Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus.

It is well established that estrogen and progesterone are critical endogenous hormones that are essential for implantation and pregnancy in females. However, the distribution of estrogen receptor α (ERα) and progesterone receptor (PR) in female reproductive tracts is elusive. Herein, we report that after serial treatments with pregnant mare's serum gonadotrophin (PMSG) with or without anti-PMSG (AP), mice could regulate the distribution of ERα and PR in the murine ovary, oviduct and uterus and the level of estradiol in serum. ERα and PR regulation by PMSG and anti-PMSG was estrous cycle-dependent and critical for promoting the embryo-implantation period. Furthermore, our results suggested that AP-42 h treatment is more effective than the other treatments. In contrast, other treatment groups also affected the distribution of ERα and PR in mouse reproductive tracts. Thus, we found that anti-PMSG has the potential to restore the distribution of ERα and PR, which could effectively reduce the negative impact of residual estrogen caused by the normal superovulation effect of PMSG in mice.

Inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulatory effect of human chorionic gonadotropin (HCG) and equine chorionic gonadotropin (ECG).

We examined the inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulating activity of human chorionic gonadotropin (HCG) and equine CG (ECG). Five TBAb positive sera obtained from patients who had been hypothyroid but were currently on T4 treatment. The TSH binding inhibitory immunoglobulin (TBII) activities of the sera were 60-160 IU/L. Inhibition of TSH binding to the TSH receptor (TSHR) [TSH binding inhibition (TBI) activity] of HCG or ECG, and inhibition of TBAb on HCG or ECG-stimulated cAMP production were examined. Both HCG and ECG preparations showed weak TBI activity in the presence of small amounts of protein [bovine serum albumin (BSA)] but were negative in the presence of large amounts of protein [normal human serum (NHS) or BSA]. Four thousand IU/mL of HCG and ECG preparation caused cAMP production similar to 100 microU/mL of bovine (b) TSH. The inhibitory effect of TBAb on cAMP production by this amount of HCG or ECG was then examined. The inhibitory effect of TBAb on cAMP production by HCG and ECG was similar to bTSH, and TBAb positive sera with more than 40 IU/L TBII activity completely blocked cAMP production by HCG, ECG and bTSH. This suggests that common alpha -subunit of both HCG and TSH are involved in the inhibitory effect of TBAb. Previous reports demonstrated that the thyroid stimulating activity of thyroid stimulating antibody (TSAb) was blocked by deglycosylated HCG (competitive antagonist of TSH binding to TSHR). The fact and our present study suggest that TSH, HCG ECG, TSAb and TBAb have a similar binding site (alpha-subunit-mimicking binding site) on the TSH receptor.

Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption.

Polychlorinated dibenzo-p-dioxins (PCDDs) are structural analogues, which produce a similar spectrum of biological and toxicological responses in animals, albeit with differential potencies. Very consistent structure-activity relationships have been found for acute toxicity and some biochemical effects among these compounds. For the current experiments, the gonadotropin-primed immature female rat model was used to study the effect of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7, 8-pentachlorodibenzo-p-dioxin (PeCDD), and 1,2,3,4,7, 8-hexachlorodibenzo-p-dioxin (HxCDD) on ovulation. Single doses of different PCDDs and their mixture were given orally to 23-day-old rats. Gonadotropin from pregnant mare's serum (PMSG) was injected (5 IU) 24 h later to induce follicular maturation. Rats were decapitated at various times after PMSG, blood was collected, and ovarian weight was measured. Serum concentrations of 17beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PrL) were determined by radioimmunoassay. Ovulation was measured at 72 h after injection of PMSG by counting ova flushed from oviducts. PCDDs dose dependently decreased the number of ova per ovary and reduced ovarian weight gain induced by PMSG. The slopes of the dose-response curves generated by individual PCDDs and/or their mixture were similar. PMSG-induced increase in serum E2 was enhanced on the day of expected ovulation by PCDDs; in contrast, serum P4 and FSH were decreased at that same time point. PCDDs also altered the temporal pattern of serum E2, FSH, and LH but not that of PrL. Histologically the effect of all three PCDDs consisted of ova trapped in preovulatory follicles and a lack of or reduced number of corpora lutea. The results indicate that the PCDDs, tested in the present model, have the same mode of action on ovulation and the reproductive hormones, e.g., LH, FSH, P4 and E2. Furthermore, the dose responses of the individual congeners are parallel to each other and also to that of their equipotent mixture, which represent a validation of the TEQ concept for one aspect of endocrine disruption, that is for inhibition of ovulation.

