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.

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.

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.

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.

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.