of the derivatives tested exhibited low binding affinities to the estrogen receptor, with RBA values ranging between 0 to 1.2 (estradiol = 100). The mouse and rat uterine weight test revealed only low estrogenic activity for this class of compounds. None of the estradiol derivatives synthesized showed antiestrogenic activity.

Sebum-suppressing activity of the nonpolar arotinoid Ro 15-0778 in rodents.

Retinoids are known to modulate sebaceous gland activity in humans and animals. The nonpolar arotinoid Ro 15-0778 [(E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2-phenylethen yl) naphthalene] does not contain a polar end group and is devoid of the classical retinoid side effects of hypervitaminosis A. The favorable toxicological profile stimulated the evaluation of this arotinoid in animal models of sebum production. In castrated, testosterone-stimulated male rats, Ro 15-0778 is 50 times more potent than 13-cis-retinoic acid in inhibiting the production and subsequent secretion of sebum. The oral ED50 value of Ro 15-0778 is 30 micrograms/kg, while an oral dose of 0.5 mg/kg inhibited sebum secretion nearly 100%. In testosterone-stimulated female rats, Ro 15-0778 inhibits sebum secretion significantly with an oral ED50 of 140 micrograms/kg and an s.c. ED50 of 75 micrograms/kg. Ro 15-0778 was also evaluated for its ability to prevent testosterone induction of the immature hamster flank organ. The topical ED50 is 0.53 mg/kg and the oral ED50 is 38 mg/kg. This arotinoid is similarly active in mature male hamsters without testosterone treatment. In addition, the retinoid is active topically and orally in reducing the size of the gerbil abdominal sebaceous gland. The compound exhibits no antiandrogenic activity when tested in ventral prostrate and seminal vesicle assays in rats. Additionally, the compound does not have estrogenic activity when tested in the rat uterine weight assay. High doses of Ro 15-0778 in humans did not demonstrate significant sebum-suppressing activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Formulation of a potential antipregnancy vaccine based on the β-subunit of human chorionic gonadotropin (β-hCG) II. Use of compounds of the muramyl dipeptide (MDP) family as adjuvants

Earlier tests of an antipregnancy vaccine consisting of the β-subunit of human chorionic gonadotropin (β-hCG) linked by reaction with a carbodiimide reagent to tetanus toxoid (TT) and adsorbed on Al(OH) 3 resulted in antibody responses that were judged inadequate in some women. Experiments were therefore conducted to evaluate the effectiveness of additional adjuvants in increasing the antibody response. Muramyl dipeptide (MDP) and several of its analogs were formulated with the vaccine and tested in rabbits. Some of the analogs, and notably N- acetyl-normuramyl- L- alanyl- D- isoglutamine , elicited substantial increments in the ability of the antisera to bind [ 125I]hCG and in its ability to neutralize hCG in the rat uterine weight assay. The effectiveness of these peptides was greatest when formulated in a water-in-oil emulsion. Increments of 10 fold were attained using a vegetable oil as the oil component. The MDP analogs were much less effective as adjuvants when formulated in oil-in-water emulsions or in aqueous suspensions of the antigen. It is concluded that selected MDP analogs incorporated in a water-in-vegatable oil emulsion can markedly increase the circulating antibody response to the β-hCG-TT vaccine.

Comparison of Corynebacterium Parvum With Freund's Complete Adjuvant as a Potentiator of the Immune Response To β‐Human Chorionic Gonadotropin Linked to Tetanus Toxoid

Corynebacterium parvum was compared with Freund's complete adjuvant (FCA) for potentiation of the rabbit immune response to beta-human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT). With each adjuvant, antibodies to hCG were detected using passive hemagglutination. Higher antibody titers were produced by FCA-treated animals. The ability of antisera to beta-hCG-TT to neutralize the biological action of native hCG was determined by the rat uterine weight assay. Anti-beta-hCG-TT sera from C. parvum-treated rabbits were not significantly different (p greater than 0.10) from FCA potentiated anti-beta-hCG-TT sera in neutralizing hCG-induced uterine weight gain. C. parvum has potential for future application in active immunization studies of fertility regulation.

Differential antiestrogen action in the immature rat uterus: A comparison of hydroxylated antiestrogens with high affinity for the estrogen receptor

-The non-steroidal antiestrogens LY117018 and monohydroxytamoxifen are partial agonists in the 3-day immature rat uterine weight test. Both compounds stimulated increases in uterine progesterone receptor concentration (as determined by DCC assay and SDG analysis) and luminal epithelial cell height. However, LY117018 was much less estrogenic than monohydroxytamoxifen. The study also showed that the antiestrogenic effects of LY117018 and monohydroxytamoxifen could be reversed in vivo by increasing doses of estradiol. The partial uterotrophic effect of monohydroxytamoxifen and full uterotrophic effects of estradiol were both inhibited by high doses of LY117018 at an approximate dosage ratio of 1:24, w/w. This result suggests a common mechanism for the action of estradiol and the hydroxylated antiestrogens. Since LY117018 reversed the binding of [ 3H]estradiol and estrogencompetable [ 3H]monohydroxytamoxifen binding in the rat uterus in vivo, the effects of nonsteroidal antiestrogens on gross uterine wet weight can be explained by competitive interaction with estradiol via the estrogen receptor mechanism. However, the weakly estrogenic antiestrogen LY117018 was unable to inhibit estrogen-stimulated rises in luminal epithelium cell height and progesterone receptor levels. These data suggest a differential interaction by LY 117018 in the rat uterus so that only selected estrogen stimulated effects i.e., uterine wet weight, are antagonized by this particular “antiestrogenic” dose of the drug.