Rat ovarian insulin-like growth factor binding protein-4: a hormone-dependent granulosa cell-derived antigonadotropin.

OBJECTIVE: To assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (IGFBP-4) mRNA expression by gonadotropins and estrogen. METHODS: Whole ovarian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant more serum gonadotropin (PMSG) followed 48 hours later with hCG, or alternatively were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Localization of IGFBP-4 expression was assessed in the former study by in situ hybridization. Finally, the ability of human IGFBP-4 to antagonize FSH-stimulated progesterone accumulation was assessed in vitro. RESULTS: The ovarian content of IGFBP-4 transcripts increased threefold (P < .05) at 12 hours after PMSG but was near baseline at 24 and 48 hours. The abundance of IGFBP-4 mRNA increased (P < .05) again at 6 and 24 hours after hCG. The expression of IGFBP-4 was localized to granulosa cells of preantral (untreated) and small antral (12 hours after PMSG) follicles. No IGFBP-4 expression was noted in large (gonadotropin-primed) antral follicles. Hypophysectomy increased (P < .05) the ovarian content of IGFBP-4 mRNA by 1.5-fold, an effect further enhanced (1.8-fold; P < .05) by the provision of FSH and DES. In vitro studies revealed the ability of increasing concentrations (0.01-1 microgram/mL) of recombinant human IGFBP-4 to inhibit the FSH-supported accumulation of progesterone. CONCLUSION: Increased expression after administration of PMSG, hCG, and FSH/DES suggests that IGFBP-4 is a dynamic and hormonally responsive component of the ovarian cycle. The lack of expression in preovulatory follicles and its antigonadotropic actions in vitro imply that the attenuated expression of IGFBP-4 may constitute a requirement for successful follicular maturation.

Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin.

Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2-3 x 10(5) viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/micrograms DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations.

The oxidation of caffeic acid derivatives as model reaction for the formation of potent gonadotropin inhibitors in plant extracts.

Synthetic caffeic acid derivatives, substoichiometrically oxidized with KMnO4, exhibit antigonadotropic activity against pregnant mare serum gonadotropin (PMSG) to a greater degree than caffeic acid itself. Inhibitory compounds, formed after an oxidation of caffeic acid and its derivatives are bound to PMSG dependent on their concentration to result in hormone-inhibitor complexes. These PMSG-inhibitor complexes exhibited little or no biological activity, depending on the structure of the inhibitor. The substoichiometric oxidation with KMnO4 led to the corresponding unstable o-quinones as first products. The complete oxidation reaction could be divided into an initial KMnO4-dependent step followed by a manganese-catalyzed autoxidation, which was accompanied by a pronounced oxygen uptake from the solution. The HPLC analysis after an oxidation of caffeic acid derivatives led to product patterns with strong similarities to those of caffeic acid in the respective product UV spectra, suggesting the formation of compounds with similar structures.

Inhibition by indomethacin of the already accelerated secretion of inhibin and estradiol in equine chorionic gonadotropin-primed immature female rats.

In immature female rats, the secretion of ovarian inhibin and estradiol is greatly accelerated by equine chorionic gonadotropin (eCG) treatment. The present study has been carried out to determine whether or not the levels of the two hormones are inhibited by a single s.c.-injection of indomethacin (INDO) 24 h after eCG administration. The levels of ovarian hormones and gonadotropins were measured by double-antibody radioimmunoassay using 125I-labeled radioligands. The serum levels of inhibin and estradiol were considerably inhibited within 24 and 12 h, respectively, after INDO injection. In addition, the serum levels of follicle-stimulating hormone (FSH) after INDO injection remained lower than the basal levels before eCG treatment. The luteinizing hormone (LH) levels were significantly reduced within 12 h after INDO treatment. The results demonstrate that the levels of inhibin and estradiol, even in the situation where the production of both hormones is already accelerated by eCG pretreatment, are suppressed by an inhibitor of prostaglandin (PG) synthesis, suggesting that locally produced PGs may play a role in the regulation of the production of both hormones in the ovary.

[Effect of gonadotropin releasing hormone agonist (Gn-RHa) on steroidogenesis in human and rat ovaries].

The effects of Gn-RHa on rat granulosa cells and on human granulosa and luteal cells were examined. (I) In rat granulosa cells: 1) Gn-RHa inhibited the stimulating effect of PMSG on progesterone and estradiol production. 2) In a tracer experiment, Gn-RHa inhibited PMSG-stimulated progesterone production by stimulating 20 alpha-hydroxyprogesterone production. On the other hand, Gn-RHa inhibited estradiol production by inhibiting PMSG-stimulated aromatization. 3) On 20 alpha-hydroxysteroid dehydrogenase activity, it appears that Gn-RHa accelerates the maximum velocity and decreases the affinity of the enzyme to 20 alpha-hydroxyprogesterone as the substrate. (II) In man: 1) In granulosa cells during the ovulatory phase, Gn-RHa inhibited PMSG-stimulated progesterone and estradiol production. 2) In luteal cells, Gn-RHa inhibited hCG-stimulated progesterone production. 3) The affinity of Gn-RH receptor in the corpus luteum was lower than that in rat ovaries. These results suggest that in the human ovary as well as in the rat ovary, Gn-RHa might have a direct effect on the development of the follicle.

On the influence of the sugar moiety on the antigonadotropic activity of luteoline glycosides.

Luteolin-7-glucoside and luteolin-7-glucuronide were isolated from Lycopus europaeus L. (Lamiaceae) and identified by 1H- and 13C-NMR spectroscopy. Luteolin-7-glucuronide proved to be active against PMSG in vitro as well as in vivo, whereas the other flavone glycosides occurring in the plant were completely inactive.

Immunohistochemical localization and physiological regulation of carbonyl reductase in immature rat ovary.

The present study was performed to clarify the role of the ovarian carbonyl reductase (OCR) in ovarian function in immature rats. The OCR activities towards three specific substrates, 13,14-dihydro-PGF2 alpha, 4-benzoylpyridine and menadione, were photometrically and radiochemically determined in the 9,000 x g supernatants of ovaries, and OCR content was measured by Western-blot-peroxidase anti-peroxidase (PAP) analysis. Immunohistochemical localization of the enzyme in the ovary was performed by the avidin-biotin-complex (ABC) method for paraffin sections. Positive immunoreactivity with OCR antibody was observed for the theca cells and interstitial gland cells at 72 hr after pregnant mare serum gonadotropin (PMSG) treatment when ovulation was confirmed, and the granulosa cells were consistently negatively stained. The OCR activity was significantly increased by PMSG, human chorionic gonadotropin (hCG) and PMSG-hCG treatments, but estradiol and tamoxifen overcame the effect of PMSG on the enzyme activity. Moreover, estradiol enhanced the effect of hCG, but tamoxifen did not. Changes in the OCR activity well-correlated with those in the OCR content. These findings indicate that the OCR is regulated by gonadotropin and estrogen and that metabolites formed by the enzyme could be closely involved in ovarian cell function.

The mode of action of indomethacin, aspirin and melatonin on the blockage of the first ovulation in immature rat pretreated with PMSG.

The purpose of this paper was to evaluate the anti-ovulatory effects of indomethacin, aspirin and melatonin by examining the LH sensitive 13,14-dihydroprostaglandin F2 alpha forming capacity in rat ovary. When ovulation was blocked by aspirin or melatonin, the forming capacity was strongly suppressed, and these effects were reversed by hCG injection. However, the ovulation blockage by indomethacin did not accompany the inhibition of the forming capacity. These results show that aspirin and melatonin block the ovulation via the hypothalamus-pituitary level, and indomethacin acts directly on the ovary.

Inhibition of ovulation in PMSG/hCG-treated immature rats by rotenone, a specific inhibitor of mitochondrial oxidation.

Immature Wistar rats were induced to ovulate by treatment with PMSG and hCG. Control animals ovulated 43.5 +/- 0.36 ova/rat. Intraperitoneal injection of rotenone doses of 0.125, 0.25 and 0.50 mg/kg reduced the ovulation rate to 24.0 +/- 3.08, 8.0 +/- 0.88 and 1.5 +/- 0.44 ova/rat, respectively. The rotenone significantly reduced ovarian cytochrome oxidase activity and progesterone production, but not production of oestradiol or testosterone. Thyroxine treatment at a dose of 5 mg/kg s.c. reversed the rotenone inhibition of ovulation. The results suggest that an increase in mitochondrial respiration is an essential feature of the ovulation process in mammals.

Extraction of the hormonal substance from hop.

Substances that suppressed a gain in the weight of pregnant mare's serum gonadotropin (PMSG)-primed immature rat ovaries, were obtained from dried powder of the hop cone. The substances were designated F1a-I and F1a-II. In immature rats at 4 days after PMSG priming, ovarian weights decreased by 58.0 +/- 3.7 and 66.9 +/- 6.6% the control by injections with 4 mg each of F1a-I and F1a-II, respectively. Apparent M1s of the partially purified fractions were nearly 80000 (F1a-I) and 66000-80000 (F1a-II). They were water-soluble. Acidic and neutral sugars were detected in the hydrolysate of the substance.

Further studies on gonadotropin-inhibiting substances excreted in human urine.

Partial separtion of human urinary substances which have properties to suppress the ovulation induced by PMS and HCG in mice was attempted by gel filtration on Sephadex G-100 and ultrafiltration using the Amicon Diaflo membranes UM-2 and UM-10. In addition to a thermostable inhibitor which has a molecular weight more than 10,000, the presence of heat-labile inhibitor with a molecular weight less than 1,000 was newly demonstrated.

Pregnant mare's serum gonadotrophin: II. Reversal of the antifertility faculty of pregnant mare's serum gonadotrophin by using clomiphene citrate or reserpine in rats.

PIP: Pregnant mare's serum gonadotropin (PMSG) 10 IU was injected in rats on Day 5 of pregnancy. Counting of implanted blastocysts was done on Day 8 of pregnancy through a midventral incision. The corpora lutea were noted to be significantly smaller (p less than .001) than controls. Autopsies were done on Day 16 of pregnancy. In some rats, clomiphene citrate (Clomid) was used as an antiestrogenic in a dose of .3 mg/kg injected sc on Days 5, 7, and 9. In others, reserpine was injected sc in a dose of .5 mg/kg on Days 5, 7, 9, 11, and 13 of pregnancy. The single injection of PMSG resulted in complete resorption of fetuses and placentae by Day 16. The Clomid reversed the antifertility action of PMSG and maintained growth of fetuses, placentae, and corpora lutea with 100% fetal survival. It is assumed that excessive luteinizing hormone (LH), as induced by the PMSG, stimulates ovarian estrogen. Since reserpine initiates prolactin release and inhibits LH release, the direct antagonism of prolactin at the luteal level may take place by inhibiting the enzyme activity of 20alpha-hydroxysteroid dehydrogenase.^ieng

Pregnant mare's serum gonadotrophin. I. Progesterone or prolactin and the reversal of antifertility efficacy of pregnant mare's serum gonadotrophin.

A single consistent luteolytic dose of 10 IU PMSG given on day 5 of pregnancy caused complete resorption of foetuses and placentae associated with a polyfollicular ovarian state in rats. Concomitant treatment with progesterone or prolactin given concurrently with PMSG was found to overcome the antifertility efficacy of PMSG and maintained the endocrine balance favouring pregnancy maintenance. It was postulated that the PMSG-induced ovarian polyfolliculogenesis might be responsible for luteolysis of the corpus luteum gravidarum.

Inhibition of ovulation induced with PMS and HCG by a melatonin-free extract of bovine pineal powder.

Using borate buffer a substance that suppresses the ovulation induced with PMS and HCG in immature mice was obtained from acetone-defatted bovine pineal powder by an extraction method similar to that used for an extraction of a gonadotropin-inhibiting substance in urine. This gonadotropin inhibitor in the pineal powder differs from melatonin or arginine vasotocin and seems to be different from the water soluble antigonadotropic substance which has been isolated from the bovine and ovine pineal. Partial purification of the gonadotropin inhibitor was accomplished by a Sephadex G-100 column.